\\text{Prob}(E_v \\mid \\neg C_v)$. But that’s similar to saying $E_v$ and $C_v$ are correlated—they tend to occur together; and it is a well-known mantra that correlation is not causation. High candy consumption in one month is correlated with low divorce rate the next. But eating candy does not prevent divorce. The correlation is due to the fact that candy eating is correlated with being young and young people have generally not been married long enough to get divorced. So, something more needs to be said.\n\nSuppose you were able to control for all the other factors causing or preventing $E_v$.\\(^1\\) Imagine you could look in a population where all of these took some fixed value. So everyone is the same age, the same religion, is undergoing the same stress at work, has the same number of children, etc. In this population it seems reasonable to expect that if candy consumption does cause low divorce rate, it will increase the probability; and the probability will increase only if it does so since in this population there’s no other way to account for an increase in probability. Somewhat more formally, let $K_i$ represent a population where all the causes of $E_v$ other than $C_i$ take some fixed value, where $i$ ranges over all the sets of values these causes can take. Then $C_i$ causes $E_v$ in $K_i$ if and only if $\\text{Prob}(E_v \\mid C_i + K_i) > \\text{Prob}(E_v \\mid \\neg C_i + K_i)$.\n\nThis is the gist of the probabilistic theory of causation, though more details need to be ironed out. For instance, note that this characterization is relative to the population satisfying $K_i$. What then about a larger population that contains $K_i$? That is one of the points of dispute. Some philosophers and social scientists insist on keeping the relativization to the $K_i$. Others allow that $C_i$ causes $E_v$ in any population that contains $K_i$, though this can lead to it being true both that $C_i$ causes $E_v$ in a population and also prevents $E_v$ there in cases where in one subpopulation $C_i$ increases the probability of $E_v$ and in another it decreases it. Under any circumstances this kind of characterization will always be relativized to a population since different populations will have different probabilities over the same variables and also they may well have other general causal relations holding, hence different factors for determining the $K_i$s. Chapter 3 worries about taking studies that show that a policy worked—i.e. caused the targeted outcomes—in one situation as evidence that it will have the same effects elsewhere. It is just this kind of relativity of causal claims to other causal factors (those that pick out the $K_i$s) that creates this worry: whether $C_i$ causes $E_v$ or prevents it or does nothing to $E_v$ at all in a given situation can depend very much on what other causal factors are there in the situation along with $C_i$.\n\n\\(^1\\) Except those on the causal pathway between $C_i$ and $E_v$ if it exists.\n\nNote also that seen as a characterization of general level causality—as a way of providing clear unambiguous sense for the concept—the probabilistic theory of causation is not reductive since we need to refer to general causal relations in specifying what $K_i$ is and thus we refer to other general causal relations in explaining what is required for any given general causal relation to hold. It is, however, very constraining. Each of the factors in $K_i$ must satisfy a similar formula with respect to $C_i$ and the remaining factors in $K_i$ since each of these is itself meant to be a cause of $E_v$. Although this may not narrow the choice of causes for $E_v$ to a single choice, it will rule out a huge number of alternatives. And adding some information about a few factors, that they are indeed a cause of $E_v$ or that they are not, can sometimes fix the set entirely given the probabilities.\n\nThis way of characterizing causality has the advantage that it connects immediately with standard statistical methods used in the social sciences to test for causal relations. These methods are used in what are called ‘observational studies’, which means that the data come from populations in their natural settings and not from specially selected populations enrolled in experiments. In the populations under study, social scientists measure correlations or regressions between factors to begin to test whether there is a causal connection between them in that population. These are weaker notions than conditional probabilities, like $\\text{Prob}(E_v \\mid C_i)$ and $\\text{Prob}(E_v \\mid \\neg C_i)$, but are closely related. In a better test, they measure partial correlations or partial regressions, holding fixed other variables that they hope represent the other causes. This is akin to the partial conditional probabilities $\\text{Prob}(E_v \\mid C_i + K_j)$ and $\\text{Prob}(E_v \\mid \\neg C_i + K_j)$. Similar kinds of ideas are used in econometrics, in estimating the coefficients that appear in economic equations that will hopefully represent causal relations.\n\nThe two assumptions of the probabilistic theory—that an effect is always probabilistically dependent on its cause and that this kind of dependency disappears when other causes are held fixed—are also at the core of what are called ‘Bayes nets methods’ for causal inference, for which computer programmes can generate all sets of causal relations among a given set of variables that are possible, given information about probabilistic dependencies among the variables, supposing the fundamental assumptions linking causes and probabilities are satisfied.\n\nThese two assumptions are also at the core of the reasoning behind randomized controlled trials (RCTs), which are highly touted as the gold standard for causal testing in medicine and, as we see in Chapter 3, in evidence-based policy, and are being pushed throughout the social sciences now, especially in development economics. The neat thing about RCTs is that they help with one of the central problems that the statistical methods discussed so far face: that we generally don’t know what the other causal factors are and so don’t know what to hold fixed.\n\nIn an RCT, the individuals in a population enrolled in the experiment—which could be individuals, schools, countries, etc.—are randomly assigned either to the treatment group, which will be subject to the cause ($C_i$), or to the control group, which will not have the cause ($\\neg C_i$), but may perhaps receive a placebo. There should be as much masking as possible: the individuals in the experiment should not know which group they are in, nor should anyone involved in further monitoring or treatment, or in reading out the results to see if $E_v$ obtains or not, or in carrying out the statistical analysis. This is to guard against conscious or unconscious bias that may influence the results that are finally recorded. The aim is to ensure that both groups have the same distribution for all the other causal factors influencing the outcome, so that every arrangement of them—every $K_i$—has the same frequency in both groups. This aim can very rarely be achieved and moreover, we won’t know when it has been: statistics can tell us how often in the long run random assignment will produce any particular imbalance but we do not have even that kind of assessment when it comes to how well the masking has succeeded. So, as always in scientific work, we must not place too much confidence in the results of a single study, even one that has been very well conducted.\n\nIn order to see the logic of the causal inference, let us suppose, though, that the other causal factors have the same frequency in the treatment and control groups. The $\\text{Prob}(E_v)$ in the treatment group will then be an average across the probability of $E_v$ given $C_i$ in each of the subpopulations in it—i.e. each of the $K_i$. So it will be an average over $\\text{Prob}(E_v \\mid C_i + K_j)$. Similarly, the $\\text{Prob}(E_v)$ in the control is the average of the probability of $E_v$ given $\\neg C_i$, in each of the subpopulations represented there—each of the $K_i$. So it is an average over $\\text{Prob}(E_v \\mid \\neg C_i + K_j)$. By our hypothesis that the other causal factors have the same distribution in the two groups, the frequency of each $K_i$ subpopulation is the same in both. So if the probability of $E_v$ is greater in the treatment group than in the control group, that implies that for one of the $K_i$ subpopulations, $\\text{Prob}(E_v \\mid C_i + K_j) > \\text{Prob}(E_v \\mid \\neg C_i + K_j)$. It follows that there is at least one subpopulation—one $K_i$—of the experimental population in which ‘$C_i$ causes $E_v$ in $K_i$’ is true, or at least is true under the probabilistic theory’s way of characterizing causality.\n\nCan we conclude from the experiment ‘$C_i$ causes $E_v$ in the experimental population’? That depends on the decision referred to earlier about what to say about ‘$C_i$ causes $E_v$’ in a population given that $C_i$ causes $E_v$ in one of its subpopulations. What matters is that, however this decision is made, those using the claim should understand it and not read more out of it than the study supports. In particular, the higher probability in the treatment over the control group shows only that $C_i$ causes $E_v$ in some subpopulation of the population enrolled in the experiment. It may hold in specific other populations\n\nor even across most. But $C_t$ may have exactly the opposite effect on $E_v$ in some subpopulations than it does in others. Finding out whether that is true requires a great deal more social science work.\n\n2.2 Intervention and Manipulation\n\nThe manipulation view of causation revolves around the idea that causes give us effective strategies for producing effects we want, or preventing those we do not; by manipulating the cause we can manipulate the effect in a predictable way. So manipulation theories characterize general-level causation roughly this way, where again there is a dispute about the exact details of the formulation: for any two times $t$ and $t'$, ‘$C_t$ causes $E_{v'}$ in situations of type $S'$ just in case manipulating $C$ at $t$—making it bigger or smaller, or bringing it in or taking it away—is regularly followed by appropriate changes in $E$ at $t'$. For this to be true, we must be careful how these manipulations are carried out. ‘Reducing class sizes causes improved reading scores’ is true in many populations. But not if you lower teacher quality at the same time as reducing class size. Alternatively the manipulation of one factor may be followed by improvement in another without any causal connection. For instance new programmes for teaching reading may be followed by better reading scores not because the programmes cause better scores but because the teachers adopting them are regularly the better teachers or they become more enthusiastic when trying out new programmes.\n\nThis is where the concept of intervention comes in. ‘Intervention’ has been given a variety of formal definitions, but the basic idea is that an intervention is a manipulation that is done in the ‘right way’ to make the causal relation, or lack of it, apparent. The right way will be one in which neither other causes nor other preventative of the effect change, an idea we are familiar with from the probabilistic theory. Nor can any causal relations involving the effect change during the intervention. That’s because it is not a fair test of a causal relation if suddenly there are a lot of new causal relations produced between the effect and other factors that were not causes before. This includes changes in the very relation under test. One familiar way in which the requirement that causal relations involving the effect not change during intervention is violated is when the manipulation of the cause is so ham-fisted that it busts up the causal relation we are trying to test. One familiar example is when we wind up the toy soldier to see if that will make it march, but we wind too tightly and break the mechanism that makes it work.\n\nNobel-prize-winning Chicago School economist Robert Lucas claims this is frequently true with attempts to manipulate economic variables to bring about desired change. For instance, inflation, when it occurs naturally, can in the short run cause reductions in unemployment. That’s because, so the story goes, entrepreneurs mistakenly see the universal rise in prices as a real rise in prices in their sector and so hire more workers to produce more goods to meet increased demand. If, though, the government manipulates inflation to improve unemployment, entrepreneurs will recognize the rise in prices for what it is—just inflation—and will not open new jobs. The government’s very attempt to use the causal relation between inflation and unemployment will break it. Or at least so says Lucas’s model.\n\nAs with probabilistic theory, the characterization of general causation using the concept of manipulation is not reductive since the definition of an intervention, however the details are worked out, will have to refer to other general causal relations.\n\nAlso, as with the probabilistic theory, the characterization of causality in terms of manipulation is closely linked with familiar methods for testing, in this case with real non-statistical experiments where other causes are held fixed and only the cause under test is varied. Here too we are often stymied by our lack of knowledge of what these other causes are. This is the problem that plagues standard before-and-after studies. In before-and-after studies the cause is administered and we look to see if the effect changes. But, are we sure that nothing else changed at the same time as the cause? One of the tricks of social science is to find situations where we can have reasonable confidence that nothing else that could influence the effect has changed even if we do not have a catalogue of what all the other influences might be to check on them. This is the strategy that is used in what are called ‘instrumental variables’ models in economics. For instance Joshua Angrist in a classic paper uses the Vietnam era draft lottery to measure the effect of veteran status on earnings (Angrist 1990). Since whether an individual was drafted was determined on the basis of a randomly assigned number, we can be fairly confident that factors that affect earnings, like education, were equally distributed between the group of individuals who were drafted and the group of individuals who were not, as in an RCT. And so we can be fairly confident that the comparison between the average earnings in one group and the average earnings in the other tells us something about the effect of veteran status on earnings.\n\nThe manipulation theory of general-level causation is closely akin to the counterfactual theory of singular causation, which is based on the idea that one specific event, $c$, is a cause of a later event, $e$, just in case $e$ would not have occurred unless $c$ had. You can see immediately that this kind of claim will be hard to test since $c$ and $e$ are specific events that either occur or do not; so we can never observe what would have happened had things been different. There are also huge problems of formulation. There is a vast literature available in philosophy on counterfactuals and causes in which you can read more about these (see e.g. Paul 2009 and references therein).\n\nSimilar counterfactuals have come to the fore in social science lately because of the rise of the evaluation industry for social policy. As Chapter 3 explains, there is a great demand to know whether the policies we have implemented have ‘worked’: Has the policy genuinely produced the change intended? To answer that, it helps to know what would have happened otherwise. RCTs are often employed to this end. But of course they cannot tell us of a specific case whether the policy brought about the effect there, since they only look at groups. If an RCT shows more positive results in the treatment group, the group in which the policy was implemented, than in the group where it was not, we can conclude that in some of the cases in the treatment group the policy produced the effect. But we do not know which; we have no way to sort cases where the policy brought about the effect from those where something else was responsible. So for reliable evaluation of the single case, some different way of reasoning is required. This often involves process tracing which we shall discuss in the next section.\n\nNot only do we not know from the RCT for which individuals the policy worked. We do not know what sets of characteristics matter. We know that there is at least one arrangement of characteristics that individuals might have—one $K_i$—for which the policy promotes the desired outcome. We call these ‘support factors’. But we do not know which it is. This matters when it comes to putting the claims established in RCTs to use in predicting what will happen if the policy were to be implemented elsewhere. If the population elsewhere has no individuals with just the right arrangement of support factors (no individuals described by the successful $K_i$s), or not enough to make it pay, the policy will not produce the desired outcomes in the new setting. Given good reason to think that the new setting does have enough individuals with the right arrangements, we have good reason to suppose the policy will work for some individuals there. But without good reason to suppose this, the RCT results will not be much use in predicting results in the new setting.\n\nConsider for instance Oportunidades, a Mexican programme for poverty reduction. Because Oportunidades was itself designed as an RCT, there is very good evidence that it did contribute to reducing poverty where it was implemented. Does the success of Oportunidades provide evidence supporting predictions regarding what would happen were a similar programme implemented in some other population? Only if enough individuals in this target population have the right arrangement of support factors. And one cannot assume without evidence that this will be so. This point is illustrated by the case of Opportunity NYC, a poverty reduction programme modelled—and named—after Oportunidades which was implemented in New York City in 2007 but discontinued in 2010 because it failed to produce the expected effects. The success of Oportunidades in Mexico did not, by itself, provide sufficient evidence to support the prediction that Opportunity NYC would produce similar results, and the mistaken assumption that it did led to a poor policy decision.\n\n### 2.3 Mechanisms\n\nThere are three senses of the term ‘mechanism’ in play currently in work on causation in philosophy and in the social and in the biomedical sciences. These include notions of *causal process*, *invariance*, and *underlying structure*.\n\n**CAUSAL PROCESSES**\n\nFor singular causation it is widely held that a cause must be connected with its effect by some spatio-temporally continuous process between them. So tracing the causal process between them can be a good way to tell whether two events are related as cause and effect, a good way IF we know how to tell a causal process when we see one. So what distinguishes a continuous sequence of events that constitutes a causal process from one that does not? One popular philosophical account answers that in causal processes energy is transferred at each step in between. But this is not of much help in social science (see e.g. Dowe 2009).\n\nA standard procedure in social science and in policy evaluation is to break the sequence into small steps where it is easier to determine for each step whether it is a genuine cause of the next. For instance, the Global Environment Facility evaluated the effectiveness of one of its programmes, the end-goal of which was to ‘establish a long-term conservation finance mechanism to support biodiversity conservation’ in the Bwindi national park (Uganda), by determining whether this programme had an effect on four intermediate outcomes (e.g. the establishment of a Bwindi Trust) which in turn were supposed to contribute to achieving the end-goal of the program (GEF 2007: 6).\n\nProcess tracing necessarily focuses on singular causation. But it can be a tool for general causation as well. If in situations of kind $S$, an event of kind $C$ at $t$ is regularly followed by an event of kind $E$ at $t'$ and it regularly happens that the same kind of causal process connects the individual events, then we can conclude at the general level that ‘In $S$-type situations, $C_t$ causes $E_{t'}$.’ This kind of reasoning has played a significant role in the biomedical sciences. For instance, tracing the process by which the chemicals in cigarette smoke lead to lung cancer provided a central piece of the evidence that showed that smoking causes lung cancer.\n\n**INVARIANCE**\n\nSuppose we observe that in a particular kind of situation $S$ one kind of feature $F_2$ regularly changes after another, $F_1$, changes. That is mere correlation and we know that correlation does not equal causation. But suppose that $F_1$\n\nchanges by intervention. If F1 causes F2 we expect that F2 will change following the intervention in the way that it always has; but not, if F1 does not. Changing levels of candy consumption may be regularly followed by changing probabilities of divorce. But this relationship is not invariant under intervention. If we intervene to change candy consumption—‘intervene’, so the only change is in the amount of candy consumed—the divorce rate will not change in tandem. This observation has given rise to the invariance account of causality now in fashion in philosophy of science. The basic idea is that, supposing C\\textsubscript{i} and E\\textsubscript{t} are regularly associated in S, then ‘C\\textsubscript{i} causes E\\textsubscript{t} in S’ just in case the association between C\\textsubscript{i} and E\\textsubscript{t} is invariant under interventions on C\\textsubscript{i}.\n\nThis account is clearly closely connected with the manipulation account, and it shares one major drawback with that account that we have not yet discussed. The manipulation account is grounded in the idea that causes provide strategies for changing the world. Suppose we do characterize causation as invariance under intervention. Then changing C\\textsubscript{i} will be a good way to change E\\textsubscript{t}—but that follows only if C\\textsubscript{i} is changed by intervention. But interventions are hard to come by. We are in a far more powerful position to predict what will happen and to engineer what we want if we know about a regular association that is invariant under the method of implementation that we will in fact employ. This gives rise to another, different characterization of general-level causation, one that we can find in contemporary economics (see e.g. Hoover 2001: ch. 2). Roughly, if C\\textsubscript{i} and E\\textsubscript{t} are regularly associated in S, then C\\textsubscript{i} causes E\\textsubscript{t} in S relative to φ just in case the association between C\\textsubscript{i} and E\\textsubscript{t} is invariant when C\\textsubscript{i} is changed by φ, where φ can then represent the method by which we will in fact change C\\textsubscript{i}. Knowing that C\\textsubscript{i} causes E\\textsubscript{t} under this characterization of causation immediately provides an effective strategy for changing E at t’.\n\nNotice that these different versions of the invariance account can give different verdicts about whether C\\textsubscript{i} causes E\\textsubscript{t} in any given situation. This underlines the importance of being clear exactly what we mean when we make causal claims in social science.\n\n**UNDERLYING STRUCTURES**\n\nDifferent causal relations hold in different social, cultural, and economic settings. What is a required politeness in one culture can cause insult in another. Why? What causal relations hold in a social setting depends on the underlying system, the underlying social, economic, and cultural structure. It is now standard in philosophy of biology to call the structures that underlie causal relations there ‘mechanisms’, and sometimes this usage is adopted in the social sciences as well. Though there is no widely used mechanistic characterization of causation, there is widespread sense that, if we are to follow the injunction of the LSE motto, to know the causes of things, we should come to understand the underlying mechanism that make the particular causal principles that hold for a given population possible.\n\nThis is clearly of central importance in social planning and policy. Consider recent problems in child welfare in the UK, where attention tended to focus on causal processes. When a tragic child death occurs, ‘The standard response is to hold an inquiry…trying to get a picture of the causal sequence of events that ended in the child’s death…We are tracing a chain of events back in time to understand how it happened.’ So says Eileen Munro (2005: 377), author of Chapter 3 and of the 2012 UK government report on child protection. Munro recommends instead a ‘systems’ approach. That means focusing on the underlying mechanism or social structure that makes these kinds of processes possible, or even probable. As a US National Academy of Science report on building safer health systems explains, ‘The focus must shift from blaming individuals for past errors to a focus on preventing future errors by designing safety into the system’ (Kohn et al. 2000: 5).\n\nSo it is clear that it is important to study social mechanisms in social science. What is not clear is what kinds of concepts should be used to describe these mechanisms and their operation, nor what tool kit of methods will help us to understand them. This is one of the central tasks right now on which philosophy and social science intersect.\n\n### 2.4 Powers\n\nJohn Stuart Mill argued that women have the same powers of leadership and imagination as men, and we have these powers even should they be seldom displayed. Give women the same education, upbringing, situation, and opportunities as men and they will display these qualities equally with men. And he used this claim to argue for dramatic changes in the social policies and practices in play at the time regarding the role of women in society.\n\nAs with all concepts, if the concept *power* is to play an important role like this in social science or in social policy deliberation, we should be clear what is meant by it. This is a big topic in metaphysics nowadays and, to a lesser extent, in philosophy of science (see e.g. Mumford 2009). It is usual to make at least four assumptions about powers. First, a system—an object, a person, an institution—can have a power without displaying it. Second, the power will be displayed if the conditions are right. Third, what actually happens when a power is displayed depends on the setting. So what happens when a woman displays her imagination will depend on whether she is at a laboratory work bench or addressing Parliament or tending her children. Fourth, when a power operates in a situation, what happens there will depend in some intelligible way on the display of the power (as when a heavy metal object does not fall to the ground because the power of a magnet is displayed).\n\nThe most typical characterizations of powers ‘back’ define them from their displays. Roughly, A has the power to $\\alpha$ just in case when properly enabled and failing something stopping it doing so (which we sometimes express as ‘nothing interferes with the display’), A does $\\alpha$. This characterization is dramatically non-reductive because of the concepts of *enablement* and *interference*. Contrast this with an alternative: ‘A has the power to $\\alpha$ just in case there are some circumstances $C\\alpha$ such that it is a law of nature that in $C\\alpha$, A does $\\alpha'$, where $C\\alpha$ is to be filled in by some possibly very long list of features that can be characterized without any power-related notions, like ‘is schooled at Eton’, ‘learns Latin’, ‘does not play with dolls’, etc. Many powers advocates do not adopt this alternative because, they argue, there is never a list that does the job. In each actual situation there is a fact of the matter about whether the circumstances that obtain there constitute an interference and whether they are enabling, but there is nothing these facts share in common that we could fill in for $C\\alpha$ other than that they are enabling or they interfere. This is taken by many to be particularly problematic since philosophers have not succeeded in having much enlightening to say about interferences and enablers at the general level.\n\nWhy then take powers seriously? Philosophers offer a panoply of metaphysical arguments. But for social science the answer seems to lie in the usefulness of the concept, as with Mill, who argued for the centrality of the related notion of *tendency* in political economy. Another word used for much the same idea is ‘capacity’. In order to link it with our contemporary discourse, I shall use the word ‘power’ throughout for all these related notions. Mill’s model for powers comes from Newtonian mechanics. The sun has the power to attract the planets. Nothing ever interferes with it so the sun constantly displays this power (called ‘gravity’). What happens when it displays it? The planets move around it in elliptical orbits. By contrast, when the earth displays its power to attract a cannonball shot at an enemy ship, the cannonball moves along a parabola. What actually happens when the power of gravitational attraction is displayed (i.e. when the sun or earth attract other objects) depends on the setting. To make a link with our immediately previous discussion, we may think of the earth–cannonball pair as a mechanism, in the sense of an underlying structure, giving rise to the causal principle, ‘The earth displaying its power of gravitational attraction causes cannonballs to move along parabolas’; and the planetary system as a mechanism giving rise to the causal principle ‘The sun displaying its power of gravitational attraction causes the planets to move along ellipses around it’.\n\nHow do we know what happens when a power is displayed in a given setting? That’s easy, at least in principle, in Newtonian mechanics. All that’s relevant to how a power affects motions is what other powers are displayed in the situation. The display of the power is represented by what we call a force, represented by a vector. When several forces are displayed at once, we add these vectors, call the result ‘the total force, $F_{\\text{tot}}$’, and calculate the acceleration of any object in the situation by the familiar formula $F_{\\text{tot}} = ma$, where $m$ is the mass of the object. That is what we do, for instance, when we want to know how a pin will behave when a magnet attracts it upward and the earth attracts it downward, or how an electron in an atom behaves when it is attracted by both the mass of the nucleus and the charge on the protons there.\n\nThat’s all physics. Life is not so easy in social science, which may have some knowledge of powers but little knowledge of methods for calculating just what happens in different situations when the powers are displayed. Consider an economics example already mentioned, the one involving skill loss during unemployment. Earlier I suggested that economics entertains a causal principle that skill loss perpetuates high levels of unemployment. That is probably a mistaken way to look at it. It is seldom the case that skill loss is the only thing going on that affects unemployment. There are, for instance, sometimes vigorous successful government efforts to combat unemployment. In these cases skill loss may be followed by improving levels of employment, contrary to what is suggested by this causal principle. A better way to put it might be ‘Skill loss has the power to cause continuing high unemployment’. This will not lead us to expect that skill loss actually produces unemployment whenever it occurs, but only that, to understand the levels of employment that actually occur, we shall have to take the downward push of skill loss into account.\n\nSo, we return to the question, ‘How do we take it into account?’ How do we predict what happens when a socio-economic power is displayed? There is no systematic answer. This is one of the problems social scientists constantly confront in trying to put their isolated parcels of hard-won knowledge—like the knowledge of specific powers such as the power of skill loss to perpetuate unemployment—to use.\n\n### 3. Putting Causal Knowledge to Use\n\nChapter 14 in this book teaches us that the meaning we assign a scientific concept must be closely paired with our methods for finding out whether it obtains. We should have good arguments to show that the methods we employ provide a good way to find out about just what we claim we are finding out about. That message has been echoed throughout this chapter with respect to the concept of causation when it is used in the social sciences. It is not just meaning and method that must match, however. So too must use. The use to which we put our social scientific claims—the inferences we draw from them and the practices we adopt on the basis of them—should be\n\nsupported by what those claims actually say; and what they say that supports our inference should in turn be supported by our methods for deciding that what they say is true. It is no good making a very broad claim whose first half is well supported by the evidence but then draw our inferences from the second half, which is unsupported.\n\nWe have looked at a number of accounts of what causal claims say. The claims say different things because they employ different concepts of causation. These concepts may all go under the same title, ‘general causal relation’, but they require different conditions to be met in order to obtain. So different methods must be used to find out about them and different uses made of them. So, of what use is knowledge of the causes of things? Knowing causes helps us to understand how things work. But it is also supposed to be of practical value. Knowing the causes of things should help us to change the world.\n\nHere we must be cautious though. There is a tendency to too much haste, to try to read off directly from our causal claims just what we must do to bring about the change we desire. That will work with some kinds of causal knowledge but not most. Consider the manipulation concept of causation found in economics that I mentioned in discussing invariance: $C_t$ causes $E_t$ relative to $\\phi$ just in case manipulating $C_t$ by $\\phi$ is followed by $E'_t$, where $\\phi$ is one of the methods available to us to change $C$ at $t$. Suppose we know that. Then we are in a powerful position to change $E$ at $t'$.\n\nBut suppose we have established instead a different causal claim, say a manipulationist claim of the kind favoured in philosophy—$C_t$ causes $E_t$ just in case if $C_t$ changes by intervention $E_t$ changes—or in invariance-type claim—$C_t$ causes $E_t$ just in case the association between them is invariant when $C_t$ changes by intervention. That too can be of immediate use—if we have an intervention to hand, which we usually do not. We have seen the same kind of problem with knowledge of powers. Knowing about what powers a system has will not by itself tell us what to expect to happen in particular circumstances nor how to build circumstances to get what we want. Nor does knowing the causal relations, on the probabilistic theory, that hold in one population, perhaps the population enrolled in an RCT, give us immediate guidance about how to bring about change in a different population.\n\nThis does not, however, in any way make causal knowledge in social science useless. It just puts it more on a par with similar knowledge in the natural sciences, where strategies are built by splinting together a very great many different pieces of knowledge and often undertaking designated new researches and developing new theory to fill in gaps. The great nineteenth-century physicist Lord Kelvin laid the first Atlantic cable in 1866. His knowledge of causes in physics was essential to his success but it definitely did not tell him just what to do. Similarly, the large team of physicists, engineers, mathematicians, and technicians who developed the US Second World War radar took many months to do so, despite the vast repository of causal knowledge the team members brought to the project. The conclusion I propose is that the causal knowledge we work so hard to gain in the social sciences can be of immense use. But there is no reason to think it should be easier to put our social science knowledge to use than it is to use our knowledge in the natural sciences.\n\nReferences\n\nAngrist, J. (1990). ‘Lifetime Earnings and the Vietnam Era Draft Lottery: Evidence from Social Security Administrative Records’, *American Economic Review*, 80(3): 313–36.\n\nDowe, P. (2009). ‘Causal Process Theories’ in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 213–33.\n\nGarrett, D. (2009). ‘Hume’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 73–91.\n\nGEF (2007). *Case Study: Bwindi Impenetrable National Park and Mgahinga Gorilla National Park Conservation Project*. Impact Evaluation Information Document, 7. Washington, DC: Global Environment Facility, Evaluation Office.\n\nHoover, K. (2001). *Causality in Macroeconomics*. Cambridge: Cambridge University Press.\n\nKohn, L., Corrigan, J., and Donaldson, M., eds (2000). *To Err is Human: Building a Safer Health System*. Washington, DC: Committee on Quality of Health Care in America, Institute of Medicine.\n\nMumford, S. (2009). ‘Causal Powers and Capacities’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 265–78.\n\nMurro, E. (2005). ‘Improving Practice: Child Protection as a Systems Problem’, *Child and Youth Services Review*, 27: 375–91.\n\nPaul, L. A. (2009). ‘Counterfactual Theories’, in H. Beebee et al. (eds), *Oxford Handbook of Causation*. Oxford: Oxford University Press, 158–84.\n\nReiss, J. (2013). *Philosophy of Economics: A Contemporary Introduction*. London: Routledge.\n\nRoberts, C. (1995). *The Logic of Historical Explanation*. University Park, PA: Pennsylvania State University Press.\n\nRussell, B. (1912–13). ‘On the Notion of Cause’, *Proceedings of the Aristotelian Society*, 13: 1–26.\n\nFurther Readings\n\nBeebee, H., Hitchcock, C., and Menzies, P., eds. (2009). *Oxford Handbook of Causation*. Oxford: Oxford University Press.\n\nCampaner, R., and Galavotti, M. C. (2007). ‘Plurality in Causality’, in P. Machamer and G. Wolters (eds), *Thinking about Causes: From Greek Philosophy to Modern Physics*. Pittsburgh: University of Pittsburgh Press. 178–99.\n\nCartwright, N. (2007). *Hunting Causes and Using Them*. Cambridge: Cambridge University Press.\n\nMcKim, V., and Turner, S., eds (1997). *Causality in Crisis: Statistical Methods and the Search for Causal Knowledge in the Social Sciences*. South Bend, IN: University of Notre Dame Press.\n\nRusso, F. (2009). *Causality and Causal Modelling in the Social Sciences*. New York: Springer.\n\n**Index**\n\naggression 104–5, 107, 114–16, 119 \nagmatology 259 \nAnany, Roger 71–2 \narchaeology 68–77, 79–82\n\nbehavioural traits 115, 118 \nenvironmental factors 102–3, 106–7, 118 \nheritable factors 107–8 \nindividual differences 104, 116–17, 119 \npluralism in the study of 113, 115–17 \npopulation differences 115–17, 119 \npopulation-level factors 109–10, 116 \nsex differences in heritability 115\n\nbehaviour genetics: \nmolecular 105, 108, 112–13 \nquantitative 104–5, 107–8, 112\n\nBoghossian, Paul 71–3 \nboundary objects 238–9 \nBrannan, Michael 93 \nBurt, Cyril 172–3\n\nCampbell, Donald 294–5 \nCartwright, Nancy 61–2 \ncase studies 288–307 \ncombinath laboratory experiments 291–2, 297, 301, 304 \ncritiques of 292–3\n\ncategories: \ncharacterization of 267–70, 272–3, 276, 278–81, 284–5 \nprocedures for measurement 276–8, 280–1, 284–5 \nrepresentation of 271–6, 279–80, 284–5 \ncausal principles 62, 310–11, 322–3 \ncausal processes 63, 305, 319 \ncausal spaces 111–13, 115 \ncausation 102–6, 115, 273, 308–10, 312–13, 319–20, 323–5 \nmanipulation view 316–18 \nprobabilistic theory of 312–14, 324 \nsingular 317, 319 \nchoice models 185–7, 189, 191, 193, 195, 197–9, 201, 203, 205–6 \nEU models 199, 201 \ngame models 191–201, 203–5\n\nindividual decision 186–91 \ncivil war 265–73, 277–8, 284 \nethnicity 270, 282–4, 311–12 \ncivil war studies 265, 275–6 \nClark, Geoff 71, 73, 81 \nclass sizes 55–7, 61–2 \nclimate change 2, 31–46, 133–4, 253 \nadaptation 39–41, 43 \nadaptive capacity 39–41 \ncost of future generations 42, 45, 130–3 \nintegrated assessment models 38, 41 \nmitigation 41–3 \nscenarios 37–41 \nvulnerability to 39–41 \nclimate change impacts 38–9, 43 \ncollectivism 7–9, 20, 95 \ncommunity-based participatory research (CBPR) 68, 79–80 \nconcepts 1, 97, 238, 268–72, 274, 277–8, 283–4, 308–9 \nBallung 268–72, 273–8, 284 \nconventional networks 96–7, 100 \nconstraint satisfaction network 97 \nconsensus conferences 251–3, 261 \nconventions 215–18, 221–6, 229 \ncrime 103–4, 108–10, 119 \nCrombie, Alistair 288–9\n\nDasgupta, Partha 24 \nDaston, Lorraine 127, 155 \nDavies, Huw 52, 64 \ndecision theory 185, 201, 203 \ndegrees of freedom 295 \nDeleuze, Gilles 139–40 \nDeVault, Marjorie 192–3 \ndevelopmental systems theory (DST) 106, 112–14 \ndisability-adjusted life year (DALY) 276, 280–1 \ndiscipline 234–5, 237–40, 244 \ndiscount rate 42, 130–1, 133 \ndiscovery 299–300, 302, 306 \ndoctrine 234–5, 242, 244 \nDouglas, Heather 70 \nDouglas, Jack 137–8 \nDupré, John 179 \nDurkheim, Émile 136–8"}}},{"rowIdx":204695,"cells":{"text":{"kind":"string","value":"NEUROENDOCRINE REGULATION OF GASTRIC ENDOCRINE CELL FUNCTION\n\nThesis submitted in accordance with the requirements of The University of Liverpool for the degree of Doctor of Philosophy by Andrew Damian Higham B.Sc., M.B. Ch.B., M.R.C.P.\n\nMarch 1998\n\nLIVERPOOL UNIVERSITY LIBRARY\n\nLATIVX\n\nThe last decade has seen major advances in the understanding of diseases of the proximal gastrointestinal tract. Not least of these has been the recognition of the significance of *Helicobacter pylori* (*H. pylori*) gastritis in the pathogenesis of peptic ulceration, chronic atrophic gastritis and gastric carcinoma. In addition the development of proton pump inhibitors (PPI) has been heralded as a major therapeutic advance. However both *H. pylori* gastritis and PPI therapy elevate plasma gastrin concentrations and this has led to increasing interest in the physiological role of the antral hormone gastrin.\n\nIn this thesis, the role of intrinsic antral neurons in the regulation of gastrin release in response to a variety of stimuli has been examined. In addition, the effects of antral denervation on gastric emptying were clearly apparent and prompted investigation of the relationship between antral neurons and the cholecystokinin-independent mechanisms that have been well described as regulators of gastric emptying in the intact rat. It was found that antral neurons are essential for normal gastric emptying but are not required for the CCK-mediated inhibition of gastric emptying.\n\nIn addition, following antral denervation there was elevated fasting and meal stimulated plasma gastrin, the post-prandial rise in gastrin release occurring by a non-gastrin releasing peptide mediated mechanism. Moreover, non-nutrient\n\ndistension of the stomach was an adequate stimulus for gastrin release following antral denervation, suggesting that in the intact rat, antral neurons normally inhibit gastrin release to non-nutrient distension.\n\nThe effects of antral denervation on the endocrine cells of the stomach were also studied as were the effects of hypergastrinaemia on the Enterochromaffin-like (ECL) cells of the gastric corpus. Of particular relevance was the demonstration by Ashahara et al., (1996) of the *reg* gene in the ECL cells of the rat. Expression of this gene has been associated with pancreatic endocrine cell growth and differentiation. Data is presented that indicates that expression of this gene is regulated by gastrin and that it may play a role in promoting the differentiation of endocrine cells of the proximal gastrointestinal tract. Mutations of *reg* were associated with the development of gastric carcinoid (ECL cell) tumours in patients with hypergastrinaemia.\n\nOverall it is clear that the antral innervation plays a central role in the control of gastrin release and that loss of the normal innervation of the G-cell leads to fasting and meal-stimulated hypergastrinaemia. The effects of persistently elevated gastrin on other cell populations of the stomach are likely to be of increasing importance with respect to understanding the mechanisms of tumour development.\n\nThe work presented in this thesis has appeared in part in the following publications:\n\n**Papers**\n\nHigham A.D., Vaillant C., Yegen B., Thompson D.G., Dockray G.J.: Relationship between CCK and antral innervation in the control of gastric emptying in the rat. *Gut* 41:24-32 1997\n\nHigham A.D., Noble P., Thompson D.G., Dockray G.J.: Increased sensitivity of gastrin cells to gastric distension following antral denervation in the rat. *J Physiol* 503(1):169-175 1997\n\nHigham A.D., Dimaline R., Varro A., Attwood S, Armstrong G, Dockray G.J., Thompson D.G.: Atrophic gastritis and large carcinoid nodules: Octreotide suppression test predicts beneficial outcome from antrectomy. *Gastroenterology (In press)*.\n\n**Abstracts**\n\nHigham, A.D. Thompson, D.G., Dockray, G.J.: Antral denervation by benzalkonium chloride leads to gastric retention of solids in the rat. *Gut* 36(1):A52 1995.\n\nHigham, A.D. Thompson, D.G., Dockray, G.J.: Peptidergic afferent and intrinsic innervation of the rat stomach following antral denervation. *Neurogastroenterol Mot* 7:9 1995.\n\nHigham, A.D. Valliant, C. Thompson, D.G., Dockray, G.J.: Complete loss of peptidergic afferent fibre innervation of the rat stomach following antral denervation. *Reg Peptides* 57:199 1995.\n\nHigham, A.D., Thompson, D.G., Dockray, G.J.: Post-prandial gastrin release after antral denervation: no role for gastrin-releasing peptide. *J Physiol* 487:34P 1996.\n\nHigham A.D., Noble P.J., Thompson D.G., Dockray G.J. Increased gastrin release following distension after antral denervation. *J Physiol* 499: 104P 1997.\n\nHigham A., Thompson D.G., Dimaline R. & Dockray G.J.: Increased *Reg* gene expression in the rat gastric corpus following omeprazole treatment. *Reg Peptides*. 71:55 1997.\n\n**Chapters and Invited Reviews:**\n\nHigham A.D.: Clinical endocrinology of gastric function. *Surgery* (in press) 1997.\n\nStatement of Originality\n\nAll of the data in this thesis that is presented as results has arisen wholly from my own work apart from the following contributions from colleagues within the Department of Physiology, University of Liverpool. The tissue culture of AR42J and HIT cells was performed by Dr Lisa Bishop as was the permanent transfection of the HIT cells. The plasma CCK assay was performed by Mrs C McLean. On occasion, the preparative surgery of the gastric fistula rats was performed by myself in conjunction with P J Noble.\n\n| Abbreviation | Description |\n|--------------|-------------|\n| BAC | Benzalkonium chloride |\n| CAG | Chronic atrophic gastritis |\n| CCK | Cholecystokinin |\n| CGRP | Cacitonin gene-related peptide |\n| CRF | Chronic renal failure |\n| EC | Enterochromaffin |\n| ECL | Enterochromaffin-like |\n| EGF | Epidermal growth factor |\n| GRP | Gastrin-releasing peptide |\n| HGF | Hepatocyte growth factor |\n| LOH | Loss of heterozygosity |\n| MEN1 | Type I multiple endocrine neoplasia syndrome |\n| NO | Nitric oxide |\n| PA | Pernicious anemia |\n| PAP | Pancreatitis associated protein |\n| PPI | Proton pump inhibitor |\n| PSP | Pancreatic stone protein |\n| PTP | Pancreatic thread protein |\n| SP | Substance P |\n| TGF-α | Transforming growth factor alpha |\n| VIP | Vasoactive intestinal polypeptide |\n| ZE | Zollinger Ellison syndrome |\n\n# Contents\n\n## Chapter 1\n\n### Introduction\n\n- Historical review of gastrointestinal peptide physiology: 2\n- Innervation of the stomach: 5\n- Neuroendocrine regulation of gastric emptying: 15\n- Neuroendocrine regulation of gastric acid secretion: 16\n- Clinical significance of hypergastrinaemia: 22\n- Gastrin, endocrine cell growth and differentiation in the stomach and pancreas: 26\n- Aims and objectives: 34\n\n## Chapter 2\n\n### Materials and methods\n\n- Materials: 37\n- Methods: 39\n\n## Chapter 3\n\n### Chemical denervation of antral neurons\n\n- Introduction: 57\n- Methods: 59\n- Results: 60\n- Discussion: 69\n- Summary: 72\n\nChapter 4\n\nAntral denervation and gastric emptying\n\nIntroduction ................................................................. 75\nMethods .................................................................. 77\nResults .................................................................... 78\nDiscussion ................................................................. 85\nSummary .................................................................. 88\n\nChapter 5\n\nAntral denervation and G-cell function\n\nIntroduction ................................................................. 91\nMethods .................................................................. 93\nResults .................................................................... 94\nDiscussion ................................................................. 105\nSummary .................................................................. 110\n\nChapter 6\n\nControl of reg expression in rat corpus mucosa and AR42J cells\n\nIntroduction ................................................................. 112\nMethods .................................................................. 113\nResults .................................................................... 114\nDiscussion ................................................................. 124\n\nChapter 7\n\nReg gene expression in human gastric mucosa\n\nIntroduction 131\nMethods 133\nResults 136\nDiscussion 145\nSummary 152\n\nChapter 8\n\nConclusion and Implications\n\nConclusions 155\nImplications and future studies 156\n\nChapter 9\n\nBibliography 162\n\nAcknowledgements 188\n\nChapter 1\n\nIntroduction\n\nAt the beginning of this century two schools of thought were prominent with regard to the mechanisms that controlled visceral function. Langley provided evidence that responses to feeding were mediated by nervous reflexes (Langley, 1898) and his conclusions were supported by the work of Sherrington (1899) and Pavlov (1910). Set against this were the experiments of Bayliss and Starling (1902) and Edkins (1905) who proposed that blood borne agents (named secretin and gastrin respectively) were responsible for stimulating digestive secretions from the pancreas and stomach. Although Bayliss and Starling (1899) previously had reported the reciprocal effects of the vagal and splanchnic innervation on gastric motility, it took almost forty years before it was considered that the nervous and endocrine systems might interact closely to regulate visceral functions such as gastric acid secretion (Uvnas 1942).\n\nWhilst the work of Bayliss and Starling was readily accepted, Edkins’ proposal that an antral hormone stimulated gastric acid secretion remained unconfirmed until the work of Komarov in the late 1930’s and of Grossman et al., a decade later (1948). Meanwhile several groups attempted to purify to homogeneity secretin, the hormone proposed by Bayliss and Starling to stimulate pancreatic secretion in response to a meal. This was achieved in 1961 by Jorpes and Mutt who isolated secretin and at the same time Gregory and Tracy isolated and characterised the peptide hormone gastrin (Gregory and Tracy, 1964). Within a few years it was shown conclusively that gastrin was produced by and localised within a defined\n\npopulation of endocrine cells in the gastric antral mucosa (McGuigan, 1968). Not surprisingly at the time, gastrin was considered to be strictly a gastrointestinal hormone. Furthermore, the similarities between gastrin and secretin seemed to confirm the prevailing view that peptides acted as classical hormones.\n\nAlmost coincidentally however, Burnstock et al., (1963) described neurotransmission in the gut that was mediated by a nonadrenergic and noncholinergic agent. Morphological studies of the myenteric plexus suggested the existence of peptide-containing nerve fibres (Baumgarten et al., 1970; Cook and Burnstock 1976). Subsequently, considerable evidence has been provided that has generated widespread support for the idea that peptides act as neurotransmitters in neurons of the enteric, sympathetic, parasympathetic and central nervous systems.\n\nAlthough von Euler and Gaddum as early as 1931, had suggested that agents with identical biological activity were present in extracts of both gut and brain, the work of Vanderhaeghen et al., (1975) raised again the idea that a peptide previously considered to be a classic gut hormone (gastrin) might be present within neurons of the central nervous system. The technical and analytical advances in the last two decades have now established fundamental new concepts in the distribution of regulatory peptides. It is now clear that any one peptide may have a specific function at a specific site, determined by the cell type responsible for its synthesis, mode of release and subsequent tissue distribution, but the same peptide may occur at another independent site within the body and have a separate function and mode of action determined again by the local environment. Hence peptides such as\n\ncholecystokinin (CCK) and gastrin, previously considered as hormones may act elsewhere as neurotransmitters or paracrine mediators, the converse also being true.\n\nWith the general acceptance of similarity between the spectrum of peptides that function as hormones and as neurotransmitters, it is perhaps less surprising that there is such close interaction between neurons and endocrine cells in the regulation of visceral functions such as gastric acid secretion. The main objective of the studies described in this thesis was to determine the role of antral neurons in the control of antral G-cell responses to physiological stimuli. The experimental model of antral denervation that was developed to investigate the role of intrinsic neurons in the regulation of the G-cell produced chronic hypergastrinaemia and delayed gastric emptying. This prompted further studies on the role of antral neurons in the regulation of gastric emptying and the effects of sustained elevations in plasma gastrin on endocrine cell gene expression with particular relevance to enterochromaffin-like (ECL) cell growth and carcinoid tumour development.\n\nThe following account will describe the normal gastric innervation and its role in regulating G cell function and gastric emptying, review the physiology of gastrin and the pathophysiology of hypergastrinaemia in particular the development of ECL cell tumours, review the evidence for the role of gastrin in endocrine cell growth and differentiation and finally introduce putative regulators of cell growth and differentiation in gastroentero-pancreatic endocrine cells.\n\nLangley (1921) first recognised the importance of the intrinsic nervous system of the gut, considering it a separate division of the autonomic nervous system in addition to the sympathetic and parasympathetic divisions. Considerable evidence in support of this has been presented (Cooke, 1987; Furness and Costa, 1987; Wood, 1987) and it is clear that processes such as absorption, secretion and coordinated motor activity may be regulated wholly by the enteric nervous system. The extrinsic innervation of the gut interacts with enteric neurons exerting an overriding influence when necessary so that gut function may be integrated with the prevailing needs of the body as a whole. Extrinsic neuronal inputs are supplied by vagal and spinal nerve trunks each providing both afferent and efferent nerve fibres.\n\ni) Vagal innervation of the stomach\n\nThe majority of nerve fibres in the vagi are afferent fibres having their nerve cell bodies in the nodose ganglia. These afferent fibres have their peripheral endings within the muscle and mucosal layers of the gut wall and act as mechano-, thermo- and chemo-receptors conveying signals to the central nervous system (Andrews, 1986; Blackshaw and Grundy, 1990; El Ouazziani and Mei, 1979; 1891). Almost all vagal afferent fibres are unmyelinated (Andrews, 1986) but a small population of nerve fibres within the vagus are small diameter myelinated fibres believed to be of sympathetic origin (Lundberg et al., 1976).\n\nUsing retrograde tracing and immunohistochemistry it has been shown that vagal afferent fibres projecting from the anterior wall of the stomach have their cell bodies in the left nodose ganglion and those that project from the posterior gastric wall have their cell bodies in the right nodose ganglion (Dockray et al., 1988; Green 1988). A number of peptides have been identified in vagal afferent fibres including substance P (SP) (Katz and Karten, 1980; Lundberg et al., 1978; 1983; 1985) CCK (Mantyh and Hurt, 1984), vasoactive intestinal polypeptide (VIP) (Lundberg, 1978; Said and Rosenberg, 1976) and gastrin (Dockray et al., 1981). Despite this variety, the majority of peptide-containing vagal nerve fibres project to the thoracic viscera (Lundberg, 1983; Mulderry 1985; Cadieux 1986) such that less than 10% of gastric vagal afferent fibres are peptidergic (Green, 1988).\n\nThe vagus also carries efferent fibres to the stomach but these account for only 10% of the total number of vagal fibres. The majority of vagal efferent fibres are excitatory cholinergic fibres that are involved with the normal regulation of gastric tone as well as secretion of gastrin and gastric acid. In addition however, there are also inhibitory pathways such as those causing release of nitric oxide (NO) (Desai et al., 1991) and VIP (Grider et al., 1985).\n\nii) Spinal afferent innervation of the stomach\n\nSimilar to the vagal innervation of the stomach, spinal afferent neurons for the most part are unmyelinated (Kuo et al., 1982). Gastric spinal afferents have their nerve cell bodies in the thoracolumbar dorsal root ganglia (Green and Dockray, 1988). Conversely however, there is no lateralisation of innervation such that\n\nretrograde tracing from one surface of the stomach labels equal numbers of nerve cell bodies in the right and left dorsal root ganglia. The spinal efferent innervation of the stomach is approximately 95% adrenergic and projects from cell bodies contained within the coeliac ganglion (Macrae et al., 1986).\n\nSeveral different peptides have been identified within spinal afferent neurons but the most important are calcitonin gene-related peptide (CGRP) (Gibson et al., 1984; Lee et al., 1987; Rosenfeld et al., 1983) and substance P (Hokfelt et al., 1976; Price 1985; Seybould and Elde, 1980). CGRP is of primary interest because of its specific distribution within the stomach and is relevant to the work contained within this thesis. A brief review of the physiology of CGRP is given below before describing the intrinsic innervation of the stomach.\n\n**Discovery and distribution of CGRP:**\n\nThe existence of CGRP was predicted on the basis of the discovery of a novel cDNA sequence corresponding to an mRNA transcript that was shown to arise from alternative splicing of the gene encoding calcitonin (Amara et al., 1982; Rosenfeld et al., 1983). Subsequently a related peptide β-CGRP was isolated (Amara et al., 1985; Steenbergh et al., 1985) whose primary gene does not encode for calcitonin. The distribution of CGRP within the stomach has important implications. CGRP-immunoreactive fibres may be found in all layers of the stomach (Clague et al., 1985; Green and Dockray 1988; Sternini et al., 1987) but there are no intrinsic neurons or endocrine cells that contain CGRP in the rat stomach. In fact in rodent stomach almost all CGRP is contained within spinal\n\nafferent extrinsic nerve fibres (Green and Dockray, 1988; Sternini et al., 1987; Varro et al., 1988). This makes CGRP a useful marker for an intact spinal afferent gastric innervation.\n\n**Biological actions of CGRP in the stomach:**\n\nIt is well established that CGRP acts as a peripheral neurotransmitter from spinal afferent nerve terminals (Holzer, 1992). Much of the available experimental evidence points to a role for CGRP in gastric mucosal protection. Hence, installation of noxious chemicals into the gastric lumen results in increased mucosal damage if the spinal afferent innervation has been lesioned (Holzer and Sametz, 1986) or the action of CGRP is blocked by immunoneutralisation (Forster and Dockray, 1991). Conversely, stimulation of CGRP release protects against mucosal damage (Holzer and Lippe, 1988; Holzer et al., 1989; 1990) induced by installation of noxious chemicals.\n\nThe gastroprotective effect of CGRP acts partly by increasing gastric mucosal blood flow possibly by the release of NO (Lambrecht et al., 1993). In addition however, CGRP has been shown to be a potent inhibitor of gastric acid secretion (Tache et al., 1984; Pappas et al., 1986; Helton et al., 1989), a factor that also contributes to the gastroprotective effects of this peptide. The inhibition of acid secretion is mediated in part by release of somatostatin from D cells of the gastric corpus (Dunning and Taborsky, 1987; Inui et al., 1991). In addition it has been shown that CGRP regulates somatostatin synthesis by corpus D cells at the level of gene transcription (Sandvik et al., 1993).\n\niii) Intrinsic innervation of the stomach\n\nThe intrinsic nerve supply of the gastrointestinal tract, collectively termed the enteric nervous system, comprises two main plexi of nerves described first by Meissner (1857) and Auerbach (1864). Meissner described a submucous plexus and Auerbach a plexus of nerves lying between the circular and longitudinal muscle layers generally referred to as the myenteric plexus. The structure and distribution of the enteric nervous system were described in detail by Gaskell (1886) and the functional significance of the enteric nervous system as a separate branch of the autonomic nervous system was proposed later by Langley (1921).\n\nStudies of the anatomy of intrinsic gut neurons were facilitated greatly by the techniques of myotomy and myomectomy coupled with immunohistochemistry first described by Furness and Costa (1979). The variety and colocalisation of neuropeptides present in enteric neurons, their projections and putative actions have received much interest over the past two decades (for review see Dockray 1994; Furness and Costa 1987). Generally speaking, the tachykinin/substance P family of peptides are present in intrinsic excitatory neurons (Katayama and North, 1978; Bartho et al., 1982; Bartho and Holzer, 1985; Holzer, 1989) and the VIP/peptide histidine isoleucine family is present in intrinsic inhibitory neurons (Brookes et al., 1991; Ekblad et al., 1987; Shultzberg et al., 1989; Grider, 1989). Gastrin-releasing peptide (GRP) is present in secretor motor neurons and this is of particular relevance as GRP-containing neurons stimulate the release of gastrin from antral G\n\ncells (Bunnett et al., 1985; Kovacs et al., 1995; Varner et al., 1991). In the lesioning experiments described later in this thesis, substance P, VIP, GRP and CGRP were assayed as markers of different classes of intrinsic or extrinsic neurons to enable confirmation of denervation. There follows a brief review of substance P, VIP and GRP in relation to the enteric nervous system.\n\n**Substance P**\n\nSubstance P (SP) was the first gut neuropeptide to be discovered. Although extracts of horse intestine had been shown to stimulate atropine resistant contraction of rabbit ileum as early as 1931 (von Euler and Gaddum, 1931), SP was isolated and sequenced first from the bovine hypothalamus (Chang and Leeman, 1970; Chang and Leeman, 1971). The identical peptide was shown later to be present in small intestine (Carraway and Leeman, 1979). Evidence for its neuronal localisation was suggested by Ehrenpreis and Pernow (1953) who reported reduced levels of SP bioactivity in the aganglionic segment of colon affected by Hirschsprung’s disease. Lembeck (1953) suggested that SP was located within the primary afferent neurons on the basis of findings of higher concentrations of peptide in dorsal compared with ventral spinal roots. Although there is compelling evidence that a subset of primary afferent neurons contain both SP and CGRP (Gibbins et al., 1985; Green and Dockray, 1988), lesioning experiments using capsaicin, have shown that in the rat stomach and small intestine the majority (>90%) of SP is present in intrinsic neurons (Holzer et al., 1980).\n\nThe presence of SP within primary afferent neurons has prompted studies that suggest that SP released from these neurons may act as a central neurotransmitter of pain and may play a role as a peripheral transmitter during inflammatory reactions in the gut (Dockray, 1994; Holzer, 1988; Sharkey, 1992). The predominant localisation of SP within intrinsic gut neurons has directed several groups towards identifying its possible role within the gut. There is evidence that SP directly stimulates intestinal smooth muscle contraction (Taylor and Bywater, 1986) and that SP release is involved in the contraction that occurs on the oral side of a distending bolus as part of the peristaltic reflex (Bartho et al., 1982; Holzer, 1989; Yokayama and North, 1983). In the rat colon, SP release appears to mediate only the noncholinergic contraction that occurs with high pressure distension as this could be inhibited by immunoneutralisation (Grider, 1989b).\n\nIn addition to the direct effects of SP on smooth muscle, there is also evidence that release of SP may stimulate other myenteric plexus neurons (Yan and Youther, 1982; Fosberg et al., 1984). Regardless of the precise physiological role of SP in the control of motility, intrinsic SP neurons generally can be considered to be excitatory and SP is the most useful marker of this class of neuron.\n\n**Vasoactive intestinal polypeptide**\n\nVasoactive intestinal polypeptide (VIP) was discovered by Said and Mutt by screening of extracts of gut and lung for vasodilator activity (Said and Mutt, 1970; 1972). Vasodilatation induced by VIP is only one manifestation of its ability to relax smooth muscle. In the gut VIP is localised to a population of myenteric neurons that\n\nproject distally and innervate the circular muscle (Brookes et al., 1991; Ekblad et al., 1987), and to a population of submucosal neurons that project to the mucosa (Schultzberg et al., 1980; Costa and Furness, 1983). There is evidence from immunoneutralisation studies that VIP may mediate the descending relaxation of the peristaltic reflex (Grider and Makhlouf, 1986; Grider et al., 1985; Grider, 1989a). In addition, VIP may be one of the final mediators of the vagovagal, non-adrenergic, non-cholinergic relaxation of the stomach (Faherenkug et al., 1978; Ito et al., 1988), and play a role in the control of gastric emptying (Forster et al., 1991; D’Amato et al., 1992).\n\nSubmucosal VIP-containing neurons that project to the mucosa stimulate secretion from enterocytes (Cooke, 1992). In terms of gastric endocrine cell function, VIP has been shown to inhibit gastric acid secretion by stimulating the release of somatostatin from mucosal D cells (Saffouri et al., 1984). From the above description therefore, in contrast to SP, VIP can be considered as a marker of inhibitory myenteric plexus neurons.\n\n**Gastrin-releasing peptide**\n\nIn 1971, Espramer and Anastasi isolated bombesin from amphibian skin (Anastasi et al., 1971). A major effect of this peptide was stimulation of acid secretion that was shown in the dog to be mediated by gastrin (Bertaccini et al., 1974). The mammalian counterpart of bombesin was identified using C-terminally directed antibodies to the amphibian peptide (Walsh et al., 1979). Coincidentally, McDonald and co-workers discovered that extracts of the non-antral porcine\n\nstomach were a potent stimulus to gastrin release (McDonald et al., 1978; McDonald et al., 1979). They proposed that the peptide responsible was gastrin-releasing peptide (GRP) and subsequently the C-terminal sequence of GRP and bombesin have been shown to be almost identical. However, GRP is not strictly the mammalian equivalent of amphibian bombesin as amphibia have separate genes encoding GRP and bombesin (Nagalla et al., 1992).\n\nIn the mammalian gut, GRP-immunoreactivity is found exclusively within neurons (Dockray et al., 1979). The majority of GRP immunoreactive fibres are present in the mucosa with some fibres present in the circular muscle and myenteric plexus. (Dockray et al., 1979). Most GRP-containing neurons have their cell bodies in the myenteric plexus and project their axons distally (Costa et al., 1984; Ekblad et al., 1984). There is good evidence that the major physiological role of this neuropeptide in the stomach is to stimulate gastric acid secretion via release of gastrin from antral G cells (Bunnett et al., 1985; Hildebrand et al., 1991; Hirschowitz and Molina, 1983; Kovacs et al., 1995).\n\nA schematic representation of the major classes of neuron present within the gastric antrum is shown in Figure 1.1.\n\nFigure 1.1: Schematic representation of the major classes of intrinsic and extrinsic neurons that innervate the gastric antrum in the rat and are susceptible to lesioning by serosal application of the neurotoxin BAC. The shaded area depicts the likely depth of penetration of BAC. LM - longitudinal muscle; CM - circular muscle; SM - submucosa. Excitatory (SP) and inhibitory (VIP) myenteric neurons are shown and receive inputs from spinal afferent (CGRP) and vagal nerve fibres. GRP is present predominantly in secretomotor nerves that supply the mucosa (see text for full details).\n\nIn recent years a substantial body of evidence has emerged detailing the interactions between the intestinal hormone cholecystokinin (CCK) and vago-vagal reflexes that together mediate the delayed gastric emptying in response to fat- and protein-rich meals (Dockray, 1988; Mayer, 1994). Exogenous CCK inhibits gastric emptying in both rat (Conorver et al., 1988; Green et al., 1988) and man (Chey et al., 1969; Liddle et al., 1986). Protein-rich meals are known to stimulate secretion of endogenous CCK (Liddle et al., 1984; 1986) and delay gastric emptying (Green et al., 1988; Moran and McHugh, 1981), an effect that is reversed by specific CCK-A receptor antagonists (Green et al., 1988; Liddle et al., 1989). In addition there is good evidence that vagal afferent fibres express CCK-A receptors (Corp et al., 1993; Moriarty et al., 1997) and that the CCK-mediated delay in gastric emptying is initiated by stimulation of vagal afferent fibres (Raybould and Tache, 1988; Forster et al., 1990; Forster et al., 1991; Holzer et al., 1994). Furthermore, the efferent limb of the reflex that results in relaxation of the gastric body also requires an intact vagus nerve (Raybould and Tache, 1988; Raybould et al., 1987). Vagal efferent relaxation of the gastric corpus is mediated by non-adrenergic and non-cholinergic neurotransmission that probably involves both NO (Desai et al., 1991; Forster et al., 1991; D’Amato et al., 1992) and VIP (Forster et al., 1991; D’Amato et al., 1992).\n\nIn addition to the CCK-vagal pathway that regulates gastric relaxation in response to feeding, gastric emptying may also be affected by spinal afferent innervation, since lesioning of spinal afferent neurons by capsaicin pre-treatment\n\nreduces the emptying rate of liquid test meals (Forster et al, 1990; Forster and Dockray, 1992).\n\nOverall the rate of gastric emptying depends on the pressure gradient between the duodenum and stomach and the resistance to flow across the pylorus and antrum (Mayer, 1994). The content of the ingested meal determines the rate of emptying and CCK-mediated vagovagal reflexes play an important role in delaying gastric emptying by relaxation of the gastric corpus. The importance of antropyloric motility in regulating gastric emptying has received less attention although the pulsatile manner of delivery of gastric content to the duodenum can be attributed to the phasic motor pattern of the antrum (Houghton et al., 1988; Malbert et al., 1991; Prather et al., 1993). There is some evidence that CCK may act on the distal stomach both by a direct action on smooth muscle and indirectly via release of neurotransmitters (Scheurer et al., 1983; Yamagishi and Debas, 1978). However the relationships between CCK and antral neurons in the regulation of gastric emptying has not been examined previously in any detail and represents one of the objectives of the studies presented in this thesis.\n\n**Neuroendocrine Regulation of Gastric Acid Secretion.**\n\nThe G cells, D cells and ECL cells of the stomach together with the extrinsic and intrinsic innervation of the stomach, provide an intricate regulatory mechanism for the control of gastric pH (Fig 1.2).\n\nFigure 1.2: Following meal ingestion, intraluminal pH rises and somatostatin (SOM) release from the D cell is inhibited. The G cell secretes gastrin that leads to histamine release from the ECL cell and H⁺ release from the parietal cell. Vagal neurons also stimulate the parietal cell (1.2a). As luminal pH then falls, antral D cells release SOM that inhibits further gastrin release. Spinal afferent neurons regulate SOM release from corpus D cells. SOM inhibits the ECL cell and parietal cell (1.2b). See text for full description.\n\nG cells\n\nThese cells are present in the antrum of the stomach and are also found in lesser numbers in the proximal duodenum (McGuigan, 1968; Solcia et al., 1975). Their primary function is to synthesise, store and release the amidated 17 amino acid peptide gastrin. G cells process the prohormone precursor peptide progastrin by sequential steps involving sulfation, phosphorylation, cleavage, C-terminal amidation or glycine extension and further cleavage (for review see Dockray et al., 1996). The acid secretory activity of gastrin is dependent on the presence of the amidated carboxyl terminus (Soll et al., 1984; Takeuchi et al., 1980). Alternative forms of gastrin, extended at their C-terminus by glycine also have been reported to possess biological activity other than stimulation of acid secretion, namely stimulation of expression of the gene encoding the H⁺-K⁺-ATPase present in parietal cells (Kaise et al., 1995). Furthermore, recent studies suggest that amidated gastrin, glycine extended gastrin and progastrin itself are all putative growth factors (Seva et al., 1994; Singh et al., 1996; Wang et al., 1996).\n\nG cells have a luminal brush border and secrete gastrin in response to the presence of protein and amino-acids within the gastric lumen (Dockray and Gregory, 1989; Walsh, 1994). In keeping with these observations are the reduction in gastrin mRNA reported with fasting and increases in gastrin mRNA abundance seen with feeding (Wu et al., 1991). In addition G cells respond to changes in luminal pH although this may be indirect, being mediated by changes in somatostatin release from adjacent antral D cells (Karnik et al., 1989). G cells\n\nexpress several different receptors on their basolateral membrane including receptors for gastrin releasing peptide (Vigna et al., 1990; Weigart et al., 1992).\n\n**ECL cells**\n\nThese cells are present only in the gastric corpus and their main function is to synthesise histamine by the decarboxylation of the amino acid histidine (Rubin and Schwartz, 1979). This reaction is catalysed within the cell cytosol by the enzyme histidine decarboxylase (Kubota et al., 1984). Histamine is then transported into secretory vesicles probably by the vesicular monoamine transporter VMAT$_2$ (Merickel and Edwards., 1995). Unequivocal evidence for the stimulation of acid secretion by histamine was provided by the discovery of H$_2$ receptor antagonists and their ability to block acid secretion (Black et al., 1972). Gastrin stimulates the ECL cell to release histamine (Chuang et al., 1992) and if the gastrin stimulus is sustained, ECL cells increase their production and storage capacity of histamine, in part by upregulating HDC and VMAT$_2$ gene expression (Dimaline and Struthers, 1996; Swarovsky et al., 1994). The secretion of histamine by the ECL cell can be inhibited by release of somatostatin from corpus D cells (Chuang et al., 1993). Finally ECL cells increase in number in response to continued gastrin stimulation (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995).\n\n**D cells**\n\nD cells are present in both the antrum and corpus of the stomach. In both sites, their function is to synthesise, store and release somatostatin (Dockray et al., 1996). Antral D cells have a luminal brush border and respond to changes in pH in\n\nthe stomach (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988). In contrast, corpus D cells do not have a luminal brush border and somatostatin mRNA abundance in corpus D cells is much less affected by changes in luminal pH that occur with feeding or treatment with omeprazole (Sandvik et al., 1993). It appears that corpus D cell somatostatin gene transcription is regulated primarily by neurohumoral influences of which the most significant may be those of the CGRP-containing spinal afferent neurons (Sandvik et al., 1993).\n\n**Regulation of acid secretion**\n\nThe parietal cells are responsible for the release of acid ($\\text{H}^+$) into the gastric lumen. They can be stimulated to release $\\text{H}^+$ by cholinergic neurons and by histamine (Black et al., 1972; Black and Shankley, 1987; Kabekar et al., 1969; Main and Pearce, 1982; Soll, 1978). Gastrin receptors have been demonstrated on parietal cells (Soll et al., 1984) suggesting that gastrin also may act directly on these cells to induce acid secretion. As intragastric pH rises (such as with ingestion of a meal) antral G cells release gastrin in response to a variety of subsequent stimuli. These include rising luminal pH (Dockray et al., 1991), increased cholinergic stimulation (Kabekar et al., 1969; Main and Pearce, 1982), release of GRP from non-cholinergic neurons (Saffouri et al., 1984; Schubert et al., 1992), the presence of intraluminal protein and amino-acids (Chiba et al., 1980; Saffouri et al., 1980; Walsh, 1994), and a reduction in somatostatin release from adjacent antral D cells that are themselves inhibited by the rising luminal pH (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988).\n\nGastrin passes via the circulation to the ECL cell and stimulates these cells to release histamine (Chuang et al., 1992). ECL cells may also respond to the increased cholinergic drive that accompanies meal ingestion (Maine and Pearce, 1982; Soll, 1982). Histamine released from ECL cells acts via parietal cell H\\textsubscript{2} receptors causing H\\textsuperscript{+} release (Black et al., 1972, Black and Shankley, 1987). This co-ordinated response provides the acid pH required for gastric digestion by pH dependent enzymes and as digestion and gastric emptying proceeds, returns gastric pH to its normal basal level (Fig 1.2a).\n\nAs the gastric pH falls, antral D cells respond by releasing somatostatin that in turn inhibits gastrin release from G cells (Gustavsson and Lundquist, 1978; Holst et al., 1987; Schubert et al., 1988). As a result of reduced intraluminal pH, gastrin levels fall, permitting somatostatin release from corpus D cells which in turn prevents further secretion of histamine from the ECL cell and H\\textsuperscript{+} from the parietal cell (Fig. 2b). The spinal afferent innervation of the corpus may also stimulate corpus D cell somatostatin release as it has been shown to functionally regulate corpus rather than antral D cell somatostatin gene expression (Sandvik et al., 1993).\n\nIt may be readily appreciated from the above description that disorders of gastric acid secretion are likely to be associated with elevated plasma gastrin levels. Hence, primary disorders of the G cell or ectopic gastrin secretion that result in hypergastrinaemia will result in increased acid output. Conversely, decreased acid output from whatever cause will result in elevated plasma gastrin as antral G cells\n\nrespond to the high intragastric pH. There follows a discussion of conditions associated with hypergastrinaemia and the related pathophysiology of the ECL cell.\n\n**Clinical significance of chronic hypergastrinaemia**\n\nFrom a clinical viewpoint the major conditions of the stomach that are associated with altered endocrine cell function are encompassed by the conditions associated with hypergastrinaemia (Figure 1.3). The commonest of these are the conditions associated with *H pylori* infection with or without concurrent treatment with a proton pump inhibitor (PPI). However, Zollinger Ellison syndrome (ZE) and pernicious anaemia are the conditions associated with the highest and most sustained elevations in plasma gastrin. In these conditions ECL cell (carcinoid) tumours have been described (see below) and it appears that it is the trophic action of gastrin on the ECL cell that is of pathophysiological importance. The elevations of plasma gastrin in *H pylori* or with PPI therapy are of lesser magnitude and rarely sustained and ECL cell tumours have not been reported. However, *H pylori* infected patients treated with a PPI may develop significant elevations in plasma gastrin and ECL cell hyperplasia has been documented in up to a third of such patients taking a PPI for five years (Eiselle et al., 1997). Although hyperplasia precedes dysplasia and neoplasia, the time course of this progression is not known and it may take several years before carcinoid tumours develop. Hence carcinoid tumours developing in patients rendered hypergastrinaemic by *H pylori* and PPI therapy remains a remote possibility.\n\n| Associated Condition | Representative Range of Plasma Gastrin* (pM) | Causal mechanism for hypergastrinaemia |\n|----------------------|---------------------------------------------|----------------------------------------|\n| Pernicious Anaemia (PA) | 200 - >1000 | Atrophic gastritis leads to achlorhydria and secondary rise in plasma gastrin |\n| Zollinger Ellison (ZE) Syndrome | Normal - >1000 | Functioning gastrin secreting tumour usually of the duodenum or pancreas causes increased acid output. |\n| Chronic Renal Failure. (CRF) | Normal - 600 | Kidneys are the major site of breakdown of circulating gastrins. |\n| Proton Pump Inhibitor Treatment (PPI) # | Normal - 350 | Reduces acid output by direct action on the parietal cells’ H⁺/K⁺ ATPase. |\n| H. pylori infection | Normal - 200 | a) antral gastritis reduces antral somatostatin with loss of inhibition of gastrin release by gastric acid. \nb) Corpus gastritis leads to atrophy and hypochlorhydria |\n\nFigure 1.3: Conditions associated with hypergastrinaemia and the primary causal mechanism for the elevated plasma gastrin.\n\n* Amidated gastrin; normal fasting <30pM; normal post-prandial <90pM.\n\n# depends on duration of treatment and presence of *H pylori*. (6-8 week treatment with PPI raises plasma gastrin up to twofold).\n\nECL cell (Carcinoid) tumours and Hypergastrinaemia.\n\nChronic atrophic gastritis (CAG) with or without pernicious anemia (PA) is the commonest cause of ECL cell hyperplasia and in 5-10% of patients there is a progression to gastric carcinoid tumors (Borch et al., 1985; Bordi et al., 1991; 1995). The achlorhydria of CAG leads to hypergastrinemia which is generally accepted to be a stimulus to ECL cell growth (Bordi et al., 1995; Creutzfeld, 1988; Hakanson and Sundler, 1990; Solcia et al., 1986). The natural history of these tumors in man has been extensively reviewed (Modlin et al., 1995; Rindi, 1995; Solcia et al., 1991; Thomas et al., 1994), prompted at least in part by the demonstration that in rodents hypergastrinemia secondary to long-term treatment with H$_2$-receptor antagonists or proton pump inhibitors, may lead to gastric carcinoid tumors (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995).\n\nEnterochromaffin-like cell tumors associated with CAG are usually small (<1cm), may be single or multiple, are most often present incidentally in patients over the age of 60, and rarely progress to metastatic disease (Bordi et al., 1995; Solcia et al., 1991; Modlin et al., 1995). This has led to conservative management for the majority of patients with or without endoscopic screening. Some authors have advocated twice daily subcutaneous injections of the long acting somatostatin analogue octreotide (Ferraro et al., 1996; Bordi et al., 1993). This therapy has been shown to reverse ECL cell hyperplasia in patients with CAG without ECL cell tumors, and to inhibit gastrin release from the antral gastrin (G-) cell (Ferraro et al., 1996; Bordi et al., 1993). Surgical antrectomy to remove G-cells is also effective in\n\nreducing plasma gastrin levels and is reported to result in complete resolution of small ECL cell tumors (Hirschowitz et al., 1992; Modlin et al., 1995; Scully et al., 1997). In contrast, the management of large ECL cell tumours (>2cm) associated with CAG is less well defined. Antrectomy has been advocated since it may induce tumor regression, or at least halt metastatic spread (Eckhauser et al., 1988; Scully et al., 1997) However, reports of a metastasis rate of >20% for primary tumours that are >2cm in diameter, together with the demonstration that in a few cases the tumors fail to regress following antrectomy, has persuaded others to recommend total gastrectomy (Borch et al., 1985; Thomas et al., 1995).\n\nAlthough all PA patients are hypergastrinaemic and the majority show ECL cell hyperplasia some other factor(s) must also be involved in the progression of hyperplasia to neoplasia as the incidence of carcinoid tumours in PA patients is 5-10% (Borch et al., 1985; Bordi et al., 1991; 1995). Azzoni has suggested that the expression of BCL-2 by hyperplastic ECL cells may be of importance as it may prolong exposure to other oncogenic factors (Azzoni et al., 1996). However relatively little is known of the underlying genetic risk factors for ECL cell tumour development in CAG.\n\nIn the sporadic form of ZE the incidence of carcinoid tumours is similar to that in PA. In contrast patients with ZE as part of the type I multiple endocrine neoplasia syndrome (MEN1) have a much higher incidence of carcinoid tumour development (up to 35%) presumably due to an inherited susceptibility to endocrine cell tumour development (Lehy et al., 1992). The recent positional cloning of the\n\nMEN1 gene (Chandrasekharappa et al., 1997) has facilitated the study of abnormalities of this gene in the development of carcinoid tumours. Loss of heterozygosity (LOH) at the MEN1 locus could be demonstrated in the majority of carcinoid tumours arising in patients with ZE as part of the MEN1 syndrome (Debelenko et al., 1997). However very few gastric carcinoids arising from CAG had associated LOH at the MEN1 gene locus suggesting other genes may be important in the pathogenesis of these tumours. Data is presented in this thesis that is in favour of a potential candidate gene, reg, being of importance to the development of carcinoid tumours. Before reviewing the available information on the reg family of genes, the role of gastrin in the differentiation and growth of endocrine cells of the stomach and pancreas is discussed.\n\nGastrin, endocrine cell growth and differentiation in the stomach and pancreas.\n\nIn addition to the well established role of amidated gastrin in regulating gastric acid output, there is an increasing body of evidence to suggest that gastrin also regulates the differentiation of some gut endocrine cells and the growth and overall numbers of certain endocrine and epithelial cell populations within the stomach.\n\nThe first direct evidence of the trophic effect of amidated gastrin came from the observation that pentagastrin administration to rats increased the functioning\n\nparietal cell mass thereby contributing to the long term increase in acid output. (Crean et al., 1969). Prior to this, gland atrophy following antrectomy had been reported (Lees and Grandjean, 1958), indirectly suggesting a trophic role for gastrin in maintaining the acid secreting mucosa. Gastrin also stimulates the growth of ECL cells (Bordi et al., 1995; Tielemans et al., 1990), a subject that has received much interest due to the theoretical risk of ECL cell tumour development in patients receiving long term pharmacological acid suppression.\n\nMore recent studies using transgenic mice clearly demonstrate the importance of amidated gastrin acting via the CCK-B receptor in the maintenance of normal cell populations within the gastric mucosa. Hence, overexpression of amidated gastrin leads to increased proliferation of precursor cells in the proliferative zone of the gastric mucosa and increased parietal and ECL cell numbers (Wang et al., 1993). Conversely, targeted disruption of the CCK-B receptor produced marked gastric mucosal atrophy with reductions in the numbers of parietal and ECL cells (Langhans et al., 1997; Nagata et al., 1996). Similarly, targeted disruption of the gastrin gene resulted in a marked reduction in ECL cell and parietal cell number but did not affect the proliferative index in the stomach (Koh et al., 1997). The latter study is of particular interest as it highlights the importance of gastrin in regulating the differentiation of gastric parietal and ECL cells independent of the degree of stem cell proliferation.\n\nThere is also good evidence that gastrin is responsible for the development in the embryo of endocrine cells of the pancreas (Wang et al., 1993). In the case of\n\npancreatic endocrine cell development candidate genes have been proposed to be involved in the growth and differentiation of islet β-cells. (Terazono et al., 1988; Otonkoski et al., 1994; Watanabe et al., 1994). The *reg* gene was first isolated by Terazono and colleagues in 1988 by differential screening of a rat regenerating pancreatic islet cDNA library. This gene has been renamed *reg Iα*, following the discovery of several other homologous genes that appear to comprise a multigene family.\n\n**The *reg* gene family**\n\nThe rat *reg Iα* cDNA contained a single open reading encoding for a 165 aminoacid protein, of which the first 21 aminoacids appeared to constitute a signal peptide. The human homologue of rat *reg Iα* was identified by screening of a human pancreatic cDNA library with a 60 base pair complement of the rat cDNA (Terazono et al., 1988). Human *reg Iα* encodes a 166 aminoacid protein with a 22 aminoacid signal peptide. Human *reg Iα* normally is expressed in the pancreas, gastric mucosa and kidney; ectopic expression of *reg* has been described in tumours of the colon and rectum (Watanabe et al., 1990). The homology between rat and human *reg* cDNA was 75% (68% in AA sequence) including similar homology in the 5’ untranslated region. Perhaps of significance was the observation that the position of all seven cysteine residues within the predicted protein were conserved. Subsequent cloning of the respective genes indicated another common feature, each consisting of 6 exons and 5 introns. These two features are common to all members of the *reg* gene family.\n\nFollowing the cloning and sequencing of *reg* it became clear that the previously described pancreatic stone protein (PSP) was a truncated product of the same gene (Watanabe *et al.*, 1990). In fact PSP consists of 144 amino acids (23-166 of the parent *reg*) and has been renamed PSP$_I$. A shorter variant also has been identified and is 11 amino acids shorter (34-166 of the parent *reg*). This protein is identical to the pancreatic thread protein (PTP) also referred to as PSP$_{II}$ (Watanabe *et al.*, 1990).\n\nOther members of the human *reg* family that have been identified so far, include *reg Iβ* (Moriizumi *et al.*, 1994), *reg* related sequence (Watanabe *et al.*, 1990) and pancreatitis associated protein (Iovanna *et al.*, 1991; Morizumi *et al.*, 1993). Confusingly, pancreatitis associated protein (PAP) has an identical aminoacid sequence to peptide 23 (Katsumata *et al.*, 1995) and HIP, a protein that was first identified by its expression ectopically in primary hepatocellular carcinoma (Lasserre *et al.*, 1992). These members of the gene family are tandemly ordered in a 95kbp DNA region of human chromosome 2p12 as determined by fluorescence in situ hybridisation (Miyashita *et al.*, 1995).\n\nThe murine *reg* gene family has also been studied but reveals unexpected differences. The murine homologue of *reg Iα* has been identified and localised to chromosome 12 (Unno *et al.*, 1993). A second member of the family *reg II* has also been cloned. Although the gene consists of 5 introns and 6 exons with conservation of all cysteine residues, the whole sequence is <60% homologous to *reg I*, has no human homologue and maps to mouse chromosome 3 (Unno *et al.*, 1993). Further\n\nmembers of the mouse *reg* gene family have recently been described. Hence murine *reg IIIα*, *reg IIIβ* and *reg IIIγ* bear reasonable homology to each other but represent a separate gene group and are localised on Chromosome 6 (Narushima et al., 1997). Unlike other members of the *reg* family, the *reg III* genes are preferentially expressed in the intestine and are expressed only at low levels in regenerating pancreatic islets.\n\nThe available evidence suggests that members of the *reg* gene family are expressed within endocrine cells of the stomach and pancreas. In particular, *reg Iα* (*reg*) expression appears to be markedly upregulated during regeneration following surgical resection (Terazono et al., 1988; or mucosal ulceraton induced by waster immersion stress (Ashahara et al., 1997) or treatment with non-steroidal anti-inflammatory drugs (Kawanami et al., 1997). However, the potential relationship between gastrin and expression of the *reg* gene has not been explored.\n\n**Reg gene expression and endocrine cell growth and differentiation**\n\nThe *reg* gene was first proposed as a regulator of pancreatic islet cell growth following its isolation from regenerating islets (Terazono et al., 1988). In mature adult pancreas, *reg* expression is limited to acinar cells. The Reg protein is secreted into pancreatic juice where it has been suggested to act as an inhibitor of pancreatic stone formation (Patard et al., 1996; Bertrand et al., 1996). During islet cell regeneration the Reg protein also appears to be produced by endocrine cells as it has been shown to colocalise almost exclusively with insulin in β-cell secretory granules (Terazono et al., 1990).\n\nOther models of islet cell growth such as hyperplasia following surgical wrapping of the pancreas (Zenilman et al., 1996) and islet cell regeneration following growth suppression by insulinoma (Miyaura et al., 1991), have shown a consistent relationship between expression of reg and increasing islet cell number. None of these studies attempted to determine whether reg may act as a growth factor or as a promoter of differentiation.\n\nOtonkoski and colleagues (1994b) proposed that reg expression was a marker of proliferation rather than differentiation based on observations using nicotinamide to stimulate differentiation and hepatocyte growth factor (HGF) to stimulate proliferation in 7 day cultures of human foetal pancreatic cells. Reg expression was significantly increased by HGF. However, the effects of HGF are not purely trophic. In fact, the in vitro mitogenic effect of HGF is limited to the first three to four days of culture (Beattie et al., 1996). Thereafter, HGF has been shown to increase the formation of islet cell clusters in 6 day cultures of human foetal pancreatic cells and increase their insulin content (Otonkoski 1994a). Thus HGF appears to stimulate both growth and differentiation in this model. HGF also stimulates the differentiation of AR42J cells into insulin secreting cells (Mashima et al., 1996). The pancreatic cell line AR42J has recently been shown to express reg (Zenilman et al., 1996) and can be manipulated by appropriate culture conditions to differentiate towards either acinar or endocrine cell phenotype (Christophe, 1994; Mashima et al., 1996). Interestingly, differentiation towards an acinar cell phenotype is associated with reduced reg expression (Zenilman et al., 1997).\n\nIn 1994, Watanabe et al., described the amelioration of diabetes (induced surgically in rats by 90% pancreatectomy) by daily administration of recombinant rat Reg protein. Administration of Reg protein led to a significant increase in islet cell size, insulin positive β-cell number and amelioration of diabetes. They also demonstrated a threefold increase in the incorporation of $^{3}$H-thymidine into cultured rat islet cells exposed to recombinant rat Reg protein. Autoradiography demonstrated a limited increase in the proliferative index of insulin positive cells from 0.1% in controls to 0.2% in Reg treated cultures. Based on these experiments, Watanabe et al., claimed that the trophic effects of Reg protein explain its ability to stimulate β-cell regeneration and ameliorate diabetes.\n\nIn contrast, Smith et al., (1994) argue against reg playing a role in islet cell growth from experimental evidence employing a model of selective β-cell growth induced by glucose infusion for 96hrs. Such a procedure produces a 50% increase in the rat islet β-cell mass with a fivefold increase in the proliferative index of islet β-cells (Bonner-Weir et al., 1989). Using this model Smith et al., (1994) could find no evidence of increased reg expression during or after the glucose infusion. Isolated rat islets in culture however, do respond to glucose exposure, both by increased reg expression and increased $^{3}$H-thymidine incorporation (Francis et al., 1992).\n\n**Reg expression in endocrine cells of the stomach**\n\nIn 1996, Ashahara and colleagues reported the presence of reg in the endocrine cells of the rat stomach. In fact, by using the techniques of in situ\n\nhybridisation and immuno-histochemistry they clearly demonstrated that the large majority of *reg*-positive cells were the ECL cells. Furthermore, *reg* mRNA abundance was upregulated during the period of mucosal repair following stress ulceration induced by water immersion. Plasma gastrin concentrations and acid output were not measured. Nevertheless the presence of *reg* in ECL cells and its upregulation during ECL cell regeneration suggest that *reg* may play a role in the growth and/or differentiation of the gastric ECL cell.\n\nIn summary, *reg Iα* is a major member of the *reg* family and is expressed during pancreatic islet cell regeneration and in the rat gastric ECL cell. The protein product of the gene stimulates the formation of mature endocrine cells in the regenerating pancreas. Expression of *reg* is associated with growth and differentiation of endocrine cells but its exact function as trophic factor or promoter of differentiation remains to be determined. The ectopic expression of *reg* and related family members in tumours of the gastrointestinal tract and liver justifies the further studies required to elucidate the exact role of the *reg* gene and its protein product. The possibility that the *reg* gene is involved in the development of gastric carcinoid tumours in patients with hypergastrinaemia is examined in this thesis.\n\nAims and Objectives\n\n1. To develop a model of localised denervation of the gastric antrum in the rat in order to study the role of intrinsic antral neurons in the regulation of gastric function.\n\n2. To examine the effects of lesioning of antral denervation on gastric emptying in the rat to test the hypothesis that intrinsic neurons of the gastric antrum regulate gastric emptying and mediate the actions of extrinsic reflexes already known to influence gastric emptying.\n\n3. To examine the effects of antral denervation on the control of gastrin release in the rat in order to test the hypothesis that antral neurons regulate the responses of the G-cell to feeding and gastric distension.\n\n4. To investigate *reg* gene expression in the pancreatic cell line AR42J in order to test the hypothesis that gastrin and EGF may regulate *reg* expression.\n\n5. To determine the relationship between *reg* expression and hypergastrinaemia in both rat and man to test the hypothesis that gastrin stimulation of ECL cell function and growth are associated with upregulation of *reg* expression.\n\n6. To investigate the possibility that mutations of the *reg* gene (a putative regulator of endocrine cell differentiation) are associated with carcinoid tumour development as a consequence of chronic hypergastrineamia.\n\nChapter Two\n\nMaterials and Methods\n\nMaterials\n\nAll laboratory chemicals that can be considered as in routine use were obtained from either BDH chemicals, Poole U.K., or from Sigma Chemicals limited, Poole, U.K.. The materials used for routine molecular biology experiments that were not available from the above companies were obtained from either Gibco GBRL, Renfrewshire, Scotland, or from Severn Biotech, Worcester, U.K.. All radioactive materials were obtained from Amersham Life Sciences, Buckinghamshire, U.K.. Other specific items are listed below.\n\nDrugs/Chemicals:\n\n- Benzalkonium chloride: Sigma Chemicals Ltd.\n- Bovumin: First link, West Midlands, U.K.\n- BW2258U89: Dr J Leban, Burroughs Wellcome, U.S.A.\n- Capsaicin: Sigma Chemicals Ltd.\n- Diazepam (Valium): Roche products Ltd., Derbyshire U.K.\n- Enrofloxacin: Bayer Health Business Group, Suffolk, U.K.\n- Fentanyl/Fluanisone (Hypnorm): Janssen Animal Health, Buckinghamshire, U.K.\n- L-740093: Merk Sharp and Dohme, Harlow, U.K.\n\nPeptides:\n\nGastrin Peninsula Laboratories, St Helens, UK.\n\nporcine GRP\\textsubscript{14-28} Bachem (uk) Ltd., Saffron Walden, U.K.\n\nrat Tyr\\textsuperscript{0}\\alpha-CGRP\\textsubscript{28-37} Bachem (uk) Ltd., Saffron Walden, U.K.\n\nSubstance P Peninsula Laboratories, St Helens, UK.\n\nporcine VIP Peninsula Laboratories, St Helens, UK\n\nRestriction endonucleases:\n\nApa I Promega, Southampton, U.K.\n\nNot I Gibco GBRL, Renfrewshire, Scotland\n\nNru Gibco GBRL, Renfrewshire, Scotland\n\nSpe I Gibco GBRL, Renfrewshire, Scotland\n\nXba Promega, Southampton, U.K.\n\nCloning Vectors:\n\npGEMT-Easy Vector Promega, Southampton, U.K.\n\npcDNA3.1 ZEO Invitrogen, De Schelp 12, Netherlands\n\nMethods\n\nAnimals\n\nAdult Wistar rats (approximately 250g) were kept on a 12hr light/dark cycle and fed standard laboratory rat chow. Rats were fasted overnight before operations and refeeding experiments but allowed free access to water. Rats were anaesthetized by i.m. injection of a mixture of fentanyl citrate (0.32 mg.kg$^{-1}$), fluanisone (4 mg.kg$^{-1}$) and diazepam (2mg.kg$^{-1}$). Post-operatively, rats were given buprenorphine (0.1mg.kg$^{-1}$) for analgesia, antibiotic prophylaxis (enrofloxacin 5mg.kg$^{-1}$) and 10ml of saline by subcutaneous injection.\n\nBenzalkonium chloride treatment\n\nThe stomach was exteriorised through a midline upper abdominal incision. The serosal surface of the region to be denervated was covered with a layer of gauze cut to size. Benzalkonium chloride (0.02%, 0.1%, or 0.5% w/v) dissolved in 0.9% NaCl was painted onto the gauze, which was kept moist with the BAC solution for 3 minutes, and then removed. The serosal surface of the stomach was then rinsed thoroughly with normal saline and returned to the peritoneal cavity and the incision closed in layers. The two faces of the stomach were treated separately; in some experiments only the anterior face of the antrum was treated. A similar method was used for denervation of the corpus employing a strip of gauze (approximately 5mm wide) placed over the corpus alongside the border of the glandular and non-glandular regions. In order to selectively lesion the afferent innervation of the stomach, the same experimental approach was employed using\n\nthe sensory neurotoxin capsaicin (1% w/v in olive oil) in place of BAC. Control animals underwent a sham operation with serosal application of normal saline (or olive oil). Animals were allowed to recover for at least 11 days before experiments.\n\n**Refeeding experiments**\n\nControl and BAC-treated rats were fasted for 24hrs on wire-bottomed cages, and were then either maintained as fasted controls or allowed to refeed for 30 minutes. In some experiments rats were injected subcutaneously 30 minutes before refeeding with either a GRP antagonist BW2258U89 at a dose of 2mg.kg$^{-1}$ (Moody et al., 1995) or with vehicle (0.14M saline). Rats were killed by cervical dislocation, decapitated and trunk blood was taken for assay of plasma gastrin. Samples of antral and corpus mucosa were taken for mRNA extraction (see below) and the stomach content was removed. Gastric retention was determined by the dry weight of solid material in the stomach.\n\n**Gastric fistula rats**\n\nRats were treated by application of either BAC or saline to the antral serosa as described above and a small stainless steel Gregory cannula was installed in the corpus as previously described (Dimaline et al., 1986). The cannula was exteriorised through a midline stab incision and the incision closed in layers. Rats began training in Bollman cages on the tenth post operative day.\n\nGastric emptying studies\n\nGastric fistula rats were allowed at least 11 days to recover from surgery before experiments. Prior to experiments the stomach was washed with warm 0.14M NaCl to remove solids. On the experimental day, the emptying of test solutions (saline, 50mM HCl, 4.5% peptone, containing phenol red, 60mg.l\\(^{-1}\\)) instilled into the stomach via the gastric fistula, was determined as previously described (Forster et al., 1990).\n\nGastric distension studies\n\nPrior to experiments the stomach was washed extensively via the gastric cannula with warm 0.14M NaCl to remove solids. Rats were left for at least 90 minutes before experiments began. Plasma gastrin responses were determined after gastric distension with the following test solutions: 2% (w/v) methyl cellulose (2%MC); 2%MC at pH6 (50mM ammonium acetate); 2% MC with 50mM HCl; 4.5% w/v peptone. In each case the stomach was distended by connecting the cannula to a reservoir of test solution and elevating the reservoir to 5cm H\\(_2\\)O pressure for 30mins. Immediately following distension, rats were killed by cervical dislocation and decapitation and 1.5ml of trunk blood was then taken for gastrin radioimmunoassay. Samples of antrum from control and BAC-treated rats were taken to confirm denervation.\n\nMeasurement of acid output\n\nRats fitted with a gastric cannula were trained in Bollman cages to permit the required handling of the cannula before acid output could be measured. Prior to experiments, rats were fasted overnight. On the day of experiment the stomach was\n\nwashed extensively with warm 0.14M NaCl to remove solids. Rats were left for at least 90 minutes before experiments began. Gastric acid output was determined by collecting gastric secretion over four sequential 15 min periods and titrating to pH 7.0 with 20mM NaOH using a pH autotitrator as previously described (Dimaline, et al., 1986).\n\n**Omeprazole treatment**\n\nRats were given omeprazole (400μmol.kg\\(^{-1}\\)) by gavage under light Halothane anaesthesia. Rats were fed *ad libitum* and received omeprazole daily for five days. On the fifth day rats were killed by cervical dislocation and decapitation and 1.5ml of trunk blood was taken for gastrin radioimmunoassay. Samples of gastric corpus were taken for total RNA extraction (see below).\n\n**Radioimmunoassay**\n\nThe neuropeptide content of tissues was determined after extraction with boiling water (0.1 g.ml\\(^{-1}\\)), homogenizing and centrifuging and re-extraction of the pellet with 0.5M acetic acid, as previously described (Hutchison et al., 1981). Water or acid extracts were assayed at the appropriate dilution for the following peptides: Gastrin releasing peptide (GRP) was assayed using antibody 1078, with porcine GRP 14-28 as standard and iodinated Tyr\\(^1\\) COOH-terminal decapeptide of bombesin as label, as previously described (Dockray et al., 1979). Substance P was assayed with antibody L83, using undecapeptide substance P as standard and \\(^{125}\\)I-Bolton Hunter labelled substance P as label. Vasoactive intestinal polypeptide (VIP) was assayed using antibody L25, with porcine VIP as standard and \\(^{125}\\)I labelled VIP as label as previously described (Hutchison\n\nCalcitonin gene related peptide (CGRP) was assayed with antibody L273, using rat Tyr\\textsuperscript{0}a-CGRP 28-37 as standard and the same peptide labelled with \\textsuperscript{125}I as label as previously described (Varro et al., 1988). The concentration of amidated gastrins in plasma gastrin was assayed using antibody L-2, using synthetic human non-sulphated G-17 as standard and for labelling with \\textsuperscript{125}I as previously described (Dockray et al., 1991). The standard conditions for each assay are shown in Figure 2.1\n\n**Extraction of RNA**\n\n**Extraction of Tissue RNA:**\n\nTissue mRNA was extracted and quantified as described previously (Dimaline et al., 1991). Samples of antral or corpus mucosa were homogenised in 3mls of 4M guanadinium isothiocyanate buffer containing 25mM sodium acetate pH 6.0 and 0.84% (vol/vol) β-mercaptoethanol and frozen at -80°C until required. After thawing at room temperature, samples were pipetted onto a 5.7M caesium chloride cushion (1.3ml) in an ultracentrifuge tube. Samples were centrifuged overnight at 41,100 rpm to pellet the RNA. The mRNA pellet was dissolved in 300µl of Tris-EDTA (TE) buffer with 0.2% SDS and precipitated with 0.3M sodium acetate under 100% ethanol overnight.\n\n**Extraction of Cell RNA**\n\nRNA extraction from cultured cells was performed using the Quickprep™ total RNA extraction kit (Pharmacia Biotech, Herts, U.K.) according to the manufacturers instructions. The media (2ml) was removed from cultured cells and replaced by 900µl of a mixture of lithium chloride buffer, β-mercaptoethanol and extractoin buffer. Cells were\n\n| Peptide Assay | Antibody/Dilution | BUFFER | INCUBATION | SEPARATION |\n|---------------|-------------------|--------|------------|------------|\n| | | | | Ch:Dx:MPr* |\n| GASTRIN | L2 - 1:100,000 | Na⁺Bicarbonate pH 8.4, 0.5% Bovumin, 10mM Na⁺ Azide | 72hrs : 4 °C | 10 : 1 : 0.5 |\n| CGRP | L271 - 1:80,00 | 50mM PO₄, pH 7.4, 0.5% Bovumin, 0.25% EDTA, 0.14M NaCl | 72hrs : 4 °C | 10 : 1 : 1 |\n| GRP | 1078 - 1:80,000 | Na⁺Bicarbonate pH 8.4, 0.5% Bovumin, 10mM Na⁺ Azide | 48hrs : 4 °C | 10 : 1 : 0.5 |\n| SUBSTANCE P | L-83 - 1:400,000 | 50mM PO₄, pH 7.4, 0.5% Bovumin, 0.14M NaCl, 10mM Na⁺ Azide | 48hrs : 4 °C | 10 : 1 : 0.5 |\n| VIP | L-25 - 1:40,000 | 20mM PO₄, pH 6.0, 0.4% BSA, 0.1% EDTA, 10mM Na⁺ Azide, 0.025% w/v polybrene | 48hrs : 4 °C | 10 : 1 : 0.5 |\n\nFig 2.1 Conditions for each radioimmunoassay are provided in the table. *For separation of the assay a mixture of charcoal dextran and milk protein (Marvel) in the above ratio (g/100ml) was added to each tube.\n\nresuspended in the solution by scraping and then pipetted into a universal container. The cells were then homogenised and the lysate transferred to a 1.5ml eppendorf containing 600µl of caesium trifluoroacetate solution and left to stand on ice for ten minutes. RNA was pelleted by centrifugation at 15000rpm for 15 minutes in a microcentrifuge. The RNA pellet was washed thoroughly, allowed to dry and resuspended in 10-20µl of diethyl pyrocarbonate-treated (DEPC) water before quantification. Any unused RNA was precipitated with 0.3M sodium acetate under 100% ethanol and stored at -80°C.\n\n**RNA electrophoresis**\n\nStored precipitated RNA was pelleted by centrifugation at 13000rpm for 15 minutes, washed twice in 70% ethanol/DEPC H₂O and once in 100% ultrapure ethanol. Pellets were air dried at room temperature and dissolved in 10-50µl of DEPC H₂O. RNA samples were quantified by spectrophotometer and 10µg aliquots added to denaturing buffer (formamide: 37% formaldehyde: 10 x MOPS (3-(4-Morpholino) propane sulfonic acid) in ratio 4:1.75:1 and heated to 55 °C for 15 minutes. Samples were then placed on ice and 2µl of RNA loading buffer added to each sample. The denatured RNA was then electrophoresed in a 1x MOPS/2% formaldehyde 1% agarose gel.\n\n**Northern Blot and Hybridisation**\n\nAfter electrophoresis, RNA was electroblotted to nylon membranes (Nytran 0.5) overnight in 0.5% TAE buffer (containing 0.2M Tris base, 1M acetate and 0.1MEDTA) at 4 °C. RNA was crosslinked to the nylon membrane by ultraviolet light (UV Cross Linker, Hoeffer scientific instruments, San Francisco, USA). Membranes were placed in\n\nhybridisation buffer (50% formamide containing 5 x Denhardts solution, 5 x sodium chloride sodium phosphate EDTA buffer (SSPE), 0.5% SDS and sonicated salmon sperm DNA (200μg.ml\\(^{-1}\\)) and warmed to 65 °C for 4 hours prior to the addition of \\(^{32}\\text{P}\\)-labelled cRNA probe (2x10\\(^6\\) cpml\\(^{-1}\\)). Membranes were hybridised overnight and then washed twice with 2xSSPE containing 0.1% SDS at room temperature for 20min, and once with 0.1xSSPE/0.1% SDS at 65°C for 20min. Signals were removed from membranes using boiling 0.1% SDS and membranes rehybridized with alternative specific probes as required. Signals were quantified using a Phosphor Imager.\n\n**Reverse transcription/PCR**\n\n*Reverse transcription* of RNA was performed by first annealing 1μl oligo dTs (500μg.ml\\(^{-1}\\)) to ≈50μg of total RNA by heating to 55°C for 5 minutes and cooling slowly. cDNA was generated by incubating the following reaction at 42°C for 1 hour.: oligo dT and RNA (10μl), 2μl DTT(100mM), 1μl dNTPs (25 mM), 1μl RNAsin, 4μl 5x AMV RT Buffer and 2μl AMV reverse transcriptase. The cDNA generated was purified by phenol/chloroform extraction, precipitated with 0.5M ammonium acetate and washed with 100% ethanol. The cDNA was then resuspended in 100μl of TE. 5μl of this solution was used as template for PCR.\n\nHuman *reg* cDNA was generated by reverse transcription of total RNA extracted from gastric mucosal biopsies taken from either normal corpus or from carcinoid nodules. Two different size products were generated by separate pairs of primers chosen to include either a 297bp central portion of the coding region.\n\ncontaining an *EcoR* I restrictiton site, or the full length coding sequence. The smaller fragment was used as template for riboprobe synthesis (see below) and the larger fragment (557bp) used for cloning and sequencing (see below). Primers were chosen based on the full coding sequence of the published human *reg* mRNA (Terazono *et al.*, 1988) as follows: The 297 bp fragment: sense - TCTCCTGCCTGATGTTTCTGTCTC antisense - GGCGGTTCTTTTTGGGGTCAT, The 557bp fragment: sense GATTGTTGATTTGCCCTCTTA, antisense - TCCAGCTGCCTCTAGTTTTTGAA.\n\nRat *reg* cDNA was generated by reverse transcription of total RNA extracted from rat pancreas. PCR primers were chosen to include the full coding sequence of the published rat *reg* mRNA (Terazono *et al.*, 1988) as follows: sense - AGCCTGCAGAGATTGTTGACTTTG; antisense - AGGGGGTTGACTTTGCTTTTGATA and gave a predicted product size of 651bp (Figure 2.2). Restriction digestion of the 651bp PCR product with *EcoR*1 cleaved the product into 253 & 398bp fragments.\n\n*Polymerase chain reaction* was performed under standard conditions in a final volume of 100µl containing 10x *taq* polymerase buffer (10µl), MgCl (1.5mM), dATP (0.2mM), dCTP (0.2mM), dGTP (0.2mM), dTTP (0.2mM), template cDNA (5µl), sense and antisense primers (5ng. µl$^{-1}$) and *taq* polymerase (0.5µl). 60µl of mineral oil was layered over the reaction mixture which was then placed into a thermal cycler (Hybaid, Middlesex UK). After a 5 minute denaturation at 95°C, thirty cycles consisting of annealing (1 minute at 58 °C), extension (2 minutes at 72°C) and denaturation (1 minute at 95°C) were performed. A final extension step (72 °C) was performed for ten minutes before determining the outcome by agarose gel electrophoresis.\n\nFigure 2.2 PCR products of human and rat *reg* cDNA using human carcinoid tumour RNA and rat pancreatic RNA as template for reverse transcription. The predicted sizes of the human PCR products were 557bp (lane 1) and 297bp (lane 2) depending on the primers used. The predicted size of the rat PCR product was 651bp (lane 3). See text for full details.\n\nDNA Ligation\n\nThe PCR fragments of human and rat *reg* cDNA were ligated into the pGEM-T Easy vector by incubating approximately equimolar concentrations of PCR product and vector in the presence of DNA ligase at 4°C overnight according to the manufacturer's instructions.\n\nCloning and Sequencing\n\n*Bacterial Transformation:* Chemically competent JM109 bacterial cells were stored at -80°C. Prior to transformation, cells were thawed on ice for 20 minutes before addition of 2 µl of 8.7M β-mercaptoethanol and 1 µl of DNA ligation mixture. Incubation on ice was continued for a further 30 minutes. The cells were then heated to 42°C for 45 seconds and immediately placed on ice for a further 2 minutes. Thereafter, 450 µl of SOC medium (containing 2% Tryptone, 0.5% Yeast extract, 10 mM NaCl, 2.5 mM Kcl, 10 mM MgCl₂·6H₂O and 20 mM glucose) was added and the reaction incubated at 37°C for one hour with constant gentle agitation.\n\nAfter this incubation a 100 µl aliquot was spread onto petri dishes containing 20 mls of LB/agar (1% Tryptone, 0.5% Yeast extract, 1.0% NaCl, 1.5% agar) that had been treated previously with 100 µl isopropyl-β-D-thiogalactosidase (IPTG 0.1 M), 30 µl 5-bromo-4-chloro-3-indolyl-β-D-galactosidase (XGAL 0.2 M) and 200 µl of ampicillin (10 mg.ml⁻¹). The remaining cell suspension was pelleted by centrifugation (5000 rpm for 3 minutes) and the cells resuspended in 200 µl of SOC medium. A further 100 µl aliquot\n\nof cells, now at a higher cell density, was plated onto a LB/agar plate. Plates were then incubated overnight at 37°C and selection of successfully transformed colonies was by colour selection and antibiotic resistance. Successfully transformed colonies (white) were sub-cloned onto a second LB/agar plate and into 3ml of LB medium containing ampicillin (0.05mg.ml\\(^{-1}\\)). Following a further overnight incubation at 37 °C, the amplified plasmid DNA was extracted and purified for restriction digestion (to confirm presence of the expected cDNA sequence insert) and sequencing.\n\n**Plasmid DNA extraction** Small, medium and large scale plasmid DNA extraction was performed using commercially available kits according to manufacturers instructions (Wizard Minipreps, Midipreps and Maxipreps). The protocol is similar for each with adjustments for scale depending on the yield of plasmid DNA required. In general, small scale preparations were sufficient for sequencing purposes. Medium scale preparations were used to generate templates for cRNA probe synthesis and large scale preparations were used for generating linearised plasmid DNA for permanent cell transfection (see below).\n\nFollowing overnight culture at 37 °C, bacterial cells were pelleted by centrifugation, the supernatant discarded and the cells resuspended in resuspension buffer. The cells were then lysed by addition of lysis solution (NaOH/SDS) and protein and bacterial DNA were precipitated by addition of a neutralising solution (potassium acetate). The precipitate was removed by centrifugation and the plasmid DNA extracted from the supernatant by addition of a high affinity resin. This resin with bound DNA was\n\nimmobilized to a column by vacuum suction. The column was then washed twice (80% ethanol) and the DNA eluted by addition of TE buffer (68 °C) followed by centrifugation with collection of the eluate. The DNA content and purity was measured by spectrophotometry.\n\n**Riboprobe synthesis**\n\nTemplates for generating cRNA probes for gastrin, HDC, somatostatin and GAPDH were provided by Dr R. Dimaline and contained the appropriate template as described previously (Dimaline *et al.*, 1991; 1993a; 1994). The template for generating the cRNA probes for rat and human *reg* were prepared by restriction fragment digestion of midiprep purified pGEM t-Easy vector containing the full length rat *reg* sequence or the 253bp human *reg* sequence as described above. After sequencing the vector to determine orientation of the insert, 10µg of each sample was digested with restriction enzyme *ApaI* in appropriate enzyme buffer in a volume of 20µl for 3 hours at 37°C. Complete linearisation of plasmid DNA was confirmed by gel electrophoresis. 0.5µl of template was used for riboprobe synthesis. The reaction conditions were as follows: 2µl 5x SP6 buffer, 1µl DTT(10mM), 2.5µl DNTPs (100µM each ATP, UTP, GTP), 1µl CTP (10µM), 0.5µl RNase, 0.5µl template DNA, 0.5 µl RNA polymerase (1-5 units) and 2.5µl $^{32}$P-CTP ($\\approx$ 5µM). The reaction mixture was incubated at 37°C for 1 hour and then RNase free DNase added for a further 15 minutes. The synthesised probe was purified by size exclusion and 1µl of purified probe placed in 5ml of scintillation fluid and\n\nincorporated radioactivity counted. Labelled probe was used at a concentration of $2 \\times 10^6$ cpm per ml of hybridisation buffer.\n\n**Preparation of DNA for permanent transfection**\n\nTwo sequences of the human *reg* gene were chosen for permanent transfection into the hamster insulinoma tumour cell (HIT) line. These were the full length coding sequence of the normal (wild type) *reg* and the sequence in which the first methionine residue (initiator methionine) had been mutated to a valine (ATG→GTG). Representative clones containing each sequence were prepared by small scale DNA extraction (Miniprep). The purified plasmid DNA (>600ng in 15μl) was then restriction digested with the combined endonucleases *Spe*I and *Not*I at 37 °C for 3 hours. The restriction fragment containing the *reg* sequence was then purified by gel electrophoresis (1.5% agarose). The DNA band of correct size (∼600bp) was cut from the gel and DNA removed from the gel onto a column by centrifugation (13,000rpm for 60 seconds), and eluted from the column by the addition of 20μl sterile water followed by centrifugation at 13,000rpm for 20 seconds. A small aliquot of the DNA was again gel electrophoresed to assess purity and concentration (20ng.μl$^{-1}$).\n\nEach restriction fragments was then ligated into the mammalian expression vector pcDNA3.1 Zeo. The vector circular DNA was linearised by concomitant digestion with the restriction endonucleases *Xba*I and *Not*I. (The *Xba*I restriction leaves an identical sequence overhang to that of *Spe*I used to create the insert DNA). Complete digestion by both enzymes was presumed by serial examination of control vector digests.\n\nby each restricton endonuclease separately. After 4 hours, the linearised vector was gel purified and eluted. The concentration of vector and its purity was estimated by gel electrophoresis.\n\nLigation of the wild type and mutant *reg* inserts was achieved by addition of equimolar concentrations of insert and vector DNA in the presence of DNA ligase and appropriate buffer. The ligation reaction was incubated overnight at 14°C. In addition, a control ligation reaction was also performed to ensure that complete digestion by both endonucleases occurred whilst preparing the vector DNA. This consisted of linearised vector without any insert. As the *Xba* and *Not I* restriction site overhangs are incompatible, re-ligation should not occur without the presence of insert DNA. Hence no colonies should form with cloning of the control ligation. Conversely, any colonies present from the *reg* insert ligations should all be positive for insert DNA. This selection process was required as the pcDNA3.Zeo vector does not incorporate the lac operon gene that allows for colour selection.\n\nFollowing cloning into JM109 cells, colonies from each of the wild type and mutant *reg* transformations were selected for small scale DNA preparation. The presence of a *reg* insert was shown by restriction digestion with *EcoRI* and then confirmed by sequencing. Positive clones for each sequence were then processed for large scale DNA preparation to yield $\\approx 300 \\mu l$ of concentrated DNA (3-5 $\\mu$cg.$\\mu l^{-1}$). For each permanent transfection, 10 $\\mu g$ of linearised DNA (in <20 $\\mu l$) was required. Therefore, the appropriate volume of each vector containing wild type and mutant *reg* inserts were linearised by\n\ndigeston using the restriction endonuclease *Nru*. Linearised DNA was incorporated into cultured HIT cells by electroporation (see below).\n\n**Tissue culture and permanent transfection**\n\nThe hamster insulinoma cell line HIT T15 was grown in RPMI 1640 medium supplemented with 11mM glucose, 10% v/v foetal calf serum, 2.5ug.ml\\(^{-1}\\) fungizone, 100U.ml\\(^{-1}\\) penicillin and 100µg.ml\\(^{-1}\\) streptomycin (all from GIBCO BRL, Life Technologies, Paisley, Scotland). Stably transfected cell lines expressing wild type or mutant human *reg* were produced by electroporation of \\(5 \\times 10^6\\) cells in 0.8ml at 200V and 1070µF in incomplete medium containing 10µg of linearised pcDNA *reg* construct. Cells were grown in complete RPMI in 10cm dishes for 48 hours prior to selection with zeocin (750µg/ml\\(^{-1}\\)) (Invitrogen). Six colonies expressing each construct were cloned and screened for *reg* expression by northern blot.\n\nThe rat pancreatic cell line AR42J was grown in RPMI 1640 medium supplemented with 10% v/v foetal calf serum. For experiments, cells were plated out at \\(1.5 \\times 10^6\\) cells in 5ml culture medium / 60ml dish and grown at 37°C/ 5% CO2. Following a 24 hour growth period, the cells were washed and their media replaced with incomplete RPMI with the following stimulii or appropriate vehicle (control). Human gastrin-17 (HG17), rat gly-extended gastrin-9 (G-Gly) in 50mM ammonium bicarbonate, wortmannin, mouse salivary gland extract (EGF) in RPMI, L740093 in 1x10\\(^{-6}\\)% DMSO. In all cases except for wortmannin, the cells were incubated for 24\n\nhours at 37°C / 5%CO₂ prior to RNA extraction. In the case of wortmannin, the cells were incubated for 4 hours prior to RNA extraction.\n\n**Statistical analysis**\n\nThe statistical test used for each experiment is indicated in the appropriate figure legend. In general, for the experiments involving denervation, the data was analysed using Mann Whitney U test and for other analyses Student’s t test was used.\n\nChapter Three\n\nChemical denervation of antral neurons\n\nIntroduction\n\nThe gastric emptying of liquids is dependent on the pressure gradient between the lumen of the stomach and proximal duodenum, and the resistance to flow between the two (Mayer et al., 1994). It has long been known that the rate of emptying depends on the nature of the luminal contents; thus, acid, hyperosmolal solutions, high viscosity, protein- and fat-rich meals all delay gastric emptying (Mayer et al., 1994). The emptying of liquids is pulsatile in nature and this can be attributed to phasic contraction of the pyloric antral region of the stomach (Houghton et al., 1988; Malbert et al., 1991; Prather et al., 1993). Alterations in the flow of gastric content across the antrum can be effected by changing either resistance to flow generated by antral contractions or the overall tone of the gastric body and the duodenum. There is a significant body of evidence to indicate the importance of vagal afferent innervation of the proximal gut (Raybould et al., 1988; Forster et al., 1990; Holzer et al., 1994) and vagal efferent innervation of the gastric corpus (Forster et al., 1991; D’Amato et al., 1992) in regulating delayed gastric emptying by inducing gastric corpus relaxation in response to hyperosmolal, protein-rich or fat-rich solutions in the duodenum. However, much less is known of the role of intrinsic antral neurons in the control of pyloric flow.\n\nAntral neurons however are known to innervate both the muscle layers of the antrum and the mucosal epithelial cells including endocrine cells (Gabella, 1987; Furness and Costa, 1987; Ekblad et al., 1987). The clearest example of this and the most relevant in terms of digestive physiology of the stomach is the innervation of Gcells of the gastric antrum by GRP-immunoreactive neurons that have their cell bodies in the myenteric plexus (Dockray et al., 1979; Miller et al., 1989). Systemically administered GRP has been shown to stimulate gastric acid output by the release of gastrin from G-cells in both animals and man (Varner et al., 1981; Bunnett et al., 1985). Further evidence that GRP exerts physiologically regulates the G-cell comes from the demonstration that G-cells express GRP receptors (Vigna et al., 1990) and GRP is capable of stimulating the release of gastrin from G cells in primary culture (Giraud et al., 1987).\n\nThe experiments described in this chapter were undertaken to define the anatomical lesion induced by chemical denervation of the rat antrum. This would then allow potential study of the role of the antral innervation in gastric emptying and the regulation of mucosal endocrine cell function. To this end I used the detergent benzalkonium chloride (BAC) which has already been used to lesion small intestinal myenteric neurons (Fox et al., 1983; Dahl et al., 1987). In keeping with the observations of others (Fox et al., 1983; Dahl et al., 1987) I was able to demonstrate that BAC application to the rat stomach produces a highly specific, reproducible and permanent loss of the all neurons in the treated region. Following antral denervation, the intrinsic innervation of the corpus is spared, but there was loss of the spinal afferent innervation of the gastric corpus. Conversely, when a band of proximal gastric corpus was denervated, intrinsic and extrinsic antral innervation was preserved.\n\nMethods\n\nAll operations were performed on Wistar rats. For antral denervation rats were starved overnight, but for corpus denervation rats were fed *ad libitum*. Following exposure of the stomach, BAC (0.02%, 0.1%, or 0.5% w/v) dissolved in 0.9% NaCl was painted onto gauze, which was kept in contact with the region to be treated and kept moist with the BAC solution for 3 minutes, and then removed. The two faces of the stomach were treated separately; in some experiments only the anterior face of the antrum was treated. In order to selectively lesion the afferent innervation of the stomach, the same experimental approach was employed using the sensory neurotoxin capsaicin (1% w/v in olive oil) in place of BAC. Control animals underwent a sham operation with serosal application of vehicle (normal saline or olive oil). Animals were allowed to recover for at least 11 days before experiments. Denervation was assessed by radioimmunoassay of neuropeptides in extracts of BAC-treated and adjacent tissues.\n\nResults\n\nDose-Response Relationship.\n\nInitial experiments were directed at determining the optimal dose of BAC for antral denervation. The tissue concentrations of GRP, CGRP, substance P and VIP measured by radioimmunoassay of antral extracts were reduced in a dose dependent manner 11 days after prior administration of BAC (Fig 3.1). At a concentration of 0.02% BAC had no effect; 0.1% BAC produced a 25% loss of CGRP, but following 0.5% w/v BAC all neuropeptides were depleted. The concentrations of substance P, VIP and GRP in extracts of the gastric corpus were not affected by prior application of BAC to the antrum (Fig 3.2). However, the concentration of CGRP in the corpus was significantly reduced by application of 0.5% BAC to the antrum. (Fig 3.2)\n\nTime Dependency of Responses\n\nIn order to determine the duration of responses to 0.5% BAC, neuropeptide concentrations were examined in extracts of tissues sampled 11 to 25 days after treatment. All neuropeptides assayed were depleted in antral extracts 11 days after BAC, and remained substantially reduced at all times up to 25 days (Fig 3.3). Similarly there was loss of CGRP in both antrum and corpus in rats treated with BAC on the antrum at all times examined (Fig 3.4).\n\nFig 3.1 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of antrum following serosal application to the antrum of BAC or vehicle (control). Only at 0.5% w/v BAC was there a significant loss of neuropeptides derived from both intrinsic and extrinsic afferent neurons of the antrum. (n = 6 all groups; * p<0.05, compared with control, Mann Whitney U test; in this and subsequent figures, values are means ± SEM).\n\nFig 3.2 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of the antrum 11 (n=5), 18 (n=5) and 25 (n=3) days after application of 0.5% w/v BAC or vehicle (control, n=6) to the serosa of the antrum. The loss of all neuropeptides was complete at day 11 and unchanged thereafter. (* p<0.05, compared with control, Mann Whitney U test)\n\nFig 3.3 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of corpus following serosal application to the antrum of BAC or vehicle (control). Even at 0.5% w/v BAC there was no significant loss of neuropeptides derived from intrinsic neurons of the Corpus. However at 0.5% w/v BAC there is a significant loss of corpus afferent innervation as shown by the significant reduction in corpus CGRP levels. (n = 6 all groups; * p<0.05, compared with control, Mann Whitney U test)\n\nFig 3.4 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of corpus 11 (n=5), 18 (n=5) and 25 (n=3) days after application of 0.5% w/v BAC or vehicle (control, n=6) to the serosa of the antrum. As with the results for the antrum, the selective loss of corpus CGRP after antral BAC was complete at day 11 and unchanged thereafter (* p<0.05, compared with control, Mann Whitney U test)\n\nSpecificity of Lesion\n\nTo examine the specificity of the effects described above, we studied the effect of BAC applied to a circumferential band (approximately 5mm) of the gastric corpus immediately distal to the non-glandular part of the stomach. In the BAC-treated region there was substantial depletion of all the neuropeptides studied (Fig 3.5). In the untreated corpus, and in the antrum, the tissue concentrations of VIP, substance P, CGRP and GRP were similar to control (Fig 3.5). It is worth emphasising that following BAC treatment of the corpus, antral CGRP was unaffected. Given that CGRP is present only in spinal afferent neurons, this result seemed to indicate a specific lesion in the corpus projections of these neurons induced by antral BAC treatment. To confirm this, the effects of the sensory neurotoxin capsaicin applied in a similar fashion to BAC, were examined as this neurotoxin is known to lesion only small diameter afferents leaving intrinsic innervation unaffected. Serosal application of 1% w/v capsaicin to the antrum, produced complete loss of CGRP in extracts of antrum and corpus (Fig 3.6). In contrast, neuropeptides found in intrinsic neurons (VIP and GRP) were not changed by treatment of the antrum with capsaicin (Fig 3.6).\n\nFinally, in some experiments BAC was applied to a single side of the antrum. In this case, there was depletion of neuropeptides on the treated side as already noted, but the concentrations in the untreated side of the antrum were similar to controls (Fig 3.7).\n\nFig 3.5 Neuropeptide concentrations (CGRP, GRP, substance P and VIP) in extracts of antrum, control corpus and a band of proximal corpus treated with either 0.5% BAC (hatched bars) or vehicle (open bars). There was complete loss of the neuropeptides in the band of corpus exposed to BAC but not in the remainder of the corpus, or the antrum. Note in particular the preservation of CGRP in the antrum. (* p<0.05, compared with vehicle, Mann Whitney U test; n = 6 all groups).\n\nFig 3.6 Neuropeptide concentrations (CGRP, GRP and VIP) in extracts of antrum and corpus following topical 0.1% w/v capsaicin (hatched bars), or vehicle (open bars), to the serosal surface of the antrum. The spinal afferent innervation of both the antrum and corpus was lost as shown by CGRP concentrations, while neuropeptides of the intrinsic innervation (GRP, VIP) were unaffected in both regions. (n = 4, both groups; * p<0.05, compared with vehicle, Mann Whitney U test).\n\nFig 3.7 Neuropeptide concentrations (CGRP and GRP) in extracts of either the anterior (a) or posterior (p) surfaces of the antrum (A) and ipsilateral corpus (C) following topical 0.5% w/v BAC (hatched bars) or vehicle (open bars) to the anterior serosal surface of the antrum. Neuropeptides of the intrinsic innervation (GRP, VIP) were reduced only in the BAC treated region. The spinal afferent innervation of both the antrum and corpus was lost as shown by CGRP concentrations predominantly on the side of the stomach that received antral BAC (n = 4, both groups; * p<0.05, compared with vehicle, Mann Whitney U test).\n\nThe experimental data obtained in these experiments provided substantial evidence that application of 0.5% w/v BAC to the serosal surface of the antrum for 5 minutes produced a chronic, profound and sustained loss of both the intrinsic and extrinsic afferent innervation. Although this is in keeping with the studies that have reported the effects of BAC on the rat small intestine (Fox et al., 1983; Dahl et al., 1987; Holle et al., 1990; Jodal et al., 1993), rat colon (Sato et al., 1978, Sakata et al., 1979) and opossum oesophagus (Gaumnitz et al., 1993) the duration of exposure and concentration of BAC used was different. In the previous reports in the rat, the preferred method has been that described by Fox et al. (1983) requiring 0.062% w/v BAC and an exposure time of 30 minutes for each surface of the intestine. Others have used higher concentrations of BAC (0.2%) with the same exposure time (Zuculoto et al., 1991). Fox et al. (1983) demonstrated clearly that the degree of denervation produced by BAC was concentration dependent. Extending these observations, See et al. (1988) confirmed that BAC induces myenteric denervation by disrupting the outer layers of the intestine following application to the serosa. The depth of damage to the intestine was dependent on the concentration used and the length of exposure time.\n\nIn order to reduce the time under anaesthetic, it seemed reasonable to use a higher concentration of BAC and to determine the appropriate dose by a dose response study. The effective dose to induce comprehensive denervation with a 5\n\nminute exposure time was found to be 0.5% w/v BAC as this dose significantly reduced neuropeptide levels to less than 10% of control values.\n\nThe neuropeptides assayed were GRP, CGRP, VIP and substance P. These neuropeptides are representative markers of separate populations of intrinsic and spinal afferent neurons although there is a degree of overlap. Hence GRP is a good and specific marker in the rat antrum for intrinsic myenteric neurons that are secretomotor and stimulate release of gastrin from G cells (Dockray et al., 1979). A proportion of these neurons also contain VIP which, in addition is also found separately in inhibitory motor neurons of the rat myenteric plexus (Ekblad et al., 1987). CGRP is present almost exclusively in spinal afferent neurons (Varro et al., 1988; Sternini et al., 1986; Sternini et al., 1987) in the rat although a proportion of these neurons also contain substance P (Green and Dockray, 1988). However the majority of substance P is found in intrinsic neurons of the myenteric and submucosal plexi (Holzer et al., 1980).\n\nThe distribution of neuropeptides within the wall of the stomach explains to some extent the pattern of loss of as determined by radioimmunoassay. Thus, GRP present predominantly in myenteric neurons was reduced to undetectable levels. Similarly CGRP present in spinal afferent neurons that innervate the serosa and muscle layers of the gut also was reduced to undetectable levels. However VIP and substance P are known to be present also in submucosal neurons and the latter also\n\nmay be found in inflammatory cells. Although there was a significant reduction in VIP and substance P, these peptides were still detectable in tissue extracts.\n\nThe earliest time point that denervation was assessed was 11 days and the latest time point was 27 days. Antral denervation was complete at 11 days and there was no change at the later times studied. This observation is in agreement with previously published experiments on the rat small intestine that have shown complete denervation as early as 24 hours after exposure to BAC (See et al., 1988) and persistence of denervation for up to 160 days (Zuculoto et al., 1991; See et al., 1988). In all the published studies on denervation induced by BAC it has been assumed that the effect was localised to the region of exposure. The data presented here indicate clearly that the spinal afferent innervation of the whole stomach is lesioned by antral BAC treatment. This was shown by loss of CGRP in antral and corpus tissue extracts following antral BAC.\n\nThree lines of evidence suggest that this observation was not due to inadvertent exposure of the corpus to BAC. Firstly, other neuropeptide levels in corpus tissue extracts remained unaffected by antral BAC treatment. Secondly, in the reverse experiment lesioning a circumferential band of proximal corpus did not produce a loss of antral CGRP. Thirdly, exposure to BAC of only the anterior surface of the antrum caused a significant reduction of intrinsic neuropeptides only in tissue extracts of the anterior and not posterior antrum.\n\nThe validity of the observation that lesioning antral spinal afferents removes corpus spinal afferent innervation was confirmed by using the specific small diameter afferent fibre neurotoxin capsaicin. Again exposure of antral spinal afferents to capsaicin reduced CGRP levels in antrum and corpus leaving other neurotransmitters levels unaffected. Taken together these observations suggest that CGRP-containing nerve fibres that supply the gastric corpus also supply the antrum, either by passage over the antrum or by projection of branches to both regions.\n\nFinally, confirmation of the effect of BAC on the gastric innervation described above were supported by immunohistochemical studies performed by Dr. C Vaillant. Samples of antrum and corpus were taken from rats that had either received antral BAC or BAC to a circumferential band of proximal corpus. The results of Dr. Vaillant’s studies essentially confirm the data obtained by radioimmunoassay. Hence there was a complete loss of immunoreactive nerve cell bodies and fibres only in the treated region following exposure to 0.5% BAC. However, specific staining for CGRP demonstrated that following antral application of BAC there was loss of CGRP-immunoreactive fibres from both the antrum and corpus whereas denervation of a band of corpus with BAC resulted in complete loss of immunoreactive fibres in the treated region, but not in the untreated antrum (Higham et al., 1997).\n\nSummary\n\nAntral BAC treatment effectively lesions all myenteric neurons and spinal afferent fibres of the antrum, as well as the spinal afferent innervation of the\n\nremainder of the stomach. The intrinsic innervation of the proximal stomach is unaffected. Consequently there is a loss of innervation of the antral musculature and antral mucosal endocrine cells. The primary aim of developing such a model of complete antral denervation was to study the effects that this would have on gastric motility and antral endocrine cell function. The results of these studies are presented in the next two chapters.\n\nChapter Four\n\nAntral denervation and Gastric Emptying\n\nFor many years, antro-pyloric motility has been recognised to be an important factor controlling the rate of gastric emptying (Canon 1911). More recently, the pulsatile nature of antral contractions and the resting pressure of the pyloric sphincter have been shown to be significant factors governing the delivery of gastric content to the duodenum (Houghton et al., 1988; Malbert and Ruckebusch, 1991; Prather et al., 1993; Dent et al., 1994; Mayer et al., 1994). The likely role of the intrinsic antral innervation in determining the pattern of antral motor contractions has received little attention. This has been primarily because of the lack of available methodology to selectively lesion only antral neurons.\n\nIn contrast the extrinsic innervation of the stomach has been more amenable to study but have been shown to have little long term effects on the pattern of antro-pyloric motility (Quigley and Louckes, 1951; Stoddard et al., 1973). Indeed the majority of studies on the extrinsic reflex control of gastric emptying have concentrated on duodeno-gastric reflexes that regulate gastric emptying by effecting changes in gastric corpus tone either through vagally mediated (Raybould and Tache, 1988; Raybould et al., 1987; Holzer et al., 1994) or spinal afferent pathways (Forster et al., 1990; Forster et al., 1991).\n\nHowever, BAC has been shown to lesion myenteric neurons in a defined segment of rat jejunum and disrupt motor patterns that are dependent only upon an intact intrinsic innervation (Holle and Forth, 1990). In this chapter the data presented are from experiments that have used BAC to lesion the antral innervation to determine the role of antral innervation on the control of gastric emptying. Initially the retention of solid food was assessed and compared to the effects of the lesion produced by antral application of capsaicin. Finally the gastric emptying of a variety of liquid test meals was determined in control and BAC treated rats. The results demonstrate the requirement for an intact antral innervation for normal gastric emptying.\n\nMethods\n\nAntrally denervated and capsaicin treated rats were prepared and after each experiment denervation confirmed by radioimmunoassay of appropriate neuropeptide markers (see methods and chapter 3). Gastric emptying was determined either by studies of the retention of solid material in the stomach of fasted rats or by the determination of the emptying of liquids instilled into the stomach of rats with a gastric cannula. Gastric cannulas were placed at the time of antral denervation and all rats were allowed at least 11 days to recover after surgery.\n\nFor the determination of gastric emptying of solids, rats were fasted for 48 hrs on wire bottomed cages, killed by cervical dislocation and the stomach content weighed wet and again after prolonged drying at $50^0C$. For the determination of gastric emptying of liquids in gastric fistula rats, the stomach was washed extensively with warm 0.14M NaCl to remove solids prior to experiments and then the emptying of test solutions (saline, HCl, peptone: containing phenol red, 60mg.l$^{-1}$) instilled into the stomach via the gastric fistula was determined.\n\nResults\n\nIn fasted rats previously treated with 0.5% BAC on the antrum, there was a striking retention of solids in the stomach compared to controls (Fig 4.1). The wet and dry weight of gastric content was variable in BAC treated rats but all 0.5% BAC-treated rats exhibited overtly distended stomachs (by solid, gas or liquid) after a 48 hr fast. The retention of solid was not apparent after treatment with lower concentrations of BAC (Fig 4.1). However with 0.5% BAC the retention of solids was seen at all time points examined (Fig 4.2). In 48 hr fasted BAC treated rats that were allowed to refeed for 30 minutes, the dry weight of gastric content increased further (Fig 4.3). The dry weight of gastric content was similar to refed control rats.\n\nGiven that BAC causes loss of intrinsic and extrinsic afferent innervation whereas capsaicin lesions only extrinsic afferents (see chapter 3), the latter was used to determine whether the effects of BAC might simply reflect the loss of afferent innervation. Rats that had been pre-treated with capsaicin on the antrum did not exhibit gastric retention after fasting (Fig 4.4). To confirm the importance of the whole antrum in regulating gastric emptying the effects of denervation of a circumferential band of corpus and of one antral surface was also examined. Interestingly, these lesions had little effect on gastric retention of solid (Fig 4.4)\n\nThe emptying of several different types of test meal was significantly delayed in rats previously treated with 0.5% BAC on the antrum (Fig 4.5). It is worth noting\n\nthat in control rats, the emptying of HCl and peptone was inhibited compared with saline. However, in BAC-treated rats there was no significant difference in the emptying of saline, HCl and peptone, even though all of these emptied less rapidly than the corresponding rates in control animals.\n\nIt is known that in intact rats, prior administration of peptone just before an emptying trial with peptone (ie peptone plus preload) produces an exaggerated inhibition of emptying due to enhanced CCK release (Forster and Dockray, 1992). The same effect was observed in the present control rats; in addition, however, peptone plus preload produced a significant inhibition of emptying compared with saline in BAC-treated rats (Fig 4.5). This suggests that CCK might be mediating this effect in BAC-treated rats implying an intact vagally mediated duodeno-gastric reflex. To ensure that the gastric retention in BAC-treated rats was not primarily due to enhanced CCK release, plasma CCK was measured in fasted BAC and control rats. The concentration in control rats ($1.0 \\pm 0.1$ pM) was not significantly different from that in BAC-treated rats ($1.2 \\pm 0.1$ pM).\n\nFig 4.1 Gastric retention after a 48 hr fast in control rats (open bars, n=6) and in rats treated with increasing concentrations of BAC to the antrum (hatched bars, n=6). Only at the highest dose of 0.5% w/v BAC was there significant retention of solid in the stomach (*p < 0.05, Mann Whitney U test).\n\nFig 4.2. Gastric retention at various time points following antral BAC (hatched bars, n=6) or sham operation (open bars, n=6) and after a 48 hr fast. At all time points there was a significant retention of solid within the stomach only in rats treated with BAC to the antrum (*p < 0.05, Mann Whitney U test).\n\nFig 4.3 Gastric retention in control rats (open bars, n=6) and in rats treated with 0.5% w/v BAC to the antrum (hatched bars, n=6) after a 48 hr fast and following a 30 minute refeed. There was a significant retention of solid in the stomach of fasted BAC treated rats compared to controls but after refeeding the dry weight of gastric content was similar in both groups (*p < 0.05, Mann Whitney U test).\n\nFig 4.4 Gastric retention after a 48 hr fast in control rats and rats treated with BAC either to the whole of the antrum, or to the anterior surface of the antrum only, or to a band of corpus adjacent to the non-glandular stomach, or with capsaicin to the antrum.\n\nData shown are for wet weight (hatched bars) and dry weight (open bars). Only BAC treatment of the whole antrum leads to gastric retention (*p < 0.05, Mann Whitney U test).\n\nFig 4.5 Gastric emptying rates for different liquids in gastric fistula rats pre-treated with either 0.5% w/v BAC (n=8, hatched bars) or vehicle (n=6, open bars). For all liquids shown emptying rates are slower in BAC treated rats. In control rats, peptone and HCl empty more slowly than saline, and peptone after a peptone preload empties more slowly than peptone alone. In BAC treated rats, saline, HCl and peptone empty at the same rate, but peptone after a peptone preload induces further delay in gastric emptying.\n\nThe present data provides evidence that the integrity of the intrinsic innervation of the rat antrum is obligatory for the normal emptying of solids and liquids from the rat stomach. Specifically, it was found that the loss of intrinsic antral neurons caused by the detergent BAC resulted in retention of solids, and delayed emptying of both nutritive (peptone) and non-nutritive (saline, HCl) solutions. The retention of solids and liquids could not be attributed to changes in plasma CCK, which is the hormone best known for its ability to inhibit gastric emptying (Dockray 1988) since plasma CCK was normal. In a further series of experiments performed on antrally denervated gastric fistula rats, Dr B Yegen studied the effects of the specific CCK-antagonist (Green et al., 1988) on the emptying of liquid test meals. In these experiments there was no significant difference in the emptying rates of saline, HCl and peptone after L-364,718 in BAC-treated rats compared to BAC rats that did not receive the antagonist. Interestingly, however, the delayed emptying of peptone after a preload was reversed and was comparable to the emptying of saline. It would appear therefore that the action of CCK in controlling gastric emptying can be exerted independently of the antral innervation.\n\nThe application of BAC to the antrum lesioned both the intrinsic and extrinsic afferent innervation; however loss of the latter cannot explain the retention of solids, since there was no retention in rats treated with capsaicin which lesions only extrinsic afferent fibres. Taken as a whole, therefore, the present data lead to the conclusion that\n\nthe antral innervation is essential for the gastric emptying of solid and of both nutritive and non-nutritive solutions, but not for the delaying actions of CCK on gastric emptying.\n\nThe neuroendocrine mechanisms that determine gastric emptying have attracted increasing attention in recent years. In the case of CCK such studies have suggested a primary site of action at receptors on vagal afferent nerve fibres, the activation of which evokes a vago-vagal reflex leading to relaxation of the gastric corpus (Raybould and Tache, 1988; Tache et al., 1988; Forster et al., 1990). The possible involvement of the distal stomach in mediating the effect of CCK on gastric emptying has been suggested by previous studies (Yamagishi and Debas, 1978) but little has been done to determine whether - in this case - CCK acts directly on smooth muscle or indirectly via the innervation. The findings here suggest that any effects of CCK on the innervation of the antro-pyloric region are not of central importance in its role as an inhibitor of gastric emptying.\n\nThe effect of antral denervation on emptying can be considered specific since denervation of a fully circumferential band of proximal corpus (which would also lesion vagal efferent fibres) did not lead to gastric retention. In part the retention of solids after antral denervation may be attributable to a decreased capacity of the antrum for trituration and therefore the reduction of solids to a form suitable for emptying. In addition, however, there is also plainly a marked delay in emptying of liquids. This effect is distinguishable from the reduced emptying of liquids seen after\n\nlesioning of afferent neurons by capsaicin (Forster et al., 1990; Forster et al., 1991) since it is only in antrally denervated rats that there is a substantial delay in the emptying of isosmotic saline.\n\nThe pumping action of the antrum is well known, and contributes to the episodic delivery of gastric contents to the duodenum (Malbert and Ruckebusch, 1991; Dent et al., 1994). We suggest that the co-ordination of this activity requires intrinsic antral neurons and possibly intact efferent vagal neurons and that in the absence of these neurons the antrum generates an unremitting high-resistance to the passage of both liquids and solids. In view of the non-specific effect of BAC in lesioning all neuron populations within the area of exposure (excluding the submucosal neurons) it is not possible to speculate which if any neuron population is essential to maintain normal gastric emptying. It is interesting to note however that targeted disruption of the neuronal nitric oxide synthase gene leads to a phenotype essentially indistinguishable from the BAC treated antrally denervated rat (Huang et al., 1993). NO has been shown to mediate the nonadrenergic, noncholinergic smooth muscle relaxation throughout the gastrointestinal tract (Boeckxstaens et al., 1991) including relaxation of the gastric corpus (Desai et al., 1991). In the NOS knockout mouse, the presumed loss of smooth muscle relaxation in the region of the antrum and pylorus had similar effects to those documented here.\n\nA further possibility that may explain the high pressure resistance in the BAC treated antrum is that the BAC treatment has a direct effect on the muscle itself. It has\n\nbeen apparent from early studies with BAC that following denervation of the small intestine there is significant thickening of both longitudinal and circular muscle layers of the intestine (Fox et al., 1983; See et al., 1988). In addition the muscle became spontaneously contractile with altered mechanical properties including the ability to generate three times the active stress of control smooth muscle in response to muscarinic agonists (Osinski and Bass, 1994). This was later shown to be a consequence of an inability to resequester Ca$^{2+}$ in the sarcoplasmic reticulum leading to increased intracellular calcium (Osinski and Bass, 1995). Although it has been impossible to distinguish between the direct effect of BAC and the secondary effects of loss of normal innervation on smooth muscle function, Hadzijahic et al. (1993) have compared different methods of inducing myenteric denervation (Surgical vs BAC) and inflammation (intraluminal stasis and 5% acetic acid) to determine the underlying stimulus to the increased muscle thickness. Their results together with the observation of muscle thickening in the NOS knockout mice indicate that the changes in muscle thickness at least are a consequence of denervation and not a direct effect of BAC on smooth muscle.\n\n**Summary**\n\nAntral BAC produces selective lesions of defined populations of neurons in the stomach and leads to dramatic changes in gastric emptying. After antral BAC treatment there was retention of solids in rats fasted for 48 hr. Moreover, in antrally denervated rats with a gastric fistula, the emptying of saline, acid and peptone was substantially delayed. The cholecystokinin-dependent inhibition of gastric emptying\n\nof peptone was preserved after antral BAC. Hence it appears that the innervation of the antrum is essential for normal emptying of both liquids and solids, but the inhibition of gastric emptying produced by cholecystokinin is not dependent on antral neurons.\n\nChapter Five\n\nAntral denervation and G-cell Function\n\nIntroduction\n\nThe release of the antral hormone gastrin is controlled by the luminal contents of the stomach and by neurohumoral agents acting at the basolateral membrane of the G-cell. The main luminal stimulus is the presence of protein and protein-digestion products while the main luminal inhibitory factor is gastric acid (Dockray & Gregory, 1989; Walsh, 1994). The available evidence suggests that gastric acid releases somatostatin from antral D-cells which in turn inhibits gastrin release by a paracrine mechanism (Saffouri et al., 1980; Chiba et al., 1980; Walsh, 1994). The mechanisms by which luminal nutrients stimulate the G-cell are less certain. The luminal surface of the G-cell consists of tufts of microvilli that project into the lumen and this has led to the idea that luminal nutrients might act directly on the cell (Lichtenberger et al., 1982; Delvalle & Yamada, 1990; Lichtenberger, 1982). However, it is also clear that luminal stimulation of the G-cell can be indirectly mediated by nervous pathways (Saffouri et al., 1984; Schubert et al., 1992).\n\nIt is well recognised that vagal efferent neurons stimulate gastrin secretion in the cephalic phase of digestion and in addition, there are local intrinsic nervous reflexes controlling the G-cell. There is evidence for cholinergic-muscarinic stimulation of the G-cell, particularly in the rat (Schubert & Makhlouf, 1993; Schubert et al., 1992; Saffouri et al., 1984), but in dog and man atropine typically has no effect on nerve-mediated gastrin release, or stimulates it - indicating the importance of non-cholinergic inputs to the G-cell (Dockray & Tracy, 1980; Feldman et al., 1979;\n\nSchiller *et al.*, 1980; Farooq & Walsh, 1975). The neuropeptide gastrin releasing peptide (GRP) which is well represented in antral mucosal nerve fibres (Dockray *et al.*, 1979), is thought to be the main non-cholinergic neurotransmitter stimulating the G-cell. In keeping with this, both antibodies and more recently selective antagonists to GRP have been shown to inhibit gastrin secretion in response to either luminal peptone, or electrical field stimulation of the antrum or vagal nerve stimulation (Schubert, *et al.*, 1985; Holst, *et al.*, 1990; Schubert, *et al.*, 1992). The experiments described in this chapter were performed in an attempt to define the relative importance of neural mechanisms compared to the direct stimulation by luminal factors on gastrin release. To this end I have studied the luminal factors that influence gastrin release in control and antrally denervated rats. The results indicate increased sensitivity to non-nutrient distension after antral denervation and suggest the existence of an antral inhibitory innervation in the rat that normally suppresses G-cell responses to non-nutrient distension.\n\nMethods\n\nAntrally denervated rats were prepared and after each experiment denervation confirmed by radioimmunoassay of appropriate neuropeptide markers in tissue extracts of antral muscle (see Methods). Gastric cannulas were placed at the time of antral denervation and all rats were allowed at least 11 days to recover after surgery. For the refeeding experiments, rats were fasted for 48 hrs on wire-bottomed cages and were then allowed to refeed for 30 minutes. The GRP antagonist (BW2258U89) {Moody et al Life Sci 56: 521-529} (2mg.kg$^{-1}$ in 0.5ml of 0.14M saline) or saline, were injected subcutaneously 30mins before refeeding and then trunk blood was taken for assay of plasma gastrin. Samples of antral and corpus mucosa were taken for RNA extraction for analysis of gastrin, somatostatin, HDC and GAPDH mRNA abundance by Northern blot.\n\nGastric fistula rats:\n\nAs before, (chapter 4) the stomach was washed with warm 0.14M NaCl to remove solids at least one hour prior to experiments. Gastric acid output was determined by collecting gastric secretion over four sequential 15 min periods and titrating to pH 7.0 with 20mM NaOH using a pH autotitrator. Plasma gastrin responses were determined after gastric distension with the following test solutions: 2% (w/v) methyl cellulose (2%MC); 2%MC at pH6 (50mM ammonium acetate); 2% MC with 50mM HCl; 4.5% w/v peptone. In each case the stomach was distended by connecting the cannula to a reservoir of test solution and elevating the reservoir to 5cm H$_2$O pressure for 30mins.\n\nResults\n\nBasal plasma gastrin\n\nBasal plasma gastrin concentrations were similar in control, fasted rats (7.6 ± 1 pM; Fig5.1) and control rats with a gastric fistula (11.8 ± 2 pM; Fig5.4). In BAC treated rats fasted for 48hrs plasma gastrin was significantly elevated (28.4 ± 7pM, p<0.05 vs control; Fig 5.1). In these animals there was also marked gastric retention compared to controls (see Chapter 4). It seemed likely that gastric retention in BAC-treated rats accounted for the increased plasma gastrin, since in fasted BAC-treated rats with a gastric fistula, plasma gastrin was similar to control (13.5 ± 3.2pM; Fig 5.4) when the gastric contents were removed by lavage via the cannula.\n\nRefeeding experiments\n\nWhen control rats were fed for 30min after fasting for 48hr, plasma gastrin increased approximately 3-fold. There was a comparable increase in plasma gastrin in BAC-treated rats but the absolute increase after refeeding was higher than that in control rats, due to the elevation of basal plasma gastrin concentration (Fig 5.1). In keeping with the idea that GRP mediates food-stimulated release of gastrin in the rat, I found that the GRP antagonist BW2258U89 (0.5 mg.kg⁻¹) inhibited the rise in plasma gastrin in control rats seen after refeeding for 30min following a 48 hr fast. In contrast, in BAC-treated rats, the post-prandial rise in gastrin was not dependent on GRP since there was no significant difference compared with BAC-treated rats that received vehicle in place of the GRP antagonist (Fig 5.2).\n\nFigure 5.1 Plasma gastrin in fasted and refed rats 11 days after either sham operation (open bars) or antral denervation by 0.5% BAC (hatched bars). In fasted BAC-treated rats there was elevated fasting plasma gastrin. After refeeding, plasma gastrin increased approximately three-fold in both control and BAC-treated rats. (n=>6 for each group, * p<0.05 vs control response, Mann Whitney U test).\n\nFigure 5.2 Plasma gastrin concentrations in control and antrally-denervated rats after administration of the specific GRP antagonist BW225U86 (hatched bars) or saline (open bars) 30mins prior to refeeding. In control rats, but not antrally denervated rats, the GRP antagonist significantly attenuated the post-prandial rise in plasma gastrin. (n=6 for all groups, * p<0.05 Mann Whitney U test).\n\nBasal acid output:\n\nUnder normal circumstances gastrin release is inhibited by luminal acid so that one potential explanation for the elevated plasma gastrin in BAC-treated rats was that BAC treatment of the antrum influenced acid secretion. In fact the range of basal acid output in BAC-treated rats overlapped with that in controls but the mean was approximately 3-fold lower (Fig 5.3). Complete antral denervation was required to reduce mean basal acid output as rats that received BAC only to the anterior surface of the stomach had a mean basal acid output comparable with controls.\n\nDistension experiments\n\nIn control rats distension with the non-nutritive solution 2% MC had no effect on plasma gastrin. In contrast, distension of BAC-treated rats with 2% MC produced a significant rise in plasma gastrin (Fig 5.4). Although this appeared to substantiate the view that distension stimulates gastrin release in antrally denervated rats, there was also the possibility that alterations in pH may play a part in the response given the reduced acid output in BAC-treated rats. Therefore distension experiments were performed with buffered 2% MC (acidified and pH6) and with peptone as a positive control. In control rats distension with 4.5% w/v peptone increased plasma gastrin 3-fold but distension with the non-nutritive solutions 2 % methylcellulose (2%MC), or 2%MC pH 6 or 2%MC with 50 mM HCl did not increase plasma gastrin (Fig 5.5).\n\nIn BAC-treated rats, the effects of distension with peptone and 2%MC with 50mM HCl in methylcellulose were similar to control. However distension by either 2%MC or 2%MC pH 6.0 did stimulate gastrin release, the increase in plasma gastrin being comparable to that produced by peptone (Fig 5.5).\n\nFigure 5.3 Mean basal acid output (mmol.15min$^{-1}$) in control and antrally denervated rats. Two groups of denervated rats were studied: either total antral denervation, or denervation of the anterior surface only (hemi-BAC). Each data point represents the mean acid output of four sequential 15min collections from one rat. Only in the rats with complete antral denervation was there a significant reduction in basal acid output. (* p<0.05 ANOVA).\n\nFigure 5.4 Plasma gastrin responses in control and BAC-treated rats after gastric lavage to remove residual gastric content. Rats were either left undistended (open bars) or were distended with the non-nutritive solution 2%MC for 30 minutes at a pressure of 5cm of H$_2$O (hatched bars). In BAC-treated rats but not controls, plasma gastrin was increased by distension with 2%MC. (n=6 or more for each group, * p<0.05 vs control response).\n\nFigure 5.5 Plasma gastrin responses in control (open bars) and BAC-treated rats (hatched bars). Rats were either left undistended or distended for 30 mins with either 2%MC (buffered with 50mM HCL or to pH6) or 4.5% w/v peptone. Plasma gastrin after distension with 4.5% peptone was similar in control and antrally-denervated gastric fistula rats. In BAC-treated rats, but not controls, plasma gastrin was also increased by distension with 2%MC buffered to pH6.0. Neither group showed an increase in plasma gastrin after distension with 2%MC with 50mmol HCl. (n=6 or more for each group, * p<0.05 vs control response, Mann Whitney U test).\n\nGastrin, somatostatin HDC and GAPDH mRNA abundance\n\nIt is known that gastric endocrine cell responses frequently involve altered changes in the synthesis of active products, as seen by changes in mRNA abundance (Dockray et al. 1996). To determine whether antral denervation might alter the synthesis of regulatory molecules in gastric endocrine cells Northern blots of mRNA’s encoding gastrin, somatostatin, HDC and GAPDH were analysed (Figures 5.6 & 5.7). There was no difference in the expression of GAPDH between control and BAC treated rats. In the antrum there was a significant reduction in antral mucosal somatostatin mRNA abundance in BAC-treated rats compared with controls and a similar decrease in gastrin mRNA abundance. In the corpus however, somatostatin mRNA abundance was unchanged in BAC-treated rats compared with controls, while HDC mRNA was variably increased. The latter is compatible with the sustained increase in plasma gastrin in these animals.\n\nFigure 5.6 Representative Northern blots of total antral mRNA hybridised with specific cRNA probes for gastrin (upper panel) and somatostatin (lower panel). Lanes 1-6 are control and lanes 7-12 BAC-treated rats. BAC treatment significantly lowered gastrin and somatostatin mRNA abundance. These changes were considered specific as BAC treatment had no effect on GAPDH mRNA abundance (results not shown).\n\nFig 5.7 Relative mRNA abundance in the antrum of GAPDH, gastrin and somatostatin, and in the corpus of GAPDH, somatostatin and HDC in control (open bars) and BAC-treated rats (hatched bars). The relative abundance of each mRNA species has been normalised by taking as 100% the abundance in controls. Note the abundance of the non-regulated marker GAPDH was similar in control and BAC treated rats. In the antrum BAC decreased gastrin and somatostatin mRNA (n=6 in each group; * p<0.05, Students t test).\n\nDiscussion\n\nIn previous studies that have examined the neuronal regulation of G-cell function the extrinsic innervation has been manipulated by stimulation, lesioning or pharmacological blockade (Dockray & Gregory, 1989; Walsh, 1994); the role of intrinsic neurons has been largely determined by pharmacological approaches using neurotransmitter antagonists or antibodies (Schubert et al., 1985; Schubert et al., 1992). In the present experiments antral denervation by BAC was used to lesion both the intrinsic and extrinsic innervation of the rat antrum. The subsequent experimental data support the conclusions that the post-prandial gastrin response in control rats is largely mediated by the neuropeptide transmitter GRP; in antrally denervated rats, a post-prandial gastrin response is maintained but this is no longer inhibited by a GRP antagonist. Instead, it appears that non-nutritive distension becomes a strong stimulus for the denervated G-cell and this explains for the most part the fasting and meal stimulated hypergastrinaemia seen in the antrally denervated rat. The results are unexpected given that it is generally thought that G-cell responses to distension are mediated by nervous reflexes (Dockray & Gregory, 1989; Walsh, 1994; Schubert & Makhlouf, 1993).\n\nSince gastrin release is inhibited by luminal acid, one potential explanation for the elevated plasma gastrin in antrally denervated rats might be reduced acid output. In fact there was a decrease in basal acid secretion after denervation. However, the capacity of the denervated G-cell to respond to distension was increased even when intragastric pH was buffered at pH 6.0 suggesting that G-cell responses secondary to\n\nchanges in acid output are unlikely to be important in the response to denervation. Furthermore, in undistended BAC-treated rats with a gastric fistula plasma gastrin was comparable to control rats suggesting that basal acid output was sufficient to maintain inhibition of gastrin release. In man, gastric distension is a good stimulus to acid secretion (Soares et al., 1977), but in the conscious rat, it has been reported that non-nutritive distension of the stomach inhibits acid secretion by activating a sympathetic reflex (Dimaline et al., 1986). Hence the chronic gastric retention after antral denervation may lead to inhibition of acid output by this mechanism.\n\nIn support of this argument, it is clear that after lesioning the antral innervation there was a pronounced gastric retention of solids, even after 48hr fasting. After complete emptying of the stomach in antrally denervated rats fitted with a gastric fistula, basal plasma gastrin was similar to control levels, suggesting that the hypergastrinaemia in denervated rats without a gastric cannula was attributable to the gastric retention.\n\nStudies in the isolated perfused rat stomach have provided evidence that GRP mediates the release of gastrin in response to luminal peptone (Schubert et al., 1992). The observation that the post-prandial increase in plasma gastrin concentration was blocked by a GRP antagonist suggests that a similar mechanism operates in conscious rats *in vivo*. However, the post-prandial increase in gastrin in antrally denervated rats was not blocked by a GRP antagonist suggesting that an additional mechanism comes into play after denervation. Interestingly, the gastrin response to gastric distension\n\nwith peptone in antrally denervated rats was no greater than that to non-nutritive distension. Two inferences may be drawn from this: First, that the stimulatory effect of nutrient is normally mediated by antral neurons, and second that antral neurons normally inhibit G-cell responses to non-nutrient distension. Studies in the isolated perfused rat stomach have shown that antral neurons mediate stimulation of gastrin release after distension at low pressures, and inhibition at high pressures (Schubert & Makhlouf, 1993). The latter may explain why methylcellulose at pH6 did not stimulate gastrin release in the intact stomach. The novel finding in the present experimental series is that there exists a mechanism for distension-evoked release of gastrin which is independent of the antral innervation.\n\nIt is generally thought that gastrointestinal responses to mechanical distension are mediated by nervous reflexes. Two possible mechanisms might account for the unexpected observation that the denervated G-cell responds to non-nutritive distension. First, the G-cell or a nearby cell in the antral mucosa might be mechanosensitive (which is suppressed by the antral innervation), or develops such sensitivity after denervation. In this context it has been recognised for many years that enterochromaffin cells (EC) cells are mechano-sensitive (Bulbring and Lin, 1958; Kirchgessner et al., 1992); denervated G-cells might therefore take on EC-cell like properties, or alternatively antral EC cells might regulate G-cell function for example through a paracrine mechanism. Second, non-nutritive distension of the stomach could release an endocrine factor from the gastric corpus, which increases gastrin release.\n\nfrom the denervated, but not innervated, antrum. In the dog, Debas *et al.* (1975) showed that distension of the corpus increased gastrin release from an extrinsically denervated antral pouch suggesting the existence of an oxynto-pyloric endocrine reflex. The existence of a similar reflex in the rat might explain the present findings. If so, however, it would appear that such a reflex is not normally activated in the innervated antrum.\n\nHypergastrinaemia stimulates the enterochromaffin-like (ECL) cells of the gastric corpus which are an important source of histamine. The increased HDC mRNA abundance observed in BAC-treated rats is compatible with the hypergastrinaemia of antral denervation. Increased histamine synthesis and secretion might be expected to stimulate increased acid output, and the reduced acid secretion observed would therefore appear not to be secondary to inhibition at the level of the ECL cell. In the antrum we found marginally reduced gastrin and somatostatin mRNA abundance. Gastrin synthesis is regulated both at the level of gene transcription and at the level of mRNA translation (Bate *et al.*, 1996). The present data raise the possibility that after denervation there is independent control of release of synthesis at a post-transcriptional level. Furthermore, given that somatostatin and gastrin mRNA abundance appeared to be reduced following denervation without an overall depression in gene transcription (indicated by similar abundance of GAPDH in control and BAC treated rats) it seems possible that the innervation of the antral mucosa normally may regulate transcription of specific genes in target endocrine cells. Taken together with the possibility that following denervation G-cells may themselves\n\ntake on the EC-cell property of mechanosensitivity, it would appear that antral neurons play some role in the maintenance of the normal differentiated state of the G-cell.\n\nUnfortunately the factors that regulate the differentiation of G-cells have proved difficult to study not least because of the problems of generating G-cell lines. However the mechanisms of differentiation in endocrine cells per se have been studied in pancreatic islet cells. Interestingly, gastrin is expressed in the foetal pancreas at the time of endocrine cell development (Brand and Fuller, 1988) and there is good evidence to suggest that gastrin acts in conjunction with TGF-α to promote normal pancreatic endocrine cell differentiation (Wang et al., 1993). In adult rats, following partial pancreatectomy, pancreatic islet cell regeneration occurs and is associated with expression of the Reg gene (reg) (Terazono et al., 1988) implying that reg expression may be involved in differentiation. The latter is of interest because the reg gene has been shown recently to be present in the gastric ECL cell (Ashahara et al., 1996), a cell whose gene expression is well known to be regulated by gastrin. In addition reg expression is increased during the regeneration phase following acute gastric mucosal injury (Ashahra et al., 1996).\n\nIt is plausible to suppose therefore that the remodelling of gastric mucosal function after denervation involves gastrin itself, acting in conjunction with other factors to regulate differentiation of endocrine cells, including the ECL cell. The\n\nstudies detailed in the next chapter begin to investigate the possibility that gastrin regulates *reg* expression.\n\n**Summary**\n\nIn the intact rat, luminal nutrient releases gastrin via activation of neurons secreting GRP whilst the antral innervation normally inhibits gastrin release in response to non-nutrient distension. After antral denervation, post-prandial gastrin release is maintained but by a mechanism independent of neurons secreting GRP. Moreover, gastric distension with a non-nutrient solution becomes an adequate stimulus for gastrin release. Despite reductions in gastrin and somatostatin mRNA abundance following BAC treatment, hypergastrinaemia occurred and produced changes in ECL-cell gene expression although basal acid output was reduced. The altered G-cell function that occurred after denervation and the possibility that this effected changes in gastric endocrine cell differentiation remains to be determined.\n\nChapter Six\n\nControl of reg expression in rat corpus mucosa and AR42J cells\n\nThe work of several laboratories indicates that the *reg* gene is expressed during pancreatic or gastric mucosal regeneration (Terazono et al., 1988; Miyaura et al., 1991; Ashahara et al., 1996). Since *reg* is expressed in pancreatic β cells and gastric ECL cells it is reasonable to suppose that it may be implicated in endocrine cell growth or differentiation. Gastrin is known to stimulate growth of ECL cells both in the human (Borch et al., 1985; Bordi et al., 1986; Roucayrol and Cattan, 1989; Solcia et al., 1991) and the rat (Havu 1986; Hakanson and Sundler, 1990) and is essential for normal gastric (Koh et al., 1997) and pancreatic (Wang et al., 1993) endocrine cell development. In the latter case, gastrin must act in conjunction with growth factors for normal differentiation to occur. However, the possible interactions between gastrin, growth factors and *reg* expression have not been directly studied.\n\nAR42J cells are an amphicrine cell line with the capability to differentiate into either acinar or endocrine cells depending on the tissue culture conditions (Christophe, 1994). These cells express both the gastrin/CCK-B receptor and the *reg* gene (Lambert et al., 1991; Zenilman et al., 1996), suggesting that they might be a useful system in which to investigate the relationships outlined above. The experiments performed in this chapter were undertaken first to determine whether gastrin might regulate *reg* gene expression in rat gastric corpus and second to study in the AR42J cell line some of the features of gastrin-induced *reg* expression. The results indicate that *reg* expression is regulated by gastrin acting via the CCK-B receptor. In addition,\n\nalthough EGF potentiated gastrin-stimulated *reg* expression the reported data suggest that *reg* expression is associated with endocrine cell differentiation and not growth.\n\n**Methods**\n\n**Animal Studies**\n\nRats were either fasted for 48 hrs on wire-bottomed cages or fed ad libitum and given omeprazole (400μmol.kg$^{-1}$) by gavage for 5 days. Rats fed *ad libitum* were used as controls. At the end of treatment, trunk blood was taken for assay of plasma gastrin and samples of corpus mucosa were taken for RNA extraction and analysis of HDC and *reg* mRNA abundance by Northern blot.\n\n**Tissue Culture**\n\nCulture of AR42J cells and treatment with amidated and gly-extended gastrin, L740093 (1nM), EGF (10pM-1μM) and wortmannin (5 x 10$^{-8}$M) was performed by Dr. LA Bishop. Cells were cultured at a density of 1x10$^6$ for 24 hrs in depleted media before appropriate treatment. All treatments were given for 24 hrs except for wortmannin (4 hrs). At the end of the culture period total cell mRNA was extracted by the lithium chloride precipitation method and *reg* mRNA abundance analysed by Northern blot (see Chapter 2).\n\nResults\n\nReg expression in rat gastric corpus\n\nIn keeping with previously published observations (Dimaline et al., 1993; Swarovsky et al., 1994), treatment of Wistar rats with omeprazole (400μmol.kg\\(^{-1}\\)) for five days lead to a five to ten-fold increase in plasma gastrin and an associated three-fold increase in HDC mRNA in gastric corpus mucosa, compared to rats fed ad libitum. Conversely, fasting significantly reduced plasma gastrin and HDC mRNA abundance (Fig 6.1). In addition however, omeprazole-induced hypergastrinaemia also resulted in altered reg gene expression in the gastric mucosa, the changes in magnitude of reg mRNA abundance being almost identical to those of HDC (Fig 6.1).\n\nReg expression in AR42J cells\n\nTo examine in more detail how gastrin might regulate reg gene expression, AR42J cells were cultured in the presence of increasing concentrations of both amidated (G17-NH\\(_2\\)) and glycine-extended gastrin (G-gly). Amidated and glycine-extended gastrin both produced dose-dependent increases in reg expression with G-gly being approximately 1000-fold less potent than its amidated counterpart (Fig 6.2 & 6.3). To determine the relevant gastrin receptor mediating the reg response, AR42J cells were treated with either G17-NH\\(_2\\) (10\\(^{-8}\\) M) or G-gly (10\\(^{-6}\\) M) in the presence of the specific CCK-B receptor antagonist L-740093. The increase in reg mRNA abundance stimulated by G17-NH\\(_2\\) was completely inhibited by the CCK-B receptor antagonist (Fig 6.4). The response to G-gly, although significantly attenuated by L-740093, remained increased by approximately two-fold (Fig 6.5).\n\nFigure 6.1 **upper panel**: Effect of fasting and omeprazole treatment on plasma gastrin (open bars), *reg* mRNA (hatched bars) and HDC mRNA abundance (stippled bars). Note *reg* mRNA abundance was decreased by fasting and increased by omeprazole treatment, the magnitude of the change paralleling that of HDC mRNA. (*n=6 for each group, * p<0.05 vs control response, Mann Whitney U test). **lower panel**: Representative Northern blot of *reg* mRNA abundance in 48 hr fasted (lanes 1-4), fed ad lib. (lanes 5-8) and omeprazole treated rats (lanes 9-12).\n\nFigure 6.2 Representative Northern blots for *reg* mRNA abundance in total RNA extracted from duplicate cultures of AR42J cells treated with increasing molar concentrations of either G17-NH$_2$ (upper panel) or G-gly (lower panel). Note the dose range of the response is approximately 3 log units higher for G-gly.\n\nFigure 6.3 Dose response relationship between G17-NH$_2$ (closed circles) and G-gly (open circles) and $reg$ mRNA abundance in AR42J cells. The $A_{50}$ for G17-NH$_2$ was approximately $10^{-8}$M and that for G-gly approximately $5 \\times 10^{-5}$M.\n\nFigure 6.4 Effect of $10^{-9}$M L-740093 (hatched bars) on the *reg* mRNA response to $10^{-8}$M G17-NH$_2$ (upper panel) and a representative Northern blot (lower panel) of *reg* mRNA abundance in total RNA extracted from duplicate cultures of AR42J cells treated with vehicle (ammonium bicarbonate; lanes 1&2), L-740093 (lanes 3&4), G17-NH$_2$ (lanes 5&6) and G17-NH$_2$ and L-740093 (lanes 7&8). The twenty five-fold increase in mRNA abundance produced by G17-NH$_2$ was completely reversed by L-740093.\n\nFigure 6.5 Effect of $10^{-9}$M L-740093 (hatched bars) on the *reg* mRNA response to $10^{-6}$M G-gly (upper panel) and a representative Northern blot (lower panel) of *reg* mRNA abundance in total RNA extracted from cultures of AR42J cells treated with ambic (lanes 1&2) G-gly (lanes 3&4) and G-gly and L-740093 (lanes 5-7). Although the response to G-gly was significantly attenuated by L-740093, *reg* mRNA abundance remained increased by approximately two-fold.\n\nEGF and *Reg* expression in AR42J cells\n\nThe effects of EGF on *reg* expression is shown in Fig 6.6. At the highest dose of EGF ($10^{-6}$M) there is no significant upregulation of *reg* expression even though this concentration of EGF has been shown to induce proliferation of AR42J cells. However, EGF, at a concentration of $10^{-9}$M potentiated the effects of amidated and glycine-extended gastrin on *reg* expression (Fig 6.7). Hence the ten to twenty-fold increase in *reg* expression induced by $10^{-8}$M G17-NH$_2$ was further enhanced ten-fold by EGF ($10^{-9}$M).\n\nWortmannin and *Reg* expression in AR42J cells\n\nInhibition of the PI3 kinase in cultured human foetal pancreatic cells has been shown to induce differentiation towards an endocrine cell phenotype (Ptasznik et al., 1997). Treatment of AR42J cells with the PI3 kinase inhibitor wortmannin ($5 \\times 10^{-8}$ M) for four hours, increased *reg* expression in both unstimulated and G17-NH$_2$-stimulated AR42J cells (Fig 6.8).\n\nFigure 6.6 **upper panel**: Relationship between increasing doses of EGF and *reg* mRNA abundance in AR42J cells. Results are expressed relative to mRNA expression in control cultures arbitrarily defined as 5% of a predicted maximum response. Even at $10^{-6}$M EGF there is no increase in *reg* mRNA abundance.\n\n**lower panel**: Representative Northern blot of *reg* mRNA in duplicate cultures of AR42J cells exposed to increasing concentrations of EGF.\n\nFig 6.7 **upper panel**: relationship between G17-NH$_2$ (10$^{-8}$M) or G-gly (10$^{-7}$M), EGF (10$^{-9}$M) and *reg* mRNA abundance in AR42J cells. Results have been normalised to the response to 10$^{-8}$M G17-NH$_2$ taken as 100%. The response of AR42J cells to either G-gly or EGF alone was approximately 10% of the G17-NH$_2$ response. However, EGF increased the response of AR42J cells to G17-NH$_2$ by fifteen-fold and to G-gly by seven-fold.\n\n**lower panel**: Representative Northern blots for *reg* mRNA in duplicate cultures of AR42J cells treated with G17-NH$_2$ (lanes 1&2), G-gly (lanes 3&4), EGF (lanes 5&6), G17-NH$_2$ and EGF (lanes 7&8) and G-gly and EGF (lanes 9&10).\n\nFig 6.8 **upper panel**: relationship between G17-NH$_2$ (10$^{-8}$M), wortmannin (5 x 10$^{-8}$M) and *reg* mRNA abundance in AR42J cells. The gastrin-stimulated response in *reg* mRNA is further increased two-fold by wortmannin. Note that wortmannin also stimulates *reg* mRNA expression in control cultures. **lower panel**: Representative Northern blots for *reg* mRNA in duplicate cultures of AR42J cells treated with Ambic (lanes 1&2), wortmannin (lanes 3&4), G17-NH$_2$ (lanes 5&6) and G17-NH$_2$ and wortmannin (lanes 7&8).\n\nThe data presented in this chapter provide evidence that *reg* expression in rat corpus mucosa responds to changes in plasma gastrin and in AR42J cells is regulated by amidated gastrin acting through the gastrin/CCK-B receptor. In addition, whilst EGF alone had no effect on *reg* expression it markedly potentiated gastrin stimulated *reg* mRNA abundance. Furthermore, in culture conditions that have been shown to favour differentiation of AR42J cells into insulin-secreting endocrine cells, *reg* gene expression was increased.\n\nRegulation of ECL cell gene expression by plasma gastrin has been demonstrated for a variety of genes including HDC (Dimaline *et al.*, 1991; Swarovsky *et al.*, 1994;). The observed threefold increase in HDC mRNA abundance following 5 day omeprazole treatment was in keeping with previously reported data. The presence of *reg* in rat ECL cells has been reported only recently (Ashahara *et al.*, 1996) and the results presented here demonstrate for the first time that *reg* gene expression in vivo can be regulated by plasma gastrin.\n\nThe present results contrast with those of Zenilman *et al.*, (1997) who showed that gastrin, CCK and glucagon (all 10nM) had no effect on reg mRNA abundance in AR42J cells. However, the tissue culture protocol in the experiments reported by Zenilman did not incorporate 24 hrs of culture in serum depleted media prior to the period of treatment. AR42J cells have been reported to synthesise and secrete gastrin\n\nthat may then act in an autocrine manner (Blackmore and Hirst, 1992) and this may explain the discrepancy between the above findings and the lack of effect of gastrin seen by Zenilman and colleagues. An additional explanation of the results of Zenilman et al., is the influence of the cell density of AR42J cells on *reg* gene expression. In preliminary experiments it was found that *reg* gene expression was upregulated (approximately tenfold) as cell density reached confluence. Zenilman et al. conducted their experiments on confluent cells unlike the experiments reported here.\n\nThe gastrin stimulated *reg* expression in AR42J cells appears to act via the gastrin/CCK-B receptor known to be present on these cells (Lambert et al., 1991). The reported dissociation constant for gastrin at this receptor in AR42J cells is 4nM (Zhou et al., 1992). The $A_{50}$ of the *reg* response to G17-NH$_2$ was $\\approx 10$ nM and the complete inhibition of the response by the specific gastrin/CCK-B receptor antagonist L-740093 indicates that this response is mediated via the gastrin/CCK-B receptor. The *reg* response to G-gly also was inhibited by L-740093 but only by $\\approx 80\\%$. The dose of G-gly required to increase *reg* expression was much higher than that for G17-NH$_2$ and given that L-740093 is a competitive antagonist at this receptor it seems likely that the effects of G-gly on *reg* expression also are mediated via the gastrin/CCK-B receptor. The parallel rightward displacement of the G-gly dose-response curve compared to that of G17-NH$_2$ also suggested that G-gly was acting at the same receptor as G17-NH$_2$ but with lower affinity.\n\nPerhaps by coincidence, the magnitude of change in *reg* mRNA abundance in the rat gastric mucosa was identical almost to that of HDC. Given that gastrin regulates HDC gene expression via the gastrin/CCK-B receptor (Hollande et al., 1994), this might suggest a common intracellular pathway regulating HDC and *reg* gene expression. The rat and human HDC promoter has been shown to be regulated by a protein kinase C dependent pathway (Hocker et al., 1996; Zhang et al., 1996). For the human HDC gene a palindromic sequence (5'-CCCTTTAAATAAAGGG-3') that confers transcriptional responsiveness to gastrin has been identified and defined as a gastrin response element. A similar sequence is present within the rat HDC promoter region (5'-CCCTTAAATAAGAGGG-3'). In each case the putative response element lies within 25 base pairs of the transcriptional start site. The 5'-regulatory region of the rat *reg* gene has also been described in AR42J cells (Miyashita et al., 1994). The regulatory sequence of most significance indicates a region -256 to -237 upstream of the transcriptional start site that has significant sequence homology with the consensus sequence of the pancreatic exocrine enhancer (Boulet et al., 1986). Although this would suggest a separate regulatory mechanism for *reg* gene transcription in AR42J cells, the response to gastrin was not studied. However, there does not appear to be a sequence similar to the putative gastrin response element of the rat HDC gene within the reported promoter region of the rat *reg* gene. Nevertheless the data clearly indicate that gastrin regulates *reg* expression in AR42J cells via a gastrin/CCK-B receptor mediated pathway.\n\nOther effects of gastrin on AR42J cells that have been shown to depend on a gastrin/CCK-B receptor mediated pathway include stimulation of amylase secretion (Lambert et al., 1991) and stimulation of growth (Pradel et al., 1993; Seva et al., 1994; Zenilman et al., 1997). This raises the possibility that *reg* gene expression may be part of the growth response of AR42J cells to gastrin. This seems unlikely for two reasons: Firstly, the growth promoting effects of G17-NH$_2$ on AR42J cells is relatively weak compared to that of EGF (Watson et al., 1993). Despite this, the data above demonstrate that $10^{-6}$M EGF had no effect on *reg* gene expression although this dose would be expected to produce a large trophic response. This indicates that *reg* expression, although present in AR42J cells, is not required for cell growth. Secondly, treatment of AR42J cells with the PI3 kinase inhibitor wortmannin which has been shown to inhibit cell growth and promote endocrine cell differentiation (Ptasznick et al., 1997) was found to increase *reg* gene expression. The effect of wortmannin on AR42J cell *reg* expression was not confined to unstimulated cells as it also potentiated the effects of gastrin on *reg* expression.\n\nSeveral other lines of evidence indicate that *reg* gene expression is associated with differentiation towards an endocrine cell phenotype. Treatment of AR42J cells with dexamethasone has been shown to promote differentiation of an acinar cell phenotype and concurrently reduced *reg* expression (Zenilman et al., 1997). Conversely, treatment with activin A, betacellulin or HGF stimulates differentiation of AR42J cells towards an endocrine cell phenotype (Mashima et al., 1996a; Ptasznik et al., 1997; Mashima et al., 1996b) and HGF has been shown to increase *reg* expression.\n\nin human foetal pancreatic cells (Otonkoski et al., 1994). Taken together, the experimental evidence suggests that *reg* expression in AR42J cells is associated with differentiation towards an endocrine cell phenotype. If so, then this process would be expected to be favoured by treatment with gastrin and potentiated by either EGF or wortmannin. The data are of interest as AR42J cells may provide a useful model to elucidate the intracellular pathways that mediate these effects.\n\nThe interaction between growth factors, gastrin and the differentiation of endocrine cells has been described in the mouse pancreas (Wang et al., 1993). Here at least, both are required in conjunction for normal development of the endocrine pancreas. In the stomach, gastrin does not seem to be essential for normal ECL cell development as these cells can still be found in the gastric mucosa of gastrin knockout mice (Koh et al., 1997). However, in this model ECL cell numbers were significantly reduced implying that gastrin regulates the number of mature ECL cells in the stomach perhaps by influencing the growth and differentiation of precursor cells. There are no data as yet on *reg* expression in such experimental situations but the provisional data presented thus far indicate that *reg* expression may be a marker for ECL cell function and more specifically may play a role in the maintenance of a normal ECL population within the gastric mucosa. The clinical implications of this are the subject of the next chapter.\n\nSummary\n\nReg expression in rat gastric corpus was increased by 5 day omeprazole treatment. In AR42J cells reg expression was shown to be regulated by amidated gastrin acting through the CCK-B receptor. EGF alone had no effect on reg expression but markedly potentiated the gastrin stimulated response. In culture conditions that favour differentiation of AR42J cells into insulin-secreting endocrine cells, reg gene expression was increased. The observations of others clearly indicate that growth factors acting together with gastrin may regulate endocrine cell growth and differentiation in the gastrointestinal tract. AR42J cells may provide a useful model to elucidate the intracellular pathways that mediate these effects.\n\nChapter Seven\n\nReg gene expression in human gastric mucosa.\n\nIn rodents hypergastrinemia secondary to long-term treatment with H$_2$-receptor antagonists or proton pump inhibitors (PPI) may lead to gastric carcinoid tumours (Hava, 1986; Poynter and Selway, 1991; Wangberg et al., 1995). This has prompted extensive reviews of the natural history of these tumours in man (Solcia et al., 1991; Thomas et al., 1994; Rindi, 1995; Modlin et al., 1995). It is now clear that hypergastrinaemia from whatever cause may result in hyperplasia of the gastric ECL cell. Significant ECL-cell hyperplasia has been demonstrated in one third of patients rendered hypergastrinaemic by *H pylori* infection and coincident treatment with a PPI (Eiselle et al., 1997). The majority of patients with CAG display some degree of ECL cell hyperplasia, the achlorhydria of CAG leading to hypergastrinemia that is generally accepted to be a stimulus to ECL cell growth (Creutzfeld, 1988; Hakanson and Sundler, 1990; Bordi et al., 1995). In fact, chronic atrophic gastritis (CAG) with or without pernicious anaemia is the commonest cause of enterochromaffin-like (ECL-) cell hyperplasia and in 5-10% of patients there is a progression to gastric carcinoid tumours (Borch et al., 1985; Bordi et al., 1991; Bordi et al., 1995).\n\nThe recent demonstration of *reg* gene expression in rat ECL cells together with the evidence suggesting that the reg protein may be important for pancreatic endocrine cell growth and differentiation imply a possible role for *reg* in the control of\n\nECL cell growth and differentiation. Gastrin has been shown to be important for entero-pancreatic endocrine cell development (Wang et al., 1993; Koh et al., 1997), acting in concert with growth factors such as TGF-α. In keeping with these observations, the results presented in the previous chapter showed gastrin stimulation of *reg* expression, potentiation of this response by EGF and upregulation of *reg* mRNA in AR42J cells exposed to culture conditions that favour differentiation towards an endocrine cell phenotype. The experiments performed in this chapter attempt to determine whether *reg* expression in the human gastric corpus is affected by plasma gastrin concentration and whether *reg* may have a pathophysiological role in ECL cell tumour development in patients with chronic hypergastrinaemia.\n\nHuman studies\n\nThe details of the patients that provided samples for the experimental studies described in this chapter are shown in Table 7.1 Plasma gastrin was assayed by RIA using antibody L2. Endoscopic biopsies were taken from the gastric corpus and from any corpus nodules if present from hypergastrinaemic patients that required a gastroscopy. Duplicate biopsies were sent to Dr G Armstrong (Consultant Histopathologist, Hope Hospital) for Histology. Total mRNA was extracted from biopsies and reg mRNA analysed by Northern blot and RT/PCR, cloning and sequencing (see Chapter 2). For the Northern blots, reg mRNA is expressed relative to the non-regulated mRNA for GAPDH.\n\nOctreotide Suppression Test\n\nTwo of the patients with carcinoid tumours (JH & KD) consented to an octreotide suppression test. The intended study was explained and informed consent obtained from both patients. Endoscopic biopsies were taken from ECL cell nodules and apparently normal corpus prior to and at the end of a 72hr infusion of octreotide (25mg.hr\\(^{-1}\\)). Plasma samples were taken before, during and after the octreotide infusion for assay of gastrin.\n\n| PATIENT | AGE | SEX | DIAGNOSIS | PLASMA GASTRIN (pM) | ECL Cell Polyps | Polyp size |\n|---------|-----|-----|------------------------------------------------|---------------------|-----------------|------------|\n| J.H. | 44 | F | P.A. Nodules | 850 | >50 | >1cm |\n| K.D. | 42 | M | P.A. Nodules | 600 | 2 | 1cm + <5mm |\n| K.W. | 72 | F | P.A. Nodules | 1200 | >20 | <1cm |\n| P.S. | 47 | M | Chronic Renal Failure Diabetes Mellitus | 650 | <10 | <5mm |\n| M.B. | 56 | F | Z.E./MEN1 | 280 | <10 | <5mm |\n| P.G. | 65 | F | P.A. | 450 | <10 | <5mm |\n| P.M. | 61 | F | P.A. | 400 | <10 | <5mm |\n| W.S. | 72 | M | P.A. | 480 | <10 | <5mm |\n| J.C. | 47 | M | Z.E. | 250 | Nil | |\n| T.O. | 72 | M | P.A. | 570 | Nil | |\n| D.K. | 46 | M | P.A. | 440 | Nil | |\n| H.W. | 75 | F | P.A. | 180 | Nil | |\n| B.C. | 62 | M | P.A. | 250 | Nil | |\n| S.B. | 47 | F | P.A. | 1300 | Nil | |\n| E.H. | 84 | F | P.A. | 850 | Nil | |\n| P.T. | 56 | F | Cystic fundic gland polyps | 7 | Nil | <5mm |\n| M.L. | 70 | F | Cystic fundic gland polyps | 28 | Nil | upto 1cm |\n| M.L. | 66 | F | Cystic fundic gland polyps | 20 | Nil | <5mm |\n| H.B. | 86 | F | Cystic fundic gland polyps | 14 | Nil | <5mm |\n| J.T. | 43 | F | Cystic fundic gland polyps | 20 | Nil | <5mm |\n| A.M. | 63 | F | Dyspepsia, Normal OGD | 15 | Nil | |\n| E.H. | 72 | F | Iron Deficiency, Normal OGD | 26 | Nil | |\n| E.Q. | 50 | F | Dyspepsia, Normal OGD | 13 | Nil | |\n| T.B. | 43 | M | Dyspepsia, Normal OGD | 25 | Nil | |\n| A.L. | 63 | M | Dyspepsia, Normal OGD | 12 | Nil | |\n\nTable 7.1 Details for all patients that provided tissue biopsies for either Northern blotting of *reg* mRNA abundance or RT/PCR to generate cDNA transcripts of *reg* mRNA for sequencing purposes. Hypergastrinaemic patients are shown above the control subjects that were selected on the basis of a normal oesophago-gastric-duodenoscopy (OGD) and normal plasma gastrin.\n\nPermanent cell transfection\n\nTwo constructs of human *reg* were selected for permanent transfection. These were the normal full length construct and the *reg* sequence that included a missense mutation at the initiator methionine residue (ATG→GTG). Stably transfected hamster insulinoma tumour (HIT) cell lines expressing wild type or mutant human *reg* were produced by electroporation of $5 \\times 10^6$ cells in 0.8ml at 200V and 1070μF in incomplete medium containing 10μg of linearised pcDNA *reg* construct. Cells were grown in complete RPMI in 10cm dishes for 48 hours prior to selection with zeocin ($750\\mu g/ml^{-1}$) (Invitrogen). Six colonies expressing each construct were cloned and screened for *reg* expression by northern blot.\n\nResults\n\nReg expression in human gastric corpus.\n\nReg mRNA abundance was determined in hypergastrinaemic patients and compared to that of control subjects (figure 7.1). In patients with sustained hypergastrinaemia (plasma gastrin <400pM) reg mRNA abundance was increased approximately threefold. In addition, suppression of plasma gastrin by a 72hr infusion of the somatostatin analogue octreotide, was associated with a significant downregulation of HDC (n=2) and reg mRNA (n=1) in tumour biopsies. The effect of octreotide on plasma gastrin and HDC mRNA abundance are shown for patient JH in Figure 7.2. The effect of octreotide on reg expression was determined in patient KD and is shown in Figure 7.3.\n\nReg expression in endocrine and non-endocrine cell polyps\n\nTotal RNA extracted from biopsies that were confirmed histologically to be non-endocrine in type (fundic cystic gland hyperplasia) showed a reduction in reg mRNA abundance compared to controls. Conversely, reg mRNA abundance was greatly increased in some ECL cell nodules compared to the patient’s own corpus but not in all cases. In fact the abundance of reg mRNA in ECL cell tumour biopsies was very variable (Figure 7.4) and did not relate directly to plasma gastrin concentrations.\n\nFig 7.1 *Upper panel* Reg mRNA abundance in patients with hypergastrinaemia (plasma gastrin >400pM) compared to control subjects (plasma gastrin <100pM). There is a threefold increase in reg mRNA in patients with hypergastrinaemia (n = 10 for each group; * p<0.05, compared with control, Mann Whitney U test). *Lower panel* Representative Northern blot of reg mRNA in controls (lane 1-4) and hypergastrinaemic patients (lanes 5-8) demonstrating the increased reg mRNA in hypergastrinaemic corpus.\n\nFig 7.2 *Upper panel* Effect of octreotide infusion on plasma concentrations of amidated gastrin in patient JH. Plasma gastrin concentrations decreased during the infusion but remained above the upper limit of normal (indicated by dashed horizontal lines). The dose of octreotide is indicated by the black bar graph. *Lower panel* Northern blot for HDC mRNA in total RNA extracted from biopsies of carcinoid nodule (lane 1) and corpus (lane 2) pre-octreotide and from carcinoid nodule (lane 3) and corpus (lane 4) post-octreotide. HDC mRNA was reduced in both nodule and corpus following the octreotide infusion.\n\nFig 7.3 Upper panel Effect of octreotide infusion on plasma concentrations of amidated gastrin in patient KD. Plasma gastrin concentrations decreased during the infusion falling to within the normal range (indicated by dashed horizontal lines). The dose of octreotide is indicated by the black bar graph. Lower panel a) Northern blot for reg mRNA in total RNA extracted from biopsies of carcinoid nodule (lane 1) and corpus (lane 2) pre-octreotide and from carcinoid nodule (lane 3) and corpus (lane 4) post-octreotide. Reg mRNA was reduced in carcinoid nodule following the octreotide infusion. b) Northern blot of the same membrane for GAPDH mRNA.\n\nFig 7.4 Upper panel Reg mRNA abundance in fundic cystic gland polyps (FCGP n=5) compared to controls (n=6). Reg mRNA was reduced by $\\approx$50% in the non-endocrine polyps (* p<0.05 Mann Whitney U test). To the right of the axis break is shown the reg mRNA abundance in ECL cell tumours in individual patients (initials on X axis) relative to their own corpus reg mRNA abundance normalised to 100 arbitrary units. Note the wide variation in tumour reg mRNA abundance Lower panel Representative Northern blot for reg mRNA in total RNA extracted from biopsies of normal corpus (lanes 1,3 and 5) non-endocrine cell polyps (lanes 2,4 and 6) hypergastrinaemic corpus (lane 7) and ECL cell nodule (lanes 8).\n\nReg Sequence and ECL cell tumours\n\nThe cDNA sequence of clones derived from RT/PCR of reg mRNA was determined in six hypergastrinaemic patients without ECL cell tumours and five patients with tumours. At least two clones were sequenced and data on the full sequence length obtained. All patients without ECL cell tumours had a normal reg sequence. Three of the five patients with nodules had at least one mutation of their reg sequence in either corpus or nodule. The sequence information from patients with ECL cell nodules is presented in Table 7.2. JH had multiple presumably acquired mutations identified from the biopsies of her nodules. PS had a single mutation identified in both corpus and nodule suggesting a germ line mutation. Interestingly, this mutation also occurred in JH and immediately appeared to suggest a pathophysiological mechanism. The mutation was a point mutation in the codon for the initiator methionine (ATG → GTG) and this would result in either the failure of initiation of protein translation or initiation of protein translation at a suitable upstream methionine residue. If protein translation commenced at the next upstream methionine, this would result in loss of the first eight amino acids of the signal peptide with a failure of the protein translated to enter the secretory pathway (Fig 7.5).\n\nTable 7.2 Sequence data from cDNA transcripts of *reg* mRNA extracted from hypergastrinaemic patients with ECL cell tumours. Three of the five patients with nodules had at least one mutation in their *reg* gene identified from either nodule or corpus. All patients without nodules had normal *reg* sequence. PS had a single mutation identified from both nodule and corpus suggesting a germ line mutation. Patient JH had multiple, presumably acquired mutations identified from her tumour RNA. Note the same mutation in the initiator methionine residue occurred in both JH and PS.\n\nFig 7.5 Upper panel Hydrophobicity plot of normal Reg protein showing the hydrophobic tail of the signal peptide (bold line) and below this the first ten amino acids of the protein sequence. Lower panel Initiator methionine mutant reg showing the point mutation resulting in the initiator methionine residue being substituted by valine. Above this is shown the predicted hydrophobicity plot assuming protein translation occurs at the next upstream methionine. Note loss of the membrane spanning hydrophobic signal peptide.\n\nFig 7.6 Northern blot of *reg* mRNA abundance in 5 clones of HIT cells permanently transfected with mutant *reg* (lanes 1-5) and 6 clones of HIT cells permanently transfected with wild type *reg* (lanes 6-11). Although there is variability, all clonal cell lines express *reg* mRNA.\n\nThe data presented in this chapter demonstrate for the first time that *reg* expression in the human gastric corpus is increased in patients with hypergastrinaemia and may be suppressed in carcinoid tumour nodules by a 72hr infusion of octreotide. Perhaps of more significance was the demonstration of an association between mutations of *reg* and the development of carcinoid tumours in patients with hypergastrinaemia.\n\nIt is now generally accepted that gastrin has a trophic action on the ECL cell and high circulating levels of gastrin promotes ECL cell hyperplasia. Hypergastrinaemia induced by H$_2$-receptor antagonists leads to the progressive development of ECL cell hyperplasia and gastric carcinoid tumours in rodents (Hava 1986; Poynter and Selway, 1991; Wangberg et al., 1995). Anti-secretory therapy in man may lead to significant ECL cell hyperplasia (Eiselle et al., 1997) but no cases of carcinoid tumour have been reported. This may be because the hypergastrinaemia induced by such therapy is rarely high enough or sustained. Conversely, conditions associated with persistent and large elevations in plasma gastrin are associated with carcinoid tumour development.\n\nSeveral genes expressed by ECL cells, including those for HDC, VMAT2 and chromogranin A, are upregulated by an elevated plasma gastrin (Dimaline et al., 1993a; Dimaline and Struthers, 1996; Dimaline et al., 1993b). The ability of gastrin to\n\nstimulate new protein synthesis and growth of ECL cells can be blocked by specific gastrin/CCK-B receptor antagonists or by somatostatin and its analogues (Dimaline and Sandvik, 1991; Bordi et al., 1993; Ferraro et al., 1996). The data presented here confirm that HDC gene expression within the human gastric corpus mucosa was increased in patients with hypergastrinaemia and may be downregulated by a 72hr infusion of octreotide. Reg mRNA was downregulated in carcinoid nodule but not in normal corpus by octreotide (see below). In both cases, plasma gastrin fell during octreotide treatment although in the case of JH, plasma gastrin remained elevated above the upper limit of normal. Despite this there was profound depression in ECL cell function in the face of elevated plasma gastrin. This observation suggests that in addition to reducing the stimulus to ECL cell function mediated by gastrin, octreotide may itself exert direct inhibitory effects on ECL cell function. The ability of octreotide to influence gene expression within carcinoid tumour tissue suggests that this tissue is still under normal regulatory control.\n\nIn patient KD, reg expression changed in parallel with HDC. There was a marked increase in reg mRNA abundance in the patient’s carcinoid nodule compared to his corpus mucosa and in his nodule at least, this was reduced by the octreotide infusion. Perhaps surprisingly, octreotide appeared to increase reg mRNA in gastric corpus. One possible explanation for this may be the variation in tissue sampling (see below). The elevated reg mRNA in gastric carcinoid tissue seen in KD was clearly not the situation for all patients with nodules as the abundance of reg mRNA in nodule\n\ntissue was highly variable between patients. There may be several reasons for this. In rodents it has been suggested that as carcinoid tumours develop and progress, they develop autonomy and become less dependent on gastrin as a trophic stimulus. The variability in *reg* expression in patients perhaps may indicate patients at a different stage of their disease. Alternatively, the degree of inflammation in the surrounding corpus was not taken into account. The ability of growth factors to potentiate the gastrin stimulated *reg* mRNA response also may explain the observed variability in nodule *reg* mRNA abundance. Hence, perhaps as CAG progresses and the parietal cell mass falls, the degree of inflammation also subsides. Tissue sampling introduces another unknown variable although this maybe more relevant to corpus mRNA as macroscopic nodules represent reproducible sites to biopsy. Although biopsies were taken from corpus mucosa that appeared macroscopically normal through an endoscope, the presence of microscopic carcinoids within biopsies that were processed for corpus mRNA cannot be excluded. This probably explains not only the variability in *reg* expression between patients but also the somewhat surprising increase in corpus *reg* mRNA in KD after his octreotide infusion. Furthermore, part or all of the increase in corpus *reg* mRNA in patients with hypergastrinaemia may be explained by an increase in ECL cell number as a consequence of hyperplasia and also due to concurrent atrophy of other mucosal cell types in patients with CAG.\n\nCarcinoid tumours occur most commonly in patients with CAG, but are also associated with ZE and CRF. The incidence of carcinoid nodules in CAG is\n\napproximately 5% and is similar to the incidence of carcinoid tumour occurring in the sporadic form of ZE syndrome. The incidence of carcinoid nodules in ZE associated with MEN1 syndrome rises to $\\approx 30\\%$ (Lehy et al., 1992). This suggests a genetic predisposition to carcinoid tumour development in this patient subgroup and is in keeping with the recently published observations of (Debelenko et al., 1997). They demonstrated that 75% of ZE patients with MEN1 syndrome have loss of heterozygosity at the MEN1 gene locus with deletion of the wild type allele. The MEN1 gene has recently been sequenced (Chandrasekharappa et al., 1997) and is the tumour suppressor gene that carries the inherited risk for developing endocrine tumours that are the feature of MEN1 syndrome. Of interest also however, was the observation of a low incidence of loss of heterozygosity at the MEN1 gene locus (1 of 6 patients) in patients with CAG and carcinoid nodules (Debelenko et al., 1997). This implies that another gene may be responsible for the risk of tumour development in these patients.\n\nThere is a growing body of evidence to suggest that the *reg* gene may play a role in the growth and differentiation of entero-pancreatic endocrine cells. It seemed reasonable to examine therefore whether mutations of *reg* may be associated with the development of carcinoid tumours in patients with hypergastrinaemia. The sequence data presented show that in 3 of 5 patients with hypergastrinaemia and carcinoid tumours there was at least one identifiable mutation in *reg* whereas in all hypergastrinaemic patients without carcinoid nodules the wild type *reg* coding\n\nsequence was found. This identifies an association between mutations of *reg* and a risk of developing carcinoid tumours in the presence of hypergastrinaemia. However, a significant number of normal *reg* sequences were also obtained from both the corpus and the nodules of patients with tumours. Again this may reflect sampling error as each biopsy of tumour tissue will also have normal mucosal cells within it. To overcome this, microscopic dissection of fresh biopsy material to obtain clonal tumour cells will be required. It is also likely that the *reg* gene is not the only genetic mutation required or necessary to develop carcinoid tumours just as mutations of menin do not appear to be solely responsible for the development of carcinoid tumours in ZE associated with MEN1 (Debelenko *et al.*, 1997).\n\nIt seems likely that both acquired and germ line mutations of *reg* may occur in association with carcinoid tumours. Patient JH had multiple mutations of *reg* in several clones sequenced indicating multiple acquired mutations. Patient PS had only one mutation of his *reg* but it occurred in three of four sequences in both his corpus and nodule. This is suggestive of a germ line mutation. Interestingly the mutation identified in PS also occurred in patient JH and occurred at the methionine residue that initiates protein synthesis. The point mutation of A → G resulted in a loss of the initiator methionine, it being replaced by a valine residue. The possible results of this mutation include; i) a complete failure of protein translation, ii) protein translation occurs at the next upstream methionine that is “in frame”, iii) protein translation occurs at the most appropriate methionine residue that may be “out of frame”. In the case of the first and last instance either no protein or a\n\ndifferent protein will be produced with an expected loss of the normal function of *reg*. In the case of the second instance the protein produced will be missing eight amino acids of the N-terminal hydrophobic signal peptide. This should result in failure of the protein product to enter the secretory pathway as the signal peptide will be too short to span the hydrophobic membrane domain. The subsequent protein product may then either be degraded within the cytosol or act intracellularly. This could result theoretically in either a loss or gain in function.\n\nAccording to the scanning model of mRNA translation proposed by Kozac (1978 and 1989), the sequence immediately adjacent to the initiator methionine codon determine the likelihood of protein translation occurring. The most important requirement is for a purine at position -3. In the absence of a purine at position -3, a G at +4 is essential for efficient translation (Kozak, 1989 & 1991; Iida and Masuda, 1996). Examination of the transfected mutant *reg* sequence fails to identify any alternative ATG codon with a surrounding sequence that meets these criteria. This would suggest that translation is unlikely to occur at an alternative start site and if it does occur at the next upstream methionine residue then translation will be inefficient. To confirm that the initiator methionine mutant *reg* resulted in failure of the translated protein product to enter the secretory pathway, constructs of human *reg* were inserted into a mammalian expression vector (pcDNA3-ZEO$^+$). The constructs were then permanently transfected into the hamster insulinoma tumour cell line (HIT) by Dr L Bishop. Cells were cultured\n\nFig 7.7 Photomicographs of HIT cells permanently transfected with either wild type (a) or mutant (b) *reg* constructs and stained sequentially with monoclonal anti-human *reg* antibody (1:2000; gift from Dr J Wand, Boston USA) and FITC conjugated horse anti mouse antibody (1:50). Note, only in the cells expressing wild type *reg* is there clearly discernible staining within the golgi apparatus and secretory granules. (x 400 approx., Courtesy of Colin Blackmore)\n\nunder zeocin selection and surviving cell lines were confirmed as expressing the *reg* gene by Northern blot of total mRNA extracted from $10^6$ cells (Fig 7.6). Clonal cell lines were then stained immuno-histochemically with a monoclonal anti-human reg antibody followed by FITC rabbit-anti-human secondary antibody. Intracellular distribution of reg protein was determined by immunofluorescence microscopy. (This work was performed by C. Blackmore, Dept of Physiology, Liverpool). The results of this work demonstrate that in the cell lines expressing wild type *reg* the protein enters the secretory pathway with clear staining of the Golgi apparatus and secretory granules (see photomicrograph 7.7a). In contrast, in cells expressing mutant *reg* there is no discernible pattern of staining (see photomicrograph 7.7b). Although this suggests that the protein either is not produced or is rapidly degraded, it remains possible that the shortened signal peptide remains attached to the N-terminal of the protein product and prevents the anti-human reg antibody from recognising the normal N-terminus of the reg protein. The specificity of the human reg antibody is being determined.\n\n**Summary**\n\nThe *reg* gene is known to be present in the human stomach and there is an increasing body of evidence to suggest that it may play a role in the growth and differentiation of endocrine cells in the gut including the ECL cell. Gastrin is also known to influence enteropancreatic endocrine cell differentiation and has a trophic effect on the ECL cell. The data presented demonstrate that plasma gastrin influences *reg* gene expression in human gastric corpus mucosa. In addition, the development of\n\ngastric carcinoid tumours in patients with hypergastrinaemia is associated with mutations of the *reg* gene. This raises the possibility of identifying patients at risk of carcinoid tumour development in the presence of sustained hypergastrinaemia.\n\nChapter Eight\n\nConclusions and Implications\n\nThe main objectives of the work contained within this thesis were to examine the role of antral neurons in the control of gastric emptying and the mechanisms that regulate gastrin release from G cells of the antrum, using a model of antral denervation in the rat. In addition, the significance of *reg* gene expression and carcinoid tumour development was examined by studying the relationships between gastrin and *reg* expression in the ECL-cells of the rat, in human gastric corpus and in a rat pancreatic cell line (AR42J).\n\n**Conclusions**\n\n1. Serosal application of BAC to the gastric antrum effectively lesions all myenteric and spinal afferent fibres of the antrum and the spinal afferent innervation of the gastric corpus. The intrinsic innervation of the proximal stomach is unaffected. Such chemically induced denervation provides a model for experimental studies of the intrinsic innervation of antral muscle and the antral mucosal endocrine cells of which the major cell of interest is the G cell.\n\n2. In the antrally denervated rat there is retention of solid within the stomach that is apparent after a 48hr fast. In antrally denervated rats fitted with a gastric fistula, the emptying of liquid test meals such as saline, acid and peptone was delayed. However, the CCK-dependent inhibition of gastric emptying of peptone was preserved after antral denervation by BAC. It appears that innervation of the antral musculature is\n\nessential for normal gastric emptying of liquids and solids but not CCK-mediated inhibition of gastric emptying.\n\n3. In the intact rat, antral neurons normally inhibit gastrin release in response to non-nutrient distension of the gastric corpus and luminal nutrient produces gastrin release via activation of neurons that release GRP. Following antral denervation, post-prandial gastrin release was maintained but was no longer dependent on either antral neurons or release of GRP. Moreover, non-nutrient distension of the stomach becomes an adequate stimulus for gastrin release.\n\n4. In AR42J cells *reg* mRNA expression was stimulated, in a dose-dependent manner, by amidated gastrin acting through the CCK-B receptor. *Reg* expression in rat and human gastric corpus was also dependent on plasma gastrin concentrations. Mutations of *reg* (a putative regulator of differentiation of gut endocrine cells) were associated with gastric carcinoid tumour development in patients with hypergastrinaemia. A major limitation of the studies thus far are that mRNA only has been measured and quantification of the protein product has not been performed.\n\n**Implications and future studies**\n\nMyenteric denervation by BAC was described first in the rat colon (Sato *et al.*, 1988), where its serosal application produced a phenotype similar to that of Hirschsprung’s disease. The effects of small intestinal denervation by BAC have also\n\nbeen described both by serosal application (myenteric plexus ablation) and luminal application (submucosal plexus ablation). The particular advantages that have been described for BAC are that it can be applied to a strictly defined area of the gut and its application lesions only the myenteric intrinsic neurons and the extrinsic innervation of the region when applied to the serosal surface. Clearly, the effects of BAC on extrinsic afferent fibres may be more widespread depending on the anatomy of innervation. Nevertheless the lesion produced relies upon the ability of intestinal smooth muscle to recover whilst neuronal loss persists. There is a significant inflammatory element to the lesion during the first 7 days (See et al., 1988). Thereafter, histologically inflammation subsides but muscle hypertrophy begins, although active stress generation by hypertrophied muscle does not exceed normal values until after the second week postoperatively (Herman and Bass, 1990).\n\nMost of the studies performed in this thesis used animals towards the end of their second post-operative week. However, the possible confounding factors of continued inflammation and antro-pyloric muscle hypertrophy could conceivably contribute to some of the observations described. In addition, because of a lack of a neuropeptide marker for extrinsic vagal fibres, no attempt was made to determine the extent of vagal damage induced by BAC. Although antral BAC treatment almost certainly disrupts the antral vagal innervation, the preservation of CCK-mediated delay in gastric emptying suggests that the duodenal vagal afferent and gastric vagal efferent fibres were intact.\n\nInflammation of the gastric mucosa is known to result in increased levels of growth factors such as TGF-α (Polk et al., 1992). Growth factors such as TGF-α and EGF have been shown to both stimulate gastrin release and inhibit acid secretion (Konturek et al., 1984; Rhodes et al., 1986). Furthermore, chronic gastric retention and delayed gastric emptying are well recognised complications of hypertrophic pyloric stenosis. In fact, chronic gastric retention is also associated with fasting and meal-stimulated hypergastrinaemia in the myenteric neuropathy that occurs as a result of *Trypanosomal* infection (Oliveira et al., 1980; Troncon et al., 1984).\n\nPerhaps of more immediate relevance may be the mucosal inflammation produced by *H.pylori*. There have been reports that antral gastritis due to *H.pylori* may also induce loss of mucosal nerve fibres and hence lead to denervation (Stead et al., 1996). Interestingly, and in keeping with the observations here, *H. pylori* infection produces basal and meal stimulated increases in plasma gastrin (Levi et al., 1989; McKoll et al., 1989; Tarnasky et al., 1993) and reduces antral somatostatin mRNA levels (Kaneko et al., 1990; Moss et al., 1992) although gastrin mRNA apparently is unaffected. The exaggerated response to GRP in *H. pylori* infection (Graham et al., 1991; McKoll and El-Omar, 1995) is in keeping with increased sensitivity seen following chronic denervation. The potential role of inflammation and growth factors in the BAC-treated rat was not examined.\n\nOf more concern however, may be the potential relationship between trophic factors, elevated plasma gastrin and the growth of gastric mucosal cells given the association between *H. pylori* and the development of gastric tumours. Certainly, the\n\nmarked potentiation by EGF of the gastrin-stimulated expression of *reg* in AR42J cells is preliminary evidence for a similar potentiation occurring in gastric mucosal endocrine cells or their precursors. The wide variability of *reg* expression in hypergastrinaemic patients may indicate different degrees of mucosal inflammation, independent of the level of their plasma gastrin and was a variable that was not controlled for in these studies.\n\nFinally, the role of gastrin and growth factors in the control of differentiation of gastric epithelial (endocrine and non-endocrine) cells is likely to become of increasing importance given the relationship between these factors, PPI therapy, *H. pylori* gastritis and gastric malignancy. The possible role of *reg Iα* as a tumour suppressor gene for endocrine cells of the stomach remains to be clarified but further studies of the *reg* gene family are likely to provide new insights into the cellular and molecular mechanisms that regulate the fundamental processes that lead to the differentiation and maintenance of cell phenotype. The role of *reg Iα* in the stomach and in AR42J cells provides a model system for testing the following hypotheses:\n\ni. expression of the *reg* gene is regulated by gastrin,\n\nii. expression of the *reg* gene influences the progressive differentiation of precursor endocrine cells towards in the gastric mucosa,\n\niii. mutations of *reg* result in failure of differentiation and may cause endocrine cell tumour development in the gastric mucosa.\n\nThe following provides an experimental outline of the future studies that will test these hypotheses:\n\ni) Does gastrin directly control *reg* expression?\n\na) Using the same techniques of tissue culture, mRNA extraction and Northern blot it would be possible to study gastrin receptor mediated events in AR42J cells using recognised gastrin receptor agonists, antagonists and inhibitors. The effects of somatostatin on *reg* expression could also be examined.\n\nb) Gastrin response elements in the promoter region of human *reg* could be identified by transfection of AR42-J cells with different constructs of the promoter region coupled to a reporter luciferase gene. Exact localisation of the relevant sequences would then require scanning mutagenesis and DNA footprinting.\n\nii) Is an intact *reg* essential for normal differentiation of an endocrine cell?\n\na) The ability of *reg* to induce differentiation will be assessed. The ability of recombinant reg protein to induce differentiation in responsive precursor cell lines will be studied and in addition, transfection of undifferentiated but responsive cells with wild type *reg* will also be performed. Expression of markers of differentiation will be assessed following stimulation with gastrin.\n\nb) Immunohistochemical markers of differentiation (CGA, HDC) and proliferation (PCNA, MIB) will be correlated with *reg* expression in precursor stem cells and mature ECL cells in patients with and without hypergastrinaemia.\n\niii Does a *reg* mutation produce abnormal physiology?\n\na) The normal biosynthesis of the Reg protein will be assessed *in vitro* in gastric biopsies by pulse chase experiments incorporating $^{35}$S methionine. Comparisons will be made between patients with and without mutations of *reg*.\n\nb). The processing of wild type and mutant *reg* by Hamster Insulinoma Tumour (HIT) cells will be assessed by morphological and biosynthetic analysis. We have already permanently transfected wild type and mutant *reg* into HIT cells that are capable of processing and secreting transfected proteins.\n\nc). 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(1996) Pancreatic thread protein is mitogenic to pancreatic-derived cells in culture. Gastroenterology 110: 1208-1214.\n\nZENILMAN ME, PERFETTI R, SWINSON K, MAGNUSON T & SHULDINER MD. (1996) Pancreatic regeneration (reg) gene expression in a rat model of islet hyperplasia. Surgery 119: 576-584.\n\nZHANG Z, HOCKER M, KOH T & WANG TC. (1996) The human histidine decarboxylase promoter is regulated by gastrin and phorbol 12-myristate 13-acetate through a downstream cis-acting element. J Biol Chem 271: 14188-14197.\n\nZHOU W, POVOSKI SP, LONGNECKER DS & BELL RH. (1992) Novel expression of gastrin (cholecystokinin-B) receptors in azaserine-induced rat pancreatic carcinoma: receptor determination and characterisation. Cancer Res 52: 6905-6911.\n\nZUCOLOTO S, SILVA JC, OLIVIERA SM & MUCCILLO G. (1991) The chronological relationship between the thickening of smooth muscle, epithelial cell proliferation and myenteric neural denervation in the rat jejunum. Cell Prolif 24: 15-20.\n\nAcknowledgements\n\nI would like to acknowledge Professor Graham Dockray for his supervision of my project and for his advice, support and encouragement over the last three years. I also wish to thank Professor David Thompson and Drs. Rod Dimaline and Andrea Varro for their help and collaboration. I am grateful for the technical assistance of Mrs Cath MacLean, Mark Houghton and particularly David Trafford whose patience and willingness to help seemed infinite. I also would like to recognise the MRC for their financial support of this research.\n\nOn a personal note, I wish to offer special thanks to my colleagues in the Department of Physiology (University of Liverpool) for their helpful advice and friendship and in particular to Lisa Bishop, PJ Noble, Atsushi Murai, Peter Djali and Colin Blackmore each of whom in their own way have made the last three years unforgettable."}}},{"rowIdx":204696,"cells":{"text":{"kind":"string","value":"AN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING'S OHIO BASIC CODE, 2017 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF Amanda Village, OHIO, AND DECLARING AN EMERGENCY.\n\nWHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.\n\nWHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.\n\nWHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.\n\nNOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF Amanda Village, OHIO:\n\nSection 1. American Legal Publishing's Ohio Basic Code, 2017 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2017 Edition.\n\nSection 2. One copy of American Legal Publishing's Ohio Basic Code, 2017 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as \"Exhibit A\".\n\nSection 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2017 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:\n(A) The enactment of the Ohio Basic Code, 2017 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.\n\n(B) The repeal provided above shall not affect:\n(1) The grant or creation of a franchise, license, right, easement or privilege;\n(2) The purchase, sale, lease or transfer of property;\n(3) The appropriation or expenditure of money or promise or guarantee of payment;\n(4) The assumption of any contract or obligation;\n(5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;\n(6) The levy or imposition of taxes, assessments or charges;\n(7) The establishment, naming, vacating or grade level of any street or public way;\n(8) The dedication of property or plat approval;\n(9) The annexation or detachment of territory;\n(10) Any legislation enacted subsequent to the adoption of this ordinance.\n(11) Any legislation specifically superseding the provision of the Ohio Basic Code.\n\nSection 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.\n\nSection 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.\n\nDate Passed: 2-6-17\n\nAttest:\n\nMayor\n\nClerk of the Legislative Authority\n\nNotice is hereby given that on the 6th day of February, 2017, there was enacted by the Legislative Authority of the Municipality of Amanda Village, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2017 Edition, as the Code of Ordinances for the Municipality of Amanda Village, Ohio.”\n\nA summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.\n\n**TITLE I: GENERAL PROVISIONS**\n\n**Chapter 10: General Provisions**\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 10.01 | Short titles |\n| 10.02 | Definitions |\n| 10.03 | Rules of construction |\n| 10.04 | Revivor; effect of amendment or repeal |\n| 10.05 | Construction of section references |\n| 10.06 | Conflicting provisions |\n| 10.07 | Severability |\n| 10.08 | Reference to offices |\n| 10.09 | Errors and omissions |\n| 10.10 | Ordinances repealed |\n| 10.11 | Ordinances unaffected |\n| 10.12 | Ordinances saved |\n| 10.13 | Application to future ordinances |\n| 10.14 | Interpretation |\n| 10.15 | Amendments to code; amendatory language |\n| 10.16 | Statutory references |\n| 10.17 | Preservation of penalties, offenses, rights and liabilities |\n| 10.18 | Determination of legislative intent |\n| 10.99 | General penalty |\n\n**TITLE III: ADMINISTRATION**\n\n**Chapter 30: General Provisions**\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 30.01 | Application of Title III |\n| 30.02 | Qualifications; oaths |\n| 30.03 | Bonds of officers and employees; amount |\n| 30.04 | Additional bond; where bonds recorded and kept |\n| 30.05 | Approval of bonds |\n| 30.06 | Sufficiency of form of bond |\n| 30.07 | Filling vacancies in offices |\n| 30.08 | Public records available |\n| 30.09 | Records Commission |\n| 30.10 | Meetings of public bodies to be open; exceptions; notice |\n| 30.11 | Municipal officers may attend conference or convention; expenses |\n| 30.12 | Residency requirements prohibited; exceptions |\n\n**Chapter 31: Executive Authority**\n\n**General Provisions**\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 31.001 | Executive power; where vested |\n\n**Mayor**\n\n31.015 Term of Mayor; power and duties \n31.016 General duties of the Mayor \n31.017 Communications to the Legislative Authority \n31.018 Protest against excess of expenditures \n31.019 Supervision of conduct of officers \n31.020 Annual report to the Legislative Authority \n31.021 Mayor to file charges against delinquent officers \n31.022 Vacancies in office of Mayor \n31.023 Disposition of fines and other moneys\n\n**Clerk**\n\n31.040 Election, term, qualifications of the Clerk \n31.041 Powers and duties of Clerk \n31.042 Books and accounts; merger of offices \n31.043 Seal of Clerk \n31.044 Combined offices of Clerk and Treasurer; Fiscal Officer\n\n**Treasurer**\n\n31.060 Election, term, qualifications of the Treasurer \n31.061 Accounts of Treasurer \n31.062 Powers and duties \n31.063 Quarterly account; annual report \n31.064 Receipt and disbursement of funds \n31.065 Duty of delivering money and property\n\n**Street Commissioner**\n\n31.080 Qualifications \n31.081 General duties \n31.082 Assistants\n\n**Other Officials**\n\n31.100 Legal counsel \n31.101 Administrator \n31.102 Board of Trustees of Public Affairs \n31.103 Fire Engineer, Engineer and Superintendent of Markets\n\n---\n\n**Chapter 32: Legislative Authority**\n\n**General Provisions**\n\n32.001 Members of the Legislative Authority; election; terms of office \n32.002 President Pro Tempore; employees \n32.003 Vacancy when President Pro Tempore becomes Mayor \n32.004 Qualifications of members of the Legislative Authority \n32.005 Compensation and bonds of municipal officers and employees \n32.006 Vacancy \n32.007 Judge of election and qualification of members; quorum and special meetings \n32.008 Rules; journal; expulsion of members \n32.009 Meetings \n32.010 General powers \n32.011 Failure to take oath or give bond \n32.012 Notice when new bond required \n32.013 Care, supervision and management of public institutions\n\nContracts, Bids and Proceedings\n\n32.025 Contracts by the Legislative Authority or Administrator\n32.026 Bids and proceedings\n32.027 Alterations or modifications of contract\n32.028 Contract restrictions\n32.029 Award to lowest responsive and responsible bidder\n\nOrdinances and Resolutions\n\n32.040 Ordinances and resolutions as evidence\n32.041 Passage procedure\n32.042 Style of ordinances\n32.043 Subject and amendment of ordinances and resolutions\n32.044 Authentication and recording of ordinances and resolutions\n32.045 Publication of ordinances and resolutions; proof of publication and circulation\n32.046 Notice for proposed amendments to the municipal Charter\n32.047 Times of publication required\n32.048 Publication and certification of ordinances in book form\n32.049 Adoption of technical ordinances and codes\n32.050 Certificate of Clerk as to publication\n32.051 Publication when no newspaper published in municipality\n32.052 Effect of not making publication\n32.053 Ordinances providing for appropriations or street improvements; emergency ordinances\n\nInitiative and Referendum\n\n32.070 Initiative petitions\n32.071 Referendum petitions\n32.072 More than one ordinance required; application of subchapter\n32.073 Presentation of petitions\n32.074 Copy of proposed ordinance or measure to be filed with Clerk\n32.075 Words to be printed in red\n32.076 Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election\n32.077 Itemized statement by petition circulator\n32.078 Prohibited practices relative to petitions\n32.079 Accepting premiums for signing\n32.080 Threats in securing signatures\n32.081 Application of subchapter if Charter adopted\n\nChapter 33: Judicial Authority\n\nGeneral Provisions\n\n33.01 Jurisdiction in ordinance cases and traffic violations\n33.02 Powers of Mayor and Mayor's Court Magistrate in criminal matters\n33.03 Duties of Mayor and Mayor's Court Magistrate; fees; office; seal\n33.04 Mayor's Court Magistrate\n33.05 Powers to suspend driver's license in OVI cases\n\nContempt of Court\n\n33.20 Summary punishment for contempt\n33.21 Acts in contempt of court\n33.22 Hearing\n33.23 Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons\n33.24 Right of accused to bail\n33.25 Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order\n33.26 Imprisonment until order obeyed\n33.27 Proceedings when party released on bail fails to appear\n\n33.28 Release of prisoner committed for contempt\n33.29 Judgment final\n33.30 Alternative remedy\n\nChapter 34: Police Department\n\nSection\n\n34.01 Marshal and Police Chief synonymous\n34.02 Appointment of Marshal\n34.03 Deputy marshals and police officers\n34.04 Auxiliary police units\n34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment\n34.06 Removal proceedings; suspension; appeals\n34.07 General powers\n34.08 Powers and duties of Marshal\n34.09 Disposition of fines and penalties\n34.10 Property recovered by police\n34.11 Disposition to claimant\n34.12 Sale of unclaimed property; disposition of proceeds\n34.13 Expenses of storage and sale; notice\n34.14 Contracts for police protection; nonresident service without contract\n34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders\n\nChapter 35: Fire Department\n\nSection\n\nGeneral Provisions\n\n35.01 Municipal fire regulations; fire department\n35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters\n35.03 Schooling of officers and firefighters of fire department\n35.04 Legislative Authority may purchase engines and equipment\n35.05 Buildings for department\n35.06 Records\n35.07 Maximum consecutive hours for firefighters on duty\n35.08 Investigation of cause of fire\n35.09 Right to examine buildings, premises, and vehicles\n35.10 Burning buildings for firefighting instruction or research\n35.11 Impersonating fire safety inspector\n35.12 Standards for equipment\n35.13 Persons entitled to be known as firefighters\n35.14 Firefighting and emergency services agreements\n35.15 Regulation of construction in fire limits\n\nVolunteer Firefighters’ Dependents Fund Board\n\n35.30 Definitions\n35.31 Establishment\n35.32 Membership; vacancies\n35.33 Election and term of members\n35.34 Organization; rules and regulations; roster\n35.35 Compensation and expenses of Board; legal advisor\n\nChapter 36: Civil Actions Against the Municipality\n\nSection\n\n36.01 Definitions\n36.02 Nonliability of municipality; exceptions\n36.03 Defenses and immunities\n36.04 Limitation of actions\n36.05 Damages\n36.06 Satisfaction of judgments\n36.07 Provision of employees’ defense; consent judgments\n36.08 Liability insurance\n36.09 Certain actions unaffected\n36.10 Certain charges against municipal officers filed with Probate Judge; proceedings\n\nTITLE VII: TRAFFIC CODE\n\nChapter 70: General Provisions\n\nGeneral Provisions\n\n70.01 Definitions\n70.02 Compliance with order of police officer\n70.03 Emergency vehicles to proceed cautiously past red or stop signal\n70.04 Exceptions generally; emergency, public safety and coroner vehicles exempt\n70.05 Persons riding or driving animals upon roadways\n70.06 Prohibitions against pedestrians and slow-moving vehicles on freeways\n70.07 Use of private property for vehicular travel\n70.08 Names of persons damaging real property by operation of vehicle to be provided to owner\n70.09 Limited access highways; barriers along; vehicles to enter and leave at designated intersections\n70.10 Through highways\n70.11 Officer may remove ignition key\n70.12 Removal of vehicles after accidents\n\nTraffic-Control Devices\n\n70.30 Obeying traffic-control devices\n70.31 Signal lights\n70.32 Signals over reversible lanes\n70.33 Ambiguous or non-working traffic signals\n70.34 Pedestrian-control signals\n70.35 Unauthorized signs and signals prohibited\n70.36 Alteration, defacement, or removal prohibited\n70.37 Unauthorized possession or sale of devices\n70.38 Signal preemption devices; prohibitions\n70.99 Penalty\n\nChapter 71: Licensing Provisions\n\nMotor Vehicle Licensing\n\n71.01 Display of license plates or validation stickers; registration\n71.02 Improper use of noncommercial motor vehicle\n71.03 Operating motor vehicle ordered immobilized; forfeiture\n71.04 Operation or sale without certificate of title\n71.05 Display of certificate of registration\n71.06 Use of unauthorized plates\n71.07 Operating without dealer or manufacturer license plates\n\nDriver’s Licenses\n\n71.20 Prohibited acts\n71.21 Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license\n71.22 License required as driver or commercial driver on public or private property; nonresident exemption\n71.23 Employment of a minor to operate a taxicab prohibited\n71.24 Restriction against owner lending vehicle for use of another\n71.25 Suspension of driver’s licenses; license suspended by court of record\n71.26 Display of license\n71.27 Prohibition against false statements\n71.28 Driving under suspension or in violation of license restriction\n\n71.29 Operating motor vehicle or motorcycle without valid license\n71.30 Driving under OVI suspension\n71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension\n71.32 Failure to reinstate license\n\n**Commercial Driver's Licenses**\n\n71.45 Definitions\n71.46 Use of actual gross weight in lieu of rating\n71.47 Prohibited acts\n71.48 Prerequisites to operation of commercial motor vehicle\n71.49 Physical qualification to operate commercial motor vehicles\n71.50 Criminal offenses\n71.51 Application of federal regulations\n71.52 Employment of drivers of commercial vehicles\n\n71.99 Penalty\n\n**Chapter 72: Traffic Rules**\n\n**General Provisions**\n\n72.001 Lanes of travel upon roadways\n72.002 Driving through safety zone\n72.003 Vehicles traveling in opposite directions\n72.004 Rules governing overtaking and passing of vehicles\n72.005 Permission to overtake and pass on the right\n72.006 Driving to left of center line\n72.007 Prohibition against driving upon left side of roadway\n72.008 Hazardous zones\n72.009 One-way highways and rotary traffic islands\n72.010 Rules for driving in marked lanes\n72.011 Space between moving vehicles\n72.012 Divided roadways\n72.013 Rules for turns at intersections\n72.014 U-turns and turning in roadway prohibited\n72.015 Starting and backing vehicles\n72.016 Turn and stop signals\n72.017 Hand and arm signals\n\n**Right-of-Way**\n\n72.030 Right-of-way at intersections\n72.031 Right-of-way when turning left\n72.032 Right-of-way at through highways; stop signs; yield signs\n72.033 Stop at sidewalk area; stop signs on private roads and driveways\n72.034 Right-of-way on public highway\n72.035 Pedestrian on sidewalk has right-of-way\n72.036 Right-of-way of public safety vehicles\n72.037 Funeral procession has right-of-way\n72.038 Pedestrians yield right-of-way to public safety vehicle\n72.039 Pedestrian on crosswalk has right-of-way\n72.040 Right-of-way yielded to blind person\n72.041 Right-of-way yielded by pedestrian\n\n**Pedestrians**\n\n72.055 Pedestrian movement in crosswalks\n72.056 Pedestrian walking along highway\n72.057 Prohibition against soliciting rides; riding on outside of vehicle\n72.058 Pedestrian on bridge or railroad crossing\n72.059 Persons operating motorized wheelchairs\n\n72.060 Intoxicated or drugged pedestrian hazard on highway\n72.061 Operation of electric personal assistive mobility devices\n\n**Grade Crossings**\n\n72.075 Stop signs at grade crossings\n72.076 Driving vehicle across railroad grade crossing\n72.077 Vehicles required to stop at grade crossings\n72.078 Slow-moving vehicles or equipment crossing railroad tracks\n\n**School Buses**\n\n72.090 Regulations concerning school buses\n72.091 Violation of regulations; report; investigation; citation; warning\n72.092 Restrictions on the operation of school buses\n72.093 School bus inspection\n72.094 School bus not used for school purposes\n72.095 Licensing by Department of Public Safety\n72.096 Registration and identification of school buses\n72.097 School bus marking\n72.098 Flashing light signal lamps\n72.099 Occupant restraining device for operator\n\n**Prohibitions**\n\n72.115 Obstruction and interference affecting view and control of driver\n72.116 Occupying travel trailer while in motion\n72.117 Driving upon closed highway prohibited\n72.118 Driving upon sidewalk area or paths exclusively for bicycles\n72.119 Obstructing passage of other vehicles\n72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle with caution\n72.121 Driving over unprotected fire hose\n72.122 Placing injurious material on highway or depositing litter from motor vehicle\n72.123 Transporting child not in child-restraint system prohibited\n72.124 Occupant restraining devices\n72.125 Use of engine brakes prohibited\n72.126 Operating motor vehicle while wearing earphones or earplugs\n72.127 Chauffeured limousines and livery services\n72.128 Operating traction engine upon improved highway\n72.129 Cracking exhaust noises; peeling out\n72.130 Shortcutting across private property\n72.131 Texting while driving prohibited\n72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited\n\n**Chapter 73: Motor Vehicle Crimes**\n\n**General Provisions**\n\n73.01 Driving under the influence of alcohol or drugs\n73.02 Implied consent\n73.03 Physical control of vehicle while under the influence\n73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs\n73.05 Reckless operation of vehicles\n73.06 Reckless operation off streets and highways; competitive operation\n73.07 Operator to be in reasonable control\n73.08 Immobilizing or disabling device violation\n73.09 Street racing defined; prohibited on public highways\n73.10 Speed limits\n73.11 Slow speed or stopping\n73.12 Emergency vehicles excepted from speed limitation\n73.13 Speed regulations on bridges\n73.14 Presenting false name or information to officer\n\n73.15 Prohibition against resisting officer\n73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles\n\n**Stopping After Accident**\n\n73.30 Failure to stop after accident\n73.31 Stopping after accident on other than public roads or highways\n73.32 Accident involving damage to realty\n73.33 Failure to report accident\n\nChapter 74: Equipment and Loads\n\n**Equipment**\n\n74.01 Unsafe vehicles, prohibition against operation\n74.02 Bumpers on motor vehicles\n74.03 Lighted lights required\n74.04 Headlights\n74.05 Tail lights and illumination of rear license plate\n74.06 Red reflectors required\n74.07 Safety lighting of commercial vehicles\n74.08 Stoplight regulations\n74.09 Obscured lights on vehicles\n74.10 Red light or flag required\n74.11 Lights on parked vehicles\n74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery\n74.13 Spotlight and auxiliary driving lights\n74.14 Cowl, fender, and back-up lights\n74.15 Two lights displayed\n74.16 Headlights required\n74.17 Lights of less intensity\n74.18 Number of lights permitted; red and flashing lights\n74.19 Standards for lights on snow removal equipment and oversize vehicles\n74.20 Flashing lights permitted for certain types of vehicles\n74.21 Lights and sign on transportation for preschool children\n74.22 Focus and aim of headlights\n74.23 Brake equipment; specifications\n74.24 Brake fluid\n74.25 Minimum standards for brakes and components\n74.26 Horns, sirens, and warning devices\n74.27 Mufflers; excessive smoke or gas\n74.28 Rearview mirrors\n74.29 Windshields and wipers\n74.30 Solid tire requirements\n74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material\n74.32 Directional signals\n74.33 Installation and sale of seat safety belts required; definition\n74.34 Requirements for extra signal equipment\n74.35 Display of warning devices on disabled vehicles\n74.36 Requirements for vehicles transporting explosives\n74.37 Studded tires; seasonal use permitted\n74.38 Safety inspection decals for buses\n74.39 Air bags\n\n**Loads**\n\n74.50 Permit required to exceed load limits\n74.51 Limitation of load extension on left side of vehicle\n74.52 All loads shall be properly secured\n74.53 Towing requirements; exception to size and weight restrictions\n74.54 Weighing of vehicle; removal of excess load\n74.55 Operation of vehicle exceeding weight limits prohibited\n\n74.56 Load limits\n74.57 Maximum width, height, and length\n74.58 Statement of gross vehicle weight\n74.59 Wheel protectors required on heavy commercial vehicles\n74.60 Liability for damages; prosecution; application of monies\n74.61 Weight exceptions for certain vehicles\n\nChapter 75: Bicycles, Motorcycles and Off-Road Vehicles\n\nGeneral Provisions\n\n75.01 Bicycles; application of Title VII\n75.02 Operation of motorized bicycle\n75.03 Rules for bicycles, motorcycles, and snowmobiles\n75.04 Prohibition against attaching bicycles and sleds to vehicles\n75.05 Riding bicycles; motorcycles abreast\n75.06 Equipment of bicycles\n\nSnowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles\n\n75.25 Definitions\n75.26 Equipment\n75.27 Code application; prohibited operation\n75.28 Permitted operation\n75.29 Licensing requirements of operator\n75.30 Maintenance of vehicles for hire\n75.31 Accident reports\n75.32 Impounding of vehicle\n75.33 Local control within police power\n75.34 Registration of vehicles\n75.35 Certificate of title; prohibitions\n\nChapter 76: Parking Regulations\n\nSection\n\n76.01 Prohibition against parking on highways\n76.02 Condition when motor vehicle left unattended\n76.03 Police may remove illegally parked vehicle\n76.04 Parking prohibitions\n76.05 Parking near curb; privileges for persons with disabilities\n76.06 Parking on private property in violation of posted prohibition\n76.07 Selling, washing or repairing vehicle upon roadway\n76.08 Truck loading zones\n76.09 Bus stops and taxicab stands\n76.10 Parking in alleys and narrow streets; exceptions\n76.11 Registered owner prima facie liable for unlawful parking\n76.12 Waiver\n\nTITLE IX: GENERAL REGULATIONS\n\nChapter 90: Animals\n\nAnimals Running at Large\n\n90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings\n90.02 Confining animal found at large; publication of notice; lien\n90.03 Unavoidable escapes\n90.04 Fees\n90.05 Rabies quarantine orders of Mayor\n90.06 Interfering with enforcement of quarantine orders\n90.07 Dogs may be killed for certain acts\n\nOffenses Relating to Animals\n\n90.20 Abandoning animals\n90.21 Injuring animals\n90.22 Poisoning animals\n90.23 Cruelty to animals; cruelty to companion animals\n90.24 Animal fights\n90.25 Trapshooting\n90.26 Loud dog\n90.27 Dog tags\n90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior\n90.29 Failure to register dog or dog kennel\n90.30 Hindering the capture of unregistered dog\n90.31 Unlawful tag\n90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog\n90.33 Retail sale and transportation of dogs\n90.34 Coloring rabbits or baby poultry; regulation of sale and display\n90.35 Jacklighting prohibited\n90.36 Restrictions on dog ownership for certain convicted felons\n\n90.99 Penalty\n\nChapter 91: Fireworks, Explosives, Fire Prevention\n\nFireworks and Explosives\n\n91.01 Definitions\n91.02 Possession, sale, and use of fireworks\n91.03 Permit to use fireworks\n91.04 Manufacturing or wholesale sale without a license; prohibitions\n91.05 Purchasers to comply with law; unauthorized purchases\n91.06 Exhibition without a license; prohibitions\n91.07 Unauthorized transportation or shipping\n91.08 Application of subchapter\n91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines\n91.10 Safety requirements for fireworks showroom structures\n91.11 Storage of explosives\n91.12 Blasting permit\n\nFire Prevention\n\n91.30 Removal of flammable materials or obstructions\n91.31 Protective appliances\n91.32 Compliance with order\n91.33 Waste receptacles\n91.34 Hotel to have fire warning device producing visible signal\n91.35 Fire suppression systems\n91.36 Violations of State Fire Code prohibited\n91.37 Posting arson notices in hotels, motels and other places\n91.38 Negligent burning\n91.39 Spreading alarm of unfriendly fire\n91.40 Unvented heaters\n\nOpen Burning\n\n91.55 Definitions\n91.56 Relations to other prohibitions\n91.57 Open burning in restricted areas\n91.58 Permission and notice to open burn\n\n91.99 Penalty\n\nChapter 92: Intoxicating Liquors\n\nSection\n\n92.01 Definitions\n92.02 Exemptions from chapter\n92.03 Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises\n92.04 Restrictions on sale of beer and liquor\n92.05 Permit required; activities prohibited without permit\n92.06 Illegal transportation prohibited\n92.07 Open container prohibited; exception\n92.08 Underage person shall not purchase intoxicating liquor or beer\n92.09 Prohibitions; minors under 18 years; low-alcohol beverages\n92.10 Alcohol vaporizing devices prohibited\n92.11 Misrepresentation to obtain alcoholic beverage for a minor prohibited\n92.12 Misrepresentation by a minor under 21 years\n92.13 Sale to underage persons prohibited\n92.14 Posting of card\n92.15 Good faith acceptances of spurious identification\n92.16 Consumption in motor vehicle prohibited\n92.17 Hours of sale or consumption\n92.18 Obstructing search of premises prohibited\n92.19 Illegal possession of intoxicating liquor prohibited\n92.20 Sale or possession of diluted liquor and refilled containers prohibited\n92.21 Keeping place where beer or intoxicating liquors are sold in violation of law\n92.22 Intoxicating liquors shall not be sold in brothels\n92.23 Use of intoxicating liquor in a public dance hall prohibited; exceptions\n92.24 Poisonously adulterated liquors\n92.25 Tavern keeper permitting rioting or drunkenness\n92.26 Notice of action to prohibit liquor business\n92.27 Procedure when injunction violated\n92.28 Liquor transaction scans\n92.29 Affirmative defenses\n\n92.99 Penalty\n\nChapter 93: Nuisances\n\nSection\n\nGeneral Provisions\n\n93.01 Application of the chapter\n93.02 Definitions\n93.03 Nuisances generally; injunctions; violation; contempt\n93.04 Maintaining certain nuisances\n93.05 Collection of cost of abating dangerous property condition; injunction; rehabilitation\n93.06 Trimming of trees and shrubbery to prevent obstruction\n\nSeptic Tanks, Cesspools, and Refuse\n\n93.20 Location of privy vaults, cesspools, and septic tanks\n93.21 Unsanitary vaults\n93.22 Removal of contents of vault\n93.23 Deposit of dead animals, offal upon land or water\n93.24 Defiling spring or well prohibited\n93.25 Dumping of refuse in municipality prohibited\n93.26 Abandoned refrigerators\n93.27 Discarding litter prohibited\n93.28 Power of municipality to fill or drain land\n\nWeeds and Litter on Private Property\n\n93.40 Keeping down weeds\n93.41 Notice to owner to cut noxious weeds, remove litter; service\n93.42 Fees for service and return\n93.43 Procedure when owner fails to comply with notice\n93.44 Written return to County Auditor; amount as a lien upon property\n\nUnclean Habitations\n\n93.60 Permitting unclean habitations\n93.61 When habitations are deemed unsanitary\n93.62 Order for abatement or vacation of premises\n93.63 Enforcement of vacation order by Fire Chief or Police Chief\n93.64 Enforcement through court proceedings\n\n93.99 Penalty\n\nChapter 94: Streets and Sidewalks\n\nGeneral Provisions\n\n94.01 Conditions precedent to improving streets\n94.02 Opening permit required\n94.03 Application and cash deposit\n94.04 Restoration of pavement\n94.05 Barriers around excavations\n94.06 Warning lights\n94.07 Sidewalk construction by the municipality\n94.08 Unloading on street or sidewalk\n94.09 Street or sidewalk obstruction\n94.10 Materials on street or sidewalk\n94.11 Duty to keep sidewalks in repair and clean of ice and snow\n94.12 Ramped curbing for persons with disabilities\n94.13 Flagpole along right-of-way\n94.14 Altering or injuring marker or monument\n\nConstruction and Repair\n\n94.25 Construction and repair may be required\n94.26 Resolution of necessity\n94.27 Notice to construct or repair\n94.28 Assessments of costs against owner\n94.29 Proceedings may include different owners\n94.30 Making and levying assessments\n\nChanges in Streets\n\n94.40 Change of name, vacating or narrowing streets by petition\n94.41 Change of name, vacating or narrowing streets without petition\n94.42 Notice; exception\n94.43 Publication of notice\n94.44 Effect of order of vacation\n94.45 Effect on public utility easements\n\n94.99 Penalty\n\nChapter 95: Unclaimed and Abandoned Vehicles\n\nSection\n\n95.01 Impounding motor vehicle on private property; requirements\n95.02 Impounding abandoned motor vehicle on public property; notice; disposition\n95.03 Disposition of vehicle ordered into storage\n95.04 Disposition of abandoned junk motor vehicles\n95.05 Abandonment of junk motor vehicle prohibited\n95.06 Junk motor vehicle; order to cover or remove; notice; exceptions\n\nChapter 96: Watercraft\n\nSection\n\n96.01 Definitions; applicability\n96.02 Failure to comply with law enforcement order; fleeing\n96.03 Duty upon approach of law enforcement vessel\n96.04 Flashing lights prohibited; exceptions\n\n96.05 Siren prohibited; exceptions\n96.06 Regulations for operation of powercraft of more than ten horsepower\n96.07 Restrictions on child operators; duty of supervisory adult\n96.08 Reckless operation; maintaining sufficient control; wakes restricted\n96.09 Unsafe conditions\n96.10 Marking of bathing and vessel areas\n96.11 Mooring prohibited in certain areas\n96.12 Operating under influence of alcohol or drugs prohibited\n96.13 Implied consent\n96.14 Incapacitated operators prohibited\n96.15 Water skiing confined to ski zones\n96.16 Observer required when towing skier\n96.17 Water skiing after dark prohibited\n96.18 Personal flotation device required for towed person\n96.19 Ski jumps prohibited\n96.20 Permit for special water events\n96.21 Sale of single celled inflatable vessels prohibited\n96.22 Sitting, standing, walking on moving vessels restricted\n96.23 Engine warm-up required\n96.24 Personal flotation devices for children under ten\n96.25 Operation without personal flotation devices prohibited\n96.26 Distress signal or flag required\n96.27 Anchor requirements\n96.28 Specification for fire extinguishers\n96.29 Backfire flame control device required\n96.30 Ventilation requirement on powercraft\n96.31 Abandonment of junk vessels or outboard motors\n96.32 Exhaust muffler required; noise levels; exceptions\n96.33 Safety equipment on rental vessels\n96.34 Capacity plate\n96.35 Littering prohibited\n96.36 Dwellings; sanitary systems\n96.37 Prima facie evidence of negligence\n96.38 Requirements for operating personal watercraft\n96.39 Numbering\n96.40 Registration\n96.41 Tags indicating expiration date; attachment of identification number\n96.42 Altering of serial numbers; false information prohibited\n96.43 Accident reports\n96.44 Enforcement\n96.45 Firearms offenses; signaling devices\n96.46 Tampering with navigation aid or vessel prohibited\n96.47 Certificate of title; exceptions\n96.48 Manufacturer's or importer's certificate\n96.49 Prohibitions relating to certificates of title\n96.50 Permanently displayed hull identification number\n\n96.99 Penalty\n\nTITLE XI: BUSINESS REGULATIONS\n\nChapter 110: General Provisions\n\nSection\n\n110.01 Licenses required to engage in certain businesses; exceptions\n110.02 Application for license\n110.03 Issuance of license\n110.04 Date and duration of license\n110.05 License not transferable\n110.06 License certificate to be displayed\n110.07 Revocation or suspension\n110.08 Appeal and review\n\n110.99 General penalty for Title XI\n\nChapter 111: Taxicabs\n\nSection\n\n111.01 Definitions\n111.02 Certificate of public convenience and necessity required\n111.03 Application for certificate\n111.04 Issuance of certificate\n111.05 Liability insurance required\n111.06 License fees\n111.07 Transfer of certificates and licenses\n111.08 Suspension and revocation of certificates\n111.09 Taxicab driver's license\n111.10 Application for driver's license\n111.11 Examination of applicant; motor vehicle operator's permit required\n111.12 Police investigation of applicant; traffic and police record\n111.13 Consideration of application\n111.14 Issuance of license; duration; annual fee\n111.15 Display of license\n111.16 Suspension and revocation of license\n111.17 Failure to comply with federal, state and municipal laws\n111.18 Vehicle equipment and maintenance\n111.19 Designation of taxicabs\n111.20 Taximeter and display of rates required\n111.21 Number of passengers allowed\n111.22 Articles left in vehicles\n111.23 Vehicles from other municipalities\n111.24 Receipts\n111.25 Refusal of passenger to pay legal fare\n111.26 Solicitation, acceptance and discharge of passengers\n111.27 Open stands; use\n111.28 Taxicab service\n111.29 Manifests\n111.30 Records and reports of holders\n111.31 Police department; duty to enforce chapter\n111.32 Disposition of vehicle license fees\n\nChapter 112: Peddlers, Itinerant Merchants, and Solicitors\n\nSection\n\n112.01 Definitions\n112.02 License requirement\n112.03 Application procedure\n112.04 Standards for issuance\n112.05 Revocation procedure\n112.06 Standards for revocation\n112.07 Appeal procedure\n112.08 Exhibition of identification\n112.09 Municipal policy on soliciting\n112.10 Notice regulating soliciting\n112.11 Duty of solicitors\n112.12 Uninvited soliciting prohibited\n112.13 Time limit on soliciting\n\nChapter 113: Commercial Amusements\n\nSection\n\n113.01 Bowling; billiards and pool\n113.02 Circuses, carnivals, shows and other such entertainment\n113.03 Deposit required\n113.04 License fee for public entertainment or exhibition\n113.05 License fee may be waived for civic interest\n\nChapter 114: Tattooing and Body Piercing Services\n\nSection\n\n114.01 Definitions\n114.02 Prohibitions\n114.03 Application for license; fees; issuance\n\n114.04 Inspection of facilities\n114.05 Suspension or revocation of license\n114.06 Consent for performing procedures on persons under 18\n114.07 Prohibitions relating to persons under 18\n114.08 Defenses to violations\n114.09 Training standards; records; safety and sanitation; equipment\n114.10 Application of local regulation on chapter\n\n114.99 Penalty\n\nTITLE XIII: GENERAL OFFENSES\n\nChapter 130: General Provisions\n\nSection\n\n130.01 Application of Title XIII\n130.02 Definitions\n130.03 Classification of offenses\n130.04 Common law offenses abrogated\n130.05 Rules of construction\n130.06 Limitation of criminal prosecutions\n130.07 Requirements for criminal liability; voluntary intoxication\n130.08 Culpable mental states\n130.09 Organizational criminal liability\n130.10 Personal accountability for organizational conduct\n130.11 Attempt\n130.12 Complicity\n130.13 Presumption of innocence; proof of offense; affirmative defense\n130.14 Battered woman syndrome\n130.15 Delinquency adjudications deemed convictions\n130.16 Criminal law jurisdiction\n130.17 Disposition of unclaimed or forfeited property held by Police Department\n130.18 Imposing sentence for misdemeanor\n130.19 Multiple sentences\n130.20 Apprehension, detention, or arrest of persons on bond\n130.21 Self defense: limitations on duty to retreat prior to using force\n\n130.99 Penalty for Title XIII\n\nChapter 131: Offenses Against Property\n\nSection\n\n131.01 Definitions\n131.02 Arson; determining property value or amount of physical harm\n131.03 Criminal damaging or endangering; vehicular vandalism\n131.04 Criminal mischief\n131.05 Damaging or endangering aircraft or airport operations\n131.06 Criminal trespass; aggravated trespass\n131.07 Tampering with coin machines\n131.08 Theft\n131.09 Unauthorized use of a vehicle\n131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property\n131.11 Passing bad checks\n131.12 Misuse of credit cards\n131.13 Making or using slugs\n131.14 Prima facie evidence of purpose to defraud\n131.15 Tampering with records\n131.16 Securing writings by deception\n131.17 Defrauding creditors\n131.18 Receiving stolen property\n131.19 Value of stolen property\n131.20 Degree of offense when certain property involved\n131.21 Injuring vines, bushes, trees, or crops\n131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property\n\n131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud\n131.24 Injury to property by hunters\n131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent\n131.26 Forgery of identification cards\n131.27 Criminal simulation\n131.28 Personating an officer\n131.29 Trademark counterfeiting\n131.30 Diminishing or interfering with forfeitable property\n131.31 Recording credit card, telephone or Social Security numbers\n131.32 Prosecutions for theft of utilities\n131.33 Motion picture piracy\n\nChapter 132: Offenses Against Public Peace\n\nSection\n\n132.01 Riot\n132.02 Failure to disperse\n132.03 Justifiable use of force to suppress riot\n132.04 Disorderly conduct\n132.05 Disturbing a lawful meeting\n132.06 Misconduct at an emergency\n132.07 Telecommunications harassment\n132.08 Inducing panic\n132.09 Making false alarms\n132.10 Inciting to violence\n132.11 Unlawful display of law enforcement emblem\n132.12 Impersonating a peace officer\n132.13 Safety of crowds attending live entertainment performances\n132.14 Misconduct involving a public transportation system\n\nChapter 133: Sex Offenses\n\nSection\n\n133.01 Definitions\n133.02 Unlawful sexual conduct with a minor\n133.03 Sexual imposition\n133.04 Public indecency\n133.05 Voyeurism\n133.06 Polygraph examinations for victims: restrictions on use\n133.07 Procuring\n133.08 Soliciting; loitering to engage in\n133.09 Prostitution\n133.10 Disseminating matter harmful to juveniles\n133.11 Displaying matter harmful to juveniles\n133.12 Deception to obtain matter harmful to juveniles\n133.13 Rules of evidence\n133.14 Declaratory judgment\n133.15 Injunction; abatement of nuisance\n133.16 Unlawful operation of viewing booths depicting sexual conduct\n133.17 Juveniles on the premises of adult entertainment establishments prohibited\n133.18 Sexually oriented businesses; illegal operation and activity\n133.19 Unlawful advertising of massage\n\n133.99 Sentencing for sexually oriented offenses; sexual predators; registration\n\nChapter 134: Gambling Offenses\n\nSection\n\n134.01 Definitions\n134.02 Prohibitions against gambling; exception\n134.03 Operating a gambling house\n134.04 Public gaming\n134.05 Cheating\n134.06 Regulations concerning operation of licensed bingo game\n134.07 Records to be kept\n134.08 Requirements for bingo game operators\n134.09 Bingo games for amusement only\n\n134.10 Prohibitions where instant bingo game is conducted\n134.11 Raffle drawings\n134.12 Instant bingo other than at bingo sessions\n134.13 Restrictions on owner or lessor of location at instant bingo\n134.14 Skill-based amusement machines; prohibited conduct\n\nChapter 135: Offenses Against Persons\n\nSection\n\n135.01 Definitions\n135.02 Negligent homicide\n135.03 Vehicular homicide; vehicular manslaughter\n135.04 Assault; negligent assault\n135.05 Injury to persons by hunters\n135.06 Menacing; aggravated menacing; menacing by stalking\n135.07 Unlawful restraint\n135.08 Criminal child enticement\n135.09 Coercion\n135.10 Bigamy\n135.11 Unlawful abortion; failure to perform viability testing\n135.12 Abortion trafficking\n135.13 Nonsupport of dependents\n135.14 Endangering children\n135.15 Interference with custody; interference with support orders\n135.16 Domestic violence\n135.17 Hazing prohibited\n135.18 Contributing to unruliness or delinquency of a child\n135.19 Failure to provide for functionally impaired person\n135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation\n135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like\n135.22 Ethnic intimidation\n135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state\n135.24 Adulteration of food\n135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans\n135.26 Nonsmoking areas in places of public assembly\n135.27 Spreading contagion\n135.28 Abuse of a corpse\n135.29 Unlawful collection of bodily substances\n\nChapter 136: Offenses Against Justice and Administration\n\nSection\n\n136.01 Definitions\n136.02 Falsification\n136.03 Compounding a crime\n136.04 Failure to report a crime\n136.05 Failure to aid a law enforcement officer\n136.06 Obstructing official business\n136.07 Obstructing justice\n136.08 Resisting arrest\n136.09 Having an unlawful interest in a public contract\n136.10 Soliciting or receiving improper compensation\n136.11 Dereliction of duty\n136.12 Interfering with civil rights\n136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities\n136.14 False report of child abuse or neglect\n136.15 Assaulting police dog or horse, or assistance dog\n136.16 Disclosure of confidential peace officer information\n136.17 Intimidation of crime victim or witness\n136.18 Using sham legal process\n136.19 Making false allegation of peace officer misconduct\n136.20 Misuse of 9-1-1 system\n136.21 Failure to disclose personal information\n\nChapter 137: Weapons Control\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 137.01 | Definitions |\n| 137.02 | Carrying concealed weapons |\n| 137.03 | Using weapons while intoxicated |\n| 137.04 | Improperly handling firearms in a motor vehicle |\n| 137.05 | Possessing criminal tools |\n| 137.06 | Failure to secure dangerous ordnance |\n| 137.07 | Unlawful transactions in weapons |\n| 137.08 | Underage purchase of firearm or handgun |\n| 137.09 | Pointing and discharging firearms and other weapons |\n| 137.10 | License or permit to possess dangerous ordnance |\n| 137.11 | Possession of an object indistinguishable from a firearm in a school safety zone |\n| 137.12 | Possession of deadly weapon while under detention |\n| 137.13 | Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession |\n| 137.14 | Defaced firearms |\n\nChapter 138: Drug Offenses\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 138.01 | Definitions |\n| 138.02 | Trafficking in controlled substances; gift of marihuana |\n| 138.03 | Drug possession offenses |\n| 138.04 | Possessing drug abuse instruments |\n| 138.05 | Permitting drug abuse |\n| 138.06 | Illegal cultivation of marihuana |\n| 138.07 | Abusing harmful intoxicants |\n| 138.08 | Illegal dispensing of drug samples |\n| 138.09 | Federal prosecution bar to municipal prosecution |\n| 138.10 | Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle |\n| 138.11 | Laboratory report required |\n| 138.12 | Counterfeit controlled substances |\n| 138.13 | Use, possession, or sale of drug paraphernalia |\n| 138.14 | Controlled substance or prescription labels |\n| 138.15 | Possession, sale and disposal of hypodermics |\n| 138.16 | Controlled substance schedules |\n| 138.17 | Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates |\n| 138.18 | Pseudoephedrine sales |\n\nTITLE XV: LAND USAGE\n\nChapter 150: General Provisions\n\nSection\n\nParks and Recreation\n\n| Section | Description |\n|---------|-------------|\n| 150.01 | Recreation Board |\n| 150.02 | Board of Park Trustees |\n\nPlanning and Zoning\n\n| Section | Description |\n|---------|-------------|\n| 150.15 | Planning Commission |\n| 150.16 | Board of Zoning Appeals |\n\nPARALLEL REFERENCES\n\nOhio Legislative History References – Master Table\n\nINDEX\n\nThis summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of Amanda Village, Ohio.\n\nSigned: \nMayor \nClerk of the Legislative Authority\n\nCERTIFICATION OF CODIFIED ORDINANCES\n\nWe, Mark A Moore, Mayor, and Carrie A Ayers, Clerk of the Legislative Authority, of the Municipality of Amanda Village, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of Amanda Village, Ohio.\n\nMark A Moore\nMayor\n\nClerk of the Legislative Authority\n\nOrdinance to set rates for outside of village limits\n\nAmend Ordinance #1-2014\n\nWhereas the legislative authority deems it necessary to amend water rates.\n\nWhereas the village would like to set rates for outside the village limits.\n\nWhereas the legislative authority would like to set a 20% monthly surcharge for water on any tap outside the village limits.\n\nWhereas the legislative authority would like to set a 20% monthly surcharge for sewer on any tap outside the village limits.\n\nNow therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby amend the water rates listed above. To add the surcharge fees for water and sewer for any tap outside of the village limits. This surcharge will be a 20% surcharge on water and sewer. That this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote: 4 yes 0 no\n\nAttest: Carrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\nYeas: 4 nays: 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\n1st 4-3-17\n2nd Read 5-1-17\n3rd Wnd 5-1-17\n\nORDINANCE TO AMEND PROPERTY MAINTENANCE CODE\n\nWHEREAS THE VILLAGE COUNCIL DESIRES TO AMEND ORDINANCE 02-2013 THE INTERNATIONAL PROPERTY MAINTENANCE CODE\n\nWHEREAS THE LEGISLATIVE AUTHORITY DEEMS IT NECESSARY TO ADD ADDITIONAL PROVISIONS TO THE EXISTING CODE\n\nWHEREAS THE ATTACHED EXHIBITS B, C, AND D ARE TO BE AMENDED TO THE EXISTING CODE 02-2013\n\nNOW THEREFORE, LET IT BE ORDAINED BY THE VILLAGE OF AMANDA, OHIO:\n\nTHAT THE VILLAGE COUNCIL AUTHORIZES AMENDMENTS TO THE INTERNATIONAL PROPERTY MAINTENANCE CODE 02-2013, WITH THE ADDITIONS OF EXHIBIT B, C, AND D\n\nTHE ORDINANCE SHALL BE IN EFFECT AND BE IN FULL FORCE FROM AND AFTER THE EARLIEST PERIOD ALLOWED BY LAW.\n\nMark A. Moore\nMAYOR\n\nVOTE: YES 5 NO 0\n\nCarrie Ayers\nFISCAL OFFICER\n\nTHE THREE READING REQUIREMENT WAS WAIVED / NOT WAIVED:\n\nYEAS: 5 NAYS: 0\n\nFISCAL OFFICER\n\nTHE UNDERSIGNED, FISCAL OFFICER OF THE LEGISLATIVE AUTHORITY, DOES HEREBY CERTIFY THAT THE FOREGOING LEGISLATION WAS POSTED IN NOT LESS THAN FIVE (5) PUBLIC PLACES, AS DETERMINED BY THE LEGISLATIVE AUTHORITY FOR THE PERIOD OF NOT LESS THAN FIFTEEN (15) DAYS PRIOR TO THE EFFECTIVE DATE THEREOF.\n\nCarrie Ayers\nFISCAL OFFICER\n\n6-5-17 1st Reading\n\nSECTION 302\nEXTERIOR\nPROPERTY AREAS\n\n302.13 STORAGE OF JUNK VEHICLES AND OTHER MOTOR VEHICLES:\n\nDEFINITIONS\n\n(A) \"Junk motor vehicles\" means any motor vehicle which meets any three of the following requirements:\n 1. Is five years old or older;\n 2. Is extensively damaged, such as damage including but not limited to any of the following: missing wheels, tires, motor, or transmission;\n 3. Is not able to be legally operated upon a public street, road, or highway for any reason, whether mechanical, operational, or otherwise;\n 4. Does not have validly issued license plates and a valid registration which allows it to be operated legally upon public streets, roads, or highways;\n 5. Has a fair market value of fifteen hundred dollars ($1,500.00) or less;\n 6. Is not running and not operable (\"operable\" meaning able to be started and driven under its own power).\n\n(B) A junk motor vehicle left in the open for 72 hours is declared a nuisance and shall be abated in accordance with provisions of this chapter.\n\n(C) \"Motor vehicle\" means every vehicle propelled or drawn by power other than muscular power, except motorized bicycles, road rollers, traction engines, power shovels, power cranes, and other equipment used in construction work and not designed for or employed in general highway transportation, hole-digging machinery, well-drilling machinery, ditch-digging machinery, farm machinery, and trailers designed and used exclusively to transport a boat between a place of storage and a marina, or in and around a marina, when drawn to towed on a street or highway for a distance of no more than ten miles.\n\nand at a speed of twenty-five miles per hour or less\n(See ORC. Section 4511.OI(B).\n\n(D) \"In the open\" shall be defined as not garaged.\n\nPROHIBITONS.\n\n(A) No person shall willfully permit a junk motor vehicle to remain in the open on private property which the person owns, occupies, or controls after receipt of an order to remove the junk motor vehicle. The order shall state that a hearing appealing the order to remove the junk motor vehicle maybe had, shall describe the vehicle to be removed, and shall be served by a Code Enforcement Officer or Police Officer.\n\n(B) If any recipient of an order to remove a junk motor vehicle shall fails to appeal the same to the Mayor in writing within ten (10) days after the receipt of the order, it shall be conclusively presumed to establish the junk motor vehicle as a nuisance and the junk motor vehicle shall be removed immediately upon order by a code official designated by the Mayor, the Fire Department, Police Department, or their designee. The fact that a junk motor vehicle is left on private property without the filing of an appeal by the recipient of an order to remove a junk motor vehicle is prima facie evidence of willful failure to comply with the order.\n\n(C) No person shall leave a junk motor vehicle for any period of time on private property to which such person does not have the right of possession without the authorization of the person having the right of possession of such property.\n\nCOLLECTOR'S VEHICLE\n\nA collector's vehicle as defined by O.R.C. Section 4501.01 is a junk motor vehicle for purposes of this section, regardless if it is licensed or unlicensed, if the collector's vehicle meets the definition of a junk motor vehicle as set forth herein.\n\nSTORAGE AND DISPOSAL OF JUNK MOTOR VEHICLES.\n\n(A) After a motor vehicle has been determined to be a junk motor vehicle, as provided herein, the Health Department, Fire Department: Code Enforcement Officer, Police Officer, or their designee shall immediately cause the junk motor vehicle to be removed from the property where the junk motor vehicle is located. The place of storage for any junk motor vehicle which has been removed by this section shall be designated by the Mayor.\n\n(B) Any junk motor vehicle which has been stored by the Police Department pursuant to this chapter, shall be disposed of in accordance with the procedures set forth in O.R.C. Section 4513.63.\n\n**APPEALS**\n\n(A) The Board of Zoning Appeals (BZA) Shall hear any appeals within thirty (30) days of the filing of an appeal by the recipient of an order to remove a junk motor vehicle left in the open. The BZA shall rule whether or not the motor vehicle is a junk motor vehicle left in the open. If the vehicle is declared by the BZA to be a junk motor vehicle left in the open, the BZ shall order the removal of the particular vehicle by the Police Department.\n\n(B) Any appeal from a decision of the BZA shall be made pursuant to O.R.C. Section 2506.\n\n**EXCEPTIONS**\n\nThis chapter shall not apply to vehicles stored inside a garage, in a licensed towing service, in a licensed motor vehicle salvage facility, in a licensed scrap processing yard, in a licensed auto repair garage yard, in a licensed paint spray shop yard, in a licensed gasoline station yard, or in a licensed vehicle dealership yard, or any other lawful storage area not within the public view as prescribed by O.R.C. Section 4737.09.\n\n**PENALTY**\n\nWhoever fails to remove a junk motor vehicle left in the open, after receipt of a proper order to remove the same, shall be subject to the penalties provided here in. Each junk motor vehicle left in the open in violation here of shall constitute a separate offense: Every twenty (20) days that this section is violated shall constitute a separate offense.\n\nSECTION 302\nEXTERIOR PROPERTY AREAS\n\n302.10 MISCELLANEOUS PROVISIONS:\n\n(A) All exterior parts of every dwelling or structure, including exterior walls, parapet walls, decorative additions, chimneys and all other exterior structures, either above or below the roof line, shall be maintained in a safe condition, weather-tight and so as to resist decay or deterioration.\n\n(B) Any dwelling, structure or accessory building whose exterior surface is bare, deteriorated, ramshackle, tumble-down, decaying, disintegrating or in poor condition shall be repaired or razed.\n\n(C) All buckled, rotted or decayed walls, doors, windows, porches, floors, steps, railing trim and their missing members shall be replaced and put in good condition. All replacements shall match and conform to current design or be replaced completely.\n\n(D) All exterior wood or unfinished surface shall be sealed and painted or the surface covered with other protective coating or treated to prevent rot and decay and to conform and match the existing paint or surface covering and original design or replacement thereof. All exterior walls and surfaces shall be properly protected against the weather where such are defective or lack weather protection, including lack of paint or surface covering or have weathered due to lack of proper protective coating.\n\n(E) Any dwelling structure or secondary or appurtenant structure whose exterior surface is deteriorated, decaying, disintegrating, or whose exterior surface has weathered with dirt and grime or has been impaired through the peeling or flaking of paint or other protective coating, and which exterior surfaces consist of an area of twenty-five percent (25%) or more of the external surface of the structure, shall be repaired, repainted or recovered with an approved protective coating or surface. All exterior surfaces shall be replaced or repaired in good condition preparatory to painting or coating. All bare exterior surfaces, which are flaking or crumbling shall be replaced or sealed in a good and workmanlike manner. All new or repaired bare surfaces shall be painted or coated. All exterior surfaces weathered\n\nwith dirt or grime, which are peeling or flaking, shall be painted or covered with approved protective coating or surface.\n\n(F) All repairs shall be completed in a timely manner consistent with accepted construction practices.\n\n(G) All dwelling structures and the premises thereof shall be maintained free from sources of breeding, harborage and infestation by insects, vermin or rodents.\n\n(H) No owner, operator or resident agent of any premises shall maintain or permit to be maintained at or on the exterior areas of any such premises any condition which deteriorates or debases the appearance of the neighborhood, reduces property values in the neighborhood, adversely alters the appearance and general character of the neighborhood, creates a fire, safety or health hazard or which is a public nuisance. Such conditions include but are not limited to the following:\n\n1. Broken or dilapidated fences, walls or other structures;\n2. Out-of-use or non-usable appliances and machinery;\n3. Rugs, rags or other materials hung on lines or in other places on the premises, which materials are not being used for general household or housekeeping purposes;\n4. Broken, dilapidated or unusable furniture, mattresses or other household furniture, broken glass, plastic materials, paints, miscellaneous coverings and/or any other materials, including those described in this section, placed at or on the premises in such a manner as to be patently unsightly, grotesque or offensive to the senses;\n5. No unregistered motor vehicles and/or junk vehicles shall be parked on any property for more than thirty (30) days;\n6. Lawns and landscaping shall be so maintained so as not to constitute a blighting or deteriorating effect on the neighborhood. Grass brush, briers, burrs shall be maintained at a height not to exceed twelve (12) inches. This provision shall not apply to vegetable gardens, bushes, vines, shrubs or trees that produce food for human consumption;\n7. No building materials or materials, earth, sand or dirt intended for use in landscaping, gardening or construction shall be left standing open or covered upon any premises for a period of time exceeding two (2) months without specific written authorization from the Village Zoning Department.\n\n(I) New construction initiated on the exterior of any structure or surrounding yard, after the effective date of the adoption of this provision, shall be completed within one (1) year. In the event of unforeseeable delays in construction, the Code Enforcement Officer is authorized to extend the one (1) year time period for an additional time period of six (6) months or less.\n\nSection 302 Exterior Property Areas\n\n302.11 STORAGE OF CERTAIN MATERIALS DECLARED A NUISANCE:\n\n(A) Definitions\n\n1. Automobile parts\" means and includes any portion or parts of any motor driven vehicle as detached from the vehicle as a whole.\n\n2. Motor vehicle in an inoperative condition\" means and includes any style or type of motor-driven vehicle used or useful for the conveyance of persons or property which is unable to move under its own power due to defective or missing parts, and which have remained in such condition for a period of not less than 30 consecutive days.\n\n3. Motor vehicle unfit for further use\" means and includes any style type of motor-driven vehicle used for the conveyance of persons or property, which is in a dangerous condition, has defective or missing parts, or is in such a condition generally as to be unfit for further (1se as a conveyance.\n\n4. Refuse\" embraces only such matter as was either in fact noxious or has been refused and abandoned by its owner as worthless.\n\n5. Rubbish\" means and includes wire, chips, shavings, bottles, broken glass, crockery, tin, cast or wooden ware, boxes, rags, dead weeds, paper circulars, handbills, boots, shoes, ashes or any waste material other than garbage or offal.\n\n6. Scrap metal\" means and includes pieces of or parts of steel, iron, tm, zinc, copper, aluminum, or any alloy thereof, whether covered with porcelain\n\nor any other material, whether intact or in parts, which has served its usefulness in its original form and can no longer be used or useful for its originally intended purpose.\n\n7. \"Used building materials\" means and includes any materials, such as wood, stone, brick, cement block, asphalt, blacktop, concrete, construction debris, or any composition thereof, used or useful in the erection of any building or structure, which have been used previously for such erection or construction, by the same persons or by any other person.\n\n(B) Storage of Certain Materials Declared a Nuisance.\n\nIt shall be unlawful and is hereby declared a nuisance for any reason to store, place or allow to remain, automobile parts, motor vehicles in an inoperative condition, motor vehicles unfit for further use, refuse, rubbish, scrap metal, or used building materials, as defined herein, on any lot, lots, parts of lots, or parcel of land, within the corporate limits if the Village.\n\n(C) Order for Removal.\n\n(1) In the event of a violation of division (b) of this section, the Mayor or Property Maintenance Code Officer shall give notice to the owner, occupant or person having charge of the premises upon which the violation occurs to cease such violation. Such notice shall be in writing and shall be served upon the owner, occupant or person having charge of the premises either personally or at the usual place of residence of such owner, occupant or person having charge of such premises or by registered or certified mail addressed to such person's last known place of residence.\n\n(2) If the person served with the notice as provided in division (1) fails to cause such violation to cease within ten days of the date upon which the notice w\n\nissued, he or she shall be subject to the penalties provided herein, and a separate offense shall be deemed committed upon each day during or on which the violation occurs or continues beyond such ten-day period; provided that no additional notice of violation is required to be given.\n\n(D) Exception\n\nNotwithstanding the provisions of division (b) of this section, it shall not be unlawful for any person to purchase used building materials and place or store them on any lot, lots parts of lots, or parcel of land, when such materials are stored within the confines of a closed garage, or building or are to a used by the purchaser or owner in later construction on the same lot or any lot owned or controlled by such person; provided that such materials shall not remain on the lot, lots, parts of lots, or parcel of land, for a period of more than 30 days, unless the construction or erection planned for the use of the materials has commenced; and provided further that such materials are used or consumed in the construction or removed from the premises within a period of four months from the time the materials are first placed on the lot, lots, parts of lots or parcel off and. It shall be unlawful for any person or persons to move any materials so stored or placed to another location within the village for the purpose of avoiding the intent of this section, except that any such materials may be moved to another lot, lots, parts of lots or parcel of land, when the same have been sold to a bona fide purchaser for value for such purchaser's own use.\n\nSECTION 302\nEXTERIOR\nPROPERTY AREAS\n\n302.13 STORAGE OF JUNK VEHICLES AND OTHER MOTOR VEHICLES:\n\nDEFINITIONS:\n(A) \"Junk motor vehicles\" means any motor vehicle which meets any three of the following requirements:\n 1. Is five years old or older;\n 2. Is extensively damaged, such as damage including but not limited to any of the following: missing wheels, tires, motor, or transmission;\n 3. Is not able to be legally operated upon a public street, road, or highway for any reason, whether mechanical, operational, or otherwise;\n 4. Does not have validly issued license plates and a valid registration which allows it to be operated legally upon public streets, roads, or highways;\n 5. Has a fair market value of fifteen hundred dollars ($1,500.00) or less;\n 6. Is not running and not operable (\"operable\" meaning able to be started and driven under its own power);\n\n(B) A junk motor vehicle left in the open for 72 hours is declared a nuisance and shall be abated in accordance with provisions of this chapter.\n\n(C) \"Motor vehicle\" means every vehicle propelled or drawn by power other than muscular power, except motorized bicycles, road rollers, traction engines, power shovels, power cranes, and other equipment used in construction work and not designed for or employed in general highway transportation, hole-digging machinery, well-drilling machinery, ditch-digging machinery, farm machinery, and trailers designed and used exclusively to transport a boat between a place of storage and a marina, or in and around a marina, when drawn to towed on a street or highway for a distance of no more than ten miles.\n\nand at a speed of twenty-five miles per hour or less\n(See ORC. Section 451I.Ol(B).\n\n(D) \"In the open\" shall be defined as not garaged.\n\nPROHIBITIONS.\n\n(A) No person shall willfully permit a junk motor vehicle to remain in the open on private property which the person owns, occupies, or controls after receipt of an order to remove the junk motor vehicle. The order shall state that a hearing appealing the order to remove the junk motor vehicle maybe had, shall describe the vehicle to be removed, and shall be served by a Code Enforcement Officer or Police Officer.\n\n(B) If any recipient of an order to remove a junk motor vehicle shall fails to appeal the same to the Mayor in writing within ten (10) days after the receipt of the order, it shall be conclusively presumed to establish the junk motor vehicle as a nuisance and the junk motor vehicle shall be removed immediately upon order by a code official designated by the Mayor, the Fire Department, Police Department, or their designee. The fact that a junk motor vehicle is left on private property without the filing of an appeal by the recipient of an order to remove a junk motor vehicle is prima facie evidence of willful failure to comply with the order.\n\n(C) No person shall leave a junk motor vehicle for any period of time on private property to which such person does not have the right of possession without the authorization of the person having the right of possession of such property.\n\nCOLLECTOR'S VEHICLE\n\nA collector's vehicle as defined by O.R.C. Section 4501.01 is a junk motor vehicle for purposes of this section, regardless if it is licensed or unlicensed, if the collector's vehicle meets the definition of a junk motor vehicle as set forth herein.\n\nSTORAGE AND DISPOSAL OF JUNK MOTOR VEHICLES.\n\n(A) After a motor vehicle has been determined to be a junk motor vehicle, as provided herein, the Health Department, Fire Department: Code Enforcement Officer, Police Officer, or their designee shall immediately cause the junk motor vehicle to be removed from the property where the junk motor vehicle is located. The place of storage for any junk motor vehicle which has been removed by this section shall be designated by the Mayor.\n\n(B) Any junk motor vehicle which has been stored by the Police Department pursuant to this chapter, shall be disposed of in accordance with the procedures set forth in O.R.C. Section 4513.63.\n\nAPPEALS\n\n(A) The Board of Zoning Appeals (BZA) Shall hear any appeals within thirty (30) days of filing an appeal by the recipient of an order to remove a junk motor vehicle left in the open. The BZA shall rule whether or not the motor vehicle is a junk motor vehicle left in the open.\n\nIf the vehicle is declared by the BZA to be a junk motor vehicle left in the open, the BZ shall order the removal of the particular vehicle by the Police Department.\n\n(B) Any appeal from a decision of the BZA shall be made pursuant to O.R.C. Section 2506.\n\nEXCEPTIONS\n\nThis chapter shall not apply to vehicles stored inside a garage, in a licensed towing service, in a licensed motor vehicle salvage facility, in a licensed scrap processing yard, in a licensed auto repair garage yard, in a licensed paint spray shop yard, in a licensed gasoline station yard, or in a licensed vehicle dealership yard, or any other lawful storage area not within the public view as prescribed by O.R.C. Section 4737.09.\n\nPENALTY\n\nWhoever fails to remove a junk motor vehicle left in the open, after receipt of a proper order to remove the same, shall be subject to the penalties provided here in. Each junk motor vehicle left in the open in violation here of shall constitute a separate offense. Every twenty (20) days that this section is violated shall constitute a separate offense.\n\nORDINANCE TO ADOPTING AN UPDATED VERSION OF THE (2018) INTERNATIONAL PROPERTY MAINTENANCE CODE, AS AMENDED, TOGETHER WITH ALL UPDATED REVISIONS OF SAID CODE.\n\nWHEREAS THE VILLAGE COUNCIL DESIRES TO AMEND ORDINANCE 02-2013 THE INTERNATIONAL PROPERTY MAINTENANCE CODE TO ADD ALL REVISIONS AND UPDATED VERSIONS;\n\nWHEREAS THE VILLAGE COUNCIL DESIRES TO ADOPT THE UPDATED 2018 INTERNATIONAL PROPERTY MAINTENANCE CODE;\n\nWHEREAS THE LEGISLATIVE AUTHORITY DEEMS IT NECESSARY TO ADD ADDITIONAL PROVISIONS TO THE EXISTING CODE IN ORDER TO MAINTAIN THE SAFETY AND WELLBEING OF THE CITIZENS;\n\nNOW THEREFORE, LET IT BE ORDAINED BY THE VILLAGE OF AMANDA, OHIO:\n\nTHAT THE VILLAGE COUNCIL AUTHORIZES AMENDMENTS AND MOVES TO ADOPT THE 2018 INTERNATIONAL PROPERTY MAINTENANCE CODE,\n\nTHE ORDINANCE SHALL BE IN EFFECT AND BE IN FULL FORCE FROM AND AFTER THE EARLIEST PERIOD ALLOWED BY LAW.\n\nMAYOR\n\nVOTE: YES 5 NO 0\n\nCARRIE AYERS\nFISCAL OFFICER\n\nTHE THREE READING REQUIREMENT WAS WAIVED / NOT WAIVED:\n\nYEAS: 5 NAYS: 0\n\nFISCAL OFFICER\n\nTHE UNDERSIGNED, FISCAL OFFICER OF THE LEGISLATIVE AUTHORITY, DOES HEREBY CERTIFY THAT THE FOREGOING LEGISLATION WAS POSTED IN NOT LESS THAN FIVE (5) PUBLIC PLACES, AS DETERMINED BY THE LEGISLATIVE AUTHORITY FOR THE PERIOD OF NOT LESS THAN FIFTEEN (15) DAYS PRIOR TO THE EFFECTIVE DATE THEREOF.\n\nFISCAL OFFICER\n\nTo: Village Officials\n\nRe: Subscription to the Ohio Basic Code, 2018 Edition\n\nThank you for your subscription to the *Ohio Basic Code, 2018 Edition*. This model Code of Ordinances has been adopted by the Village as their own Code, and the 2018 Edition replaces any previous editions of the Village Code. Please note, however, that if you have received a looseleaf “Title 17: Municipal Regulations,” this **Title 17 is not affected by this 2018 Edition and should be retained**. Please mail any Village ordinances to update your Title 17 to our office at your earliest convenience.\n\nEnclosed you will also find a sample adopting ordinance, a summary of new matter (for publication notification), and a certification of codified ordinances, all provided for your convenience in adopting the *Ohio Basic Code, 2018 Edition*.\n\nWe are pleased to provide the *Ohio Basic Code, 2018 Edition*, and we look forward to continuing to serve Ohio villages.\n\nVery truly yours,\n\nTodd Paul Myers, J.D.\nVice President, Major Client Services\nemail@example.com\n\nCERTIFICATION OF CODIFIED ORDINANCES\n\nWe, ________________, Mayor, and ________________, Clerk of the Legislative Authority, of the Municipality of ________________, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of ________________, Ohio.\n\n__________________________\nMayor\n\n__________________________\nClerk of the Legislative Authority\n\nAN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING’S OHIO BASIC CODE, 2018 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF ________, OHIO, AND DECLARING AN EMERGENCY.\n\nWHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.\n\nWHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.\n\nWHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.\n\nNOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF ________, OHIO:\n\nSection 1. American Legal Publishing’s Ohio Basic Code, 2018 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2018 Edition.\n\nSection 2. One copy of American Legal Publishing’s Ohio Basic Code, 2018 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as “Exhibit A”.\n\nSection 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2018 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:\n(A) The enactment of the Ohio Basic Code, 2018 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.\n\n(B) The repeal provided above shall not affect:\n (1) The grant or creation of a franchise, license, right, easement or privilege;\n (2) The purchase, sale, lease or transfer of property;\n (3) The appropriation or expenditure of money or promise or guarantee of payment;\n (4) The assumption of any contract or obligation;\n (5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;\n (6) The levy or imposition of taxes, assessments or charges;\n (7) The establishment, naming, vacating or grade level of any street or public way;\n (8) The dedication of property or plat approval;\n (9) The annexation or detachment of territory;\n (10) Any legislation enacted subsequent to the adoption of this ordinance.\n (11) Any legislation specifically superseding the provision of the Ohio Basic Code.\n\nSection 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.\n\nSection 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.\n\nDate Passed: 2-5-17\n\nAttest:\n\nMayor\n\nClerk of the Legislative Authority\n\nNotice is hereby given that on the 5th day of Feb., 2018, there was enacted by the Legislative Authority of the Municipality of Village of Avon, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2018 Edition, as the Code of Ordinances for the Municipality of Village of Avon, Ohio.”\n\nA summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.\n\nTITLE I: GENERAL PROVISIONS\n\nChapter 10: General Provisions\n\nSection\n\n10.01 Short titles\n10.02 Definitions\n10.03 Rules of construction\n10.04 Revivor; effect of amendment or repeal\n10.05 Construction of section references\n10.06 Conflicting provisions\n10.07 Severability\n10.08 Reference to offices\n10.09 Errors and omissions\n10.10 Ordinances repealed\n10.11 Ordinances unaffected\n10.12 Ordinances saved\n10.13 Application to future ordinances\n10.14 Interpretation\n10.15 Amendments to code; amendatory language\n10.16 Statutory references\n10.17 Preservation of penalties, offenses, rights and liabilities\n10.18 Determination of legislative intent\n10.99 General penalty\n\nTITLE III: ADMINISTRATION\n\nChapter 30: General Provisions\n\nSection\n\n30.01 Application of Title III\n30.02 Qualifications; oaths\n30.03 Bonds of officers and employees; amount\n30.04 Additional bond; where bonds recorded and kept\n30.05 Approval of bonds\n30.06 Sufficiency of form of bond\n30.07 Filling vacancies in offices\n30.08 Public records available\n30.09 Records Commission\n30.10 Meetings of public bodies to be open; exceptions; notice\n30.11 Municipal officers may attend conference or convention; expenses\n30.12 Residency requirements prohibited; exceptions\n\nChapter 31: Executive Authority\n\nSection\n\nGeneral Provisions\n\n31.001 Executive power; where vested\n\n**Mayor**\n\n31.015 Term of Mayor; power and duties \n31.016 General duties of the Mayor \n31.017 Communications to the Legislative Authority \n31.018 Protest against excess of expenditures \n31.019 Supervision of conduct of officers \n31.020 Annual report to the Legislative Authority \n31.021 Mayor to file charges against delinquent officers \n31.022 Vacancies in office of Mayor \n31.023 Disposition of fines and other moneys\n\n**Clerk**\n\n31.040 Election, term, qualifications of the Clerk \n31.041 Powers and duties of Clerk \n31.042 Books and accounts; merger of offices \n31.043 Seal of Clerk \n31.044 Combined offices of Clerk and Treasurer; Fiscal Officer\n\n**Treasurer**\n\n31.060 Election, term, qualifications of the Treasurer \n31.061 Accounts of Treasurer \n31.062 Powers and duties \n31.063 Quarterly account; annual report \n31.064 Receipt and disbursement of funds \n31.065 Duty of delivering money and property\n\n**Street Commissioner**\n\n31.080 Qualifications \n31.081 General duties \n31.082 Assistants\n\n**Other Officials**\n\n31.100 Legal counsel \n31.101 Administrator \n31.102 Board of Trustees of Public Affairs \n31.103 Fire Engineer, Engineer and Superintendent of Markets\n\n---\n\n**Chapter 32: Legislative Authority**\n\n**General Provisions**\n\n32.001 Members of the Legislative Authority; election; terms of office \n32.002 President Pro Tempore; employees \n32.003 Vacancy when President Pro Tempore becomes Mayor \n32.004 Qualifications of members of the Legislative Authority \n32.005 Compensation and bonds of municipal officers and employees \n32.006 Vacancy \n32.007 Judge of election and qualification of members; quorum and special meetings \n32.008 Rules; journal; expulsion of members \n32.009 Meetings \n32.010 General powers \n32.011 Failure to take oath or give bond \n32.012 Notice when new bond required \n32.013 Care, supervision and management of public institutions\n\n### Contracts, Bids and Proceedings\n\n32.025 Contracts by the Legislative Authority or Administrator \n32.026 Bids and proceedings \n32.027 Alterations or modifications of contract \n32.028 Contract restrictions \n32.029 Award to lowest responsive and responsible bidder \n\n### Ordinances and Resolutions\n\n32.040 Ordinances and resolutions as evidence \n32.041 Passage procedure \n32.042 Style of ordinances \n32.043 Subject and amendment of ordinances and resolutions \n32.044 Authentication and recording of ordinances and resolutions \n32.045 Publication of ordinances and resolutions; proof of publication and circulation \n32.046 Notice for proposed amendments to the municipal Charter \n32.047 Times of publication required \n32.048 Publication and certification of ordinances in book form \n32.049 Adoption of technical ordinances and codes \n32.050 Certificate of Clerk as to publication \n32.051 Publication when no newspaper published in municipality \n32.052 Effect of not making publication \n32.053 Ordinances providing for appropriations or street improvements; emergency ordinances \n\n### Initiative and Referendum\n\n32.070 Initiative petitions \n32.071 Referendum petitions \n32.072 More than one ordinance required; application of subchapter \n32.073 Presentation of petitions \n32.074 Copy of proposed ordinance or measure to be filed with Clerk \n32.075 Words to be printed in red \n32.076 Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election \n32.077 Itemized statement by petition circulator \n32.078 Prohibited practices relative to petitions \n32.079 Accepting premiums for signing \n32.080 Threats in securing signatures \n32.081 Application of subchapter if Charter adopted \n\n### Chapter 33: Judicial Authority\n\n#### General Provisions\n\n33.01 Jurisdiction in ordinance cases and traffic violations \n33.02 Powers of Mayor and Mayor’s Court Magistrate in criminal matters \n33.03 Duties of Mayor and Mayor’s Court Magistrate; fees; office; seal \n33.04 Mayor’s Court Magistrate \n33.05 Powers to suspend driver’s license in OVI cases \n\n#### Contempt of Court\n\n33.20 Summary punishment for contempt \n33.21 Acts in contempt of court \n33.22 Hearing \n33.23 Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons \n33.24 Right of accused to bail \n33.25 Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order \n33.26 Imprisonment until order obeyed \n33.27 Proceedings when party released on bail fails to appear\n\n33.28 Release of prisoner committed for contempt\n33.29 Judgment final\n33.30 Alternative remedy\n\nChapter 34: Police Department\n\nSection\n\n34.01 Marshal and Police Chief synonymous\n34.02 Appointment of Marshal\n34.03 Deputy marshals and police officers\n34.04 Auxiliary police units\n34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment\n34.06 Removal proceedings; suspension; appeals\n34.07 General powers\n34.08 Powers and duties of Marshal\n34.09 Disposition of fines and penalties\n34.10 Property recovered by police\n34.11 Disposition to claimant\n34.12 Sale of unclaimed property; disposition of proceeds\n34.13 Expenses of storage and sale; notice\n34.14 Contracts for police protection; nonresident service without contract\n34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders\n\nChapter 35: Fire Department\n\nGeneral Provisions\n\nSection\n\n35.01 Municipal fire regulations; fire department\n35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters\n35.03 Schooling of officers and firefighters of fire department\n35.04 Legislative Authority may purchase engines and equipment\n35.05 Buildings for department\n35.06 Records\n35.07 Maximum consecutive hours for firefighters on duty\n35.08 Investigation of cause of fire\n35.09 Right to examine buildings, premises, and vehicles\n35.10 Burning buildings for firefighting instruction or research\n35.11 Impersonating fire safety inspector\n35.12 Standards for equipment\n35.13 Persons entitled to be known as firefighters\n35.14 Firefighting and emergency services agreements\n35.15 Regulation of construction in fire limits\n\nVolunteer Firefighters’ Dependents Fund Board\n\nSection\n\n35.30 Definitions\n35.31 Establishment\n35.32 Membership; vacancies\n35.33 Election and term of members\n35.34 Organization; rules and regulations; roster\n35.35 Compensation and expenses of Board; legal advisor\n\nChapter 36: Civil Actions Against the Municipality\n\nSection\n\n36.01 Definitions\n36.02 Nonliability of municipality; exceptions\n36.03 Defenses and immunities\n36.04 Limitation of actions\n36.05 Damages\n36.06 Satisfaction of judgments\n36.07 Provision of employees’ defense; consent judgments\n36.08 Liability insurance\n36.09 Certain actions unaffected\n36.10 Certain charges against municipal officers filed with Probate Judge; proceedings\n\nTITLE VII: TRAFFIC CODE\n\nChapter 70: General Provisions\n\nGeneral Provisions\n\n| Section | Description |\n|---------|-------------|\n| 70.01 | Definitions |\n| 70.02 | Compliance with order of police officer |\n| 70.03 | Emergency vehicles to proceed cautiously past red or stop signal |\n| 70.04 | Exceptions generally; emergency, public safety and coroner vehicles exempt |\n| 70.05 | Persons riding or driving animals upon roadways |\n| 70.06 | Prohibitions against pedestrians and slow-moving vehicles on freeways |\n| 70.07 | Use of private property for vehicular travel |\n| 70.08 | Names of persons damaging real property by operation of vehicle to be provided to owner |\n| 70.09 | Limited access highways; barriers along; vehicles to enter and leave at designated intersections |\n| 70.10 | Through highways |\n| 70.11 | Officer may remove ignition key |\n| 70.12 | Removal of vehicles after accidents |\n\nTraffic-Control Devices\n\n| Section | Description |\n|---------|-------------|\n| 70.30 | Obeying traffic-control devices |\n| 70.31 | Signal lights |\n| 70.32 | Signals over reversible lanes |\n| 70.33 | Ambiguous or non-working traffic signals |\n| 70.34 | Pedestrian-control signals |\n| 70.35 | Unauthorized signs and signals prohibited |\n| 70.36 | Alteration, defacement, or removal prohibited |\n| 70.37 | Unauthorized possession or sale of devices |\n| 70.38 | Signal preemption devices; prohibitions |\n| 70.99 | Penalty |\n\nChapter 71: Licensing Provisions\n\nMotor Vehicle Licensing\n\n| Section | Description |\n|---------|-------------|\n| 71.01 | Display of license plates or validation stickers; registration |\n| 71.02 | Improper use of noncommercial motor vehicle |\n| 71.03 | Operating motor vehicle ordered immobilized; forfeiture |\n| 71.04 | Operation or sale without certificate of title |\n| 71.05 | Display of certificate of registration |\n| 71.06 | Use of unauthorized plates |\n| 71.07 | Operating without dealer or manufacturer license plates |\n\nDriver’s Licenses\n\n| Section | Description |\n|---------|-------------|\n| 71.20 | Prohibited acts |\n| 71.21 | Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license |\n| 71.22 | License required as driver or commercial driver on public or private property; nonresident exemption |\n| 71.23 | Employment of a minor to operate a taxicab prohibited |\n| 71.24 | Restriction against owner lending vehicle for use of another |\n| 71.25 | Suspension of driver’s licenses; license suspended by court of record |\n| 71.26 | Display of license |\n| 71.27 | Prohibition against false statements |\n| 71.28 | Driving under suspension or in violation of license restriction |\n\n71.29 Operating motor vehicle or motorcycle without valid license\n71.30 Driving under OVI suspension\n71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension\n71.32 Failure to reinstate license\n\n**Commercial Driver's Licenses**\n\n71.45 Definitions\n71.46 Use of actual gross weight in lieu of rating\n71.47 Prohibited acts\n71.48 Prerequisites to operation of commercial motor vehicle\n71.49 Physical qualification to operate commercial motor vehicles\n71.50 Criminal offenses\n71.51 Application of federal regulations\n71.52 Employment of drivers of commercial vehicles\n71.99 Penalty\n\n**Chapter 72: Traffic Rules**\n\n**General Provisions**\n\n72.001 Lanes of travel upon roadways\n72.002 Driving through safety zone\n72.003 Vehicles traveling in opposite directions\n72.004 Rules governing overtaking and passing of vehicles\n72.005 Permission to overtake and pass on the right\n72.006 Driving to left of center line\n72.007 Prohibition against driving upon left side of roadway\n72.008 Hazardous zones\n72.009 One-way highways and rotary traffic islands\n72.010 Rules for driving in marked lanes\n72.011 Space between moving vehicles\n72.012 Divided roadways\n72.013 Rules for turns at intersections\n72.014 U-turns and turning in roadway prohibited\n72.015 Starting and backing vehicles\n72.016 Turn and stop signals\n72.017 Hand and arm signals\n\n**Right-of-Way**\n\n72.030 Right-of-way at intersections\n72.031 Right-of-way when turning left\n72.032 Right-of-way at through highways; stop signs; yield signs\n72.033 Stop at sidewalk area; stop signs on private roads and driveways\n72.034 Right-of-way on public highway\n72.035 Pedestrian on sidewalk has right-of-way\n72.036 Right-of-way of public safety vehicles\n72.037 Funeral procession has right-of-way\n72.038 Pedestrians yield right-of-way to public safety vehicle\n72.039 Pedestrian on crosswalk has right-of-way\n72.040 Right-of-way yielded to blind person\n72.041 Right-of-way yielded by pedestrian\n\n**Pedestrians**\n\n72.055 Pedestrian movement in crosswalks\n72.056 Pedestrian walking along highway\n72.057 Prohibition against soliciting rides; riding on outside of vehicle\n72.058 Pedestrian on bridge or railroad crossing\n72.059 Persons operating motorized wheelchairs\n\n72.060 Intoxicated or drugged pedestrian hazard on highway\n72.061 Operation of electric personal assistive mobility devices\n72.062 Operation of personal delivery device on sidewalks and crosswalks\n\n**Grade Crossings**\n\n72.075 Stop signs at grade crossings\n72.076 Driving vehicle across railroad grade crossing\n72.077 Vehicles required to stop at grade crossings\n72.078 Slow-moving vehicles or equipment crossing railroad tracks\n\n**School Buses**\n\n72.090 Regulations concerning school buses\n72.091 Violation of regulations; report; investigation; citation; warning\n72.092 Restrictions on the operation of school buses\n72.093 School bus inspection\n72.094 School bus not used for school purposes\n72.095 Licensing by Department of Public Safety\n72.096 Registration and identification of school buses\n72.097 School bus marking\n72.098 Flashing light signal lamps\n72.099 Occupant restraining device for operator\n\n**Prohibitions**\n\n72.115 Obstruction and interference affecting view and control of driver\n72.116 Occupying travel trailer while in motion\n72.117 Driving upon closed highway prohibited\n72.118 Driving upon sidewalk area or paths exclusively for bicycles\n72.119 Obstructing passage of other vehicles\n72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle with caution\n72.121 Driving over unprotected fire hose\n72.122 Placing injurious material on highway or depositing litter from motor vehicle\n72.123 Transporting child not in child-restraint system prohibited\n72.124 Occupant restraining devices\n72.125 Use of engine brakes prohibited\n72.126 Operating motor vehicle while wearing earphones or earplugs\n72.127 Chauffeured limousines and livery services\n72.128 Operating traction engine upon improved highway\n72.129 Cracking exhaust noises; peeling out\n72.130 Shortcutting across private property\n72.131 Texting while driving prohibited\n72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited\n\n**Chapter 73: Motor Vehicle Crimes**\n\n**General Provisions**\n\n73.01 Driving under the influence of alcohol or drugs\n73.02 Implied consent\n73.03 Physical control of vehicle while under the influence\n73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs\n73.05 Reckless operation of vehicles\n73.06 Reckless operation off streets and highways; competitive operation\n73.07 Operator to be in reasonable control\n73.08 Immobilizing or disabling device violation\n73.09 Street racing defined; prohibited on public highways\n73.10 Speed limits\n73.11 Slow speed or stopping\n73.12 Emergency vehicles excepted from speed limitation\n73.13 Speed regulations on bridges\n\n73.14 Presenting false name or information to officer\n73.15 Prohibition against resisting officer\n73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles\n\n**Stopping After Accident**\n\n73.30 Failure to stop after accident\n73.31 Stopping after accident on other than public roads or highways\n73.32 Accident involving damage to realty\n73.33 Failure to report accident\n\n**Chapter 74: Equipment and Loads**\n\n**Equipment**\n\n74.01 Unsafe vehicles, prohibition against operation\n74.02 Bumpers on motor vehicles\n74.03 Lighted lights required\n74.04 Headlights\n74.05 Tail lights and illumination of rear license plate\n74.06 Red reflectors required\n74.07 Safety lighting of commercial vehicles\n74.08 Stoplight regulations\n74.09 Obscured lights on vehicles\n74.10 Red light or flag required\n74.11 Lights on parked vehicles\n74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery\n74.13 Spotlight and auxiliary driving lights\n74.14 Cowl, fender, and back-up lights\n74.15 Two lights displayed\n74.16 Headlights required\n74.17 Lights of less intensity\n74.18 Number of lights permitted; red and flashing lights\n74.19 Standards for lights on snow removal equipment and oversize vehicles\n74.20 Flashing lights permitted for certain types of vehicles\n74.21 Lights and sign on transportation for preschool children\n74.22 Focus and aim of headlights\n74.23 Brake equipment; specifications\n74.24 Brake fluid\n74.25 Minimum standards for brakes and components\n74.26 Horns, sirens, and warning devices\n74.27 Mufflers; excessive smoke or gas\n74.28 Rearview mirrors\n74.29 Windshields and wipers\n74.30 Solid tire requirements\n74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material\n74.32 Directional signals\n74.33 Installation and sale of seat safety belts required; definition\n74.34 Requirements for extra signal equipment\n74.35 Display of warning devices on disabled vehicles\n74.36 Requirements for vehicles transporting explosives\n74.37 Studded tires; seasonal use permitted\n74.38 Safety inspection decals for buses\n74.39 Air bags\n\n**Loads**\n\n74.50 Permit required to exceed load limits\n74.51 Limitation of load extension on left side of vehicle\n74.52 All loads shall be properly secured\n74.53 Towing requirements; exception to size and weight restrictions\n74.54 Weighing of vehicle; removal of excess load\n\n74.55 Operation of vehicle exceeding weight limits prohibited\n74.56 Load limits\n74.57 Maximum width, height, and length\n74.58 Statement of gross vehicle weight\n74.59 Wheel protectors required on heavy commercial vehicles\n74.60 Liability for damages; prosecution; application of monies\n74.61 Weight exceptions for certain vehicles\n\nChapter 75: Bicycles, Motorcycles and Off-Road Vehicles\n\nSection\n\nGeneral Provisions\n\n75.01 Bicycles; application of Title VII\n75.02 Operation of motorized bicycle\n75.03 Rules for bicycles, motorcycles, and snowmobiles\n75.04 Prohibition against attaching bicycles and sleds to vehicles\n75.05 Riding bicycles; motorcycles abreast\n75.06 Equipment of bicycles\n\nSnowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles\n\n75.25 Definitions\n75.26 Equipment\n75.27 Code application; prohibited operation\n75.28 Permitted operation\n75.29 Licensing requirements of operator\n75.30 Maintenance of vehicles for hire\n75.31 Accident reports\n75.32 Impounding of vehicle\n75.33 Local control within police power\n75.34 Registration of vehicles\n75.35 Certificate of title; prohibitions\n\nChapter 76: Parking Regulations\n\nSection\n\n76.01 Prohibition against parking on highways\n76.02 Condition when motor vehicle left unattended\n76.03 Police may remove illegally parked vehicle\n76.04 Parking prohibitions\n76.05 Parking near curb; privileges for persons with disabilities\n76.06 Parking on private property in violation of posted prohibition\n76.07 Selling, washing or repairing vehicle upon roadway\n76.08 Truck loading zones\n76.09 Bus stops and taxicab stands\n76.10 Parking in alleys and narrow streets; exceptions\n76.11 Registered owner prima facie liable for unlawful parking\n76.12 Waiver\n\nTITLE IX: GENERAL REGULATIONS\n\nChapter 90: Animals\n\nSection\n\nAnimals Running at Large\n\n90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings\n90.02 Confining animal found at large; publication of notice; lien\n90.03 Unavoidable escapes\n90.04 Fees\n90.05 Rabies quarantine orders of Mayor\n90.06 Interfering with enforcement of quarantine orders\n90.07 Dogs may be killed for certain acts\n\nOffenses Relating to Animals\n\n90.20 Abandoning animals\n90.21 Injuring animals\n90.22 Poisoning animals\n90.23 Cruelty to animals; cruelty to companion animals\n90.24 Animal fights\n90.25 Trapshooting\n90.26 Loud dog\n90.27 Dog tags\n90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior\n90.29 Failure to register dog or dog kennel\n90.30 Hindering the capture of unregistered dog\n90.31 Unlawful tag\n90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog\n90.33 Retail sale and transportation of dogs\n90.34 Coloring rabbits or baby poultry; regulation of sale and display\n90.35 Jacklighting prohibited\n90.36 Restrictions on dog ownership for certain convicted felons\n90.37 Sexual conduct with an animal\n90.99 Penalty\n\nChapter 91: Fireworks, Explosives, Fire Prevention\n\nFireworks and Explosives\n\n91.01 Definitions\n91.02 Possession, sale, and use of fireworks\n91.03 Permit to use fireworks\n91.04 Manufacturing or wholesale sale without a license; prohibitions\n91.05 Purchasers to comply with law; unauthorized purchases\n91.06 Exhibition without a license; prohibitions\n91.07 Unauthorized transportation or shipping\n91.08 Application of subchapter\n91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines\n91.10 Safety requirements for fireworks showroom structures\n91.11 Storage of explosives\n91.12 Blasting permit\n\nFire Prevention\n\n91.30 Removal of flammable materials or obstructions\n91.31 Protective appliances\n91.32 Compliance with order\n91.33 Waste receptacles\n91.34 Hotel to have fire warning device producing visible signal\n91.35 Fire suppression systems\n91.36 Violations of State Fire Code prohibited\n91.37 Posting arson notices in hotels, motels and other places\n91.38 Negligent burning\n91.39 Spreading alarm of unfriendly fire\n91.40 Unvented heaters\n\nOpen Burning\n\n91.55 Definitions\n91.56 Relations to other prohibitions\n91.57 Open burning in restricted areas\n91.58 Permission and notice to open burn\n91.99 Penalty\n\nChapter 92: Intoxicating Liquors\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 92.01 | Definitions |\n| 92.02 | Exemptions from chapter |\n| 92.03 | Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises |\n| 92.04 | Restrictions on sale of beer and liquor |\n| 92.05 | Permit required; activities prohibited without permit |\n| 92.06 | Illegal transportation prohibited |\n| 92.07 | Open container prohibited; exception |\n| 92.08 | Underage person shall not purchase intoxicating liquor or beer |\n| 92.09 | Prohibitions; minors under 18 years; low-alcohol beverages |\n| 92.10 | Alcohol vaporizing devices prohibited |\n| 92.11 | Misrepresentation to obtain alcoholic beverage for a minor prohibited |\n| 92.12 | Misrepresentation by a minor under 21 years |\n| 92.13 | Sale to underage persons prohibited |\n| 92.14 | Posting of card |\n| 92.15 | Good faith acceptances of spurious identification |\n| 92.16 | Consumption in motor vehicle prohibited |\n| 92.17 | Hours of sale or consumption |\n| 92.18 | Obstructing search of premises prohibited |\n| 92.19 | Illegal possession of intoxicating liquor prohibited |\n| 92.20 | Sale or possession of diluted liquor and refilled containers prohibited |\n| 92.21 | Keeping place where beer or intoxicating liquors are sold in violation of law |\n| 92.22 | Intoxicating liquors shall not be sold in brothels |\n| 92.23 | Use of intoxicating liquor in a public dance hall prohibited; exceptions |\n| 92.24 | Poisonously adulterated liquors |\n| 92.25 | Tavern keeper permitting rioting or drunkenness |\n| 92.26 | Notice of action to prohibit liquor business |\n| 92.27 | Procedure when injunction violated |\n| 92.28 | Liquor transaction scans |\n| 92.29 | Affirmative defenses |\n| 92.99 | Penalty |\n\nChapter 93: Nuisances\n\nSection\n\nGeneral Provisions\n\n| Section | Description |\n|---------|-------------|\n| 93.01 | Application of the chapter |\n| 93.02 | Definitions |\n| 93.03 | Nuisances generally; injunctions; violation; contempt |\n| 93.04 | Maintaining certain nuisances |\n| 93.05 | Collection of cost of abating dangerous property condition; injunction; rehabilitation |\n| 93.06 | Trimming of trees and shrubbery to prevent obstruction |\n\nSeptic Tanks, Cesspools, and Refuse\n\n| Section | Description |\n|---------|-------------|\n| 93.20 | Location of privy vaults, cesspools, and septic tanks |\n| 93.21 | Unsanitary vaults |\n| 93.22 | Removal of contents of vault |\n| 93.23 | Deposit of dead animals, offal upon land or water |\n| 93.24 | Defiling spring or well prohibited |\n| 93.25 | Dumping of refuse in municipality prohibited |\n| 93.26 | Abandoned refrigerators |\n| 93.27 | Discarding litter prohibited |\n| 93.28 | Power of municipality to fill or drain land |\n\nWeeds and Litter on Private Property\n\n| Section | Description |\n|---------|-------------|\n| 93.40 | Keeping down weeds |\n| 93.41 | Notice to owner to cut noxious weeds, remove litter; service |\n| 93.42 | Fees for service and return |\n| 93.43 | Procedure when owner fails to comply with notice |\n| 93.44 | Written return to County Auditor; amount as a lien upon property |\n\nUnclean Habitations\n\n93.60 Permitting unclean habitations\n93.61 When habitations are deemed unsanitary\n93.62 Order for abatement or vacation of premises\n93.63 Enforcement of vacation order by Fire Chief or Police Chief\n93.64 Enforcement through court proceedings\n\n93.99 Penalty\n\nChapter 94: Streets and Sidewalks\n\nGeneral Provisions\n\n94.01 Conditions precedent to improving streets\n94.02 Opening permit required\n94.03 Application and cash deposit\n94.04 Restoration of pavement\n94.05 Barriers around excavations\n94.06 Warning lights\n94.07 Sidewalk construction by the municipality\n94.08 Unloading on street or sidewalk\n94.09 Street or sidewalk obstruction\n94.10 Materials on street or sidewalk\n94.11 Duty to keep sidewalks in repair and clean of ice and snow\n94.12 Ramped curbing for persons with disabilities\n94.13 Flagpole along right-of-way\n94.14 Altering or injuring marker or monument\n\nConstruction and Repair\n\n94.25 Construction and repair may be required\n94.26 Resolution of necessity\n94.27 Notice to construct or repair\n94.28 Assessments of costs against owner\n94.29 Proceedings may include different owners\n94.30 Making and levying assessments\n\nChanges in Streets\n\n94.40 Change of name, vacating or narrowing streets by petition\n94.41 Change of name, vacating or narrowing streets without petition\n94.42 Notice; exception\n94.43 Publication of notice\n94.44 Effect of order of vacation\n94.45 Effect on public utility easements\n\n94.99 Penalty\n\nChapter 95: Unclaimed and Abandoned Vehicles\n\nSection\n\n95.01 Impounding motor vehicle on private property; requirements\n95.02 Impounding abandoned motor vehicle on public property; notice; disposition\n95.03 Disposition of vehicle ordered into storage\n95.04 Disposition of abandoned junk motor vehicles\n95.05 Abandonment of junk motor vehicle prohibited\n95.06 Junk motor vehicle; order to cover or remove; notice; exceptions\n\nChapter 96: Watercraft\n\nSection\n\n96.01 Definitions; applicability\n96.02 Failure to comply with law enforcement order; fleeing\n96.03 Duty upon approach of law enforcement vessel\n96.04 Flashing lights prohibited; exceptions\n\n| Section | Description |\n|---------|-------------|\n| 96.05 | Siren prohibited; exceptions |\n| 96.06 | Regulations for operation of powercraft of more than ten horsepower |\n| 96.07 | Restrictions on child operators; duty of supervisory adult |\n| 96.08 | Reckless operation; maintaining sufficient control; wakes restricted |\n| 96.09 | Unsafe conditions |\n| 96.10 | Marking of bathing and vessel areas |\n| 96.11 | Mooring prohibited in certain areas |\n| 96.12 | Operating under influence of alcohol or drugs prohibited |\n| 96.13 | Implied consent |\n| 96.14 | Incapacitated operators prohibited |\n| 96.15 | Water skiing confined to ski zones |\n| 96.16 | Observer required when towing skier |\n| 96.17 | Water skiing after dark prohibited |\n| 96.18 | Personal flotation device required for towed person |\n| 96.19 | Ski jumps prohibited |\n| 96.20 | Permit for special water events |\n| 96.21 | Sale of single celled inflatable vessels prohibited |\n| 96.22 | Sitting, standing, walking on moving vessels restricted |\n| 96.23 | Engine warm-up required |\n| 96.24 | Personal flotation devices for children under ten |\n| 96.25 | Operation without personal flotation devices prohibited |\n| 96.26 | Distress signal or flag required |\n| 96.27 | Anchor requirements |\n| 96.28 | Specification for fire extinguishers |\n| 96.29 | Backfire flame control device required |\n| 96.30 | Ventilation requirement on powercraft |\n| 96.31 | Abandonment of junk vessels or outboard motors |\n| 96.32 | Exhaust muffler required; noise levels; exceptions |\n| 96.33 | Safety equipment on rental vessels |\n| 96.34 | Capacity plate |\n| 96.35 | Littering prohibited |\n| 96.36 | Dwellings; sanitary systems |\n| 96.37 | Prima facie evidence of negligence |\n| 96.38 | Requirements for operating personal watercraft |\n| 96.39 | Numbering |\n| 96.40 | Registration |\n| 96.41 | Tags indicating expiration date; attachment of identification number |\n| 96.42 | Altering of serial numbers; false information prohibited |\n| 96.43 | Accident reports |\n| 96.44 | Enforcement |\n| 96.45 | Firearms offenses; signaling devices |\n| 96.46 | Tampering with navigation aid or vessel prohibited |\n| 96.47 | Certificate of title; exceptions |\n| 96.48 | Manufacturer’s or importer’s certificate |\n| 96.49 | Prohibitions relating to certificates of title |\n| 96.50 | Permanently displayed hull identification number |\n| 96.99 | Penalty |\n\n---\n\n**TITLE XI: BUSINESS REGULATIONS**\n\n**Chapter 110: General Provisions**\n\n| Section | Description |\n|---------|-------------|\n| 110.01 | Licenses required to engage in certain businesses; exceptions |\n| 110.02 | Application for license |\n| 110.03 | Issuance of license |\n| 110.04 | Date and duration of license |\n| 110.05 | License not transferable |\n| 110.06 | License certificate to be displayed |\n| 110.07 | Revocation or suspension |\n| 110.08 | Appeal and review |\n| 110.99 | General penalty for Title XI |\n\nChapter 111: Taxicabs\n\nSection\n\n111.01 Definitions\n111.02 Certificate of public convenience and necessity required\n111.03 Application for certificate\n111.04 Issuance of certificate\n111.05 Liability insurance required\n111.06 License fees\n111.07 Transfer of certificates and licenses\n111.08 Suspension and revocation of certificates\n111.09 Taxicab driver's license\n111.10 Application for driver's license\n111.11 Examination of applicant; motor vehicle operator's permit required\n111.12 Police investigation of applicant; traffic and police record\n111.13 Consideration of application\n111.14 Issuance of license; duration; annual fee\n111.15 Display of license\n111.16 Suspension and revocation of license\n111.17 Failure to comply with federal, state and municipal laws\n111.18 Vehicle equipment and maintenance\n111.19 Designation of taxicabs\n111.20 Taximeter and display of rates required\n111.21 Number of passengers allowed\n111.22 Articles left in vehicles\n111.23 Vehicles from other municipalities\n111.24 Receipts\n111.25 Refusal of passenger to pay legal fare\n111.26 Solicitation, acceptance and discharge of passengers\n111.27 Open stands; use\n111.28 Taxicab service\n111.29 Manifests\n111.30 Records and reports of holders\n111.31 Police department; duty to enforce chapter\n111.32 Disposition of vehicle license fees\n\nChapter 112: Peddlers, Itinerant Merchants, and Solicitors\n\nSection\n\n112.01 Definitions\n112.02 License requirement\n112.03 Application procedure\n112.04 Standards for issuance\n112.05 Revocation procedure\n112.06 Standards for revocation\n112.07 Appeal procedure\n112.08 Exhibition of identification\n112.09 Municipal policy on soliciting\n112.10 Notice regulating soliciting\n112.11 Duty of solicitors\n112.12 Uninvited soliciting prohibited\n112.13 Time limit on soliciting\n\nChapter 113: Commercial Amusements\n\nSection\n\n113.01 Bowling; billiards and pool\n113.02 Circuses, carnivals, shows and other such entertainment\n113.03 Deposit required\n113.04 License fee for public entertainment or exhibition\n113.05 License fee may be waived for civic interest\n\nChapter 114: Tattooing and Body Piercing Services\n\nSection\n\n114.01 Definitions\n114.02 Prohibitions\n114.03 Application for license; fees; issuance\n\n114.04 Inspection of facilities\n114.05 Suspension or revocation of license\n114.06 Consent for performing procedures on persons under 18\n114.07 Prohibitions relating to persons under 18\n114.08 Defenses to violations\n114.09 Training standards; records; safety and sanitation; equipment\n114.10 Application of local regulation on chapter\n\n114.99 Penalty\n\nTITLE XIII: GENERAL OFFENSES\n\nChapter 130: General Provisions\n\nSection\n\n130.01 Application of Title XIII\n130.02 Definitions\n130.03 Classification of offenses\n130.04 Common law offenses abrogated\n130.05 Rules of construction\n130.06 Limitation of criminal prosecutions\n130.07 Requirements for criminal liability; voluntary intoxication\n130.08 Culpable mental states\n130.09 Organizational criminal liability\n130.10 Personal accountability for organizational conduct\n130.11 Attempt\n130.12 Complicity\n130.13 Presumption of innocence; proof of offense; affirmative defense\n130.14 Battered woman syndrome\n130.15 Delinquency adjudications deemed convictions\n130.16 Criminal law jurisdiction\n130.17 Disposition of unclaimed or forfeited property held by Police Department\n130.18 Imposing sentence for misdemeanor\n130.19 Multiple sentences\n130.20 Apprehension, detention, or arrest of persons on bond\n130.21 Self defense: limitations on duty to retreat prior to using force\n\n130.99 Penalty for Title XIII\n\nChapter 131: Offenses Against Property\n\nSection\n\n131.01 Definitions\n131.02 Arson; determining property value or amount of physical harm\n131.03 Criminal damaging or endangering; vehicular vandalism\n131.04 Criminal mischief\n131.05 Damaging or endangering aircraft or airport operations\n131.06 Criminal trespass; aggravated trespass\n131.07 Tampering with coin machines\n131.08 Theft\n131.09 Unauthorized use of a vehicle\n131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property\n131.11 Passing bad checks\n131.12 Misuse of credit cards\n131.13 Making or using slugs\n131.14 Prima facie evidence of purpose to defraud\n131.15 Tampering with records\n131.16 Securing writings by deception\n131.17 Defrauding creditors\n131.18 Receiving stolen property\n131.19 Value of stolen property\n131.20 Degree of offense when certain property involved\n131.21 Injuring vines, bushes, trees, or crops\n131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property\n\n131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud\n131.24 Injury to property by hunters\n131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent\n131.26 Forgery of identification cards\n131.27 Criminal simulation\n131.28 Personating an officer\n131.29 Trademark counterfeiting\n131.30 Diminishing or interfering with forfeitable property\n131.31 Recording credit card, telephone or Social Security numbers\n131.32 Prosecutions for theft of utilities\n131.33 Motion picture piracy\n\nChapter 132: Offenses Against Public Peace\n\nSection\n\n132.01 Riot\n132.02 Failure to disperse\n132.03 Justifiable use of force to suppress riot\n132.04 Disorderly conduct\n132.05 Disturbing a lawful meeting\n132.06 Misconduct at an emergency\n132.07 Telecommunications harassment\n132.08 Inducing panic\n132.09 Making false alarms\n132.10 Inciting to violence\n132.11 Unlawful display of law enforcement emblem\n132.12 Impersonating a peace officer\n132.13 Safety of crowds attending live entertainment performances\n132.14 Misconduct involving a public transportation system\n\nChapter 133: Sex Offenses\n\nSection\n\n133.01 Definitions\n133.02 Unlawful sexual conduct with a minor\n133.03 Sexual imposition\n133.04 Public indecency\n133.05 Voyeurism\n133.06 Polygraph examinations for victims: restrictions on use\n133.07 Procuring\n133.08 Soliciting; loitering to engage in\n133.09 Prostitution\n133.10 Disseminating matter harmful to juveniles\n133.11 Displaying matter harmful to juveniles\n133.12 Deception to obtain matter harmful to juveniles\n133.13 Rules of evidence\n133.14 Declaratory judgment\n133.15 Injunction; abatement of nuisance\n133.16 Unlawful operation of viewing booths depicting sexual conduct\n133.17 Juveniles on the premises of adult entertainment establishments prohibited\n133.18 Sexually oriented businesses; illegal operation and activity\n133.19 Unlawful advertising of massage\n\n133.99 Sentencing for sexually oriented offenses; sexual predators; registration\n\nChapter 134: Gambling Offenses\n\nSection\n\n134.01 Definitions\n134.02 Prohibitions against gambling; exception\n134.03 Operating a gambling house\n134.04 Public gaming\n134.05 Cheating\n134.06 Regulations concerning operation of licensed bingo game\n134.07 Records to be kept\n134.08 Requirements for bingo game operators\n134.09 Bingo games for amusement only\n\n134.10 Prohibitions where instant bingo game is conducted\n134.11 Raffle drawings\n134.12 Instant bingo other than at bingo sessions\n134.13 Restrictions on owner or lessor of location at instant bingo\n134.14 Skill-based amusement machines; prohibited conduct\n\nChapter 135: Offenses Against Persons\n\nSection\n\n135.01 Definitions\n135.02 Negligent homicide\n135.03 Vehicular homicide; vehicular manslaughter\n135.04 Assault; negligent assault\n135.05 Injury to persons by hunters\n135.06 Menacing; aggravated menacing; menacing by stalking\n135.07 Unlawful restraint\n135.08 Criminal child enticement\n135.09 Coercion\n135.10 Bigamy\n135.11 Unlawful abortion; failure to perform viability testing\n135.12 Abortion trafficking\n135.13 Nonsupport of dependents\n135.14 Endangering children\n135.15 Interference with custody; interference with support orders\n135.16 Domestic violence\n135.17 Hazing prohibited\n135.18 Contributing to unruliness or delinquency of a child\n135.19 Failure to provide for functionally impaired person\n135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation\n135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like\n135.22 Ethnic intimidation\n135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state\n135.24 Adulteration of food\n135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans\n135.26 Nonsmoking areas in places of public assembly\n135.27 Spreading contagion\n135.28 Abuse of a corpse\n135.29 Unlawful collection of bodily substances\n\nChapter 136: Offenses Against Justice and Administration\n\nSection\n\n136.01 Definitions\n136.02 Falsification\n136.03 Compounding a crime\n136.04 Failure to report a crime\n136.05 Failure to aid a law enforcement officer\n136.06 Obstructing official business\n136.07 Obstructing justice\n136.08 Resisting arrest\n136.09 Having an unlawful interest in a public contract\n136.10 Soliciting or receiving improper compensation\n136.11 Dereliction of duty\n136.12 Interfering with civil rights\n136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities\n136.14 False report of child abuse or neglect\n136.15 Assaulting police dog or horse, or assistance dog\n136.16 Disclosure of confidential peace officer information\n136.17 Intimidation of crime victim or witness\n136.18 Using sham legal process\n136.19 Making false allegation of peace officer misconduct\n136.20 Misuse of 9-1-1 system\n136.21 Failure to disclose personal information\n\nChapter 137: Weapons Control\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 137.01 | Definitions |\n| 137.02 | Carrying concealed weapons |\n| 137.03 | Using weapons while intoxicated |\n| 137.04 | Improperly handling firearms in a motor vehicle |\n| 137.05 | Possessing criminal tools |\n| 137.06 | Failure to secure dangerous ordnance |\n| 137.07 | Unlawful transactions in weapons |\n| 137.08 | Underage purchase of firearm or handgun |\n| 137.09 | Pointing and discharging firearms and other weapons |\n| 137.10 | License or permit to possess dangerous ordnance |\n| 137.11 | Possession of an object indistinguishable from a firearm in a school safety zone |\n| 137.12 | Possession of deadly weapon while under detention |\n| 137.13 | Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession |\n| 137.14 | Defaced firearms |\n\nChapter 138: Drug Offenses\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 138.01 | Definitions |\n| 138.02 | Trafficking in controlled substances; gift of marihuana |\n| 138.03 | Drug possession offenses |\n| 138.04 | Possessing drug abuse instruments |\n| 138.05 | Permitting drug abuse |\n| 138.06 | Illegal cultivation of marihuana |\n| 138.07 | Abusing harmful intoxicants |\n| 138.08 | Illegal dispensing of drug samples |\n| 138.09 | Federal prosecution bar to municipal prosecution |\n| 138.10 | Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle |\n| 138.11 | Laboratory report required |\n| 138.12 | Counterfeit controlled substances |\n| 138.13 | Use, possession, or sale of drug paraphernalia |\n| 138.14 | Controlled substance or prescription labels |\n| 138.15 | Possession, sale and disposal of hypodermics |\n| 138.16 | Controlled substance schedules |\n| 138.17 | Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates |\n| 138.18 | Pseudoephedrine sales |\n\nTITLE XV: LAND USAGE\n\nChapter 150: General Provisions\n\nSection\n\nParks and Recreation\n\n| Section | Description |\n|---------|-------------|\n| 150.01 | Recreation Board |\n| 150.02 | Board of Park Trustees |\n\nPlanning and Zoning\n\n| Section | Description |\n|---------|-------------|\n| 150.15 | Planning Commission |\n| 150.16 | Board of Zoning Appeals |\n\nPARALLEL REFERENCES\n\nOhio Legislative History References – Master Table\n\nINDEX\n\nThis summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of __________, Ohio.\n\nSigned: _________________________ _________________________\nMayor Clerk of the Legislative Authority\n\nOrdinance #4- 2018\n\nOrdinance to Increase Trash Rates\n\nWhereas the legislative authority deems it necessary to increase trash rates.\n\nWhereas the new contract price has increased deeming it necessary to increase rates.\n\nWhereas the legislative authority would like increase residential rates from 13.47 to 15.37 and senior rates from 12.47 to 14.37\n\nWhereas the legislative authority would like increase dumpster rates 2 yd to $42.00 and 4 yd to $65.00\n\nWhereas all other trash rates will remain the same.\n\nNow therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby pass the trash rates listed above. That this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 5 yes 0 no\n\nAttest: Carrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\n\nYeas 5 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\nORDINANCE TO RESTRICT PARKING ON PARTS OF LUTZ ST.\n\nWhereas the village legislative authority wishes to restrict parking on the north side of Lutz St from Halderman St to the side doors of Midwest Fabricating to state no parking zone from 7am to 2pm.\n\nWhereas the legislative authority deems it in the best interest of the residents.\n\nNOW THEREFORE BE IT ORDAINED BY THE VILLAGE OF AMANDA, OHIO:\n\nThat the area along side of Midwest Fabricating on the north side of Lutz St. be restricted parking-- no parking zone during 7am-2pm from Halderman St to side doors of Midwest Fabricating.\n\nThat this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 5 yes 0 no\n\nAttest:\nCarrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\nYea 5 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\nORDINANCE TO RAISE EMPLOYEES SALARY\n\nWHEREAS the village Legislative Authority give employees a raise.\n\nWHEREAS a 3% increase in wages for salary and hourly employees.\n\nNOW THEREFORE, BE IT ORDAINED BY THE VILLAGE OF AMANDA, OHIO:\n\nThat the village council feel a 3% increase in salary is warranted and shall be implemented on the current pay cycle. For hourly and salary employees.\n\nThat this ordinance shall take effect and be in force from and after the earliest period allowed by law.\n\nMayor Mark A. Moore\n\nVote: Yes 5 No 0\n\nAttest:\nCarrie Ayers\nFiscal Officer\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than (5) five public places as determined by the Legislative Authority, for a period of not less than (15) fifteen days prior to the effective date thereof.\n\nFiscal Officer\n\nOrdinance to regulate all complaints\n\nWhereas the legislative authority deems it necessary to regulate incoming complaints.\n\nWhereas the legislative authority following legal counsel and Ohio Municipal League advise, would like to require any complaints coming into the village office whether for zoning, property maintenance, or any other village business identify themselves by name and address.\n\nWhereas identifying the complainant can serve to establish that complaints are made in good faith.\n\nNow therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby regulate incoming complaints. That all incoming complaints are required to identify themselves by name and address. That this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 5 yes 0 no\n\nAttest:\n\nCarrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\n\nYeas 5 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\nOrdinance to regulate all record requests\n\nWhereas the legislative authority deems it necessary to regulate incoming requests as well as public record request.\n\nWhereas the legislative authority following legal counsel and Ohio Municipal League advise, would like to require any requests coming into the village office whether for zoning, property maintenance, or any other village business identify themselves by name and address.\n\nWhereas identifying the requests can serve to establish that request are made in good faith.\n\nNow therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby regulate all incoming requests. That all incoming requests are required to identify themselves by name and address. That this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 4 yes 0 no\n\nAttest:\n\nCarrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\n\nYea 4 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\nOrdinance to Set Garbage Guidelines\n\nWhereas the Village Legislative Authority would like to set guidelines to regulate the garbage collection.\n\nWhereas the guidelines will regulate the garbage pick-up dates and times, as well as set limits how much refuse is allowed.\n\nWhereas the guidelines further address required containers, in addition to what items are suitable/not suitable for collection.\n\nNow Therefore let it be ordained by VILLAGE OF AMANDA, OHIO:\n\nThat the village Legislative Authority does hereby set Garbage Guidelines set forth in the attached appendix A, to regulate garbage collection.\n\nThat this ordinance shall take effect upon the earliest date allowed by law.\n\nMayor Mark A. Moore\n\nVote 5 yes 0 no\n\nAttest:\n\nCarrie Ayers Fiscal Officer\n\nThe three reading requirement was waived/not waived:\n\nYea 5 nays 0\n\nThe undersigned, Fiscal Officer of the Legislative Authority, does hereby certify that the foregoing legislation was posted in not less than five (5) public places, as determined by the legislative authority, for a period of not less than fifteen (15) days prior to the effective date thereof.\n\nFiscal Officer\n\n1st Reading 11/05/18\n2nd Reading 12-3-18\n\nGarbage Guidelines\n\n- Garbage is collected 1 day per week on your scheduled pick up day (Tuesday). Selective dumpsters are set up on a 2 day a week pick up the alternate day is (Friday). Pick up times runs between 6 A.M. and 5 P.M.\n\n- Garbage must be set out no earlier than the night before pick-up. Containers are to be removed the same day.\n\n- All garbage must be placed within 5 feet of curb or alley. (Do not block road or sidewalk).\n\n- Major holiday of Thanksgiving and Christmas collection will run one day behind.\n\n- City residents are limited to 8-45 gallon cans or bags or 2-96-gallon rollout containers a week. Weighing no more than 50 pounds per container. Excluding yard waste. Cardboard boxes and burning barrels are not acceptable containers. Ashe containers shall be made of metal; ash and rubbish containers shall be of a kind suitable for collection purposes and shall be of such weight that can be handled by one person.\n\n- Home cleanouts are in excess of regular trash service and will be subject to additional charges.\n\n- Brush and tree limbs, carpets, etc. must be bundled and tied in lengths no longer than 3 to 4 feet.\n\n- One large item a month at no cost. Any additional large items will be charged. 24-hour advance notice to notify drivers of large item pick-up.\n\n- Special and Hazardous waste including but not limited to construction material, rocks, dirt, metal pipe, concrete, liquids, paint, batteries, tires, hazardous chemicals, anything with Freon (refrigerators and air conditioners), or any Epa prohibited items will NOT be collected.\n\n- If animals or weather scatter trash from your trash cans it is NOT the trash haulers responsibility to clean up, Residents are responsible for cleaning their area. To avoid this make sure all refuse is in a lidded container.\n\nJanuary 2019\n\nRe: The Ohio Basic Code, 2019 Edition\n\nEnclosed please find your subscription order for the *Ohio Basic Code, 2019 Edition*. Please note that the *Ohio Basic Code, 2019 Edition*, has been designed to replace, in its entirety, any previous editions of the Ohio Basic Code.\n\nWe are pleased to provide the *Ohio Basic Code, 2019 Edition*. Should you have any questions, please do not hesitate to contact our office.\n\nVery truly yours,\n\nTodd Paul Myers, J.D.\nVice President, Major Client Services\nfirstname.lastname@example.org\n\nAN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING’S OHIO BASIC CODE, 2019 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF Village of Amanda, OHIO, AND DECLARING AN EMERGENCY.\n\nWHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified and are insufficient in form and substance for the complete preservation of the public peace, health, safety and general welfare of the municipality and for the proper conduct of its affairs.\n\nWHEREAS, American Legal Publishing Corporation publishes a Code of Ordinances suitable for adoption by municipalities in Ohio.\n\nWHEREAS, it is necessary to provide for the usual daily operation of the municipality and for the immediate preservation of the public peace, health, safety and general welfare of the municipality that this ordinance take effect at an early date.\n\nNOW, THEREFORE, BE IT ORDAINED BY THE LEGISLATIVE AUTHORITY OF THE MUNICIPALITY OF Village of Amanda, OHIO:\n\nSection 1. American Legal Publishing’s Ohio Basic Code, 2019 Edition, as reviewed and approved by the Legislative Authority, is hereby adopted and enacted. Any prior version of the Ohio Basic Code which may have been previously adopted by the municipality is hereby repealed as obsolete and is hereby replaced in its entirety by this Ohio Basic Code, 2019 Edition.\n\nSection 2. One copy of American Legal Publishing’s Ohio Basic Code, 2019 Edition, certified as correct by the Mayor and Clerk of the Legislative Authority, as required by Ohio Revised Code § 731.23, shall be kept in its initial form on file in the office of the Clerk of the municipality and retained as a permanent ordinance record of the municipality. The Clerk of the municipality is authorized and directed to publish a summary of all new matters contained in the Code of Ordinances as required by Ohio Revised Code § 731.23. Such summary is attached hereto and marked as “Exhibit A”.\n\nSection 3. All ordinances and resolutions or parts thereof which are in conflict or inconsistent with any provision of the Ohio Basic Code, 2019 Edition, as adopted in Section 1 hereof, are hereby repealed as of the effective date of this ordinance, except as follows:\n(A) The enactment of the Ohio Basic Code, 2019 Edition, shall not be construed to affect a right or liability accrued or incurred under any legislative provision prior to the effective date of such enactment, or an action or proceeding for the enforcement of such right or liability. Such enactment shall not be construed to relieve any person from punishment for an act committed in violation of any such legislative provision, nor to affect an indictment or prosecution therefor. For such purposes, any such legislative provision shall continue in full force notwithstanding its repeal for the purpose of revision and codification.\n\n(B) The repeal provided above shall not affect:\n (1) The grant or creation of a franchise, license, right, easement or privilege;\n (2) The purchase, sale, lease or transfer of property;\n (3) The appropriation or expenditure of money or promise or guarantee of payment;\n (4) The assumption of any contract or obligation;\n (5) The issuance and delivery of any bonds, obligations or other instruments of indebtedness;\n (6) The levy or imposition of taxes, assessments or charges;\n (7) The establishment, naming, vacating or grade level of any street or public way;\n (8) The dedication of property or plat approval;\n (9) The annexation or detachment of territory;\n (10) Any legislation enacted subsequent to the adoption of this ordinance.\n (11) Any legislation specifically superseding the provision of the Ohio Basic Code.\n\nSection 4. Whenever reference is made in any documents, publications, or signs of the municipality, including but not limited to traffic tickets and traffic-control signs, to a section as it existed in a former edition of the Ohio Basic Code, the reference shall extend and apply to the section referred to as subsequently amended, revised, recodified, or renumbered.\n\nSection 5. This ordinance is declared to be an emergency measure necessary for the immediate preservation of the peace, health, safety and general welfare of the people of this municipality, and shall take effect at the earliest date provided by law.\n\nDate Passed: 2-1-19\n\nAttest:\n\nMayor\n\nClerk of the Legislative Authority\n\nNotice is hereby given that on the 4th day of Feb., 2019, there was enacted by the Legislative Authority of the Municipality of Village of Avon, Ohio, an ordinance entitled “An Ordinance Approving, Adopting and Enacting American Legal Publishing’s Ohio Basic Code, 2019 Edition, as the Code of Ordinances for the Municipality of Village of Avon, Ohio.”\n\nA summary of the subjects, including all new matters contained in the Code of Ordinances, as adopted, are as follows. The majority of Basic Code provisions are based directly on state law.\n\n---\n\n**TITLE I: GENERAL PROVISIONS**\n\nChapter 10: General Provisions\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 10.01 | Short titles |\n| 10.02 | Definitions |\n| 10.03 | Rules of construction |\n| 10.04 | Revivor; effect of amendment or repeal |\n| 10.05 | Construction of section references |\n| 10.06 | Conflicting provisions |\n| 10.07 | Severability |\n| 10.08 | Reference to offices |\n| 10.09 | Errors and omissions |\n| 10.10 | Ordinances repealed |\n| 10.11 | Ordinances unaffected |\n| 10.12 | Ordinances saved |\n| 10.13 | Application to future ordinances |\n| 10.14 | Interpretation |\n| 10.15 | Amendments to code; amendatory language |\n| 10.16 | Statutory references |\n| 10.17 | Preservation of penalties, offenses, rights and liabilities |\n| 10.18 | Determination of legislative intent |\n| 10.99 | General penalty |\n\n---\n\n**TITLE III: ADMINISTRATION**\n\nChapter 30: General Provisions\n\n| Section | Title |\n|---------|--------------------------------------------|\n| 30.01 | Application of Title III |\n| 30.02 | Qualifications; oaths |\n| 30.03 | Bonds of officers and employees; amount |\n| 30.04 | Additional bond; where bonds recorded and kept |\n| 30.05 | Approval of bonds |\n| 30.06 | Sufficiency of form of bond |\n| 30.07 | Filling vacancies in offices |\n| 30.08 | Public records available |\n| 30.09 | Records Commission |\n| 30.10 | Meetings of public bodies to be open; exceptions; notice |\n| 30.11 | Municipal officers may attend conference or convention; expenses |\n| 30.12 | Residency requirements prohibited; exceptions |\n\nChapter 31: Executive Authority\n\nSection\n\n*General Provisions*\n\n31.001 Executive power; where vested\n\n**Mayor**\n\n31.015 Term of Mayor; power and duties \n31.016 General duties of the Mayor \n31.017 Communications to the Legislative Authority \n31.018 Protest against excess of expenditures \n31.019 Supervision of conduct of officers \n31.020 Annual report to the Legislative Authority \n31.021 Mayor to file charges against delinquent officers \n31.022 Vacancies in office of Mayor \n31.023 Disposition of fines and other moneys\n\n**Clerk**\n\n31.040 Election, term, qualifications of the Clerk \n31.041 Powers and duties of Clerk \n31.042 Books and accounts; merger of offices \n31.043 Seal of Clerk \n31.044 Combined offices of Clerk and Treasurer; Fiscal Officer\n\n**Treasurer**\n\n31.060 Election, term, qualifications of the Treasurer \n31.061 Accounts of Treasurer \n31.062 Powers and duties \n31.063 Quarterly account; annual report \n31.064 Receipt and disbursement of funds \n31.065 Duty of delivering money and property\n\n**Street Commissioner**\n\n31.080 Qualifications \n31.081 General duties \n31.082 Assistants\n\n**Other Officials**\n\n31.100 Legal counsel \n31.101 Administrator \n31.102 Board of Trustees of Public Affairs \n31.103 Fire Engineer, Engineer and Superintendent of Markets\n\n**Chapter 32: Legislative Authority**\n\n**General Provisions**\n\n32.001 Members of the Legislative Authority; election; terms of office \n32.002 President Pro Tempore; employees \n32.003 Vacancy when President Pro Tempore becomes Mayor \n32.004 Qualifications of members of the Legislative Authority \n32.005 Compensation and bonds of municipal officers and employees \n32.006 Vacancy \n32.007 Judge of election and qualification of members; quorum and special meetings \n32.008 Rules; journal; expulsion of members \n32.009 Meetings \n32.010 General powers \n32.011 Failure to take oath or give bond \n32.012 Notice when new bond required \n32.013 Care, supervision and management of public institutions\n\n### Contracts, Bids and Proceedings\n\n| Section | Description |\n|---------|-------------|\n| 32.025 | Contracts by the Legislative Authority or Administrator |\n| 32.026 | Bids and proceedings |\n| 32.027 | Alterations or modifications of contract |\n| 32.028 | Contract restrictions |\n| 32.029 | Award to lowest responsive and responsible bidder |\n\n### Ordinances and Resolutions\n\n| Section | Description |\n|---------|-------------|\n| 32.040 | Ordinances and resolutions as evidence |\n| 32.041 | Passage procedure |\n| 32.042 | Style of ordinances |\n| 32.043 | Subject and amendment of ordinances and resolutions |\n| 32.044 | Authentication and recording of ordinances and resolutions |\n| 32.045 | Publication of ordinances and resolutions; proof of publication and circulation |\n| 32.046 | Notice for proposed amendments to the municipal Charter |\n| 32.047 | Times of publication required |\n| 32.048 | Publication and certification of ordinances in book form |\n| 32.049 | Adoption of technical ordinances and codes |\n| 32.050 | Certificate of Clerk as to publication |\n| 32.051 | Publication when no newspaper published in municipality |\n| 32.052 | Effect of not making publication |\n| 32.053 | Ordinances providing for appropriations or street improvements; emergency ordinances |\n\n### Initiative and Referendum\n\n| Section | Description |\n|---------|-------------|\n| 32.070 | Initiative petitions |\n| 32.071 | Referendum petitions |\n| 32.072 | More than one ordinance required; application of subchapter |\n| 32.073 | Presentation of petitions |\n| 32.074 | Copy of proposed ordinance or measure to be filed with Clerk |\n| 32.075 | Words to be printed in red |\n| 32.076 | Designation of committee filing petition; public inspection of petitions; ordinances passed or repealed prior to election |\n| 32.077 | Itemized statement by petition circulator |\n| 32.078 | Prohibited practices relative to petitions |\n| 32.079 | Accepting premiums for signing |\n| 32.080 | Threats in securing signatures |\n| 32.081 | Application of subchapter if Charter adopted |\n\n### Chapter 33: Judicial Authority\n\n#### General Provisions\n\n| Section | Description |\n|---------|-------------|\n| 33.01 | Jurisdiction in ordinance cases and traffic violations |\n| 33.02 | Powers of Mayor and Mayor's Court Magistrate in criminal matters |\n| 33.03 | Duties of Mayor and Mayor's Court Magistrate; fees; office; seal |\n| 33.04 | Mayor's Court Magistrate |\n| 33.05 | Powers to suspend driver's license in OVI cases |\n\n#### Contempt of Court\n\n| Section | Description |\n|---------|-------------|\n| 33.20 | Summary punishment for contempt |\n| 33.21 | Acts in contempt of court |\n| 33.22 | Hearing |\n| 33.23 | Contempt action for failure to pay support, failure to comply or interference with a visitation order; summons |\n| 33.24 | Right of accused to bail |\n| 33.25 | Hearing on contempt; penalties; support orders; failure to withhold or deduct money pursuant to support order |\n| 33.26 | Imprisonment until order obeyed |\n| 33.27 | Proceedings when party released on bail fails to appear |\n\n33.28 Release of prisoner committed for contempt\n33.29 Judgment final\n33.30 Alternative remedy\n\nChapter 34: Police Department\n\nSection\n\n34.01 Marshal and Police Chief synonymous\n34.02 Appointment of Marshal\n34.03 Deputy marshals and police officers\n34.04 Auxiliary police units\n34.05 Offenses affecting employment of law enforcement officers; probationary period; final appointment\n34.06 Removal proceedings; suspension; appeals\n34.07 General powers\n34.08 Powers and duties of Marshal\n34.09 Disposition of fines and penalties\n34.10 Property recovered by police\n34.11 Disposition to claimant\n34.12 Sale of unclaimed property; disposition of proceeds\n34.13 Expenses of storage and sale; notice\n34.14 Contracts for police protection; nonresident service without contract\n34.15 Peace officer administering oaths; acknowledging complaints, summonses, affidavits and returns of court orders\n\nChapter 35: Fire Department\n\nSection\n\nGeneral Provisions\n\n35.01 Municipal fire regulations; fire department\n35.02 Fire Chief; Fire Prevention Officer; employment of firefighters; criminal records check for firefighters\n35.03 Schooling of officers and firefighters of fire department\n35.04 Legislative Authority may purchase engines and equipment\n35.05 Buildings for department\n35.06 Records\n35.07 Maximum consecutive hours for firefighters on duty\n35.08 Investigation of cause of fire\n35.09 Right to examine buildings, premises, and vehicles\n35.10 Burning buildings for firefighting instruction or research\n35.11 Impersonating fire safety inspector\n35.12 Standards for equipment\n35.13 Persons entitled to be known as firefighters\n35.14 Firefighting and emergency services agreements\n35.15 Regulation of construction in fire limits\n\nVolunteer Firefighters’ Dependents Fund Board\n\n35.30 Definitions\n35.31 Establishment\n35.32 Membership; vacancies\n35.33 Election and term of members\n35.34 Organization; rules and regulations; roster\n35.35 Compensation and expenses of Board; legal advisor\n\nChapter 36: Civil Actions Against the Municipality\n\nSection\n\n36.01 Definitions\n36.02 Nonliability of municipality; exceptions\n36.03 Defenses and immunities\n36.04 Limitation of actions\n36.05 Damages\n36.06 Satisfaction of judgments\n36.07 Defending and indemnifying employees\n36.08 Liability insurance\n36.09 Certain actions unaffected\n36.10 Certain charges against municipal officers filed with Probate Judge; proceedings\n\nTITLE VII: TRAFFIC CODE\n\nChapter 70: General Provisions\n\nGeneral Provisions\n\n| Section | Description |\n|---------|-------------|\n| 70.01 | Definitions |\n| 70.02 | Compliance with order of police officer |\n| 70.03 | Emergency vehicles to proceed cautiously past red or stop signal |\n| 70.04 | Exceptions generally; emergency, public safety and coroner vehicles exempt |\n| 70.05 | Persons riding or driving animals upon roadways |\n| 70.06 | Prohibitions against pedestrians and slow-moving vehicles on freeways |\n| 70.07 | Use of private property for vehicular travel |\n| 70.08 | Names of persons damaging real property by operation of vehicle to be provided to owner |\n| 70.09 | Limited access highways; barriers along; vehicles to enter and leave at designated intersections |\n| 70.10 | Through highways |\n| 70.11 | Officer may remove ignition key |\n| 70.12 | Removal of vehicles after accidents |\n\nTraffic-Control Devices\n\n| Section | Description |\n|---------|-------------|\n| 70.30 | Obeying traffic-control devices |\n| 70.31 | Signal lights |\n| 70.32 | Signals over reversible lanes |\n| 70.33 | Ambiguous or non-working traffic signals |\n| 70.34 | Pedestrian-control signals |\n| 70.35 | Unauthorized signs and signals prohibited |\n| 70.36 | Alteration, defacement, or removal prohibited |\n| 70.37 | Unauthorized possession or sale of devices |\n| 70.38 | Signal preemption devices; prohibitions |\n| 70.99 | Penalty |\n\nChapter 71: Licensing Provisions\n\nMotor Vehicle Licensing\n\n| Section | Description |\n|---------|-------------|\n| 71.01 | Display of license plates or validation stickers; registration |\n| 71.02 | Improper use of noncommercial motor vehicle |\n| 71.03 | Operating motor vehicle ordered immobilized; forfeiture |\n| 71.04 | Operation or sale without certificate of title |\n| 71.05 | Display of certificate of registration |\n| 71.06 | Use of unauthorized plates |\n| 71.07 | Operating without dealer or manufacturer license plates |\n\nDriver's Licenses\n\n| Section | Description |\n|---------|-------------|\n| 71.20 | Prohibited acts |\n| 71.21 | Permitting minor to operate vehicle prohibited; temporary instruction permit; probationary license |\n| 71.22 | License required as driver or commercial driver on public or private property; nonresident exemption |\n| 71.23 | Employment of a minor to operate a taxicab prohibited |\n| 71.24 | Restriction against owner lending vehicle for use of another |\n| 71.25 | Suspension of driver's licenses; license suspended by court of record |\n| 71.26 | Display of license |\n| 71.27 | Prohibition against false statements |\n| 71.28 | Driving under suspension or in violation of license restriction |\n\n71.29 Operating motor vehicle or motorcycle without valid license\n71.30 Driving under OVI suspension\n71.31 Driving under financial responsibility law suspension or cancellation; driving under a nonpayment of judgment suspension\n71.32 Failure to reinstate license\n\n**Commercial Driver's Licenses**\n\n71.45 Definitions\n71.46 Use of actual gross weight in lieu of rating\n71.47 Prohibited acts\n71.48 Prerequisites to operation of commercial motor vehicle\n71.49 Physical qualification to operate commercial motor vehicles\n71.50 Criminal offenses\n71.51 Application of federal regulations\n71.52 Employment of drivers of commercial vehicles\n\n71.99 Penalty\n\n---\n\n**Chapter 72: Traffic Rules**\n\n**General Provisions**\n\n72.001 Lanes of travel upon roadways\n72.002 Driving through safety zone\n72.003 Vehicles traveling in opposite directions\n72.004 Rules governing overtaking and passing of vehicles\n72.005 Permission to overtake and pass on the right\n72.006 Driving to left of center line\n72.007 Prohibition against driving upon left side of roadway\n72.008 Hazardous zones\n72.009 One-way highways and rotary traffic islands\n72.010 Rules for driving in marked lanes\n72.011 Space between moving vehicles\n72.012 Divided roadways\n72.013 Rules for turns at intersections\n72.014 U-turns and turning in roadway prohibited\n72.015 Starting and backing vehicles\n72.016 Turn and stop signals\n72.017 Hand and arm signals\n\n**Right-of-Way**\n\n72.030 Right-of-way at intersections\n72.031 Right-of-way when turning left\n72.032 Right-of-way at through highways; stop signs; yield signs\n72.033 Stop at sidewalk area; stop signs on private roads and driveways\n72.034 Right-of-way on public highway\n72.035 Pedestrian on sidewalk has right-of-way\n72.036 Right-of-way of public safety vehicles\n72.037 Funeral procession has right-of-way\n72.038 Pedestrians yield right-of-way to public safety vehicle\n72.039 Pedestrian on crosswalk has right-of-way\n72.040 Right-of-way yielded to blind person\n72.041 Right-of-way yielded by pedestrian\n\n**Pedestrians**\n\n72.055 Pedestrian movement in crosswalks\n72.056 Pedestrian walking along highway\n72.057 Prohibition against soliciting rides; riding on outside of vehicle\n72.058 Pedestrian on bridge or railroad crossing\n72.059 Persons operating motorized wheelchairs\n\n72.060 Intoxicated or drugged pedestrian hazard on highway\n72.061 Operation of electric personal assistive mobility devices\n72.062 Operation of personal delivery device on sidewalks and crosswalks\n\n**Grade Crossings**\n\n72.075 Stop signs at grade crossings\n72.076 Driving vehicle across railroad grade crossing\n72.077 Vehicles required to stop at grade crossings\n72.078 Slow-moving vehicles or equipment crossing railroad tracks\n\n**School Buses**\n\n72.090 Regulations concerning school buses\n72.091 Violation of regulations; report; investigation; citation; warning\n72.092 Restrictions on the operation of school buses\n72.093 School bus inspection\n72.094 School bus not used for school purposes\n72.095 Licensing by Department of Public Safety\n72.096 Registration and identification of school buses\n72.097 School bus marking\n72.098 Flashing light signal lamps\n72.099 Occupant restraining device for operator\n\n**Prohibitions**\n\n72.115 Obstruction and interference affecting view and control of driver\n72.116 Occupying travel trailer while in motion\n72.117 Driving upon closed highway prohibited\n72.118 Driving upon sidewalk area or paths exclusively for bicycles\n72.119 Obstructing passage of other vehicles\n72.120 Following an emergency or public vehicle prohibited; approaching stationary public safety vehicle and certain other vehicles with caution\n72.121 Driving over unprotected fire hose\n72.122 Placing injurious material on highway or depositing litter from motor vehicle\n72.123 Transporting child not in child-restraint system prohibited\n72.124 Occupant restraining devices\n72.125 Use of engine brakes prohibited\n72.126 Operating motor vehicle while wearing earphones or earplugs\n72.127 Chauffeured limousines and livery services\n72.128 Operating traction engine upon improved highway\n72.129 Cracking exhaust noises; peeling out\n72.130 Shortcutting across private property\n72.131 Texting while driving prohibited\n72.132 Use of electronic wireless communication devices by minors or probationary drivers while driving prohibited\n\n**Chapter 73: Motor Vehicle Crimes**\n\n**General Provisions**\n\n73.01 Driving under the influence of alcohol or drugs\n73.02 Implied consent\n73.03 Physical control of vehicle while under the influence\n73.04 Driving commercial vehicle with impaired alertness or ability; use of drugs\n73.05 Reckless operation of vehicles\n73.06 Reckless operation off streets and highways; competitive operation\n73.07 Operator to be in reasonable control\n73.08 Immobilizing or disabling device violation\n73.09 Street racing defined; prohibited on public highways\n73.10 Speed limits\n73.11 Slow speed or stopping\n73.12 Emergency vehicles excepted from speed limitation\n73.13 Speed regulations on bridges\n\n73.14 Presenting false name or information to officer\n73.15 Prohibition against resisting officer\n73.16 Operation restricted for mini-trucks and low-speed, under-speed, or utility vehicles\n\n**Stopping After Accident**\n\n73.30 Failure to stop after accident\n73.31 Stopping after accident on other than public roads or highways\n73.32 Accident involving damage to realty\n73.33 Failure to report accident\n\n**Chapter 74: Equipment and Loads**\n\n**Equipment**\n\n74.01 Unsafe vehicles, prohibition against operation\n74.02 Bumpers on motor vehicles\n74.03 Lighted lights required\n74.04 Headlights\n74.05 Tail lights and illumination of rear license plate\n74.06 Red reflectors required\n74.07 Safety lighting of commercial vehicles\n74.08 Stoplight regulations\n74.09 Obscured lights on vehicles\n74.10 Red light or flag required\n74.11 Lights on parked vehicles\n74.12 Lights and emblem on slow-moving vehicles; lights and reflectors on multi-wheel agricultural tractors or farm machinery\n74.13 Spotlight and auxiliary driving lights\n74.14 Cowl, fender, and back-up lights\n74.15 Two lights displayed\n74.16 Headlights required\n74.17 Lights of less intensity\n74.18 Number of lights permitted; red and flashing lights\n74.19 Standards for lights on snow removal equipment and oversize vehicles\n74.20 Flashing lights permitted for certain types of vehicles\n74.21 Lights and sign on transportation for preschool children\n74.22 Focus and aim of headlights\n74.23 Brake equipment; specifications\n74.24 Brake fluid\n74.25 Minimum standards for brakes and components\n74.26 Horns, sirens, and warning devices\n74.27 Mufflers; excessive smoke or gas\n74.28 Rearview mirrors\n74.29 Windshields and wipers\n74.30 Solid tire requirements\n74.31 Requirements for safety glass in motor vehicles; use of tinted glass or reflectorized material\n74.32 Directional signals\n74.33 Installation and sale of seat safety belts required; definition\n74.34 Requirements for extra signal equipment\n74.35 Display of warning devices on disabled vehicles\n74.36 Requirements for vehicles transporting explosives\n74.37 Studded tires; seasonal use permitted\n74.38 Safety inspection decals for buses\n74.39 Air bags\n\n**Loads**\n\n74.50 Permit required to exceed load limits\n74.51 Limitation of load extension on left side of vehicle\n74.52 All loads shall be properly secured\n74.53 Towing requirements; exception to size and weight restrictions\n74.54 Weighing of vehicle; removal of excess load\n\n74.55 Operation of vehicle exceeding weight limits prohibited\n74.56 Load limits\n74.57 Maximum width, height, and length\n74.58 Statement of gross vehicle weight\n74.59 Wheel protectors required on heavy commercial vehicles\n74.60 Liability for damages; prosecution; application of monies\n74.61 Weight exceptions for certain vehicles\n\nChapter 75: Bicycles, Motorcycles and Off-Road Vehicles\n\nSection\n\nGeneral Provisions\n\n75.01 Bicycles; application of Title VII\n75.02 Operation of motorized bicycle\n75.03 Rules for bicycles, motorcycles, and snowmobiles\n75.04 Prohibition against attaching bicycles and sleds to vehicles\n75.05 Riding bicycles; motorcycles abreast\n75.06 Equipment of bicycles\n\nSnowmobiles, Off-Highway Motorcycles, and All-Purpose Vehicles\n\n75.25 Definitions\n75.26 Equipment\n75.27 Code application; prohibited operation\n75.28 Permitted operation\n75.29 Licensing requirements of operator\n75.30 Maintenance of vehicles for hire\n75.31 Accident reports\n75.32 Impounding of vehicle\n75.33 Local control within police power\n75.34 Registration of vehicles\n75.35 Certificate of title; prohibitions\n\nChapter 76: Parking Regulations\n\nSection\n\n76.01 Prohibition against parking on highways\n76.02 Condition when motor vehicle left unattended\n76.03 Police may remove illegally parked vehicle\n76.04 Parking prohibitions\n76.05 Parking near curb; privileges for persons with disabilities\n76.06 Parking on private property in violation of posted prohibition\n76.07 Selling, washing or repairing vehicle upon roadway\n76.08 Truck loading zones\n76.09 Bus stops and taxicab stands\n76.10 Parking in alleys and narrow streets; exceptions\n76.11 Registered owner prima facie liable for unlawful parking\n76.12 Waiver\n\nTITLE IX: GENERAL REGULATIONS\n\nChapter 90: Animals\n\nSection\n\nAnimals Running at Large\n\n90.01 Dogs or other animals running at large; nuisance, dangerous or vicious dogs; hearings\n90.02 Confining animal found at large; publication of notice; lien\n90.03 Unavoidable escapes\n90.04 Fees\n90.05 Rabies quarantine orders of Mayor\n90.06 Interfering with enforcement of quarantine orders\n90.07 Dogs may be killed for certain acts\n\nOffenses Relating to Animals\n\n90.20 Abandoning animals\n90.21 Injuring animals\n90.22 Poisoning animals\n90.23 Cruelty to animals; cruelty to companion animals\n90.24 Animal fights\n90.25 Trapshooting\n90.26 Loud dog\n90.27 Dog tags\n90.28 Nuisance, dangerous and vicious dog defined; transfer of ownership certificate; form stating dog's prior behavior\n90.29 Failure to register dog or dog kennel\n90.30 Hindering the capture of unregistered dog\n90.31 Unlawful tag\n90.32 Rights of blind, deaf or hearing impaired, or mobility impaired person, or trainer with assistance dog\n90.33 Retail sale and transportation of dogs\n90.34 Coloring rabbits or baby poultry; regulation of sale and display\n90.35 Jacklighting prohibited\n90.36 Restrictions on dog ownership for certain convicted felons\n90.37 Sexual conduct with an animal\n\n90.99 Penalty\n\nChapter 91: Fireworks, Explosives, Fire Prevention\n\nSection\n\nFireworks and Explosives\n\n91.01 Definitions\n91.02 Possession, sale, and use of fireworks\n91.03 Permit to use fireworks\n91.04 Manufacturing or wholesale sale without a license; prohibitions\n91.05 Purchasers to comply with law; unauthorized purchases\n91.06 Exhibition without a license; prohibitions\n91.07 Unauthorized transportation or shipping\n91.08 Application of subchapter\n91.09 Arrest of offender; seizure and forfeiture of fireworks; distribution of fines\n91.10 Safety requirements for fireworks showroom structures\n91.11 Storage of explosives\n91.12 Blasting permit\n\nFire Prevention\n\n91.30 Removal of flammable materials or obstructions\n91.31 Protective appliances\n91.32 Compliance with order\n91.33 Waste receptacles\n91.34 Hotel to have fire warning device producing visible signal\n91.35 Fire suppression systems\n91.36 Violations of State Fire Code prohibited\n91.37 Posting arson notices in hotels, motels and other places\n91.38 Negligent burning\n91.39 Spreading alarm of unfriendly fire\n91.40 Unvented heaters\n\nOpen Burning\n\n91.55 Definitions\n91.56 Relations to other prohibitions\n91.57 Open burning in restricted areas\n91.58 Permission and notice to open burn\n\n91.99 Penalty\n\nChapter 92: Intoxicating Liquors\n\nSection\n\n| Section | Description |\n|---------|-------------|\n| 92.01 | Definitions |\n| 92.02 | Exemptions from chapter |\n| 92.03 | Restrictions applicable to sale of beer and intoxicating liquor for consumption on the premises |\n| 92.04 | Restrictions on sale of beer and liquor |\n| 92.05 | Permit required; activities prohibited without permit |\n| 92.06 | Illegal transportation prohibited |\n| 92.07 | Open container prohibited; exception |\n| 92.08 | Underage person shall not purchase intoxicating liquor or beer |\n| 92.09 | Prohibitions; minors under 18 years; low-alcohol beverages |\n| 92.10 | Alcohol vaporizing devices prohibited |\n| 92.11 | Misrepresentation to obtain alcoholic beverage for a minor prohibited |\n| 92.12 | Misrepresentation by a minor under 21 years |\n| 92.13 | Sale to underage persons prohibited |\n| 92.14 | Posting of card |\n| 92.15 | Good faith acceptances of spurious identification |\n| 92.16 | Consumption in motor vehicle prohibited |\n| 92.17 | Hours of sale or consumption |\n| 92.18 | Obstructing search of premises prohibited |\n| 92.19 | Illegal possession of intoxicating liquor prohibited |\n| 92.20 | Sale or possession of diluted liquor and refilled containers prohibited |\n| 92.21 | Keeping place where beer or intoxicating liquors are sold in violation of law |\n| 92.22 | Intoxicating liquors shall not be sold in brothels |\n| 92.23 | Use of intoxicating liquor in a public dance hall prohibited; exceptions |\n| 92.24 | Poisonously adulterated liquors |\n| 92.25 | Tavern keeper permitting rioting or drunkenness |\n| 92.26 | Notice of action to prohibit liquor business |\n| 92.27 | Procedure when injunction violated |\n| 92.28 | Liquor transaction scans |\n| 92.29 | Affirmative defenses |\n| 92.99 | Penalty |\n\nChapter 93: Nuisances\n\nSection\n\nGeneral Provisions\n\n| Section | Description |\n|---------|-------------|\n| 93.01 | Application of the chapter |\n| 93.02 | Definitions |\n| 93.03 | Nuisances generally; injunctions; violation; contempt |\n| 93.04 | Maintaining certain nuisances |\n| 93.05 | Collection of cost of abating dangerous property condition; injunction; rehabilitation |\n| 93.06 | Trimming of trees and shrubbery to prevent obstruction |\n\nSeptic Tanks, Cesspools, and Refuse\n\n| Section | Description |\n|---------|-------------|\n| 93.20 | Location of privy vaults, cesspools, and septic tanks |\n| 93.21 | Unsanitary vaults |\n| 93.22 | Removal of contents of vault |\n| 93.23 | Deposit of dead animals, offal upon land or water |\n| 93.24 | Defiling spring or well prohibited |\n| 93.25 | Dumping of refuse in municipality prohibited |\n| 93.26 | Abandoned refrigerators |\n| 93.27 | Discarding litter prohibited |\n| 93.28 | Power of municipality to fill or drain land |\n\nWeeds and Litter on Private Property\n\n| Section | Description |\n|---------|-------------|\n| 93.40 | Keeping down weeds |\n| 93.41 | Notice to owner to cut noxious weeds, remove litter; service |\n| 93.42 | Fees for service and return |\n| 93.43 | Procedure when owner fails to comply with notice |\n| 93.44 | Written return to County Auditor; amount as a lien upon property |\n\nUnclean Habitations\n\n93.60 Permitting unclean habitations\n93.61 When habitations are deemed unsanitary\n93.62 Order for abatement or vacation of premises\n93.63 Enforcement of vacation order by Fire Chief or Police Chief\n93.64 Enforcement through court proceedings\n\n93.99 Penalty\n\nChapter 94: Streets and Sidewalks\n\nGeneral Provisions\n\n94.01 Conditions precedent to improving streets\n94.02 Opening permit required\n94.03 Application and cash deposit\n94.04 Restoration of pavement\n94.05 Barriers around excavations\n94.06 Warning lights\n94.07 Sidewalk construction by the municipality\n94.08 Unloading on street or sidewalk\n94.09 Street or sidewalk obstruction\n94.10 Materials on street or sidewalk\n94.11 Duty to keep sidewalks in repair and clean of ice and snow\n94.12 Ramped curbing for persons with disabilities\n94.13 Flagpole along right-of-way\n94.14 Altering or injuring marker or monument\n\nConstruction and Repair\n\n94.25 Construction and repair may be required\n94.26 Resolution of necessity\n94.27 Notice to construct or repair\n94.28 Assessments of costs against owner\n94.29 Proceedings may include different owners\n94.30 Making and levying assessments\n\nChanges in Streets\n\n94.40 Change of name, vacating or narrowing streets by petition\n94.41 Change of name, vacating or narrowing streets without petition\n94.42 Notice; exception\n94.43 Publication of notice\n94.44 Effect of order of vacation\n94.45 Effect on public utility easements\n\n94.99 Penalty\n\nChapter 95: Unclaimed and Abandoned Vehicles\n\nSection\n\n95.01 Impounding motor vehicle on private property; requirements\n95.02 Impounding abandoned motor vehicle on public property; notice; disposition\n95.03 Disposition of vehicle ordered into storage\n95.04 Disposition of abandoned junk motor vehicles\n95.05 Abandonment of junk motor vehicle prohibited\n95.06 Junk motor vehicle; order to cover or remove; notice; exceptions\n\nChapter 96: Watercraft\n\nSection\n\n96.01 Definitions; applicability\n96.02 Failure to comply with law enforcement order; fleeing\n96.03 Duty upon approach of law enforcement vessel\n96.04 Flashing lights prohibited; exceptions\n\n96.05 Siren prohibited; exceptions\n96.06 Regulations for operation of powercraft of more than ten horsepower\n96.07 Restrictions on child operators; duty of supervisory adult\n96.08 Reckless operation; maintaining sufficient control; wakes restricted\n96.09 Unsafe conditions\n96.10 Marking of bathing and vessel areas\n96.11 Mooring prohibited in certain areas\n96.12 Operating under influence of alcohol or drugs prohibited\n96.13 Implied consent\n96.14 Incapacitated operators prohibited\n96.15 Water skiing confined to ski zones\n96.16 Observer required when towing skier\n96.17 Water skiing after dark prohibited\n96.18 Personal flotation device required for towed person\n96.19 Ski jumps prohibited\n96.20 Permit for special water events\n96.21 Sale of single celled inflatable vessels prohibited\n96.22 Sitting, standing, walking on moving vessels restricted\n96.23 Engine warm-up required\n96.24 Personal flotation devices for children under ten\n96.25 Operation without personal flotation devices prohibited\n96.26 Distress signal or flag required\n96.27 Anchor requirements\n96.28 Specification for fire extinguishers\n96.29 Backfire flame control device required\n96.30 Ventilation requirement on powercraft\n96.31 Abandonment of junk vessels or outboard motors\n96.32 Exhaust muffler required; noise levels; exceptions\n96.33 Safety equipment on rental vessels\n96.34 Capacity plate\n96.35 Littering prohibited\n96.36 Dwellings; sanitary systems\n96.37 Prima facie evidence of negligence\n96.38 Requirements for operating personal watercraft\n96.39 Numbering\n96.40 Registration\n96.41 Tags indicating expiration date; attachment of identification number\n96.42 Altering of serial numbers; false information prohibited\n96.43 Accident reports\n96.44 Enforcement\n96.45 Firearms offenses; signaling devices\n96.46 Tampering with navigation aid or vessel prohibited\n96.47 Certificate of title; exceptions\n96.48 Manufacturer’s or importer’s certificate\n96.49 Prohibitions relating to certificates of title\n96.50 Permanently displayed hull identification number\n\n96.99 Penalty\n\nTITLE XI: BUSINESS REGULATIONS\n\nChapter 110: General Provisions\n\nSection\n\n110.01 Licenses required to engage in certain businesses; exceptions\n110.02 Application for license\n110.03 Issuance of license\n110.04 Date and duration of license\n110.05 License not transferable\n110.06 License certificate to be displayed\n110.07 Revocation or suspension\n110.08 Appeal and review\n\n110.99 General penalty for Title XI\n\nChapter 111: Taxicabs\n\nSection\n\n111.01 Definitions\n111.02 Certificate of public convenience and necessity required\n111.03 Application for certificate\n111.04 Issuance of certificate\n111.05 Liability insurance required\n111.06 License fees\n111.07 Transfer of certificates and licenses\n111.08 Suspension and revocation of certificates\n111.09 Taxicab driver's license\n111.10 Application for driver's license\n111.11 Examination of applicant; motor vehicle operator's permit required\n111.12 Police investigation of applicant; traffic and police record\n111.13 Consideration of application\n111.14 Issuance of license; duration; annual fee\n111.15 Display of license\n111.16 Suspension and revocation of license\n111.17 Failure to comply with federal, state and municipal laws\n111.18 Vehicle equipment and maintenance\n111.19 Designation of taxicabs\n111.20 Taximeter and display of rates required\n111.21 Number of passengers allowed\n111.22 Articles left in vehicles\n111.23 Vehicles from other municipalities\n111.24 Receipts\n111.25 Refusal of passenger to pay legal fare\n111.26 Solicitation, acceptance and discharge of passengers\n111.27 Open stands; use\n111.28 Taxicab service\n111.29 Manifests\n111.30 Records and reports of holders\n111.31 Police department; duty to enforce chapter\n111.32 Disposition of vehicle license fees\n\nChapter 112: Peddlers, Itinerant Merchants, and Solicitors\n\nSection\n\n112.01 Definitions\n112.02 License requirement\n112.03 Application procedure\n112.04 Standards for issuance\n112.05 Revocation procedure\n112.06 Standards for revocation\n112.07 Appeal procedure\n112.08 Exhibition of identification\n112.09 Municipal policy on soliciting\n112.10 Notice regulating soliciting\n112.11 Duty of solicitors\n112.12 Uninvited soliciting prohibited\n112.13 Time limit on soliciting\n\nChapter 113: Commercial Amusements\n\nSection\n\n113.01 Bowling; billiards and pool\n113.02 Circuses, carnivals, shows and other such entertainment\n113.03 Deposit required\n113.04 License fee for public entertainment or exhibition\n113.05 License fee may be waived for civic interest\n\nChapter 114: Tattooing and Body Piercing Services\n\nSection\n\n114.01 Definitions\n114.02 Prohibitions\n114.03 Application for license; fees; issuance\n\n114.04 Inspection of facilities\n114.05 Suspension or revocation of license\n114.06 Consent for performing procedures on persons under 18\n114.07 Prohibitions relating to persons under 18\n114.08 Defenses to violations\n114.09 Training standards; records; safety and sanitation; equipment\n114.10 Application of local regulation on chapter\n\n114.99 Penalty\n\nTITLE XIII: GENERAL OFFENSES\n\nChapter 130: General Provisions\n\nSection\n\n130.01 Application of Title XIII\n130.02 Definitions\n130.03 Classification of offenses\n130.04 Common law offenses abrogated\n130.05 Rules of construction\n130.06 Limitation of criminal prosecutions\n130.07 Requirements for criminal liability; voluntary intoxication\n130.08 Culpable mental states\n130.09 Organizational criminal liability\n130.10 Personal accountability for organizational conduct\n130.11 Attempt\n130.12 Complicity\n130.13 Presumption of innocence; proof of offense; affirmative defense\n130.14 Battered woman syndrome\n130.15 Delinquency adjudications deemed convictions\n130.16 Criminal law jurisdiction\n130.17 Disposition of unclaimed or forfeited property held by Police Department\n130.18 Imposing sentence for misdemeanor\n130.19 Multiple sentences\n130.20 Apprehension, detention, or arrest of persons on bond\n130.21 Self defense: limitations on duty to retreat prior to using force\n\n130.99 Penalty for Title XIII\n\nChapter 131: Offenses Against Property\n\nSection\n\n131.01 Definitions\n131.02 Arson; determining property value or amount of physical harm\n131.03 Criminal damaging or endangering; vehicular vandalism\n131.04 Criminal mischief\n131.05 Damaging or endangering aircraft or airport operations\n131.06 Criminal trespass; aggravated trespass\n131.07 Tampering with coin machines\n131.08 Theft\n131.09 Unauthorized use of a vehicle\n131.10 Unauthorized use of property, including telecommunication property and computers; possession of municipal property\n131.11 Passing bad checks\n131.12 Misuse of credit cards\n131.13 Making or using slugs\n131.14 Prima facie evidence of purpose to defraud\n131.15 Tampering with records\n131.16 Securing writings by deception\n131.17 Defrauding creditors\n131.18 Receiving stolen property\n131.19 Value of stolen property\n131.20 Degree of offense when certain property involved\n131.21 Injuring vines, bushes, trees, or crops\n131.22 Detention and arrest of shoplifters and those committing motion picture piracy; protection of institutional property\n\n131.23 Insurance fraud; workers' compensation fraud; Medicaid fraud\n131.24 Injury to property by hunters\n131.25 Evidence of intent to commit theft of rented property; evidence of lack of capacity to consent\n131.26 Forgery of identification cards\n131.27 Criminal simulation\n131.28 Personating an officer\n131.29 Trademark counterfeiting\n131.30 Diminishing or interfering with forfeitable property\n131.31 Recording credit card, telephone or Social Security numbers\n131.32 Prosecutions for theft of utilities\n131.33 Motion picture piracy\n\nChapter 132: Offenses Against Public Peace\n\nSection\n\n132.01 Riot\n132.02 Failure to disperse\n132.03 Justifiable use of force to suppress riot\n132.04 Disorderly conduct\n132.05 Disturbing a lawful meeting\n132.06 Misconduct at an emergency\n132.07 Telecommunications harassment\n132.08 Inducing panic\n132.09 Making false alarms\n132.10 Inciting to violence\n132.11 Unlawful display of law enforcement emblem\n132.12 Impersonating a peace officer\n132.13 Safety of crowds attending live entertainment performances\n132.14 Misconduct involving a public transportation system\n\nChapter 133: Sex Offenses\n\nSection\n\n133.01 Definitions\n133.02 Unlawful sexual conduct with a minor\n133.03 Sexual imposition\n133.04 Public indecency\n133.05 Voyeurism\n133.06 Polygraph examinations for victims: restrictions on use\n133.07 Procuring\n133.08 Soliciting; loitering to engage in\n133.09 Prostitution\n133.10 Disseminating matter harmful to juveniles\n133.11 Displaying matter harmful to juveniles\n133.12 Deception to obtain matter harmful to juveniles\n133.13 Rules of evidence\n133.14 Declaratory judgment\n133.15 Injunction; abatement of nuisance\n133.16 Unlawful operation of viewing booths depicting sexual conduct\n133.17 Juveniles on the premises of adult entertainment establishments prohibited\n133.18 Sexually oriented businesses; illegal operation and activity\n133.19 Unlawful advertising of massage\n\n133.99 Sentencing for sexually oriented offenses; sexual predators; registration\n\nChapter 134: Gambling Offenses\n\nSection\n\n134.01 Definitions\n134.02 Prohibitions against gambling; exception\n134.03 Operating a gambling house\n134.04 Public gaming\n134.05 Cheating\n134.06 Regulations concerning operation of licensed bingo game\n134.07 Records to be kept\n134.08 Requirements for bingo game operators\n134.09 Bingo games for amusement only\n\n134.10 Prohibitions where instant bingo game is conducted\n134.11 Raffle drawings\n134.12 Instant bingo other than at bingo sessions\n134.13 Restrictions on owner or lessor of location at instant bingo\n134.14 Skill-based amusement machines; prohibited conduct\n\nChapter 135: Offenses Against Persons\n\nSection\n\n135.01 Definitions\n135.02 Negligent homicide\n135.03 Vehicular homicide; vehicular manslaughter\n135.04 Assault; negligent assault\n135.05 Injury to persons by hunters\n135.06 Menacing; aggravated menacing; menacing by stalking\n135.07 Unlawful restraint\n135.08 Criminal child enticement\n135.09 Coercion\n135.10 Bigamy\n135.11 Unlawful abortion; failure to perform viability testing\n135.12 Abortion trafficking\n135.13 Nonsupport of dependents\n135.14 Endangering children\n135.15 Interference with custody; interference with support orders\n135.16 Domestic violence\n135.17 Hazing prohibited\n135.18 Contributing to unruliness or delinquency of a child\n135.19 Failure to provide for functionally impaired person\n135.20 Patient abuse or neglect; patient endangerment; exceptions; false statements; retaliation\n135.21 Interference with right of person to engage in housing transactions because of race, religion, or the like\n135.22 Ethnic intimidation\n135.23 Violating a protection order, consent agreement, anti-stalking protection order or order issued by a court of another state\n135.24 Adulteration of food\n135.25 Illegal distribution of cigarettes, other tobacco products, or alternative nicotine products; transaction scans\n135.26 Nonsmoking areas in places of public assembly\n135.27 Spreading contagion\n135.28 Abuse of a corpse\n135.29 Unlawful collection of bodily substances\n\nChapter 136: Offenses Against Justice and Administration\n\nSection\n\n136.01 Definitions\n136.02 Falsification\n136.03 Compounding a crime\n136.04 Failure to report a crime\n136.05 Failure to aid a law enforcement officer\n136.06 Obstructing official business\n136.07 Obstructing justice\n136.08 Resisting arrest\n136.09 Having an unlawful interest in a public contract\n136.10 Soliciting or receiving improper compensation\n136.11 Dereliction of duty\n136.12 Interfering with civil rights\n136.13 Illegal conveyance of prohibited items onto grounds of a detention facility or other specified governmental facilities\n136.14 False report of child abuse or neglect\n136.15 Assaulting police dog or horse, or assistance dog\n136.16 Disclosure of confidential peace officer information\n136.17 Intimidation of crime victim or witness\n136.18 Using sham legal process\n136.19 Making false allegation of peace officer misconduct\n136.20 Misuse of 9-1-1 system\n136.21 Failure to disclose personal information\n\nChapter 137: Weapons Control\n\nSection\n\n137.01 Definitions\n137.02 Carrying concealed weapons\n137.03 Using weapons while intoxicated\n137.04 Improperly handling firearms in a motor vehicle\n137.05 Possessing criminal tools\n137.06 Failure to secure dangerous ordnance\n137.07 Unlawful transactions in weapons\n137.08 Underage purchase of firearm or handgun\n137.09 Pointing and discharging firearms and other weapons\n137.10 License or permit to possess dangerous ordnance\n137.11 Possession of an object indistinguishable from a firearm in a school safety zone\n137.12 Possession of deadly weapon while under detention\n137.13 Concealed handgun licenses: possession of a revoked or suspended license; additional restrictions; posting of signs prohibiting possession\n137.14 Defaced firearms\n\nChapter 138: Drug Offenses\n\nSection\n\n138.01 Definitions\n138.02 Trafficking in controlled substances; gift of marihuana\n138.03 Drug possession offenses\n138.04 Possessing drug abuse instruments\n138.05 Permitting drug abuse\n138.06 Illegal cultivation of marihuana\n138.07 Abusing harmful intoxicants\n138.08 Illegal dispensing of drug samples\n138.09 Federal prosecution bar to municipal prosecution\n138.10 Nitrous oxide: improper dispensing or distribution; possession in a motor vehicle\n138.11 Laboratory report required\n138.12 Counterfeit controlled substances\n138.13 Use, possession, or sale of drug paraphernalia\n138.14 Controlled substance or prescription labels\n138.15 Possession, sale and disposal of hypodermics\n138.16 Controlled substance schedules\n138.17 Unlawful furnishing of prescription to enable persons to be issued handicapped parking placards or license plates\n138.18 Pseudoephedrine sales\n\nTITLE XV: LAND USAGE\n\nChapter 150: General Provisions\n\nSection\n\nParks and Recreation\n\n150.01 Recreation Board\n150.02 Board of Park Trustees\n\nPlanning and Zoning\n\n150.15 Planning Commission\n150.16 Board of Zoning Appeals\n\nPARALLEL REFERENCES\n\nOhio Legislative History References – Master Table\n\nINDEX\n\nThis summary of contents has been verified and authorized for publication by the Legislative Authority of the Municipality of Village of Avon, Ohio.\n\nSigned: [Signature] Mayor\n\nClerk of the Legislative Authority\n\nCERTIFICATION OF CODIFIED ORDINANCES\n\nWe, Mark A. Mear, Mayor, and Carrie Ayers, Clerk of the Legislative Authority, of the Municipality of Village of Anarole, Ohio, pursuant to Ohio Revised Code §§ 731.23 and 731.42, hereby certify that the general and permanent ordinances of the Municipality, as revised, rearranged, compiled, renumbered as to sections, codified and printed herewith in component codes and titles are correct as and constitute the Code of Ordinances for the Municipality of Village of Anarole, Ohio.\n\n[Signature]\nMayor\n\n[Signature]\nClerk of the Legislative Authority"}}},{"rowIdx":204697,"cells":{"text":{"kind":"string","value":"Project Overview\n\nWhether traveling to school, running errands, or enjoying a stroll through the park, many people in Pekin choose to walk and bike, and many more would choose to do so if conditions were better.\n\nA safe and connected system of sidewalks, trails, and bikeways not only supports people to traveling by bike and foot, but also contributes to the safety of all road users and to the quality of life for Pekin residents.\n\nThe Pekin Bicycle and Pedestrian Master Plan will serve as the City’s vision and blueprint for creating a safer, more comfortable transportation environment for people of all ages, abilities, and backgrounds, especially for people who walk, bike, and use mobility assistance devices like wheelchairs or walkers.\n\nThis long-range planning document will fuse inspiration and ideas from Pekin residents and community stakeholders with technical analysis of current conditions and best practices in bicycle and pedestrian planning and facility design.\n\nThe final plan will include recommendations for new trails, bikeways, sidewalks, and intersection improvements, as well as supporting policies, programs, and implementation strategies. These recommendations will guide future investments in infrastructure projects and help the City secure funding from state, federal, and other sources.\n\nPlanning Inputs\n\n- Field Analysis of Current Conditions\n- Public Workshops and Survey Forms\n- Existing & Previously Planned Facilities\n- Network Quality (LTS) Analysis\n- Existing Bike/Ped Activity & Demand Analysis\n- Direction from Municipalities and MPO\n- Steering Committee & Stakeholder Input\n\nProject Timeline\n\n**PHASE 1: Existing Conditions, Needs Assessment, Visioning**\n- FEB\n- MAR\n- APR\n- MAY\n- JUN\n\nThe first phase of the plan focuses on creating a shared understanding of current conditions for walking and biking and learning from Pekin residents about community needs, issues, and aspirations relating to active transportation. The project team will establish a vision for the Bicycle and Pedestrian Master Plan and supporting goals to help bring the vision to life. These early stages in the planning process will form the foundation for identifying and prioritizing recommendations for new trails, sidewalks, bikeways, and supporting programs.\n\n**PHASE 2: Plan Recommendations & Implementation Strategies**\n- JUL\n- AUG\n- SEP\n\nThe second phase of the project focuses on developing recommendations for projects that improve walking and bicycling safety, connectivity, and access, like new sidewalks, bike lanes, and trails. The project team will also identify policy, programming, and project development strategies to support the plan’s vision and integrate walking and biking into transportation decision-making and projects.\n\n**PHASE 3: Draft Plan Development & Review**\n- OCT\n- NOV\n- DEC\n\nThe third and final phase of the project focuses on refining the plan recommendations, prioritization, and implementation strategies based on feedback from the open house public meeting and creating a draft plan document for review. After final revisions, the plan will be considered for official adoption.\n\nPekin Bicycle & Pedestrian Master Plan\n\nSeptember 28, 2023\nOpen House #2\n\nTHE VISION: The City of Pekin strives to make walking and bicycling safe, accessible, convenient, and equitable transportation options that connect people to places, foster recreational and economic development opportunities, support healthy and active living, and elevate quality of life.\n\nGOAL #1 - SAFETY: Create a safe travel environment for everyone, especially vulnerable road users like people walking, rolling, and bicycling.\n\nObjective 1.1. Improve pedestrian and bicycle safety through the installation of sidewalks, trails, dedicated bikeways, and intersection enhancements.\n\nObjective 1.2. Create a safety action plan to evaluate crash history, identify trends, and develop a high-injury network for future safety investments.\n\nObjective 1.3. Reduce fatal and serious injury crashes through proven safety countermeasures.\n\nObjective 1.4. Support safe and responsible travel through education programs and campaigns.\n\nGOAL #2 - NETWORK: Develop a complete, convenient, and interconnected active transportation network.\n\nObjective 2.1. Expand the Pekin Bike Trail to increase community access to one of the community’s most valued recreation and active transportation amenities.\n\nObjective 2.2. Target sidewalk infill, sidewalk system expansion, and crossing improvements on pedestrian priority corridors.\n\nObjective 2.3. Develop a low-stress on-street bikeway system to support access to everyday destinations throughout Pekin.\n\nObjective 2.4. Coordinate with community partners and local, regional, and state agencies to increase regional connectivity and access to destinations in neighboring communities.\n\nObjective 2.5. Develop a community-wide active transportation wayfinding system to guide people to schools, parks, Downtown Pekin, the Pekin Bike Trail, and other major destinations.\n\nObjective 2.6. Prioritize and pursue projects in a manner that balances community impact, project feasibility, and available resources and staff capacity.\n\nGOAL #3 - DESIGN: Design active transportation facilities that are accessible and comfortable for people of all ages and abilities.\n\nObjective 3.1. Apply current standards and best practices for low-stress bicycle and pedestrian facility design.\n\nObjective 3.2. Prioritize projects that address known ADA deficiencies, align with the recommendations in the ADA Transition Plan, and promote safe and accessible pathways for people with disabilities, children, seniors, and people with limited mobility.\n\nObjective 3.2. Evaluate Pekin Bike Trail crossings to improve user safety and to increase motorist awareness.\n\nGOAL #4 - POLICY: Develop policies that integrate active transportation into city decision-making processes.\n\nObjective 4.1. Develop internal procedures to consider and integrate active transportation facilities in capital improvement planning, programming, and project development.\n\nObjective 4.2. Support active transportation through revisions to the zoning ordinance, subdivision regulations, and street standards.\n\nObjective 4.3. Establish baseline community outreach and engagement protocols for transportation projects.\n\nGOAL #5 - PROGRAMS: Foster an environment that supports and embraces walking and biking through community partnerships and education, encouragement, and awareness programs.\n\nObjective 5.1. Foster collaboration and partnerships with community organizations and agencies throughout the region to identify and implement education, encouragement, and awareness programs and campaigns to increase walking and bicycling activity and safety.\n\nObjective 5.2. Develop Safe Routes to School program in partnership with Pekin Public Schools to increase walking and bicycling to school.\n\nObjective 5.3. Create an awareness campaign to raise the profile of active transportation, promote safe travel behaviors, and highlight the benefits of walking and bicycling.\n\nOnline Survey Results\n\nWalking-Related Questions\n\nHow easy is it to walk in your neighborhood and to nearby destinations?\n\n- Very Easy: 11%\n- Moderately Easy: 31%\n- Neutral: 15%\n- Moderately Difficult: 24%\n- Very Difficult: 10%\n- Other (please specify): 6%\n\nHow far do you typically travel when walking?\n\n- Less than 0.5 miles: 10%\n- 0.5 to 1 mile: 17%\n- 1 to 2 miles: 36%\n- 2 to 5 miles: 31%\n- 5 to 10 miles: 3%\n- Over 10 miles: 0%\n- N/A: 3%\n\nFor which of the following reasons do you choose to walk for work or recreation trips? (Check all that apply)\n\n- Safety/security: 23%\n- Convenience: 24%\n- Health benefits/fitness: 95%\n- Environmental benefits: 39%\n- For fun/enjoyment: 72%\n- Don't have access to a vehicle: 1%\n- Don't have access to public transit: 0%\n- Other (please specify): 4%\n\nWhich of the following barriers prevent you from walking more? (Please select all that apply)\n\n- I do not feel safe and worry about: 55%\n- I am physically unable to walk: 1%\n- I have no place to park: 68%\n- Lack of sidewalks: 18%\n- Obstruction to vision: 15%\n- Lack of lighting: 43%\n- I don't like walking: 27%\n- I don't like walking because of children: 8%\n- I don't like walking because of crime: 4%\n- I don't like walking because of weather: 16%\n- Noise: 6%\n- I walk too much as it is: 4%\n- Other (please specify): 7%\n\nPlease evaluate the following sentence based on how it represents you: \"I would like to walk more than I do now for my daily commute, errands, and other activities.\"\n\n- Agree: 42%\n- Somewhat Agree: 24%\n- Neutral: 21%\n- Somewhat Disagree: 7%\n- Disagree: 6%\n\nPlease evaluate the following sentence based on how it represents you: \"I would like to walk more than I do now for fun, exercise, and other recreational activities.\"\n\n- Agree: 78%\n- Somewhat Agree: 16%\n- Neutral: 5%\n- Somewhat Disagree: 1%\n- Disagree: 1%\n\nWhat destinations would you like to be able to walk to? (Choose all that apply)\n\n- Work: 12%\n- Bus Stop: 4%\n- School: 11%\n- Trails: 75%\n- Parks, playgrounds, and recreation centers: 75%\n- Grocery stores, local shops, and restaurants: 59%\n- Government services (city hall, police station, social services, library, office, etc.): 23%\n- Place of worship (church, synagogue, mosque, etc.): 21%\n- Other (please specify): 5%\n\nPekin Bicycle & Pedestrian Master Plan\n\nSeptember 28, 2023\nOpen House #2\n\nOnline Survey Results\n\nBicycling-Related Questions\n\nHow easy is it for you to bike in your neighborhood and to nearby destinations?\n\n- Very easy: 8%\n- Moderately Easy: 21%\n- Neutral: 24%\n- Moderately Difficult: 31%\n- Very difficult: 16%\n\nHow far do you typically travel when biking?\n\n- Less than 0.5 miles: 5%\n- 0.5 to 1 mile: 2%\n- 1 to 2 miles: 11%\n- 2 to 5 miles: 25%\n- 5 to 10 miles: 23%\n- Over 10 miles: 11%\n- N/A: 22%\n\nFor which of the following reasons do you choose to bike for work, school, or recreation trips? (Check all that apply)\n\n- Safety concerns: 28%\n- Convenience: 20%\n- Health benefits/exercise: 92%\n- Environmental benefits: 49%\n- For fun/entertainment: 72%\n- Don’t have access to a vehicle: 1%\n- Don’t have access to public transit: 1%\n- Other (please specify): 2%\n\nWhat type of cyclist best describes you? (One selection allowed)\n\n- Strong and Fearless (Comfortable biking on major streets without bike lanes): 16%\n- Enthusiastic and Confident (Comfortable biking on major streets only with bike lanes): 16%\n- Interested but Concerned (Only comfortable biking if separated from cars and/or major streets): 54%\n- No Way, No How (Not interested in biking at all): 15%\n\nPlease evaluate the following sentence based on how it represents you: “I would like to bike more than I do now for my daily commute, errands, and other activities.”\n\n- Agree: 38%\n- Somewhat Agree: 24%\n- Neutral: 21%\n- Somewhat Disagree: 6%\n- Disagree: 11%\n\nPlease evaluate the following sentence based on how it represents you: “I would like to bike more than I do now for fun, exercise, and other recreational activities.”\n\n- Agree: 63%\n- Somewhat Agree: 21%\n- Neutral: 8%\n- Somewhat Disagree: 5%\n- Disagree: 4%\n\nWhich of the following bikeway types would you like to see in Pekin? (Check all that apply)\n\n- Shared Lane: 6%\n- Painted Shoulder/Shoulder Bike Lane: 50%\n- Marked and Signed Bike Route: 49%\n- Bike Lane: 59%\n- Buffered Bike Lane: 47%\n- Separated Bike Lane (Cycle Track): 65%\n- Shared Use Path / Speed Hump: 84%\n\nWhich of the following barriers prevent you from biking more? (Please select all that apply)\n\n- I don't have time/space to ride: 11%\n- Distractions from traffic: 10%\n- I live too far from the places I need to go: 4%\n- Lack of dedicated bike lanes: 52%\n- Lack of safe places to park: 69%\n- There isn't an entire place to ride: 58%\n- Not enough parking: 32%\n- Too much traffic: 22%\n- I don't feel safe biking around pedestrians: 22%\n- Disability or other health issues: 19%\n- Weather prevents me: 17%\n- None. I like as much as I want to bike (like I currently do): 4%\n- Other (please specify): 15%\n\nWhat destinations would you like to be able to bike to? (Choose all that apply)\n\n- Work: 27%\n- Bus Stop: 3%\n- School: 12%\n- Trails: 94%\n- Parks, playgrounds, and recreation centers: 77%\n- Grocery stores, local shops, and restaurants: 55%\n- Government services (city hall, library, social services, post office, etc.): 28%\n- Place of worship (church, temple, mosque, etc.): 17%\n- Other (please specify): 6%\n\nBicycle Improvements Toolkit\n\nThere is no one-size-fits-all approach to bicycle improvements. Each bicycle facility or intersection improvement must be compatible with roadway characteristics, traffic patterns, and adjacent land uses. The following bicycle facilities and intersection improvements highlight the diversity of options available to better support bicycling in Pekin.\n\n**BICYCLE FACILITY TYPES**\n\n- **Shared Travel Lanes**\n- **Paved Shoulders**\n- **Separated Bicycle Lanes**\n- **Bicycle Wayfinding & Direction Signs**\n- **Bicycle Boulevards**\n- **Bicycle Lanes / Buffered Bicycle Lanes**\n- **Shared Use Paths & Trails**\n- **Bicycle Parking**\n\n**INTERSECTION IMPROVEMENTS**\n\n- **Bike Boxes**\n- **Bike Lane Markings/Conflict Markings/Green Paint**\n- **Bicycle Signals**\n- **Protected Intersections**\n\nPekin Bicycle & Pedestrian Master Plan\n\nSeptember 28, 2023\nOpen House #2\n\nProposed Bicycle Network\n\nPekin Bicycle & Pedestrian Master Plan\n\nCommunity Features\n- City Hall\n- Hospital\n- Library\n- Post Office\n- School\n- Place of Worship\n- Full Service Grocery Store\n- Park\n- CityLink Bus Stop\n\nBicycle Recommendations\n- Existing Trail\n- Shared Use Path\n- Side Path\n- Separated Bike Lane\n- Bike Lane\n- Bike Blvd\n- Shared Travel Lane\n- Crossing Improvements\n\nPedestrian Improvements Toolkit\n\nCreating a safe, comfortable, accessible, and attractive environment for walking does not happen overnight. It takes years to develop a high-quality pedestrian system. With the right plans and policies in place, the City of Pekin can direct public infrastructure investments and private development to support people walking, regardless of their age, ability, or use of mobility assistance devices.\n\n**NETWORK ENHANCEMENTS**\n\n- **Sidewalks**\n - New Construction\n - Sidewalk Infill & Repairs\n\n- **Street Furniture**\n - Benches\n - Planters\n\n- **Pedestrian-Scale Lighting**\n - Streetlights\n - Pathway Lights\n\n- **Shade Trees**\n - Trees along sidewalks\n - Trees in parks\n\n**INTERSECTION IMPROVEMENTS**\n\n- **Curb Extensions**\n - Curbs extended into intersections\n\n- **ADA-Accessible Curb Ramps**\n - Ramps for wheelchair access\n\n- **Mid-Block Crosswalks**\n - Crosswalks at intersections\n\n- **Pedestrian Signals & Countdown Timers**\n - Traffic signals with pedestrian signals\n\n- **Marked Crosswalks**\n - Painted crosswalks\n\n- **Raised Crosswalks**\n - Elevated crosswalks\n\n- **Leading Pedestrian Interval**\n - Traffic lights timed for pedestrians\n\n- **Median Refuge Islands**\n - Islands in the middle of intersections\n\n---\n\nPekin Bicycle & Pedestrian Master Plan\n\nSeptember 28, 2023\nOpen House #2\n\nThe ADA Transition Plan will be the primary driver of investments in sidewalks and intersection improvements.\n\nWith that in mind, the pedestrian network in the Pekin Bicycle and Pedestrian Master Plan focuses on pedestrian priority corridors.\n\nPedestrian priority corridors consist primarily of arterial and collector roadways that connect neighborhoods to retail, shopping, schools, parks, and other vital destinations.\n\nThis approach acknowledges that not all roads are created equal. Pedestrian priority corridors generally carry higher volumes of motor vehicle traffic at higher speeds, and safe and accessible pedestrian facilities are necessary to support pedestrian activity.\n\nAlso included in the pedestrian network are shared use paths, sidepaths, and the existing Pekin Bike Path.\n\nCommunity Features\n\n- City Hall\n- Hospital\n- Library\n- Post Office\n- School\n- Full Service Grocery Store\n- Place of Worship\n- Park\n- CityLink Bus Stop\n\nPedestrian Recommendations\n\n- Pedestrian Priority Corridor\n- Existing Trail\n- Shared Use Path\n- Side Path\n- Crossing Improvements\n\nProject Prioritization\n\nActive transportation infrastructure projects must compete with other capital improvements and municipal services, as well as with one another, for limited internal and external resources. In order to maximize investment and provide the greatest benefit, the City of Pekin should take a prioritized approach to invest in active transportation infrastructure and plan implementation.\n\nImpact Factors:\n- Access to schools, parks, commercial districts/hubs, transit\n- Low stress/all ages and abilities\n- Connections to existing facilities\n- Safety (addressing high-crash corridors & intersections)\n- Public support\n\nEffort Factors:\n- Project Cost\n- Project Complexity\n- Ownership\n- Coordination with Programmed Improvements\n\nImplementation Categories:\n\n| Priority | Feasibility | Description |\n|----------|-------------|-------------|\n| High | High | Long Term Improvement |\n| | | Projects for further study and evaluation. Seek grant funding to advance projects. |\n| Low | Low | Short Term Improvement |\n| | | High priority and easy to implement projects for short term development. |\n| Low | High | Opportunity Improvement |\n| | | Lower priority projects that may become an opportunity with the necessary funding or partnerships. |\n\nWhat are your Priorities?\n\nSupporting Programs\n\nEARN-A-BIKE PROGRAM\nEarn-a-Bike programs give students the opportunity to learn basic bike maintenance and bicycling skills, as well as route selection and mapping. Students who participate in these programs typically learn to build a bicycle from the ground up, and upon completion of the program receive a refurbished bike (usually the one they build), along with a helmet, bike lock, and bike lights.\n\nTRAIL USER ETIQUETTE CAMPAIGN\nStatewide, local parks and trails are available for use by bicyclists, walkers, runners, skateboarders, rollerbladers, parents with strollers, dogs, children, and, in some cases, equestrians, as well as other modes. A campaign for trail user etiquette works to ensure that all users safely and responsibly share the trail. The campaign may include media advertisements, trail signage, brochures, an “ambassador” program, etc.\n\nHAPPY TRAILS TO HEALTHY FOODS\nMany communities have developed programs that promote physical activity and healthy eating goals in improving overall public health and wellness. This important link can be highlighted in a fun and interactive manner through promoting healthy food outlets along the bicycle, pedestrian, trail, and transit network and partnering with health food providers to identify safe routes for active transportation to their locations.\n\nCAR-FREE STREET EVENTS\nCar-free street events are temporary “openings” that create a temporary park that is open to the public for walking, bicycling, dancing, etc. The purpose of the event is to encourage biking and other forms of physical activity to the general public by providing a fun, welcoming environment for activity. Car-free street events have been very successful internationally and are rapidly becoming popular in the U.S.\n\nFAMILY BIKING PROGRAMS\nFamily Biking programs help parents figure out how to safely transport children by bicycle and help children learn bicycling skills. Activities may include bicycle safety checks, a group ride or parade, “freedom from training wheels” clinics, and opportunities to try out different ways to transport children (e.g., trailers, cargo bicycles, kid seats, etc.).\n\nSAFE ROUTES TO SCHOOL PROGRAM\nThe City of Pekin can benefit from a comprehensive Safe Routes to School program that combines local facility, safety, and route planning with the development of school and travel events, like walking school buses or bike trains. Both of these strategies require a commitment and support from school leadership and staff, the City of Pekin, and families with school-aged children.\n\nSILVER SNEAKERS SENIORS PROGRAM\nInterested agencies, nonprofits, health departments and senior centers can partner to develop an active lifestyles program for senior citizens utilizing the bicycle, pedestrian, and greenways network. Activities could include adult tricycle or bicycle rides, nature walks, walks to lunch, and safety education.\n\nOUTDOOR PUBLIC ART\nOutdoor art along a bicycle, pedestrian, and trail network can bring attention to the network and attract newcomers to bicycling and walking, while also enhancing outdoor space. By adding art to trail facilities, the community is creating a unique interactive amenity for both residents and visitors. Such programs can also attract new partners, promoters, and sponsors of the active transportation network.\n\nINTERPRETIVE SIGNAGE\nInterpretive signage along a trail and greenway network serves as an education tool. Information related to the history of an area, its cultural significance, or natural features is provided on a graphically appealing sign. Topics could range from native species of plants to river currents to famous historical figures.\n\nBICYCLE FRIENDLY BUSINESS PROGRAM\nBusinesses in Pekin have an important role to play in fostering a bicycle friendly community. From supporting employees with secure bicycle storage facilities, showers, and lockers to incentivizing customers that arrive on bike with discounts or other promotions, there are many ways businesses can promote bicycling. The League of American Bicyclists Bicycle Friendly Business program will be a valuable resource, and businesses should strive to achieve recognition as a Bicycle Friendly Business.\n\nBIKE MONTH EVENTS\nCities and towns across the country participate in National Bike Month annually, during May. The League of American Bicyclists (LAB) hosts a website for event organizers. The website contains information on nationwide and local events, an organizing handbook, and promotional materials. Events can vary widely, including events to promote cycling like morning-commute energizer stations and an end-of-day rally or celebration, a group ride with the mayor, discounts at local businesses for bicycle commuters, short, themed community bicycle rides, mountain bike skills clinics, and commuter courses for adults.\n\nPERSONAL TRAVEL ENCOURAGEMENT\nPTE programs are designed to educate the public on how to approximately 10% and increase bicycling, walking and transit use within a target area. The program:\n\n- Delivers customized travel information packets;\n- Hosts fun events such as guided rides, walks, and classes; and\n- Sends trained outreach staff to farmers’ markets and other community events\n\nBICYCLE COUNT PROGRAM\nBy utilizing both a manual and automated counting practices, Pekin can better measure and understand how people travel in the community, which can provide insight into heavily utilized, and where to invest in future transportation improvements. The Pedestrian and Bicycle Information Center (PBIC) has a wealth of information and resources to help the City develop a comprehensive counting program.\n\nTARGETED TRAFFIC ENFORCEMENT\nBy focusing traffic enforcement efforts on areas of high travel around schools, senior centers, business districts, and high-crash locations, the Pekin Police Department can support bicycle and pedestrian safety, especially for the most vulnerable road users in our community.\n\nTHEMED WALKING/BIKING MAPS & GUIDES\nWalking and biking maps and guides can be used to explore the city’s rich history, unique character, and beautiful parks. The guides would be targeted to a variety of ages and abilities, offering varying routes for families, touring cyclists, and others. Themes can include history, architecture, parks, and culture and suited for both Pekin residents and visitors.\n\nTRAFFIC TICKET DIVERSION CLASS\nA diversion class is offered to those offenders of certain bicycle-related traffic violations, such as running a stop sign on a bike. It can be aimed just at bicyclists or at bicyclists and motorists, or pedestrians. In lieu of a citation and/or fine, individuals can take a one-time, free or inexpensive class.\n\nCREATE-A-COMMUTER PROGRAM\nCreate-A-Commuter programs equip people with the tools to overcome common barriers to bicycling to work, school, and other everyday destinations. A successful program often includes bicycle skills and maintenance courses, mentorship pairing with experienced cyclists, and Guaranteed Ride Home (GRH) element to provide participants with taxi, rideshare, or transit service if an unexpected need arises.\n\nBICYCLE + PEDESTRIAN RESOURCE WEBSITE\nThe City could create a website that serves as a one-stop resource for bicyclists, pedestrians, and trail users of all types. Information could include tips for commuters, route planning services, community events, message boards, and more.\n\nBICYCLE PARKING PROGRAM\nPeople traveling by bicycle rely on safe and secure bicycle parking facilities to support their trips. To incentivize bicycle parking installation, the City of Pekin should consider the development of a bicycle parking program that provides reduced-cost bicycle racks for local businesses and community destinations.\n\nKEY QUESTION: ARE THERE OTHER PROGRAM IDEAS YOU WOULD LIKE TO SEE IN PEKIN?\n(write your thoughts or place a dot beside an existing idea to show your support)\n\nLAUNCH PARTIES FOR NEW BIKEWAYS\nThe area’s cities and counties should partner with local advocacy groups to generate media attention and engage local citizens in each incremental expansion of the bikeway network. Popular launch parties in other jurisdictions have included bicycle-handling workshops, free bike mechanic services, live music, bicycle giveaways, and other activities.\n\nWe Need Your Input!\n\nWhat will make this plan a success?\n\nWhat’s your top priority or project?\n\nWhat else do we need to know?"}}},{"rowIdx":204698,"cells":{"text":{"kind":"string","value":"Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy\n\nPascal J. Lafontant \n*DePauw University*, email@example.com\n\nFollow this and additional works at: [https://scholarship.depauw.edu/bio_facpubs](https://scholarship.depauw.edu/bio_facpubs)\n\nPart of the Biology Commons\n\n**Recommended Citation**\nLafontant PJ, Behzad AR, Brown E, Landry P, Hu N, et al. (2013) Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy. *PLoS ONE* 8(8): e72388. doi:10.1371/journal.pone.0072388\n\nThis Article is brought to you for free and open access by the Biology at Scholarly and Creative Work from DePauw University. It has been accepted for inclusion in Biology Faculty publications by an authorized administrator of Scholarly and Creative Work from DePauw University.\n\nCardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy\n\nPascal J. Lafontant¹*, Ali R. Behzad², Evelyn Brown³, Paul Landry³, Norman Hu⁴, Alan R. Burns³,⁵\n\n¹ Department of Biology, DePauw University, Greencastle, Indiana, United States of America, ² Imaging and Characterization Core Lab, King Abdullah University of Science and Technology, Thuwal, Kingdom of Saudi Arabia, ³ College of Optometry, University of Houston, Houston, Texas, United States of America, ⁴ Department of Pediatrics, University of Utah School of Medicine, Salt Lake City, Utah, United States of America, ⁵ Department of Pediatrics, Baylor College of Medicine, Houston, Texas, United States of America\n\nAbstract\n\nThe zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature of the interface between the compact and spongy myocardium. Here we describe how these two distinct layers are structurally and functionally integrated. We demonstrate by transmission electron microscopy that this interface is complex and composed primarily of a junctional region occupied by collagen, as well as a population of fibroblasts that form a highly complex network. We also describe a continuum of uniquely flattened transitional cardiac myocytes that form a circumferential plate upon which the radially-oriented luminal trabeculae are anchored. In addition, we have uncovered within the transitional ring a subpopulation of markedly electron dense cardiac myocytes. At discrete intervals the transitional cardiac myocytes form contact bridges across the junctional space that are stabilized through localized desmosomes and fascia adherent junctions with adjacent compact cardiac myocytes. Finally using serial block-face scanning electron microscopy, segmentation and volume reconstruction, we confirm the three-dimensional nature of the junctional region as well as the presence of the sheet-like fibroblast network. These ultrastructural studies demonstrate the previously unrecognized complexity with which the compact and spongy layers are structurally integrated, and provide a new basis for understanding development and regeneration in the zebrafish heart.\n\nCitation: Lafontant PJ, Behzad AR, Brown E, Landry P, Hu N, et al. (2013) Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy. PLoS ONE 8(8): e72388. doi:10.1371/journal.pone.0072388\n\nEditor: Leonard Eisenberg, New York Medical College, United States of America\n\nReceived March 1, 2013; Accepted July 9, 2013; Published August 23, 2013\n\nCopyright: © 2013 Lafontant et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.\n\nFunding: PJL is supported by the Fisher and Faculty Development Funds at DePauw University. A. Burns is supported National Institutes of Health/National Eye Institute grants EY017120 and P30EY007551. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.\n\nCompeting Interests: The authors have declared that no competing interests exist.\n\n* E-mail: firstname.lastname@example.org\n\nIntroduction\n\nThe zebrafish heart consists of a spongy ventricular myocardium made of trabecular bundles projecting radially into the ventricular lumen, and of an outer compact heart layer that encases the spongy trabeculae. The proportion of spongy to compact heart varies significantly between fish and appears to strongly correlate to each species particular ecological physiology [1,2,3,4]. The hearts of fish with relatively low activity consist primarily or exclusively of a spongy myocardium with a rudimentary compact myocardium fed by deoxygenated luminal blood flow (type-1 heart). More active fish display a thicker compact myocardium invested with vessels carrying oxygenated blood (type-2 heart). The two myocardial segments have been historically and empirically considered distinct anatomical structures that form a functional unit supporting the physiological demands of fish. Because of its hypothesized importance in fish ventricular function, the characteristics of the spongy-compact interface (SCI) between the two ventricular layers have been an area of considerable interest and controversy.\n\nThe zebrafish has emerged as an important model of heart development and regeneration. The adult zebrafish heart is a type-2 heart with a compact layer perfused by capillaries. An extensive number of studies have provided insights into the molecular mechanisms underlying the development of the zebrafish ventricle [5,6,7] and its spongy trabeculae [8,9]. Other studies have explored the molecular mechanisms that orchestrate the zebrafish’s remarkable ability to regenerate [10,11]. More recently, the emergence of the mature zebrafish ventricle has been demonstrated by genetic approach to be driven by clonally-dependent cardiac myocytes, illustrating diversity within the ventricular myocyte population [12]. A number of studies have also provided insight into the ultrastructural characteristics of the developing and adult zebrafish heart [13,14,15]. However to date the nature of SCI in the zebrafish heart has received little attention.\n\nStudies in a variety of fish using different approaches have resulted in markedly diverse understandings and models of this\n\ninterface region. Early studies in the albacore tuna suggested that the two myocardial layers were attached by a connective tissue layer [16,17]. Transmission electron microscopy studies in four different fish species, not including the tuna, provided the first detailed evidence not only for connective tissue at the interface of the two myocardial layers [18,19], but of the presence of fibroblasts and a distinct set of flattened transitional cardiac myocytes occupying this complex junctional region (JR). On the other hand, in a more recent study of the sockeye salmon and rainbow trout, similar structures in the interface region were not observed [20]. Whether these studies underscore the structural diversity of the JR in fish, or reflect differences in study methodologies is not clear. The different findings in the salmon and trout heart may be indicative of the intra- and inter-species diversity in the architectural arrangements of the two layers.\n\nThe purpose of our study was to ascertain the cellular and ultrastructural nature of the interface region between compact and spongy heart of adult zebrafish using light and transmission electron microcopy (TEM). Here, we provide evidence of a complex and previously unrecognized JR containing a network of fibroblasts, a subpopulation of phenotypically transitional cardiac myocytes at the base of the spongy myocardium, and describe the spatial distribution of adherens junctions linking the spongy and compact layers. The three-dimensional structure of the JR was confirmed by serial block-face scanning electron microscopy (SBF-SEM) and computerized segmentation. To our knowledge, this is the first detailed description of the JR of the zebrafish ventricle, as well as the first ultrastructural 3D reconstruction of this region using SBF-SEM.\n\n**Materials and Methods**\n\n**Animals**\n\nZebrafish were obtained from Aquatic Research Organisms (Hampton, NH), and maintained in 10-gallon tanks, with 15 to 20 fish per tank, at 28 degrees Celsius on a 14/10 hour day/night cycle. Experimental procedures were approved by the Committee for the Care and Use of Laboratory Animals at DePauw University.\n\n**Transmission Electron Microscopy**\n\nFor studies of the myocardium in plastic sections and by transmission electron microscopy tissue samples were processed as described previously [21]. Zebrafish were euthanized using 0.2% MS Tricaine. Once all body and opercula movements had ceased, the heart was removed by grasping and pulling the aorta proximal to the bulbus arteriosus. The hearts were fixed in 100 mM cacodylate buffer containing 2.5% glutaraldehyde overnight at 4 degrees C. The hearts were washed the next day in cacodylate buffer and stored at 4 degrees C for later processing. Hearts were post-fixed in 1% tannic acid and transferred to 1% osmium tetroxide, dehydrated in an acetone series, and then embedded in Embed-812 resin (Electron Microscopy Sciences, Hatsfield, PA). Two-micron thick sagittal sections were cut and toluidine-blue stained for light microscopy analysis. Stained sections were visualized and imaged using a Nikon Optiphot (Nikon Instruments Inc., Melville, NY). For electron microscopy analysis, ultra-thin sections 100 nm thick were cut, set on single slot or 200-mesh copper or nickel grids, and imaged on a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company, Hillsboro, OR). Additional fixed and embedded hearts (n = 9) were obtained from the University of Utah and processed as previously described [14]. Briefly, these hearts were perfusion-fixed with 2.5% glutaraldehyde-2% paraformaldehyde in 0.1 M cacodylate buffer, and 4×10⁻⁴ g/kg dose of diltiazem for 4 hours. The hearts were then post-fixed in 2% osmium tetroxide, dehydrated, and embedded in Spurr’s epoxy resin. Ultra-thin sections 100 nm thick were cut and mounted on single slot or 200-mesh copper or nickel grids and imaged on a Tecnai G2 Spirit BioTWIN electron microscope (FEI Company, Hillsboro, OR).\n\n**Immunofluorescence Imaging**\n\nFollowing isolation, zebrafish hearts were cryoprotected in 30% sucrose in PBS overnight. The following day, the hearts were embedded in freezing medium (Tissue-tek OCT, Torrance, CA), kept at −80 degrees C and later sectioned using a Leica CM 1900 cryostat (Leica Microsystems, Bannockburn, IL). Ten-micron sagittal sections were fixed for 3 minutes in 4% paraformaldehyde, permeabilized with 0.5% triton X in PBS, blocked with 3% BSA for 2 hours, and immunoreacted overnight with anti-MYH1 antibody or MEF antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA), followed by Texas-Red conjugated anti-mouse or anti-rabbit secondary antibody. Next, the sections were incubated with FITC-labeled wheat germ agglutinin (WGA, Triticum vulgaris, Sigma-Aldrich, Saint Louis, MO) for 2 hours. Other sections were stained with TRITC-labeled WGA. Following Hoechst staining, sections were imaged on a Zeiss Axio Imager.M (Carl Zeiss Microscopy, Thornwood, NY) or a Leica TCS SP5 confocal microscope (Leica Microsystems, Bannockburn, IL).\n\n**Serial Block-face Scanning Electron Microscopy Imaging**\n\nTissue fixation and embedding were performed using the previously published protocols with some modifications [22]. Briefly, excised zebrafish hearts were fixed in 2.5% glutaraldehyde in 100 mM sodium cacodylate buffer at 4 degrees C overnight. The hearts were rinsed the next day in 100 mM sodium cacodylate. The hearts were post-fixed and heavy metal-contrasted with potassium ferrocyanide, osmium tetroxide, thiocarbohydrazide, uranyl acetate and lead aspartate. Next, the hearts were dehydrated in ethanol followed by acetone and embedded in Durcupan resin. Approximately 2×2 mm, 100 nm thick sections were obtained, mounted on slot grids and surveyed by TEM. Blocks with optimal tissue orientation and easily identifiable landmarks in the region of interest were selected for further trimming and imaging. Serial block-face sectioning (at 50 or 100 nm imaging intervals) and imaging were performed using a Gatan 3View system (Gatan, Pleasanton, CA) mounted in an FEI Quanta FEG 200 SEM (FEI Company, Hillsboro, OR). The SEM was operated at an accelerating voltage of 2.0 kV and 30 pascals pressure in low vacuum mode. A solid state backscatter detector was used to acquire serial section images (stacks of 800, 632, and 500 sections) from three regions of interest. Three-dimensional segmentation and reconstruction of fibroblasts and cardiac myocytes was performed using Amira 5.2 software.\n\n**Results**\n\nI. **Electron Translucent and Electron Dense Cardiac Myocytes Are Organized into a Transitional Cell Ring**\n\nTo determine the nature of the interface region between the compact and spongy heart of the adult zebrafish, we first studied the ventricle under light microscopy. Using two-micron thick toluidine blue-stained plastic embedded sections, we observed the expected organization: a relatively thin layer of the compact heart (approximately 10–20 um thick) framing the trabeculae which projected radially into the ventricular lumen (Fig. 1. A). However closer inspection revealed an unexpected number of darkly-stained elongated structures, at irregular intervals at the interface between\n\nFigure 1. Low magnification of the adult zebrafish heart by light and TEM. (A), light microscopy view of the apical region of the zebrafish ventricle showing a thin layer compact heart (CH) with trabeculae (Tr) projecting within the lumen (Lu). (B), higher magnification of the apex showing darkly stained linear structures (arrows) close to the spongy-compact interface of the ventricular myocardium. (C), low magnification TEM of compact ventricular myocardium constituted of four to five overlapping cardiac myocytes (CM) layers from epicardium (Epi) to lumen (Lu), with electron dense cells (arrow) within a complex junctional region (JR, brackets) at the interface of compact and spongy myocardium. (D), higher magnification show mitochondria (Mt) filled section of electron dense transitional cardiac myocyte (EDTCM). EDTCM are apposed to trabecular cardiac myocytes and endocardial cells (Endo) on the luminal side. EDTCMs are separated from the compact cardiac myocytes (CCM) by a junctional space, an interstitial space within the JR. (E), low magnification TEM of the JR (brackets) showing flattened electron translucent transitional cardiac myocytes (ETTCM) in contact with trabeculae and in proximity to endocardium (Endo) on the luminal side. (F), higher magnification of a flattened cells show presence of actin and myosin filaments, apposition to trabeculae and endocardial cells as well as separation from the compact myocardium by the junctional space.\n\ndoi:10.1371/journal.pone.0072388.g001\n\nFigure 2. Electron dense and electron translucent transitional cardiac myocytes interactions. (A), example of end-to-end interaction between EDTCM and electron translucent cardiac myocytes (ETTCM) forming a continuum at the spongy-compact interface. (B), higher magnification of contact area with complex adhesion junctions including desmosomes between the two cells. (C), example of interaction between mitochondria filled and actin-myosin filament (AMF) containing EDTCM cells and ETTCM via adhesion junctions (arrows). (D), higher magnification of EDTCM and ETTCM showing gap junctions (arrows), and (E), desmosomes. (F), AMF in a transitional cell. (G), higher magnification (inset in F, rotated) showing the hexagonal array of myosin in cardiac myocytes. (F) sarcomere and z-band in the same cell. AMF, actin-myosin filament; CCM, compact cardiac myocytes; CW, cardiac myocytes; EDTCM, electron dense transitional cardiac myocytes; ETTCM, electron translucent transitional cardiac myocytes; Endo, endocardium; Epi, IF, intermediate filaments; Lu, lumen; Mt, mitochondrion; Tr, trabeculae.\n\ndoi:10.1371/journal.pone.0072388.g002\n\nthe compact and spongy segments of the ventricle (Fig. 1, B). To elucidate the nature of these darkly-stained structures, we studied their characteristics and precise location within the zebrafish junctional region (JR) using transmission electron microscopy (TEM). Low magnification TEM reveals a compact myocardium of 4–5 layers of cardiac myocytes (CM) (Fig. 1, C). The compact and spongy interface is revealed as a complex JR containing a set of flattened CM as compared to the compact or trabecular CM. Ultrastructurally, the darkly stained structures appear as electron dense CM running along the adjacent base of the trabeculae (Fig. 1, D). We have called these cells: “dark cells” or electron dense transitional cardiac myocytes (EDTCM). Except for their high electron density, these cells display similar flattened and rectangular phenotypes as the transitional CM described previously [18]. In adjacent regions, the ventricle displays a similar arrangement but with electron translucent transitional cardiac myocytes (ETTCM) (Fig. 1, E). Actin-myosin filament bundles were easily identified within the ETTCM confirming their CM phenotype (Fig. 1 F), whereas the cytoskeletal contents of the EDTCM were obscured by the electron dense material contained in these cells.\n\nII. Transitional Cell Ring and Trabeculae Contacts\n\nThe EDTCM were in direct contact with the ETTCM (Fig. 2, A). Within the plane of observation, shorter segments of EDTCM alternated with longer segments of ETTCMs, and together these two cell populations appear to form a continuous ring of transitional CM within the JR. The transitional CM could be seen linked at their tapered ends through complex adherent junctions resembling fascia adherentes (Fig. 2, B). Additionally, the transitional CM were in direct contact or in close apposition to the base of CM sheets of projecting spongy trabeculae (Fig. 2, A, C), and in the intertrabecular lacunar spaces they were bounded in their luminal aspect by the endocardium (Fig. 2, C). However in contrast to their connections to trabeculae, no contacts were observed between transitional CM of the transitional ring and the adjacent endocardial cells. In some instances, EDTCM and ETTCM minimally overlapped at their tapered ends (Fig. 2, C), with connections occurring through discrete gap junctions (Fig. 2, D) and desmosomes (Fig. 2, E). Detailed observations of the cytoplasm of these cells show the presence of actin-myosin filaments oriented in different planes (Fig. 2, F). The presence of caveolae and the hexagonal array of equidistant thick myosin filaments characteristic of cardiac muscle cells can be appreciated (Fig. 2, G). Z-bands bordering sarcomeres oriented perpendicularly or obliquely in the plane of observation (Fig. 2, H) can also be seen in these cells, further confirming their cardiac myocyte phenotype. Finally the cardiac nature of these narrow width transitional cells was also confirmed using immunostaining with anti-myosin heavy chain-1 (MYH1) and anti-myocyte enhancer factor-2 (MEF) antibodies to specifically identify cardiac myocytes (Fig. 3).\n\nWe further investigated the nature of the arrangement between the transitional CM ring and the CM forming the trabeculae. Transitional CM can be found linked to trabecular myocytes through a variety of adherens junctions. These junctions included fascia adherentes (Fig. 4, A, B) that link actin-myosin filaments of adjacent cells. Various amounts of amorphous material were observed along transitional CM and trabecular CM areas of apposition (Fig. 4 C, D). However discrete electron dense areas...\n\nwith a sub-domain containing desmosomes were also observed (Fig. 4 E, F).\n\nIII. Fibroblast Network and Collagen in the Junctional Region\n\nThe transitional ring of myocytes on its abluminal side is bounded by a narrow (0.5–1 um) connective tissue space. This space separates the EDTCM and ETTCM from the adjacent CM of the compact layer, creating a junctional space within the JR (Fig. 5. A). The junctional space appears to create an extensive structural discontinuity between the spongy and the compact myocardium. It contains numerous cells arranged longitudinally and running parallel to the transitional CM ring. The absence of basement membranes, their elongated shape, and their location within this interstitial space suggest they are fibroblasts (Fig. 5. B, C). These fibroblasts contained ovoid nuclei with scant perinuclear cytoplasm that appeared mostly devoid of organelles, except for occasional vesicles and polyribosomes. In the perinuclear region of the fibroblasts, the junctional space widens to 2–3 microns. The cytoplasm of these fibroblasts is stretched bi-directionally into long thin filopodia-like processes. These fibroblasts mostly form a single layer in the junctional space with little to minimal overlap of their cytoplasm. The fibroblasts and their cytoplasmic processes fit tightly within the junctional space, however a measurable space was always observed between the fibroblast and the adjacent transitional and the compact ventricular CM.\n\nThe fibroblasts were also found in close proximity or in direct contact with parallel or perpendicularly-oriented collagen fibers (Fig. 5. B, C). At irregular intervals, terminal profiles displaying long cytoplasmic extensions were in close proximity with the membranes of long cytoplasmic processes originating from another fibroblast (Fig. 5. D). In some instances, cytoplasmic terminal ends of fibroblasts overlapped, or could be seen in contact with the same bundle of collagen. Occasionally fibroblasts terminal ends were found in close contact; however we could not unequivocally confirm specialized adhesion junctions between interacting terminal ends. Occasionally close approximation between fibroblast filopodia and CM could be seen; however no direct contact between fibroblasts and CM and no specialized myocytes-fibroblasts junctions were observed. Close observation of many of the transitional CM however revealed the presence of caveolae within the membranes orientated toward the fibroblasts within the junctional space (Fig. 5. C). Across the junctional space, the compact CM membranes were observed to have numerous caveolae facing toward the fibroblasts (Fig. 5. D, E). More examples of caveolae could be seen on the membranes of CM facing the junctional space at a distance from the fibroblast processes (Figure S1). While most fibroblast nuclei and cytoplasmic processes resided within the JR, some cytoplasmic projections could be seen invested in the compact myocardium adjacent to small vessels, suggesting that fibroblasts may not be locally restricted to the junctional space.\n\nFigure 5. Fibroblasts network in the junctional region of the zebrafish ventricle. (A), TEM of a fibroblast (Fb) cytoplasm and its thin extended process/filopodium (FP) 100 to 200 nm thick spanning the junctional space (JS) in between the transitional CM ring and the adjacent compact cardiac myocyte (CCM). Note vesicle (*) in an enlarged region (~300 nm) of the fibroblasts filopodium (FP). (B), fibroblast profile with nucleus (Nu) and its cytoplasm extending into a long thin process, and collagen fiber (CF) bundles running perpendicular to the section’s plane. (C), another fibroblast and its nucleus (Nu) and with cytoplasm extending into a long process, and collagen fiber (CF) bundles running parallel to the section’s plane. (D), fibroblast filopodial termini in close approximation. Electron dense region in the close approximation area suggestive of specialized adhesion junction (arrow). Note the presence of caveolae (*) on the membrane of the CCM facing the fibroblast in the junctional space.\n\ndoi:10.1371/journal.pone.0072388.g005\n\nFigure 6. Transitional CM ring and compact CM contacts. (A), ETTCM and Compact CM direct contacts mediated by adhesion junctions (arrows). Note the JS with fibroblast (Fb) on the right and narrow JS of the left of the contact region. (B), higher magnification of inset in (A) showing adhesion junctions associated with fascia adherentes (thick arrow), and also with desmosomes (thin arrow). Note the abundance of caveolae (*) in the transitional CM membrane in the contact region. (C), another narrow region of contact (arrow) between a transitional and a compact cardiac myocyte.\n\nforming a bridge between the two cells and adjacent Z-band in the CCM. A fibroblast process is seen on each side of the junction. Note a vesicle (V) in the fibroblast process. (D), higher magnification of the contact area (arrow) showing a desmosome associated with actin, myosin, and intermediate filaments on the ETTCM and abundant ribosomes (rb) in the CCM. (E), a tri-cellular junction between two transitional cardiac CM and one compact CM. (F), higher magnification of (E, inset) showing desmosomes between the ETTCM and each ETTCM with the compact CM (arrows). (G), discrete adhesion junction between a transitional and compact CM (arrows). (H), higher magnification of inset 1 of (F). (I), higher magnification of inset 2 of F, showing compact CM contact within an invagination of the transitional CM.\n\ndoi:10.1371/journal.pone.0072388.g006\n\nIV. Transitional Cardiac Myocytes Contacts across the Junctional Space\n\nFrom our observations, we estimated that the junctional space containing fibroblasts and collagen occupies 80 to 90% of the junctional region between compact and spongy heart, so that the majority of the CM membranes in the transitional ring are not directly linked to the compact heart. The remaining portion of the spongy-compact interface consisted of cell membrane appositions between transitional CM and the most adjacent CM of the compact heart (Fig. 6. A). These regions of apposition contained specialized adherens junctions including closely spaced desmosomes and fascia adherents (Fig. 6. B). These areas of contact between the transitional ring and compact myocardium circumscribed the junctional space and interrupted the course of the fibroblasts and their filopodia in the plane of the sections (Fig. 6. C, D). Occasionally a region of tri-cellular contact was observed, where a compact myocyte was joined at the end-to-end contact of two transitional CM through desmosomes (Fig. 6. E, F). However most transitional ring myocytes and compact CM contacts occurred through apposition of their lateral borders via desmosomes (Fig. 6. G, H), membrane protrusions, and interdigitations (Fig. 6. I).\n\nV. Serial Block-face SEM, Segmentation, and Volume Reconstruction of the Junctional Region\n\nTo better assess the three-dimensional architecture of the junctional region and confirm the three-dimensional nature of the fibroblast network, we performed serial block-face scanning electron microscopy (SBF-SEM) of the adult zebrafish heart. A preparatory survey of the fibroblast network in the JR of the zebrafish ventricle was performed by TEM in 100 nm sections from heavy metal-contrasted Durcupan-embedded hearts. The junctional region and the fibroblasts were easily identifiable. Image stacks generated with the FEI Quanta 200 FEG SEM equipped with the Gatan 3View (Fig. 7. A) were used to generate three-dimensional reconstructions of the JR architecture (Fig. 7. B, Movie S1), and confirm the 3D projection of fibroblasts in the junctional volume space. Observation of a subset of serial sections demonstrated the predicted discontinuous nature of cellular contacts between the compact heart and transitional myocytes that anchor the luminal trabeculae, and confirmed fibroblast-fibroblast contacts in the junctional space (Fig. 7. C-I, Movie S2). Segmentation of fibroblasts from serial sections followed by three-dimensional reconstruction demonstrated the sheet-like nature of fibroblasts arranged in a network occupying the junctional space of the zebrafish heart (Fig. 7. J, K). Additional segmentation of transitional and compact cardiac myocytes demonstrated contact surfaces between compact and spongy heart through the fibroblast network layer (Fig. 7. L).\n\nDiscussion\n\nThis paper documents a previously unrecognized connective tissue space located at the interface of the spongy and compact myocardial of the adult zebrafish heart. It extends our understanding of the cellular and ultrastructural nature of the ventricular JR of an important research model. The existence of a connective tissue layer in the junctional region of type-2 fish hearts has remained controversial. A continuous fibrous membrane interposed between the compact and spongy layers has been reported in the hearts of tuna [16,17], atlantic salmon and rainbow trout [23]. These observations led to the hypothesis of the connective tissue as an important adhesive substrate between the two myocardial layers. In these early studies, detailed composition of the connective tissue layer was not described. Midttun using TEM demonstrated in four type-2 fish hearts species the presence of fibroblasts and collagen fibers within a 6 to 7 um wide space existing in the junctional region [18]. By contrast in a study by Pieperhoff et al., using light and scanning electron microscopy, a connective tissue layer at the interface of the two myocardial layers in the sockeye salmon and trout was not observed [20], beyond small focal regions of extracellular matrix enrichment. Unlike the previous studies, these authors challenged the hypothesis of a connective tissue layer separating the two myocardial layers. In both Midtun’s and our studies, the junctional space and its content were not readily detected by light microscopy; however, they could be observed by transmission electron microscopy, suggesting that ultrastructural thin sections imaging may be necessary to describe the structure of the JR of fish ventricles.\n\nOur ultrastructural observations reveal a junctional space measuring for the most part less than one micrometer wide, populated by a single layer of fibroblasts organized into a network, and framed by CM belonging the compact myocardium distally and the spongy myocardium proximally. Fibroblasts are an important component of the mammalian heart and play an essential role in heart structure and physiology. They are a source of extracellular matrix molecules and growth factors, and they serve as sensors for mechanical signals [24,25,26,27,28]. While it has been suggested in early studies that collagen accumulation modulates regeneration in the zebrafish ventricle [10], fibroblasts in the fish heart have received little attention. More recently studies of cardiac injury in the zebrafish [29,30,31] and giant danio [21] demonstrated accumulation and resorption of collagen during fish ventricular regeneration, suggesting an important role for fibroblasts. Indeed the presence of activated fibroblasts was observed during ventricular remodeling in the zebrafish [31] and giant danio heart [21]. The presence of fibroblasts has previously been noted in the sub-epicardial space of fish species, including the zebrafish [14]. In the present study, the fibroblasts’ arrangement suggests a highly organized three-dimensional network exists at the interface of the compact and spongy ventricle. The function of this fibroblast network is presently unclear.\n\nFibroblast networks have been documented in a variety of organs and species, with direct fibroblast-fibroblast contacts being observed in mammalian corneas [32,33]. Fibroblast interactions suggestive of a network have also been reported in the mammalian heart [34]. Fibroblasts have also been implicated in physiological mammalian cardiac muscle growth [27] as well as in injury responses [35,36,37,38]. Our study by TEM and the reconstruction following segmentation of the SBF-SEM micrographs demonstrate that fibroblasts form a three-dimensional network in the JR of the adult zebrafish heart. In addition to fibroblastfibroblast interactions, direct CM and fibroblast contacts have also been reported in normal and diseased hearts and in culture [39,40,41]. In this study however, we did not uncover direct CM-fibroblasts contacts in the uninjured zebrafish heart. When fibroblast processes were seen in close proximity and in apparent apposition to myocytes, the intervening myocyte basement membrane was always present. Yet we cannot rule out that these contacts exist and may occur at a frequency too low to be easily...\n\ndetected. Nevertheless, we found a remarkable number of caveolae in the sarcolemma of CM bordering the junctional space and facing adjacent fibroblasts. This suggests possible mechanisms by which paracrine signaling may take place between adjacent CM and fibroblasts occupying the junctional space.\n\nThe present study also establishes that the junctional space occupies the majority of the overall SCI, and creates an extensive volume of non-contact between the two myocardial layers. Given the sparsity of the collagen fibers observed in the junctional space, we question whether in the zebrafish, the connective tissue would provide sufficient support to maintain the structural integrity of the two layers. We speculate that the connective tissue compartment may not be the primary mechanism by which adhesion between the two myocardia is maintained. Indeed, the junctional space is interrupted by contact bridges between the two layers at discrete intervals through membrane apposition of adjacent CM, and that desmosomes and fascia adherentes are frequently found within the contact regions between transitional zebrafish CM and compact CM. These findings are consistent with the demonstration of highly enriched adhesion junction molecules between the two myocardial layers observed in salmon and trout [20].\n\nAn important finding is the transitional ring of cardiac myocytes similar to that previously described by Midrunn, that underscores the phenotypic diversity of cardiac myocytes present in the zebrafish heart, suggesting specific structure-function relationship. The notion of cardiac myocyte diversity is apparent in recent work demonstrating the clonal contribution of cardiac myocytes to the emergence of the zebrafish heart [12]. In that study two populations of peripheral cardiac myocytes were described by genetic labeling and spatio-temporal characteristics: a primordial and a cortical set. The localization of the primordial layer suggests it occupies a space similar to the transitional ring population described in the present study. Consequently, it would be interesting to determine whether these two populations spatially interact or are one and the same. Another intriguing finding is the discovery of a subset of “dark” electron dense myocytes within the transitional CM ring that supports the trabeculae. To our knowledge, these electron dense cardiac myocytes have not been reported in fish or mammalian hearts. A set of dark cells was observed in the ventricle of icefish; however, they were believed to be non-contractile in nature [42]. Observations of the zebrafish electron dense cells at high magnification show clear evidence of myofibrils. In addition these cells form a continuum with adjacent electron translucent CM as well as other electron dense CM in the transitional ring and myocytes of adjacent trabeculae via adhesive junctions. These observations raise the intriguing possibility of further phenotypic specialization within the transitional CM population bordering the JS. We speculate this particular phenotypic change may be regulated by regional stress distribution within the myocardium or may reflect their metabolic state. The understanding of the functional and the molecular differences between the dark and light cells within the transitional ring warrants further study.\n\nConclusions\n\nIn conclusion, this study sheds light on the phenotypic diversity of CM at the JR of the adult zebrafish heart. It documents the existence of a fibroblast network that contributes to the architectural complexity of the region (Fig. 8). We suggest the transitional CM form the main anchoring substrate for the trabecular and compact heart. It is possible that in type-2 fish hearts, both the connective tissue layer and the adherens junctions account for the integrity of the spongy-compact interface. It is also possible that in fish, the ratio of connective tissue and direct CM adhesive contact, as well as the distribution of adherens junctions might be dependent on the species, their age and ecological\n\nphysiology. In the end it is intriguing to consider how the articulation of the compact and spongy interface progresses during development and how the re-articulation occurs during regeneration of the zebrafish heart.\n\nSupporting Information\n\nFigure S1 Transitional myocyte abluminal caveolae. (A), Junctional space (JS) with a fibroblast process (FP). (B), Higher magnification of inset in (A) showing caveolae (*) on transitional CM facing the JS. (TIF)\n\nMovie S1 Architecture of the junctional region of zebrafish heart. The sheet-like nature of fibroblasts can be appreciated. (MP4)\n\nMovie S2 Segmented fibroblasts and transitional CM. (MP4)\n\nReferences\n\n1. Sauter RM (1980) Morphological studies on the ventricle of teleost and elasmobranch hearts. J Zool, Lond 190: 259–272.\n2. Sanchez-Quintana D, Garcia-Martinez V, Climent V, Hurle JM (1995) Morphological analysis of the fish heart ventricle: myocardial and connective tissue architecture in three species. Ann Anat 177: 267–274.\n3. Sanchez-Quintana D, Hurle JM (1997) Ventricular myocardial architecture in marine fishes. Anat Rec 217: 263–273.\n4. Sauter RM (1983) Morphology and innervation of the fish heart. Adv Anat Embryol Cell Biol 89: 1–102.\n5. 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PLoS Biol 2: e329.\n\nAcknowledgments\n\nWe thank Margaret Gondo for TEM help, Lynn Bedard and Tamara Beauboeuf for editorial suggestions, Tanmoy Das and Henning Schneider for valuable discussions, the Prindle Institute for Ethics at DePauw University for dedicated space for scholarly work, and also John Eskew, Wendy Tomamichel, and Clay Higginbotham for technical help and with animal care. We thank Jayne Williams for logistical help. We thank Barry Stein of The Electron Microscopy Center and Jim Powers of the Light Microscopy Imaging Center at Indiana University-Bloomington for their help.\n\nAuthor Contributions\n\nConceived and designed the experiments: PJL A. Behzad EB PL NH A. Burns. Performed the experiments: PJL A. Behzad EB PL NH A. Burns. Analyzed the data: PJL PL A. Burns. Contributed reagents/materials/analysis tools: PJL A. Behzad EB PL NH A. Burns. Wrote the paper: PJL. Designed and performed TEM experiments: PJL A. Burns EB NH. 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Dev Cell 16: 233–244.\n28. MacKenna DA, Dolli F, Vuori K, Ruoslahri E (1998) Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts. J Clin Invest 101: 301–310.\n29. Schnabel K, Wu CC, Kurth T, Weidinger G (2011) Regeneration of cryoinjury induced necrotic heart lesions in zebrafish is associated with epicardial activation and cardiomyocyte proliferation. PLoS One 6: e18503.\n30. Gonzalez-Rosa JM, Martin V, Peralta M, Torres M, Mercader N (2011) Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish. Development 138: 1663–1674.\n31. Chabials F, Veit J, Rainer G, Jazwinska A (2011) The zebrafish heart regenerates after cryoinjury-induced myocardial infarction. BMC Dev Biol 11: 21.\n32. Haasenstiel J (1982) On the ultrastructure of the developing and adult mouse corneal stroma. Anat Embryol (Berl) 168: 291–305.\n33. Nishiida T, Yasumoto K, Otori T, Desaki J (1983) The network structure of corneal fibroblasts in the rat as revealed by scanning electron microscopy. Invest Ophthalmol Vis Sci 29: 1837–1890.\n34. Goldsmith EC, Hoffman A, Morales MO, Potts JD, Price RL, et al. (2004) Organization of fibroblasts in the heart. Dev Dyn 230: 787–794.\n35. Dobaczewski M, Frangogiannis NG (2009) Chemokines and cardiac fibrosis. Front Biosci (Schol Ed) 1: 391–405.\n36. Kohr S (2009) Myofibroblasts in diseased hearts: new players in cardiac arrhythmias? Heart Rhythm 6: 845–856.\n37. Porter KE, Turner NA (2012) Cardiac fibroblasts: at the heart of myocardial remodeling. Circ Res Ther 12: 255–278.\n38. van den Borne SW, Dirx J, Blanksteijn WM, Verjans J, Hoštůra L, et al. (2010) Myocardial remodeling after infarction: the role of myofibroblasts. Nat Rev Cardiol 7: 30–37.\n39. Chilton L, Giles WR, Smith GL (2007) Evidence of intercellular coupling between co-cultured adult rabbit ventricular myocytes and myofibroblasts. J Physiol 583: 225–236.\n40. Kohl P, Cannellini P (2007) Cardiac myocyte-nonmyocyte electrotonic coupling: implications for ventricular arrhythmogenesis. Heart Rhythm 4: 233–235.\n41. Miraglia M, Salvatore N, Rohr S (2007) Myofibroblasts induce ectopic activity in cardiac tissue. Circ Res 101: 755–758.\n42. Harrison P, Zummo G, Farina F, Tota B, Johnston IA (1991) Gross-Anatomy, Myoarchitecture, and Ultrastructure of the Heart Ventricle in the Haemoglobinless Icefish Chaenocephalus-Acraatus. Canadian Journal of Zoology-Revue Canadienne De Zoologie 69: 1339–1347."}}},{"rowIdx":204699,"cells":{"text":{"kind":"string","value":"Cellular characterization of the outgrowth and connectivity of the brain serotonin system\n\nJeroen Dudok\n\nThe research described in this thesis was conducted at the Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, The Netherlands, and was supported by a grant from the Dutch Organization of Scientific Research (NWO-MW-PIO900-01-001).\n\nPublication of this thesis was financially supported by:\n\nCNCR\nDepartment of Functional Genomics\nVrije Universiteit Amsterdam\n\nCover: Serotonergic neurons in the dorsal raphe nucleus\nPrint: Wöhrmann Print Service\nISBN: 978-90-8570-585-7\n\n© Jeroen Dudok 2010. All rights reserved. No part of this thesis may be reproduced, stored in a retrieval system, or transmitted in any form or by any means without prior permission of the author.\n\nCellular characterization of the outgrowth and connectivity of the brain serotonin system\n\nACADEMISCH PROEFSCHRIFT\n\nter verkrijging van de graad Doctor aan\nde Vrije Universiteit Amsterdam,\nop gezag van de rector magnificus\nprof.dr. L.M. Bouter,\nin het openbaar te verdedigen\nten overstaan van de promotiecommissie\nvan de faculteit der Aard- en Levenswetenschappen\nop woensdag 29 september 2010 om 15.45 uur\nin de aula van de universiteit,\nDe Boelelaan 1105\n\ndoor\n\nJacobus Johannis Dudok\ngeboren te Zierikzee\n\npromotor: prof.dr. M. Verhage\ncopromotor: dr. A.J.A. Groffen\n\nAan mijn ouders\n\n# Contents\n\n| Chapter | Title | Page |\n|---------|----------------------------------------------------------------------|------|\n| 1 | General introduction | 11 |\n| 2 | Chronic activation of the 5-HT$_2$ receptor reduces 5-HT neurite density as studied in organotypic slice cultures | 37 |\n| 3 | Dynamic vesicular trafficking of the serotonin reuptake transporter in hippocampal neurons | 57 |\n| 4 | Presynaptic localization of tryptophan hydroxylase 2-EGFP in mature hippocampal neurons | 73 |\n| 5 | The effect of a polymorphism in Piccolo on localization and trafficking dynamics of the serotonin reuptake transporter | 85 |\n| 6 | Deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality | 105 |\n| 7 | Towards a genetic approach to study serotonergic outgrowth and connectivity in vivo | 127 |\n| 8 | General discussion | 140 |\n| | Nederlandse samenvatting | 154 |\n| | List of abbreviations | 159 |\n| | Dankwoord | 161 |\n| | Curriculum Vitae | 164 |\n\nChapter 1\n\nGeneral introduction\n\nGeneral introduction\n\nThe mammalian brain is by far the most complex system in the body. The human brain consists of approximately 100 billion nerve cells (neurons), which form a highly complex wiring scheme. How this complex wiring is achieved is still poorly understood. The brain contains several clusters of cells which contain a certain neurotransmitter, such as dopaminergic cells in the substantia nigra or acetylcholinergic cells in ventral and dorsal striatum.\n\nAnother example of such clusters are the raphe nuclei in the midbrain that secrete serotonin (5-hydroxytryptamine, 5-HT). 5-HT has diverse modes of action in the central nervous system and is involved in brain development as will be described in this introduction. There are a few remarkable characteristics of the 5-HT system. First of all, the 5-HT system consists of only a few neurons (~1000 in rodents, ~0.5 million in humans) compared to the total number of neurons. Nevertheless, these neurons send projections to virtually every other brain area. It is estimated that each 5-HT neuron makes more than $1 \\times 10^6$ connections. Secondly, 5-HT is predominantly released extrasynaptically (so called volume or paracrine transmission), which implies that 5-HT acts on a slower and longer-lasting time scale compared to classical neurotransmitters like glutamate. Thirdly, emerging evidence shows that 5-HT has a role during brain development. Finally, the 5-HT system is involved in certain behavioural and psychopathological processes including anxiety, depression, aggression and obsessive-compulsive disorder (OCD).\n\nIn this chapter the main aspects of the 5-HT system will be introduced and the diverse modes of action which 5-HT has are described. First, there will be a description about the anatomical organization and development of the 5-HT system. Subsequently, the role of 5-HT in brain development will be introduced and the relation between the 5-HT system and behavioural and psychopathological processes will be discussed. Finally, the aim and scope of this thesis will be outlined.\n\nAnatomical organization of 5-HT cell clusters\n\nAfter the initial discovery of 5-HT (see box 1), it took about 20 years before the cellular distribution of 5-HT was described by Dahlstrom and Fuxe using the Falck-Hillarp technique. They showed that 5-HT cell bodies are clustered in nine cell groups close to the midline in the rhombencephalon, called the raphe nuclei. The nine nuclei are divided in two groups, the rostral and caudal group. The caudal group consists of clusters B1 to B5 and send projections predominantly in the midbrain itself and towards the spinal cord. On the other hand, the rostral\n\ncell group consists of clusters B6 to B9 and sends projections to the forebrain. The largest clusters of cells are the dorsal raphe nucleus (DRN) and the median raphe nucleus (MRN) (B7 and B8 respectively). These raphe nuclei also contain several types of non-5-HT neurons, such as GABA-ergic and noradrenergic neurons. The 5-HT neurons densely innervate virtually every brain area, with the most dense 5-HT innervations in the cortex and limbic structures including the hippocampus and basal ganglia.\n\n**Box I**\n\n**The discovery of 5-HT**\n\nIn the 1930’s Dr. Vittorio Erspamer tried to extract chemical compounds from enterochromaffin cells capable of causing smooth muscle contraction in the gut. In an extract of rabbit gastric mucosa, he identified a substance that could cause smooth muscle contraction. He called this substance enteramine, since it was an amino acid derivative (amine) found in enterochromaffin cells.\n\nIn the 1940’s another group identified a substance which had vasoconstrictive activity in serum. This serum influenced the tone, hence the name serotonin. When analyzing both substances, it was found that enteramine and serotonin were the same substance.\n\nThe first indication for a role of 5-HT in the brain came from Woolley, who showed that 5-HT and the hallucinogenic compound lysergic acid diethylamide (LSD) had comparable actions in the cortex of the cat. This eventually led to the discovery of 5-HT in the vertebrate brain like man and dog.\n\nDahlstrom and Fuxe were the first to describe the distribution of 5-HT cell bodies in the brain by histochemical detection of the 5-HT derivative indoleamine. The description of 5-HT projections from the 5-HT cell bodies towards the telencephalon and the diencephalon followed one year later. It was found that in the brain stem a relatively small population of 5-HT neurons was present, with the largest clusters in the dorsal and median raphe nuclei.\n\n**The development of the 5-HT system**\n\nIn the mouse brain, 5-HT neurons are generated around embryonic day (E) 10, and these cells are among the first neurons expressing a specific neurotransmitter. In the neural tube, 5-HT precursor cells are formed by the combined action of sonic Hedgehog, Fgf4 and Fgf8. The proper position of these 5-HT precursor cells is defined by the midbrain-hindbrain organizing centre (MHO), Shh signaling and the transcription factor Nkx2.2 (see figure 1).\n\nIn a knockout (KO) mouse for Nkx2.2, no 5-HT neurons are present in the midbrain raphe nuclei, except in the DRN, showing that Nkx2.2 is essential for 5-HT neuron differentiation (Briscoe et al., 1999). Another transcription factor that is involved in the initial development of 5-HT neurons is GATA3 (van Doorninck et al., 1999). After 5-HT precursor cells are formed, other\n\nfactors are required to establish mature 5-HT neurons. Among these factors are two transcription factors, LIM homeobox transcription factor 1 (Lmx1) and PC12 Ets factor (Pet1).\n\nLmx1b expression starts at E10.75 in the ventral part of the hindbrain including the floor plate and from E13.5 Lmx1b is present in the raphe nuclei (Ding et al., 2003). Lmx1b KO mice die within 24 hours after birth (Chen et al., 1998). Lmx1b-deficient mice lack all 5-HT neurons, even when Lmx1b was only deleted in 5-HT neurons, (Ding et al., 2003; Zhao et al., 2006). These studies show that Lmx1b is not required for the initial generation of 5-HT neurons, but is necessary for the differentiation and survival of 5-HT neurons during postnatal development.\n\nPet1 was found in a screen in PC12 cells to identify novel ETS domain transcription factors (Fyodorov et al., 1998). Pet1 expression is restricted to the raphe nuclei and precedes the expression of 5-HT by approximately half a day (Hendricks et al., 1999). Furthermore, several genes specific for 5-HT neurons, such as the rate limiting enzyme tryptophan hydroxylase (Tph) and the 5-HT reuptake transporter (SERT) have a conserved Pet1 binding site in their promoter (Hendricks et al., 1999). This suggests that Pet1 is involved in the final\n\ndifferentiation of 5-HT neurons. In a Pet1 KO mouse, there is an approximately 70% reduction in number of 5-HT neurons. The fact that there are still 30% of 5-HT neurons remaining suggests that these are either independent of Pet1 or there is another unknown transcription factor.\n\nAfter this genetic cascade, at approximately E11.5, 5-HT neurons start expressing the enzymes necessary for 5-HT synthesis (see box II) and proteins necessary for reuptake (SERT) and degradation of 5-HT (Monoamine-A (MAO-A)). Shortly after initiation of expression of these genes, 5-HT neurons start to\n\n**Box II**\n\n**Synthesis of 5-HT**\n\n5-HT is a neurotransmitter which belongs to the group of the biogenic amines, to which also the catecholamines (dopamine, adrenaline and noradrenaline) belong. All biogenic amines are derived from amino acids: the catecholamines from tyrosine, and 5-HT from tryptophan. 5-HT and the precursor tryptophan belong to a group of chemical compounds called the indoles, with a benzene ring joined to a five-member ring containing nitrogen. Tryptophan is an essential amino acid, which means that this amino acid cannot be made in the body from other amino acids and must therefore be ingested through the diet.\n\nFor synthesis of 5-HT in the brain, tryptophan is actively transported through the blood brain barrier via the large neutral amino acid transporter (LAT1). Once in the brain, tryptophan is metabolized to 5-HT using two different enzymes. First, tryptophan is hydroxylated in the 5-position to generate 5-hydroxy-L-tryptophan. This reaction is catalyzed by Tph, which is the rate limiting enzyme in 5-HT synthesis. Subsequently 5-hydroxy-L-tryptophan is converted to 5-hydroxytryptamine (5-HT) by the enzyme 5-hydroxytryptophan decarboxylase. 5-HT is stored in synaptic vesicles and large dense cored vesicles and transported into these vesicles by the enzyme Vesicular Monoamine Transporter 2 (VMAT2). After being released, 5-HT is transported back into the terminal using SERT, and either recycled or converted to 5-hydroxyindolacetic acid (5-HIAA) by MAO-A.\n\nThe rate limiting enzyme Tph has a (6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin (Tetrahydrobiopterin, BH4) binding site. BH4 is an essential cofactor for the proper functioning of Tph. Without BH4, Tph cannot hydroxylate its substrate and thus 5-HT cannot be synthesized. BH4 is also required for dopamine synthesis by acting as a cofactor for tyrosine hydroxylase. Mice deficient for the BH4 gene die within 48 hours after birth and have extremely low levels of 5-HT and dopamine.\n\nIt has long been thought that only one Tph gene was present in the mammalian brain, although in zebrafish, two Tph genes were identified. However, in a mouse deficient for the Tph gene, no difference in brain 5-HT levels was found compared to control mice. This led to the discovery of the second Tph gene, called neuronal Tph or Tph2 (Walther et al., 2003). By using immunohistochemical analysis and in situ hybridization, it has been shown that Tph1 is expressed in the periferic system in the enterochromaffin cells and in the pineal gland in the brain, whereas Tph2 is exclusively expressed in the raphe nuclei in brain and in the myenteric plexus in the gut (Patel et al., 2004; Malek et al., 2005; Gutknecht et al., 2009).\n\nrelease 5-HT and start to send axon branches towards the spinal cord and via the medial forebrain bundle towards the cortex. Only after birth the 5-HT network is fully matured.\n\nIn the human brain, 5-HT neurons are for the first time detected when the embryo is five weeks old (Sundstrom et al., 1993). At ten weeks of age, 5-HT neurons are detected in all nuclei of the reticular formation and after 15 weeks, the 5-HT neurons are clustered in the raphe nuclei (Shen et al., 1989). After birth, 5-HT levels increase during the first two years and then decline to adult levels at the age of five years (Hedner et al., 1986).\n\n**5-HT release is both synaptic and extrasynaptic**\n\nSeveral classical neurotransmitters such as GABA, acetylcholine and glutamate are released from small synaptic vesicles (SSVs) at synaptic sites with a postsynapse in close proximity. However, neurotransmitters such as dopamine and 5-HT are released not only synaptically, but there is also so-called extrasynaptic release (also called paracrine transmission or volume transmission; here there will be referred to this type of release as volume transmission). A major difference between synaptic release and volume transmission is that there is no postsynapse present, so upon release the neurotransmitters diffuse away and can mediate effects distant from the release site. In this manner volume transmission can produce paracrine effects. In contrast to synaptic release, where SSVs are only released from axonal terminals, volume transmission of 5-HT occurs also in the soma and in dendrites, the so-called somatodendritic release. In 5-HT neurons, vesicles storing 5-HT are not only present in axonal terminals, but also in somata, dendrites and axonal varicosities (Hery and Ternaux, 1981). It was shown in Retzius neurons of the leech that 5-HT is released from both SSVs and large dense core vesicles (LDCVs), where there is an average discharge of 4700 and 80,000 molecules of 5-HT for SSVs and LDCVs respectively (Bruns and Jahn, 1995). In another study of the same authors, several differences between SSV and LDCV release of 5-HT were shown. First of all there was a difference in distribution: while SSVs were exclusively found in axons, LDCVs were also found at the soma. Furthermore LDCV release was slower compared to SSV release (Bruns et al., 2000).\n\nThere are several lines of evidence for volume transmission of 5-HT, some of which are indirect. First of all it has been shown that 5-HT varicosities contain 5-HT vesicles, but lack a postsynaptic specialization in close proximity (Moukhles et al., 1997). 5-HT receptors and the SERT are localized at sites away from release sites, indicating that 5-HT diffuses over larger distances to activate these receptors (Ridet et al., 1993). The presence of SERT distant from release sites\n\nsuggests that these are required to transport 5-HT back into the cells to terminate the action of 5-HT in the extracellular space. Secondly, it has been shown by carbon fiber amperometry that upon stimulation an increase of 5-HT levels in the extracellular medium occurs (Bunin and Wightman, 1998). This suggests that upon stimulation, not all 5-HT is buffered by synaptic receptors and transporters, but diffuses away into the extracellular medium (Bunin and Wightman, 1998).\n\nIn contrast to extrasynaptic release, synaptic release occurs predominantly from SSVs. Apart from vesicular release of 5-HT, there is also evidence for non-vesicular release of 5-HT using a carrier-mediated mechanism (Levi and Raiteri, 1993).\n\nThe ratio between volume transmission and synaptic transmission of 5-HT depends on the location in the brain. In the DRN itself, 5-HT release is predominantly paracrine, and here somatodendritic 5-HT release activates 5-HT\\textsubscript{A} autoreceptors which are present at the somata and dendrites. These activated receptors activate a G protein-activated inwardly rectifying K\\textsuperscript{+} (GIRK) current. In target areas of the 5-HT system, the ratio between volume transmission and synaptic transmission is still under debate. However, there are electron microscopy studies that quantified the percentage of 5-HT varicosities which formed a complex with a postsynaptic specialization. This revealed that ~25%, 48% and 38% of 5-HT varicosities formed a synaptic complex in rat hippocampus, suprachiasmatic nuclei and supraoptic nuclei of the hypothalamus respectively (Oleskevich and Descarries, 1990; Boulaiach et al., 1994). It is noteworthy that 5-HT is not released exclusively from 5-HT neurons. During development, thalamocortical fibers transiently express SERT, capture 5-HT and use it as a borrowed transmitter (Lebrand et al., 1996). In the striatum, dopamine transporters can take up 5-HT and subsequently co-release dopamine and 5-HT from dopaminergic terminals (Zhou et al., 2005a).\n\n**5-HT has several effects on synaptic transmission**\n\n5-HT not only functions to activate postsynaptic receptors, but also has diverse functions in the presynapse. Several studies were performed on invertebrates, such as crayfish, lamprey and aplysia. It was observed at the crayfish neuromuscular junction that a brief exposure to 5-HT resulted in increased release of glutamate (Dixon and Atwood, 1989a). Injection of a protein kinase A inhibitor or an adenylate cyclase inhibitor abolished this effect, suggesting that the facilitatory effect of 5-HT required the action of both the phosphatidylinositol and adenylate cyclase second messenger pathway (Dixon and Atwood, 1989b). In a study by Wang and colleagues, they made use of the styryl dye FM1-43, a fluorescent dye which is taken up by endocytosis following vesicle release, thus\n\nspecifically labeling recycled vesicles (Betz and Bewick, 1992). Using this approach, application of 5-HT at the crayfish neuromuscular junction did not affect the rate of recycling. Instead, 5-HT increased the number of vesicles which are available for release, the ready-releasable pool (RRP) of vesicles (Wang and Zucker, 1998).\n\nIn contrast to this stimulatory effect in the crayfish neuromuscular junction, 5-HT has an inhibitory action on synaptic transmission in the lamprey giant synapse (Buchanan and Grillner, 1991). This effect of 5-HT is likely mediated via G protein βγ subunits, since injection of these subunits mimic the effect of 5-HT (Blackmer et al., 2001). Furthermore the presynaptic protein SNAP25 is involved in this effect, since injection of Botulimum toxin A, which cleaves SNAP25, abolished the effect of 5-HT (Gerachshenko et al., 2005). Again using FM-dyes it was demonstrated that the inhibitory effect of 5-HT on the lamprey giant synapse is mediated via a change in vesicle fusion properties since 5-HT seemed to prevent full fusion of vesicles (Photowala et al., 2006).\n\nIn aplysia the effect of 5-HT on the gill-withdrawal reflex is well studied. In the 70’s it was shown for the first time that local application of 5-HT sensitized the gill-withdrawal reflex. This effect was also shown after application of cyclic adenosine monophosphate (cAMP), suggesting that this effect of 5-HT is mediated via an increase in cAMP (Brunelli et al., 1976). Both 5-HT and cAMP can close single potassium channels in aplysia sensory neurons, resulting in an increased transmitter release (Siegelbaum et al., 1982). This K⁺ channel, called the S (serotonin)-type K⁺ channel is modulated by 5-HT, cAMP and protein kinase A (PKA) (Shuster et al., 1985). Using calcium imaging on isolated synapses in culture, it was shown that 5-HT application results in a calcium influx in the terminal (Eliot et al., 1993). A brief single pulse of 5-HT resulted in short-term facilitation lasting minutes, whereas five subsequent pulses of 5-HT over 1.5 hours resulted in long-term facilitation lasting more than one day (Montarolo et al., 1986). 5-HT does not only affect release characteristics at single synapses, but upon application also activates silent presynaptic terminals. This results in the generation of new functional release sites within hours after application (Kim et al., 2003). This 5-HT-related growth of new synapses requires Cdc42, N-WASP and PAK (Udo et al., 2005).\n\nIn summary, 5-HT affects synaptic transmission in several ways. In crayfish neuromuscular junctions, 5-HT increases synaptic transmission by increasing the size of the RRP of vesicles. In the lamprey giant synapse 5-HT inhibits transmission by affecting single vesicle release kinetics. Finally, in aplysia sensory neurons, application of 5-HT can induce both short term facilitation and long term facilitation via an increase in transmitter release and the formation of new functional release sites.\n\n5-HT has a role during development in maturation of the brain.\n\nAn increasing amount of evidence points towards a role of 5-HT in brain development and maturation. Additionally, several studies show that 5-HT has an important role during early embryogenesis. 5-HT is present at early stages in sea urchins, chick embryos and rodents (Emanuelsson et al., 1988; Buznikov, 1991). Application of a 5-HT antagonist to sea urchin embryos significantly delayed cell division, implicating a role in the regulation of cell division (Renaud et al., 1983). In sea urchin blastulae, 5-HT, but also dopamine and noradrenaline stimulate ciliary activity. Application of these monoamines was associated with changes in intracellular calcium and adenyl cyclase activity (Soliman, 1983). In the chick embryo, 5-HT is synthesized by the notochord (Wallace, 1982). Monoamine inhibitors cause malformation in the chick embryo such as neural tube defects. Exposure of mouse embryos to 5-HT resulted in craniofacial malformations and increased cell death in craniofacial and cardiac mesenchyme (Yavarone et al., 1993); (Shuey et al., 1993). Several studies in rodents used injections of either p-chlorophenylalanine (PCPA) to inhibit Tph or the 5-HT-specific neurotoxin 5,7-dihydroxytryptamine. Injection of PCPA in mouse embryos from E12-E17 resulted in subtle alterations in pyramidal and non-pyramidal neuronal populations (Vitalis et al., 2007). 5,7-dihydroxytryptamine was injected at the day of birth in mice embryos in the medial forebrain bundle. These injected mice had a decreased 5-HT innervation towards the cortex and hippocampus and male mice showed a decreased novelty-induced exploration, suggesting an anxiety-like phenotype (Hohmann et al., 2007).\n\nKO mice have shown to be a valuable tool in studying the role of the 5-HT system in development. KO mice for tryptophan hydroxylase 1 (Tph1), the rate limiting enzyme involved in periferic 5-HT synthesis, lack periferic 5-HT (Walther et al., 2003). Although these KO mice do not have an overt phenotype, approximately 80% of pups born from homozygous mothers are smaller and show developmental abnormalities compared to embryos born from heterozygous or wildtype mothers (Cote et al., 2007).\n\nThis phenotype was only observed in Tph1 KO embryos born from Tph1 KO mothers, showing that maternal 5-HT is crucial for normal embryonic development. Tryptophan hydroxylase 2 (Tph2) is the Tph isoform that is exclusively expressed in the raphe nuclei (Gutknecht et al., 2009). Deletion of Tph2 does not result in an overt phenotype, nor does deletion of both Tph1 and Tph2, although in another study a reduced viability in these mice was observed (Savelieva et al., 2008; Alenina et al., 2009). Thus it appears that maternal 5-HT is indispensable for proper embryonic (brain) development, but 5-HT synthesis in the embryo is not.\n\n5-HT as a neurotrophic factor\n\nIn addition to the role of 5-HT in (early) neurodevelopment, 5-HT also functions as a neurotrophic factor. Application of 5-HT to buccal ganglion neuron 19 in helisoma caused a cessation in filopodial motility and an inhibition of neurite outgrowth (Haydon et al., 1984). Application of 5-HT results in a rise in intracellular calcium levels in the growth cone which probably links directly to the inhibition in outgrowth (McCobb et al., 1988). Application of 5-HT to 5-HT neurons also resulted in decreased outgrowth (Whitaker-Azmitia and Azmitia, 1986). In contrast to this inhibitory effect of 5-HT on growth of 5-HT neurons, 5-HT acts as a stimulatory signal on thalamic neurons which display more processes per cell and increased length of processes upon 5-HT administration (Lieske et al., 1999).\n\nMouse models with an impaired 5-HT system\n\nSince the introduction of the knockout technique in mice, several genes of the 5-HT system have been assessed for their effect on development and behaviour. The most important genes with striking effects on brain development or behaviour will be mentioned here. Mice lacking the MAO-A gene involved in the degradation of 5-HT to 5-hydroxyindolacetic acid (5-HIAA) display increased levels of 5-HT (Cases et al., 1996). These mice have a lack of barrels in the somatosensory cortex that is restored by application of PCPA, suggesting that this effect is due to excess levels of 5-HT (Cases et al., 1996). Additionally, mice lacking MAO-A display increased aggressive behaviour (Cases et al., 1995).\n\nOn the contrary, deleting the Vesicular Monoamine Transporter 2 (VMAT2) which is necessary for 5-HT packaging in vesicles, results in mice with brain 5-HT levels of ~1% compared to control (Wang et al., 1997; Fon et al., 1997). These KO mice are significantly smaller compared to wildtype littermates and the majority dies within a few days after birth. 5-HT fibers are almost completely absent in the cortex at that age. VMAT2 heterozygotes display prolonged immobility times in the forced swim test, suggesting depressive-like behaviour (Fukui et al., 2007). This behaviour was normalized by the antidepressant imipramine.\n\nDeletion of the SERT which is essential for 5-HT reuptake results in mice with increased extracellular 5-HT levels (Mathews et al., 2004). These mice have a reduced number of 5-HT neurons in the DRN, a reduced firing rate of these neurons and these mice display behavioural alterations (Lira et al., 2003). Notably, these behavioural effects were mimicked by applying the SERT blocker fluoxetine to young control mice between postnatal day 4 and 21 (Ansorge et al., 2004).\n\nIn addition to these genes involved in 5-HT synthesis, packaging, breakdown or reuptake, several 5-HT receptor genes have also been ablated in mice (for an overview of 5-HT receptors see Box III). The 5-HT\\textsubscript{1A} receptor acts as a heteroreceptor on non-5-HT neurons, but also as a 5-HT autoreceptor in the DRN. Mice with a deletion of the 5-HT\\textsubscript{1A} receptor spent less time in the center of the open field in the open field test and avoid the open arms in the elevated plus maze, suggesting that these mice display an increased anxiety-related behaviour (Parks et al., 1998; Ramboz et al., 1998). A very elegant study demonstrated that this effect on behaviour in the 5-HT\\textsubscript{1A} receptor KO occurs during development. In this study a second mouse line was made in which the expression of the 5-HT\\textsubscript{1A} receptor could be restored specifically in the forebrain. Rescue of 5-HT\\textsubscript{1A} receptor expression in the forebrain was sufficient to restore normal anxiety-related behaviour (Gross et al., 2002). In contrast, deletion of the 5-HT\\textsubscript{1A} receptor during development and rescue of the receptor after postnatal day 21 did not restore normal anxiety-related behaviour (Gross et al., 2002). This clearly shows that the 5-HT\\textsubscript{1A} receptor is required during development to establish normal anxiety-related behaviour.\n\n**Box III**\n\n**5-HT receptors**\n\nAt least 14 different 5-HT receptor subtypes are known which are divided into 6 different groups. The first and largest group of 5-HT receptors is the 5-HT\\textsubscript{1} group consisting of the 1A, 1B, 1D, 1E, 1F and 1P receptors. All receptors in this group are coupled to G proteins, and activation results in a decrease in cAMP levels. The most important receptor of this group is the 5-HT\\textsubscript{1A} receptor, which is expressed in the raphe nuclei on 5-HT cell bodies and functions as a 5-HT autoreceptor. Binding of 5-HT to the 5-HT\\textsubscript{1A} receptor results in activation of a GIRK channel which results in hyperpolarization of the 5-HT neuron; in this way, the 5-HT\\textsubscript{1A} receptor is involved in fine-tuning 5-HT release.\n\nTo the 5-HT\\textsubscript{2} receptor group belong the 2A, 2B and 2C receptors. These receptors are also coupled to G proteins, and activation of these receptors results in an increase of inositol 3-phosphate (IP3). The 5-HT\\textsubscript{3} receptor is the only 5-HT receptor which is a (Na\\textsuperscript{+}/K\\textsuperscript{+}) ion channel. The 5-HT\\textsubscript{4} and 5-HT\\textsubscript{6,7} receptors are coupled to Gs proteins and activation results in an increase of cAMP. Finally, activation of the 5-HT\\textsubscript{5A,B} receptor results in cAMP decrease. For the majority of these 5-HT receptors there are agonists and antagonists known. For example, the 5-HT\\textsubscript{1A} receptor is agonized by 8-OH-DPAT whereas it is antagonized by WAY-100635.\n\nMice lacking the 5-HT\\textsubscript{1B} receptor display an increased aggressive-related behaviour: they have a shorter latency to attack in the resident intruder paradigm (Saudou et al., 1994). The 5-HT innervation is increased in the hippocampus and the amygdala of these mice, possibly contributing to the aggressive phenotype of these mice (Ase et al., 2001). Another receptor known to\n\nbe localized in the DRN is the 5-HT\\textsubscript{2C} receptor (Clemett et al., 2000). This receptor is localized on GABA-ergic neurons and activation of this receptor results in a decreased firing of 5-HT neurons (Boothman et al., 2006). Mice that lack the 5-HT\\textsubscript{2C} receptor are overweight and suffer from seizures (Tecott et al., 1995). Moreover, studies have been performed to show that these mice display compulsive-like behaviour (Chou-Green et al., 2003).\n\nAn additional study showed that 5-HT\\textsubscript{2C} KO mice display decreased inhibition in the conflict anxiety paradigm (Weisstaub et al., 2006). This decreased inhibition was rescued by selective restoration of the 5-HT\\textsubscript{2C} receptor in the cortex, suggesting that 5-HT\\textsubscript{2C} receptors in the DRN are not involved in this effect (Weisstaub et al., 2006).\n\nThus, 5-HT receptor KO mice do not exhibit overt developmental defects, but anxiety- or aggressive-related behaviour of these animals is affected as assessed in various behavioural paradigms.\n\n**The 5-HT system in relation to psychopathological processes**\n\n**Anxiety & depression**\n\nThe notion that the 5-HT system could be involved in psychopathological conditions such as major depression and anxiety-related disorders came only after the discovery that drugs used to treat these conditions exerted their function (partially) via the 5-HT system. In the late 50’s, compounds such as iproniazide and G22355 (later named imipramine) were prescribed to patients suffering from tuberculosis. However, serendipitously it was found that these compounds were also effective in treating depression (Bloch et al., 1954; Kuhn, 1958). Only several years after it was found that these drugs had an antidepressant response, the mode of action of these drugs was elucidated; iproniazide inhibited the degradation of mono-aminergic compounds (5-HT, noradrenaline and dopamine), whereas imipramine blocked the reuptake of 5-HT and noradrenaline into the terminal. These findings led to the postulation of the monoamine hypothesis of depression in the late 60’s, which stated that a decreased concentration of 5-HT and noradrenaline in the extracellular space might be responsible for depression. It was poorly understood, however, why there was such a discrepancy between the immediate modes of action of these compounds in contrast to the delayed therapeutic effects. Also, it was not clear why only $\\sim$2/3 of all patients responded to these drugs.\n\nThese considerations abandoned the monoamine hypothesis and the so-called network hypothesis of depression was postulated. This hypothesis states that depression results from an alteration in certain neuronal networks, and antidepressants restore these networks, which explains the time lag between\n\nstarting antidepressant administration and the onset of therapeutic actions. Evidence for this hypothesis, however, is still limited and mostly indirect. Human studies using functional magnetic resonance imaging (fMRI) or positron emission tomography (PET) imaging on patients suffering from major depression have shown volume reductions in cortex and hippocampus. Although farfetched, one could argue that this reflects a decreased network density.\n\nThe last few years a new hypothesis was proposed, largely based on the work by Santarelli and colleagues: the neurogenesis hypothesis. It had already been shown that treatment with antidepressants increased adult neurogenesis in the dentate gyrus (Malberg et al., 2000; Perera et al., 2007). Santarelli and colleagues showed that blocking neurogenesis in the dentate gyrus by X-irradiation blocked the behavioural effects of the antidepressant fluoxetine (Prozac) (Santarelli et al., 2003). In other words neurogenesis is required for the behavioural effects of antidepressants. However, several studies have shown that neurogenesis is not involved in the etiology of depression.\n\nThus, although it is clear that there is a link between the 5-HT system and anxiety and depression, it is still far from clear what this exact link is.\n\n**Aggression**\n\nAggressive behaviour is defined as violent and impulsive behaviour often resulting in breaking the law. 5-HT is one of the neurotransmitters which is implicated in the etiology of aggression. A mutation in the MAO-A gene caused aggression-related abnormal behaviour (impulsive aggression, arson, attempted rape, and exhibitionism) in five males of a large kindred (Brunner et al., 1993). Several studies have shown a reduced concentration of the 5-HT metabolite 5-HIAA in cerebrospinal fluid (CSF) of individuals suffering from aggressive behaviour (Asberg et al., 1976; Coccaro et al., 1997). In rhesus monkeys, there was a negative relation found between high aggression and CSF 5-HIAA levels (Higley et al., 1992). A number of studies implicate the 5-HT$_2$ receptor subtypes in aggressive behaviour. A study by Winstanley and colleagues showed that 5-HT depletion increased the number of premature responses in the 5-choice serial reaction time task in rats (Winstanley et al., 2004). Also, in this study it was found that application of a 5-HT$_{2A}$ antagonist decreased impulsive behaviour, whereas application of a 5-HT$_{2C}$ antagonist had the opposite effect (Winstanley et al., 2004). A clinical study showed that antipsychotic drugs which had anti-aggressive properties were 5-HT$_{2A}$ antagonists (Krakowski et al., 2006). PET imaging studies show an increased 5-HT$_{2A}$ receptor binding in aggressive patients or in borderline personality disorder patients (Siever et al., 2002; Soloff et al., 2007). These abovementioned studies indicate increased 5-HT$_{2A}$ receptor sensitivity in aggressive behaviour patients.\n\nAutism\n\nAutism is a behavioural disorder with unknown etiology, although impaired brain development may underlie this disorder. The link between autism and 5-HT was made after the discovery that autistic patients had elevated platelet 5-HT levels (Boullin et al., 1970; Ritvo et al., 1970). Additionally, depletion of the 5-HT precursor tryptophan resulted in a deterioration in autistic patients (McDougle et al., 1993). Chugani and colleagues used alpha-[11C]methyl-L-tryptophan as a tracer for PET to measure 5-HT synthesis. They showed that in autistic boys there is a decreased 5-HT synthesis in frontal cortex, thalamus, and dentate nucleus of the cerebellum, although there is an increased 5-HT synthesis in the contralateral dentate nucleus (Chugani et al., 1997). Possibly increased levels of 5-HT during early brain development result in the loss of 5-HT terminals which could explain why several drugs which show great efficacy in treating autism enhance 5-HT neurotransmission (Brodkin et al., 1997; Fatemi et al., 1998). More evidence for this hypothesis is the fact that prenatal exposure to cocaine, which is a releaser of 5-HT, is a risk factor for developing autism (Davis et al., 1992). Furthermore, several studies using the 5-HT agonist 5-methoxytryptamine during development in rodents show that this results in autistic-like behaviour (Winslow and Insel, 1990; Kahne et al., 2002).\n\nPolymorphisms in genes involved in the 5-HT system\n\nSERT\n\nIn several genes involved in the 5-HT system, polymorphisms and short nucleotide polymorphisms (SNPs) have been found. An example of a gene in which polymorphisms have been found which correlates with psychopathological processes is SERT. Soon after the cloning of the human SERT, several polymorphisms have been found (Furlong et al., 1998; Battersby et al., 1999). These include two SNPs in the promoter region, a variable number of tandem repeats polymorphism in intron 2, and rare Ile-425-Val and Pro-339-Leu SNPs which affects function and surface expression (Kilic et al., 2003; Prasad et al., 2005). One polymorphism has been found in the promoter region. This polymorphism, a 44 bp insertion has been called the 5-HTT gene-linked polymorphic region, 5-HTTLPR. The two polymorphisms, called the short (s) and long (l) variant have been shown to modulate SERT transcriptional activity (Lesch et al., 1996). In a luciferase assay in human placental choriocarcinoma cells it was shown that the l variant has approximately a threefold higher transcriptional activity compared to the s variant (Heils et al., 1996). Additionally, cells expressing the l/l variant of the SERT displayed a twofold\n\nhigher uptake rate of 5-HT compared to cells expressing the s/s variant. In human postmortem brain material, reduced mRNA concentrations of SERT were found in the raphe nuclei from subjects having the s/s variant of the SERT promoter polymorphism. This shows that both in vitro and in vivo, the s/s variant is associated with a decreased SERT expression and reduced 5-HT reuptake (Little et al., 1998).\n\nIn 1996, the first study was published which demonstrated a link between the SERT promoter polymorphism and anxiety, aggression and depression traits (Lesch et al., 1996). Since then, several studies have demonstrated a link between the s/s promoter variant and aggression, anxiety and depression. Moreover, several studies show that individuals carrying the s/s variant have a poorer response to antidepressant treatment than individuals carrying the l/l or l/s variant (Smeraldi et al., 1998; Zanardi et al., 2000).\n\n**MAO-A**\n\nMAO is involved in the degradation of 5-HT to 5-HIAA, and there are 2 isoforms encoded by different genes, MAO-A and MAO-B. MAO-A has a higher affinity for 5-HT than MAO-B. These genes are both localized on the X chromosome (Lan et al., 1989). In humans a point mutation in the MAO-A gene has been found in the 8th exon, resulting in a truncated MAO-A protein. The family in which the point mutation was identified suffered from abnormal behaviour including disturbed regulation of impulsive aggressiveness (Brunner et al., 1993). In the promoter region of MAO-A a 30 bp repeat polymorphism was found. The low-activity 3-repeat allele was associated with antisocial behaviour in a German population (Samochowiec et al., 1999). In the rhesus macaque, a repeat polymorphism in the promoter affected transcription of the MAO-A gene and was associated with aggressive behaviour (Newman et al., 2005).\n\n**Tph2**\n\nTph1 and Tph2 are the rate limiting enzymes for 5-HT synthesis. Tph1 is expressed in the enterochromaffin cells in the gut and in the pineal gland, whereas Tph2 is the neuronal form and exclusively expressed in the raphe nuclei (Patel et al., 2004; Zill et al., 2004a). In Tph2 a C1473G mutation, resulting in a Pro-447-Arg substitution results in a reduction in 5-HT synthesis. BALB/cJ mice, which are homozygous for the 1473G mutation, have a 50 to 70% reduction in 5-HT synthesis in the frontal cortex resulting in ~40% reduction in 5-HT content in the frontal cortex (Zhang et al., 2004). In the human Tph2 gene, a G1463A SNP resulting in a Arg441His replacement was identified which, when transfected in PC12 cells, resulted in a ~80% loss of function in 5-HT synthesis.\n\n(Zhang et al., 2005). In a cohort of 87 patients suffering from major depression, 9 were found to carry this SNP, while among 219 controls, only 3 subjects had this SNP (Zhang et al., 2005). Knockin mice carrying this human mutation displayed behavioural abnormalities (Beaulieu et al., 2008). In contrast to SNPs found in exons, also SNPs in introns of the Tph2 gene have been found. In intron 5, a SNP has been found which was associated with major depression and suicide (Zill et al., 2004b; Harvey et al., 2004).\n\nAs in the SERT promoter, also in the Tph2 promoter polymorphisms have been found. One of these polymorphisms, rs4570625, has been associated with increased amygdala reactivity to emotional stimuli (Canli et al., 2005). Two polymorphisms, rs4570625 and rs11178997, were linked to anxiety disorder and major depression (Zhou et al., 2005b). As shown by a luciferase assay in 5-HT neurons, the polymorphism rs11178997 significantly reduced Tph2 transcriptional activity by 22% (Scheuch et al., 2007).\n\n**Conclusions**\n\n5-HT is a transmitter that has several modes of action in the central nervous system. First of all, 5-HT has a role during brain development and maturation. Secondly, due to the volume transmission, 5-HT can influence several aspects of behaviour, depending on the receptor(s) to which 5-HT binds. 5-HT can also regulate synaptic transmission. KO mice for the 5-HT\\textsubscript{1A}, 5-HT\\textsubscript{1B} or SERT gene display altered anxiety-related, depression-related and aggression-related behaviour. Finally, polymorphisms in several genes functioning in the 5-HT system are implicated in depression, anxiety, aggression and autism.\n\n**Aim and outline of this thesis**\n\nIn this thesis the outgrowth and connectivity of the brain 5-HT system is studied. To study these aspects we used a number of different approaches. *First* of all, we used organotypic slice cultures to study the outgrowth of the 5-HT system in vitro. *Secondly*, we used neuronal cultures to study trafficking of two components of the 5-HT system, SERT and Tph2. *Thirdly*, we investigated whether a SNP in a presynaptic gene which associates with major depressive disorder, affects trafficking of the SERT. *Fourthly*, we used a genetic approach in mice to study the effect of a silenced 5-HT system on mouse development. *Finally*, we describe our approach to generate a mouse model in which the 5-HT system is labeled with fluorescent reporter genes to study the outgrowth and connectivity in vivo.\n\nIn chapter 2, we used organotypic slice cocultures of the dorsal raphe nucleus and the hippocampus as a target area. We have used this model system to study the outgrowth of the 5-HT system in vitro. Moreover, we investigated whether this system could be used to assess the effect of pharmacological manipulations on the outgrowth of the 5-HT system.\n\nIn chapter 3, we tagged SERT with the red fluorescent protein mCherry, and expressed this protein in hippocampal neurons to investigate the trafficking dynamics of SERT.\n\nIn chapter 4, we tagged Tph2 with EGFP and took the same approach as in chapter 3 to investigate the subcellular distribution and trafficking of Tph2 in neurons.\n\nIn chapter 5, we studied whether knockdown of the presynaptic gene Piccolo affects SERT trafficking and incorporation into the membrane. A SNP in the C2A domain of this protein was found which associated with major depressive disorder. We expressed these two variants and investigated whether there is a difference in SERT localization. Also, we investigated whether knockdown of Piccolo affects SERT trafficking.\n\nIn chapter 6, we used a genetic approach to silence the 5-HT system. 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Verhage$^1$\n\n$^1$Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n$^a$Department of Anatomy and Neurosciences, Vrije Universiteit Medical Center, Amsterdam, the Netherlands\n$^b$Present address: Kavli Institute for Systems Neuroscience and Centre for the Biology of Memory, Norwegian University of Science and Technology NTNU, Trondheim, Norway\n\nPublished in Brain Research (2009) Dec 11;1302:1-9\n\nAbstract\n\nThe serotonin system densely innervates the brain and is implicated in psychopathological processes. Here we studied the effect of serotonin and serotonin pharmacological compounds on the outgrowth of serotonergic projections using organotypic slice co-cultures of hippocampus and dorsal raphe nuclei. Immunohistochemical analysis showed that several serotonergic neurites had grown into the target slice within 7 days in culture, after which the neurite density stabilized. These projections expressed the serotonin-synthesizing enzyme tryptophan hydroxylase and the serotonin transporter and contained several serotonin positive varicosities that also accumulated presynaptic markers. Chronic application of a 5-HT$_2$ agonist reduced the serotonergic neurite density, without effects on survival of serotonergic neurons. In contrast, application of a 5-HT$_{1A}$ agonist or the serotonin transporter inhibitor fluoxetine did not affect serotonergic neurite density. We conclude that serotonergic connectivity was reproduced in vitro and that the serotonin neurite density is inhibited by chronic activation of the 5-HT$_2$ receptor.\n\nIntroduction\n\nThe serotonin (5-hydroxytryptamine (5-HT)) system has its cell bodies clustered in the midbrain raphe nuclei and sends abundant projections to virtually every brain area (Hornung, 2003; Rubenstein, 1998). The 5-HT system influences a wide variety of physiological processes, such as sleep-wake rhythm, feeding, sexual behaviour and nociception (Jacobs and Azmitia, 1992). Moreover, the 5-HT system is implicated in several psychopathological processes, such as anxiety, aggression, obsessive compulsive disorder and depression (Castren, 2005).\n\nDuring brain development, the 5-HT neurons are one of the first groups of cells expressing a specific neurotransmitter (Lauder, 1990; Gaspar et al., 2003). Accumulating evidence shows that alterations in brain 5-HT levels during development could result in alterations in brain development and behavioural alterations. 5-HT reuptake transporter (SERT) knockout (KO) mice have an increase in extra neuronal 5-HT but a reduction in overall brain 5-HT levels (Bengel et al., 1998; Mathews et al., 2004). They exhibit behavioural alterations, reduced number of 5-HT neurons in the dorsal raphe nuclei (DRN) and reduced firing of 5-HT neurons in the DRN (Lira et al., 2003). These behavioural alterations observed in SERT KO mice can be mimicked by application of the\n\nSERT inhibitor fluoxetine during development between postnatal day 4 and 21 (Ansorge et al., 2004).\n\nIn contrast to reduction in brain 5-HT content after disruption of the SERT gene, there is a significant increase in brain 5-HT levels after inactivation of the Monoamine Oxidase-A (MAO-A) gene (Cases et al., 1996). The chronic elevation of 5-HT levels during brain development results in lack of barrels in the barrel cortex (Cases et al., 1996). These data suggest that either reductions in brain 5-HT levels or excess 5-HT levels during a critical period in the development of the brain can lead to abnormalities in brain development and behavioural alterations. In vitro experiments have shown that 5-HT can inhibit the outgrowth of neurites or even cause growth cone collapse on 5-HT neurons (Haydon et al., 1984; Koert et al., 2001). Whether alterations in brain 5-HT levels during development of the brain result in alterations in 5-HT outgrowth, branching or connectivity, or whether the behavioural alterations observed are a result of alterations in the 5-HT outgrowth and connectivity is currently unknown. Organotypic slice cultures of the DRN and a target slice could be a valuable reduced model system to study 5-HT outgrowth in vitro.\n\nTherefore the aim of this study was to investigate whether organotypic slice co-cultures of DRN and hippocampus, a method that has previously been characterized, can be used to study 5-HT outgrowth and the effect of pharmacological manipulations in vitro (Papp et al., 1995; Guthrie et al., 2005). We show that within seven days of culturing several 5-HT neurites have grown into the hippocampal slice. Using these slice co-cultures, we studied whether chronic application of 5-HT pharmacological compounds affects the outgrowth of 5-HT neurites from the DRN. To this end we used a 5-HT\\textsubscript{1A} receptor agonist, a 5-HT\\textsubscript{2} receptor agonist and the SERT inhibitor fluoxetine since it has been shown before, also on non-5-HT cells, that (chronic) activation of a 5-HT\\textsubscript{1A} or 5-HT\\textsubscript{2} receptor or blockade of the SERT can affect (5-HT) outgrowth or synaptogenesis (Wilson et al., 1998; Kondoh et al., 2004; Fricker et al., 2005; Zhou et al., 2006). We show here that chronic application of a 5-HT\\textsubscript{2} agonist results in a reduction in 5-HT neurite density. We conclude that these organotypic slice co-cultures can be used to study 5-HT outgrowth and the effect of 5-HT pharmacological compounds on this outgrowth in vitro.\n\n**Results**\n\n*Development and outgrowth of 5-HT neurons in organotypic slice co-cultures*\n\nTo study the outgrowth of 5-HT neurites ex vivo, we used organotypic slice co-cultures of the DRN and a hippocampal slice. We positioned the slices close to each other to allow 5-HT neurites to grow into the hippocampal slice. Within\n\nseven days of culturing the slices flattened out and several non-neuronal cells migrated away from the slices (Fig. 1).\n\n**Figure 1. Overview of the culturing method**\nThe DRN and hippocampal slice were positioned close to each other to allow 5-HT neurites to grow into the hippocampal slice. After culturing for 7 days, the slices flatten and several cells (e.g. macrophages) migrate away from the slices. Scale bar: 500 µm.\n\nWe performed immunohistochemistry for 5-HT to investigate whether 5-HT neurons survived in the slices. This showed that in several DRN slices 5-HT neurons were present, which resembled the topology of 5-HT neurons in the DRN in vivo (Fig. 2A). However, there were also DRN slices which contained only few 5-HT neurons, possibly resulting from a variation in the dissection of the DRN slice. A Z-stack of 5-HT cell somata showed that these cells have several primary neurites which contain numerous varicosities (Fig. 2B). In the growth cones of 5-HT neurites, 5-HT was also detected (Fig. 2C). Next, we focussed on the expression of tryptophan hydroxylase (Tph) and SERT, two markers for the 5-HT system. In 5-HT immunopositive cell bodies and in the 5-HT neurites and varicosities Tph was expressed (Fig. 2D,E). We found that all the 5-HT neurites were positive for SERT labelling, with the highest level of SERT staining on the soma and in the 5-HT varicosities (Fig. 2F,G). Moreover, SERT was also expressed in a punctate pattern in the 5-HT growth cones. (Fig. 2H). In vivo, 5-HT is released from varicosities in 5-HT axons and dendrites and 5-HT release is predominantly paracrine (Bunin and Wightman, 1999). In the slices, the 5-HT neurites contain several round and fusiform shaped varicosities (Fig. 3A,B).\n\nFigure 2. 5-HT neurons survive in the slice and grow out several 5-HT neurites\n\n(A) A DRN slice cultured for 7 days which is stained for 5-HT shows that several 5-HT cells are present in the slice. Note the presence of the lateral wings of the DRN (arrows) and the group of 5-HT neurons in the ventral part of the DRN (arrowhead). (B) A Z stack of two cells shows that the cells project several neurites that are 5-HT positive. (C) The growth cones also contain 5-HT, both in the core (arrowhead) and in the filopodia (arrows). (D,E) Staining for the rate limiting enzyme Tph shows that Tph is present both in the cell bodies and in the varicosities in the neurites. (F,G) The other marker for the 5-HT system, SERT, is present in 5-HT cell bodies and 5-HT neurites. (H) Moreover, there is also a punctate SERT staining in 5-HT growth cones, both in the core (arrowhead) and in the filopodia (arrows). Scale bars: 200 µm in A, 50 µm in B and D, 20 µm in F and G, 5 µm in C and E, 2 µm in H.\n\nImmunohistochemistry revealed that in these varicosities the calcium sensor Synaptotagmin and the active zone marker Bassoon are present, suggesting that the varicosities are presynaptic sites where calcium dependent 5-HT release occurs (Fig. 3C,D) (Perin et al., 1991; tom Dieck et al., 1998). This showed that in organotypic slice co-cultures of DRN and hippocampus 5-HT neurons survive and grow out neurites that contain 5-HT varicosities with immunoreactivity for all tested components of the 5-HT release machinery.\n\nDense ingrowth of 5-HT neurites into hippocampal slices\n\nTo study the outgrowth, we fixed slice cultures at different time points and quantified the 5-HT outgrowth. To this end the images were converted to binary images and the 5-HT neurite density in the slices was quantified (Fig. 4A-C).\n\nFigure 3. 5-HT neurites contain several varicosities which are positive for presynaptic markers\n\n(A) A Z-stack in the hippocampal slice shows that several thin, highly branching 5-HT neurites have grown into the slice. (B) A blow-up of the red box in A shows that these neurites contain several varicosities. (C,D) These varicosities are positive for the protein involved in calcium dependent secretion, Synaptotagmin (Syt), and for the active zone marker Bassoon. Scale bars: 50 µm in A, 10 µm in B and D and 2 µm in C.\n\nWe quantified the 5-HT neurite density as the area in the slice occupied by 5-HT neurites compared to the total area of the slice. Immunohistochemistry for 5-HT on hippocampal slices cultured without the DRN for seven days revealed that there were no 5-HT neurites present (Fig. 4E). Thus, 5-HT neurites observed in the hippocampal slice in co-cultures are the result of re-growth of 5-HT neurites from the DRN slice. In co-cultures fixed at four days in vitro (DIV) we observed that the DRN slice already contained several 5-HT neurites, and the first 5-HT neurites started to grow into the hippocampal slice (Fig. 4F,I). At DIV7, also the\n\nhippocampal slice contained several 5-HT neurites (Fig. 4G,J). Quantification of the 5-HT neurite density in the slices at DIV14 and DIV21 revealed that this was not significantly different from DIV7 (Fig. 4D,H; DIV7 DRN 8.42 ± 0.8%, HIP 6.31 ± 0.84% n=16; DIV14 DRN 9.49 ± 1.42%, HIP 5.12 ± 0.9% n=12; DIV21 DRN 9.26 ± 1.16%, HIP 6.61 ± 1.43% n=4). We investigated whether there is regional variation in the 5-HT neurite distribution in the hippocampus. At DIV4 the first 5-HT neurites which start growing into the hippocampal slice do not appear to display a preference for a certain region. (Fig. 4K). At DIV7, 5-HT neurites have grown in a uniform distribution into the hippocampus, and there are no regions which contain a higher density of 5-HT neurites than other regions (Fig. 4L). Thus, the initial outgrowth of the 5-HT neurites occurs in the first 7 days, after which the network stabilizes and the homogenous distribution suggests that 5-HT neurites do not have any preferential target region within the hippocampal slice in vitro.\n\n**The effect of 5-HT pharmacological compounds on the outgrowth of 5-HT projections**\n\nFinally, we studied whether this culture model system can be used to study the effect of chronic application of 5-HT pharmacological compounds on the outgrowth of 5-HT neurites. There are several 5-HT receptors which are expressed in both the DRN and the hippocampus.\n\n**Figure 4 next page. Dense ingrowth of 5-HT neurites into hippocampal slices within seven days of culturing**\n\nAn original image of a hippocampal slice co-cultured with a DRN slice shows that there is abundant ingrowth of 5-HT neurites, but there is also a lot of red background staining. (B) When this image is converted to a binary image using the steps described in experimental procedures, the 5-HT neurites are positively labelled (black), whereas the background is removed (white). (C) A blow up of part of the binary image shows that all 5-HT neurites are detected, and the red background is removed. (D) Quantification of the 5-HT neurite density (Y-axis) in the DRN and hippocampal slices at DIV7, DIV14 and DIV21. (E) A hippocampal slice cultured without the DRN contains only background staining, and no 5-HT neurites. (F,I) Maximal 5-HT ingrowth of the slices is reached in the first seven days of culturing. At DIV4, the DRN slice already contains several 5-HT neurites, and the first 5-HT neurites start to grow into the hippocampal slice. (G,J) At DIV7, both the DRN and hippocampal slice contain abundant 5-HT neurites. (H) At DIV21 the 5-HT neurite density in the slices has not changed. (K,L) No regional variation in 5-HT neurite density in the hippocampal slice at DIV4 (K) and DIV7 (L). Figures I and J are a blow up of the black box in F and G respectively. Figures K and L are blow ups of the hippocampal slices in figure F and G respectively. Scale bars: 500 µm in A, B, E, F, G, H, I, J, K and L, 20 µm in C.\n\nThese include the 5-HT\\textsubscript{1A} receptor, the 5-HT\\textsubscript{2} receptor and the 5-HT\\textsubscript{7} receptor (Gustafson et al., 1996; Clemett et al., 2000; Xu and Pandey, 2000; Bickmeyer et al., 2002; Garcia-Alcocer et al., 2006). Since it has been shown before, also on non-5-HT cells, that (chronic) activation of a 5-HT\\textsubscript{1A} or 5-HT\\textsubscript{2} receptor or blockade of the SERT can affect (5-HT-ergic) outgrowth or synaptogenesis, we decided to use a 5-HT\\textsubscript{1A} receptor agonist 8-hydroxy-2-(di-n-propylamino)tetraline (8-OH-DPAT), a 5-HT\\textsubscript{2} receptor agonist (+)-2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI hydrochloride) and the SERT inhibitor fluoxetine (Wilson et al., 1998; Kondoh et al., 2004; Fricker et al.,\n\nWe chronically applied the pharmacological compounds in 10 µM concentrations during the first seven days of outgrowth. First of all, we studied the effect of chronic blockade of the SERT by fluoxetine. Fluoxetine did not affect 5-HT neurite density (Fluoxetine: DRN: 8.44 ± 0.72%, HIP: 6.12 ± 1.12% n=10) compared to control condition (Fig. 5A,B,E,F). Subsequently, we tested the effect of chronically activating the 5-HT\\textsubscript{1A} and 5-HT\\textsubscript{2} receptor, two receptors which are present on 5-HT neurons. Moreover, it has been shown previously that activation of these receptors can affect outgrowth of Purkinje cells (Kondoh et al., 2004). First we investigated the effect of the 5-HT\\textsubscript{1A} receptor agonist 8-OH-DPAT. Activation of the autoreceptor by 5-HT or an agonist results in a reduction in 5-HT release (Quick, 2003). However, 8-OH-DPAT did not affect 5-HT neurite density (DRN 10.67 ± 0.79%, HIP 7.21 ± 0.9% n=9) (Fig. 5 C,E,F).\n\n**Figure 5. Chronic application of a 5-HT\\textsubscript{2} agonist reduces 5-HT neurite density in the slices**\n\n(A) In control condition, after seven days of culturing several 5-HT neurites have grown into the slices. (B) Chronic blockade of the SERT with the SERT inhibitor fluoxetine did not affect 5-HT neurite density in the slices. (C) Chronic activation of the 5-HT\\textsubscript{1A} receptor with 8-OH-DPAT also did not affect 5-HT neurite density in the slices. (D) However, chronic activation of the 5-HT\\textsubscript{2} receptor with DOI significantly reduced 5-HT neurite density both in the DRN and hippocampal slice. (E,F) Quantification of 5-HT neurite density in the DRN slice and hippocampal slice, respectively. Scale bar: 500 µm. * p< 0.05, ** p<0.01, *** p<0.001.\n\nNext, we investigated the effect of chronic 5-HT\\textsubscript{2} receptor activation. Activation of the 5-HT\\textsubscript{2} receptor results in an increased protein kinase C activity and release of calcium from internal stores. We chronically activated the 5-HT\\textsubscript{2} receptor by applying the 5-HT\\textsubscript{2} receptor agonist DOI hydrochloride. Chronic application of the 5-HT\\textsubscript{2} receptor agonist DOI hydrochloride resulted in a significantly decreased 5-HT neurite density (Fig. 5 D,E,F) (DRN 2.94 ± 1.01%, HIP 2.29 ± 1.05% n=5). To exclude the possibility that pharmacological treatment affected the survival of 5-HT neurons, we counted the number of 5-HT cells per group. This showed that treatment with fluoxetine, 8-OH-DPAT or DOI hydrochloride did not affect the number of 5-HT neurons in the slices (data not shown). Hence, changes in viability of 5-HT neurons cannot explain the effect of DOI hydrochloride. We conclude that these organotypic slice co-cultures can be used to study the 5-HT outgrowth and ingrowth into a target area in vitro and that chronic activation of the 5-HT\\textsubscript{2} receptor results in a decreased 5-HT neurite density.\n\n**Discussion**\n\n*An organotypic co-culture model system to study 5-HT outgrowth ex vivo*\n\nWe used organotypic slice co-cultures to study the outgrowth of the 5-HT system ex vivo. Neurite projections of 5-HT neurons grew into the slices and at 7 days a dense network had formed with many serotonergic boutons that had accumulated presynaptic markers. The regional differences in 5-HT innervation observed in the hippocampus in vivo were not reproduced ex vivo and it has been observed that fewer synapses form in slices than in vivo (see discussion in Papp et al., 1995). However, also in vivo the majority of the 5-HT release sites (70-80%, (Oleskevich and Descarries, 1990), are not classical synapses, i.e. with a juxtaposed postsynaptic specialization. Hence, the in vivo connectivity of the 5-HT system appears to depend largely on non-synaptic release. Therefore, despite some discrepancies compared to the in vivo situation, the slice co-culture system is an excellent, reduced model system to unravel the basic principles that orchestrate 5-HT neurite outgrowth and connectivity. Such a reduced system is a valuable supplementation of in vivo studies, which are often complicated by the exceptionally complex and dynamic connectivity of the 5-HT system, with for instance large fluctuations in innervation over the course of a few days (transient hyper-innervation, see (Fujimiya et al., 1986; D’Amato et al., 1987).\n\n*Chronic activation of the 5-HT\\textsubscript{2} receptor decreased 5-HT neurite density*\n\nWe found that chronic application of a 5-HT\\textsubscript{2} agonist decreased the neurite density of 5-HT neurites. Since DOI hydrochloride treatment did not affect 5-HT neuron survival, 5-HT mediated cytotoxicity cannot explain the decrease in 5-HT\n\nneurite density. Hence, the decreased neurite density appears to be the result of a specific effect of the activation of the 5-HT$_2$ receptor on the neurites (decreased outgrowth/increased pruning). In contrast, chronic activation of the 5-HT$_{1A}$ receptor did not mimic the effects of 5-HT, although the 5-HT$_{1A}$ receptor has often been implicated in shaping 5-HT connectivity (Gross et al., 2000; Gross et al., 2002). Activation of somatodendritic 5-HT$_{1A}$ receptors results in hyperpolarisation and a decrease in 5-HT release (Quick, 2003). However, chronic activation results in rapid desensitization (Assie et al., 2006) which may explain why we do not observe an effect of chronic 5-HT$_{1A}$ receptor activation. On the other hand, 5-HT$_2$ receptor activation increases intracellular calcium and activates protein kinase C (Tamir et al., 1992). This suggests that (chronic) activation of the 5-HT$_2$ receptor affects outgrowth, since elevation of calcium levels in growth cones reduces outgrowth (Gomez and Spitzer, 1999). Since DOI hydrochloride has a comparable affinity for the 5-HT$_{2A}$ and 5-HT$_{2C}$ receptor subtype, based on these data we cannot conclude which of the two subtypes is involved in the effect of DOI hydrochloride.\n\nHowever, although the 5-HT$_{2A}$ receptor is widely expressed throughout the brain, the receptor seems not to be expressed in the DRN (Xu and Pandey, 2000). In contrast, the 5-HT$_{2C}$ receptor is present in the DRN (Clemett et al., 2000; Serrats et al., 2005). Moreover, it has been shown previously, that activation of the 5-HT$_{2C}$ receptor in the DRN results in a reduction in 5-HT neuron firing (Boothman et al., 2006). Therefore, based on the anatomical distribution we speculate that the effect of DOI hydrochloride is predominantly mediated via the 5-HT$_{2C}$ receptor. Fluoxetine did not affect 5-HT neurite density. It could be expected that fluoxetine application mimics a 5-HT$_{1A}$ receptor agonist, i.e. SERT blockade results in an increase in extracellular 5-HT which could activate 5-HT$_{1A}$ autoreceptors. However, possibly upon application of fluoxetine extracellular 5-HT is rapidly diluted in the culture medium in vitro, and therefore extracellular 5-HT will not reach high enough concentration levels to mimic the effect of a 5-HT$_{1A}$ receptor agonist. Also chronic fluoxetine treatment in vivo failed to produce effects similar to chronic 5-HT application (Zhou et al., 2006). In fact, chronic fluoxetine enhanced neurite density in this case (Zhou et al., 2006).\n\n**Conclusions**\n\nWe have shown here that in DRN – hippocampus organotypic slice co-cultures 5-HT projections grow into the target slice. After four days of culturing in the DRN slice already several 5-HT projections were present, and the first 5-HT projections started to grow into the target slice. Within seven days of culturing also the target slice contained several 5-HT neurites. These 5-HT neurites,\n\ncontained several varicosities, which are presumably sites of 5-HT release, since presynaptic markers were present in these varicosities. Finally, we have shown that this culture model system can be used to study the effect of pharmacological manipulations on the outgrowth of 5-HT neurites, and that application of a 5-HT$_2$ agonist results in a reduction in 5-HT neurite density in the slices.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Laboratory animals*\n\nWildtype C57BL/6 mouse fetuses were obtained from caesarean section at embryonic day 18 or postnatal day 1. Animals were housed and bred according to institutional, Dutch and U.S. governmental guidelines.\n\n*Pharmacological compounds and antibodies*\n\nMouse monoclonal anti-Tph antibody, which detects both Tph1 and Tph2, was obtained from Sigma-Aldrich and used in a 1:1000 dilution. Rabbit anti 5-HT polyclonal and mouse anti-SERT monoclonal were obtained from Immunostar/Diasorin and used in a 1:1000 dilution. Bassoon and Synaptotagmin monoclonal antibodies were used in a 1:500 and 1:1000 dilutions respectively and obtained from Stressgen. As secondary antibodies Alexa593-conjugated goat anti rabbit and Alexa488-conjugated goat anti mouse were used in a 1:1000 dilution (Molecular probes). 8-OH-DPAT and DOI hydrochloride were obtained from Sigma-Aldrich. Fluoxetine was obtained from Tocris.\n\n*Organotypic slice co-cultures*\n\nOrganotypic slice cultures were made as follows. After decapitation of the fetuses, the heads were immediately transferred to ice-cold dissection Gey’s balanced salt solution (dGBSS, Invitrogen, supplemented with 0.65 g/l glucose and 200 µM kynurenic acid). For isolation of a DRN slice, the brains were dissected in dGBSS and the midbrain cut into 400 µm thick slices using a McIlwain tissue chopper (Mickle Engineering, Gomshall, UK). Individual slices were separated in dGBSS and the hindbrain slice containing the rostral DRN was identified by visual inspection. The DRN was dissected out using a dissection knife. The entire hippocampus was dissected out of the brain and individual slices (400 µm) were sagitally cut perpendicular to the hippocampus’ longitudinal axis. The hippocampal and DRN slices were allowed to recuperate in dGBSS at 4°C for 60 minutes. A hippocampal and DRN slice were cultured in close proximity on a poly-D-lysine coated 12x24 glass coverslip (O. Kindler GmbH & Co. Mikroskopische Gläser, Freiburg, Germany) in a plasma clot (chicken plasma, Cocalico Biologicals Inc. Reamstown, U.S.) which was coagulated with thrombin (Merck, Darmstadt, Germany). Culturing medium\n\nconsisted of 50% BME Hanks, 25% Hanks balanced salt solution and 25% horse serum supplemented with 1.3 g/L glucose and 200 µM glutamine. For culturing 700 µL of culturing medium was added to the slices in a Nunc flat-bottomed tube. The slices were incubated in a roller drum at 36°C. After the first week, medium was changed twice a week.\n\n**Pharmacological treatments**\nAll pharmacological compounds were dissolved in ddH$_2$O, filter sterilized and aliquots stored at -80°C. After DIV1 10 µM of the pharmacological compound was added to the slices. Every day a new aliquot of pharmacological compound was added to the slices. In control slices only vehicle was added (sterile ddH$_2$O). At DIV7 the slices were fixed, processed and analyzed as described below.\n\n**Immunohistochemistry**\nSlices were fixed for 20 minutes in 4% paraformaldehyde dissolved in phosphate buffered saline. After fixation the slices were washed three times five minutes in phosphate buffered saline (PBS) and nonspecific binding was blocked by incubating the slices in PBS containing 0.1% Triton X-100 and 2% normal goat serum for two hours. The slices were incubated overnight with the primary antibodies diluted in PBS containing 0.1% Triton X-100 supplemented with 2% normal goat serum at 4°C. After incubation in the primary antibodies, the slices were washed three times two hours in PBS on a shaking platform. The secondary antibodies were diluted in PBS supplemented with 2% normal goat serum and incubated for one hour. After incubation in the secondary antibodies, the slices were washed again three times two hours in PBS and mounted in Dabco-Mowiol (Sigma) on glass coverslips. All reactions were carried out at room temperature unless otherwise stated. No labeling was observed when omitting the primary antibodies.\n\n**Confocal analysis**\nConfocal analysis of the slices was performed on a LSM 510 microscope (Carl Zeiss b.v. Weesp, the Netherlands) and a 63x Plan-Neofluar lens (Numerical aperture 1.4, Carl Zeiss). To excite the Alexa 488 antibody a HeNe1 laser was used and for excitation of the Alexa 593 antibody a HeNe2 laser was used. For analysis of the 5-HT neuron morphology and the 5-HT neurites, Z-stacks of 1 µm were made. Images were analyzed and further processed in Zeiss CLSM software.\n\n**Histological quantification of 5-HT neurite density**\nQuantification of 5-HT immunopositive neurites and cells was performed using an MCID Elite imaging system (Imaging Research Inc., Ont., Canada). Images of the immunostained co-cultures were digitized using an objective magnification\n\nof 20x on a Leica DM/RBE photo-microscope with a Sony (DXC-950P, 640 x 512 pixels) camera using epifluorescence microscopy. The 5-HT-immunopositive neurites were segregated from background using several point operators and spatial filters combined in an algorithm designed to detect local changes in relative optical density. Briefly, images underwent histogram equalization and smoothing (low-pass filter, kernel size 7x7). The unfiltered image was subtracted from the smoothed image, followed by a series of steps to optimize the processed image and make it a suitable measuring template for detecting objects the size and shape of 5-HT neurites or cell bodies. This algorithm was preferred over relative optical density thresholding since it does not involve an observer-dependent operation. Finally, a line was manually drawn around the DRN or hippocampal slice and the number of positive pixels (i.e. pixels representing 5-HT neurites) compared to the total number of pixels was calculated. The number of 5-HT neurons was counted manually in the slices.\n\nData analysis\nIn order to analyze differences in neurite density between different pharmacological treatments, ANOVA was used with the Bonferonni test for post hoc analysis. Data shown are mean values ± standard error of the mean (SEM). 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Synapse 12:155-168.\n\ntom Dieck S, Sammarti-Vila L, Langnaese K, Richter K, Kindler S, Soyke A, Wex H, Smalla KH, Kampf U, Franzer JT, Stumm M, Garner CC, Gundelfinger ED (1998) Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J Cell Biol 142:499-509.\n\nWilson CC, Faber KM, Haring JH (1998) Serotonin regulates synaptic connections in the dentate molecular layer of adult rats via 5-HT1a receptors: evidence for a glial mechanism. Brain Res 782:235-239.\n\nXu T, Pandey SC (2000) Cellular localization of serotonin(2A) (5HT(2A)) receptors in the rat brain. Brain Res Bull 51:499-505.\n\nZhou L, Huang KX, Kecojevic A, Welsh AM, Koliatsos VE (2006) Evidence that serotonin reuptake modulators increase the density of serotonin innervation in the forebrain. *J Neurochem* 96:396-406.\n\nChapter 3\n\nDynamic vesicular trafficking of the serotonin reuptake transporter in hippocampal neurons\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1} and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\n\\textit{In preparation}\n\nAbstract\n\nThe serotonin (5-HT) reuptake transporter (SERT) has an important role in 5-HT transmission by terminating the action of 5-HT in the synaptic cleft. Here we address the question how SERT is transported in neuronal cells. We studied the trafficking of SERT in hippocampal neurons by tagging SERT with a fluorescent protein. We show that SERT displays a punctate distribution in axons and a more uniform distribution in dendrites. The majority of SERT puncta do not co-localize with the synaptic marker Synapsin, showing that SERT is predominantly localized extra-synaptically. SERT is transported in secretory vesicles as SERT co-localizes with the secretory vesicle marker tissue plasminogen activator (tPA). Live cell imaging revealed a highly dynamic trafficking of SERT along the neurites. Approximately 35% of the vesicles moved, with an average velocity of $\\sim$1.2 µm/s. SERT vesicles do not display a preference for direction. We conclude that SERT, tagged with a fluorescent protein, displays a highly dynamic character in hippocampal neurons. These data are important to achieve a better understanding of the dynamics of treatment with 5-HT reuptake inhibitors.\n\nIntroduction\n\nThe projections of the 5-HT system abundantly innervate several brain areas. 5-HT is released both synaptically and extra-synaptically. After being released, 5-HT is cleared from the extracellular space by the SERT. Since this protein regulates the termination of the action of 5-HT, it is very important in regulating efficacy of 5-HT transmission. Moreover, SERT is the primary target for the selective serotonin reuptake inhibitors (SSRIs) class of antidepressants, several tricyclic antidepressants, and may mediate the effects of amphetamines such as 3,4-methylenedioxy-N-methylamphetamine (MDMA).\n\nSERT is a sodium and chloride neurotransmitter symporter and belongs to the solute carrier family 6, to which also transporters for dopamine, noradrenaline/adrenaline, glycine and GABA belong (Chen et al., 2004). In the adult brain, SERT is expressed in 5-HT neurons, but during development also transiently in the cortex, hippocampus, thalamocortex and the lateral geniculate nucleus (Lebrand et al., 1996; Zhuang et al., 2005). In 5-HT neurons, SERT is predominantly localized in axons but also in the cytoplasm of the soma and dendrites (Zhou et al., 1998; Tao-Cheng and Zhou, 1999). In axons SERT is not only associated with varicosities, sites where 5-HT is released, but also with the plasma membrane of the axons (Zhou et al., 1998).\n\nSeveral lines of evidence indicate that 5-HT has a role during brain development. In mice, deletion of SERT results in a 60-80% reduction in total\n\nbrain tissue 5-HT level, although there is a 7-8 fold increase in extracellular 5-HT levels in the striatum and substantia nigra (Fabre et al., 2000; Mathews et al., 2004). Analysis of emotional behaviour of these mice revealed that deletion of SERT results in abnormalities in anxiety-related behaviour (Holmes et al., 2003a; Holmes et al., 2003b). Additionally, blockade of SERT during development with the SSRIs fluoxetine, clomipramine or citalopram results in mice which display altered emotional behaviour in adulthood (Ansorge et al., 2004; Ansorge et al., 2008). These studies show that SERT is important in establishing normal emotional behaviour during development, presumably by regulating the extracellular concentration of 5-HT in the brain.\n\nThe level of SERT present at the cell membrane at synaptic boutons determines the rate of 5-HT reuptake and hence the efficacy of 5-HT transmission. Therefore, there is extensive regulation of the level of cell-surface expressed SERT. The trafficking and regulation of SERT cell-surface expression has been studied in many different cell types, including HEK293 cells, iC11 cells, basophilic leukemia 2H3 cells and 5-HT neurons (Zhu et al., 2004; Lau et al., 2008a; Lau et al., 2008b). A study using biotinylated SERT in 5-HT neurons showed that citalopram reduced the amount of cell surface-expressed SERT (Lau et al., 2008b) However, live cell imaging to investigate the trafficking dynamics of SERT has not been studied in neurons before.\n\nHere we investigated the trafficking of SERT in hippocampal neurons. To this end we expressed SERT tagged with the fluorescent protein mCherry in hippocampal neurons. SERT displays a polarized distribution, as in axons SERT is distributed in a punctate manner, whereas in dendrites SERT is present in a uniform distribution. SERT is transported in secretory vesicles through the neurites, as it co-localized with the secretory vesicle marker tPA. Live cell imaging revealed that SERT displays a highly dynamic vesicular trafficking. Analysis of direction of movement showed that SERT does not display a preference for a direction of movement. In future experiments, live cell imaging of mCherry-SERT can be used to address whether 5-HT reuptake inhibitors affect the highly dynamic SERT trafficking.\n\n**Results**\n\n*Localization of mCherry-SERT in hippocampal neurons differs between axons and dendrites*\n\nTo study the trafficking we tagged the N-terminus of SERT with the red fluorescent protein mCherry, which is an improved variant of monomeric red fluorescent protein in terms of maturation and photostability (Shaner et al., 2004). Tagging the N-terminus of SERT with a fluorescent protein does not interfere with the function of SERT (Schmid et al., 2001; Just et al., 2004;\n\nWe used hippocampal neurons that do not express SERT, although it is expressed transiently in a subpopulation of hippocampal neurons during development (Narboux-Neme et al., 2008).\n\nThe expression of mCherry-SERT in dissociated hippocampal neurons was analyzed between 9-12 days in vitro (DIV) using live cell imaging and immunocytochemistry on fixed neurons. This revealed that the whole neuron was extensively labelled with mCherry-SERT (Fig. 1A). Immunocytochemistry for the dendritic marker Microtubule Associated Protein 2 (MAP2) was used to discern between the dendrite and axon (Fig. 1B,C). SERT localization differed between axons and dendrites. In dendrites SERT is predominantly uniformly distributed where only occasionally a puncta of SERT is observed. However in axons, SERT displays a more punctate distribution suggesting that SERT is transported in vesicles through the axon (Fig. 1D,E,F). Alternatively, the punctate pattern may reflect local accumulation of SERT in varicosities. To further explore whether these puncta are varicosities or vesicles, we used immunocytochemistry and live cell imaging.\n\n**Figure 1. Distribution of mCherry-SERT differs between axons and dendrites**\n\n(A-C) Expression of mCherry-SERT in a hippocampal neuron (DIV11). (A) mCherry-SERT is present in all the neurites and the cell body. (B) MAP2 labelling to discern between axons and dendrites. (C) Merged image. (D-F) Blow up of the white box in C. (D) mCherry-SERT distribution differs between axons and dendrites, as in the axon mCherry-SERT displays a punctate distribution, whereas in dendrites mCherry-SERT displays a more uniform distribution where only occasionally a mCherry-SERT puncta is observed. (E) MAP2 labelling. (F) Merged image. Scale bars in C and F are 10 µm.\n\nSERT is transported in secretory vesicles\n\nSince SERT displayed a punctate distribution in neurites, we investigated whether these puncta are secretory vesicles. To this end mCherry-SERT was co-expressed with tPA-GFP, which is transported in secretory vesicles in hippocampal neurons (Lochner et al., 1998; Silverman et al., 2005). In neurons expressing both mCherry-SERT and tPA-GFP, the majority of SERT puncta co-localized with tPA-GFP (Fig. 2A-F). Therefore, these data strongly suggest that SERT is transported in secretory vesicles along the axons in hippocampal neurons.\n\nFigure 2. mCherry-SERT is transported in secretory vesicles\n\n(A-C) Co-expression of mCherry-SERT and tPA-GFP in a hippocampal neuron (DIV11). (A) mCherry-SERT displays a punctate distribution. (B) tPA-GFP is transported in secretory vesicles. (C) The majority of mCherry-SERT puncta co-localize with tPA-GFP, suggesting that mCherry-SERT is transported in secretory vesicles. (D-F) Blow-up of the white box in C. (D) mCherry-SERT expression. (E) tPA-GFP expression. (F) Merged image. Arrows show the co-localization between mCherry-SERT and tPA-GFP. Scale bars in C and F are 10 μm.\n\nTo investigate whether vesicles of SERT co-localized with presynaptic terminals, we performed immunocytochemistry for the synaptic marker Synapsin on neurons transfected with mCherry-SERT. Although several mCherry-SERT puncta are in close vicinity of Synapsin puncta (Fig. 3 A-F), only few mCherry-SERT puncta co-localized with Synapsin. Thus, mCherry-SERT is transported in secretory vesicles and several of those vesicles localize to the vicinity of presynaptic terminals.\n\nFigure 3. Several mCherry-SERT vesicles are localized in the vicinity of presynaptic terminals\n\n(A-C) Expression of mCherry-SERT in a hippocampal neuron (DIV11). (A) mCherry-SERT expression, (B) Synapsin labelling, (C) Merged image. Several mCherry-SERT vesicles are localized in the vicinity of presynaptic terminals, and some co-localized with the presynaptic marker Synapsin. (D-F) Blow up of the white box in C. (D) mCherry-SERT expression, (E) Synapsin labelling, (F) Merged image. Arrows in F show mCherry-SERT vesicles which are localized in the vicinity of presynaptic terminals. Arrowhead shows a mCherry-SERT vesicle which co-localizes with a Synapsin puncta. Scale bars in C and F are 10 µm.\n\nFigure 4 next page. Trafficking dynamics of mCherry-SERT secretory vesicles\n\nLive cell imaging of mCherry-SERT trafficking dynamics in hippocampal neurons (DIV 9-11). (A) Two frames 5 seconds apart (green is 1 s, red is 5 s) from a 1 minute movie reveal the different modes of trafficking of mCherry-SERT. There are mCherry-SERT vesicles which move in the anterograde direction (arrow), in the retrograde direction (arrowhead) or are stationary (yellow puncta, asterisks). (B) Corresponding kymograph of part of the neurite displays the different trafficking modes of mCherry-SERT. Vesicle 1 is a stationary vesicle, vesicle 2 moves in the anterograde direction, whereas vesicle 4 moves in the retrograde direction. Vesicle 3 is a vesicle which pauses for a while and then continues moving. Vesicle numbers are shown in the individual images taken 10 seconds apart and vesicle trajectories are highlighted in the kymograph. (C) No difference between average anterograde and retrograde velocity as can be observed in the histogram of the average velocity. (D) No difference between average velocity of mCherry-SERT vesicles which move in the anterograde or retrograde direction or which display bidirectional movements. (E) Percentage moving vesicles does not differ between vesicles which move anterogradely, retrogradely or bidirectionally. (F) Also, percentage pausing time for moving vesicles is not different between vesicles which move anterogradely, retrogradely or bidirectionally. Scale bars in A and B are 10 µm.\n\nSERT trafficking is highly dynamic\n\nWe analyzed the trafficking dynamics of SERT using live-cell imaging of hippocampal neurons expressing mCherry-SERT. We observed rapid movements of mCherry-SERT vesicles in both the anterograde and retrograde direction. Additionally, also stationary mCherry-SERT puncta were observed (Fig. 4A). To illustrate different types of movement we show part of a neurite with 10 second time intervals. In this neurite a stationary vesicle can be observed (vesicle 1), but also a vesicle which moves in an anterograde direction (vesicle 2) and a vesicle which moves in a retrograde direction (vesicle 4). Additionally, vesicles are...\n\nobserved which are temporarily stationary but then continue moving (vesicle 3 in Fig. 4B). In the corresponding kymograph of this part of the neurite the trajectories of the numbered vesicles are shown (Fig. 4B). We quantified the direction of vesicle movement, percentage of moving vesicles, average velocity of moving vesicles and percentage pausing time of moving vesicles using the automated vesicle detection program Fluotrack (Broeke et al., 2008). First we measured the average velocity of anterogradely and retrogradely moving vesicles. The frequency histogram plotting the velocities of mCherry-SERT vesicles moving in the anterograde or retrograde direction showed that the velocity distributions for anterogradely and retrogradely moving vesicles did not differ (Fig. 4C). The average velocity of all moving vesicles was $1.22 \\pm 0.03$ µm/s. The average velocity of anterograde vesicles was $1.31 \\pm 0.05$ µm/s compared to $1.34 \\pm 0.06$ µm/s in retrograde direction. Vesicles moving bidirectional (vesicles that changed direction during imaging) moved with an average velocity of $1.12 \\pm 0.055$ µm/s (Fig. 4D). Next, we determined the percentage of puncta which moved in the anterograde or retrograde direction, moved bidirectional or were stationary. This revealed that on average $34.52 \\pm 5.31\\%$ of mCherry-SERT puncta were moving during one minute of imaging. Further analysis of fraction of moving mCherry-SERT vesicles which moved in the anterograde or retrograde direction or moved bidirectional, revealed that there was no difference (Fig. 4E, see also table 1). Finally, we calculated the pausing time for the moving vesicles, which is the fraction of time moving vesicles pause during the 1 minute movie. There was no difference in percentage pausing time between mCherry-SERT vesicles which moved anterogradely, retrogradely or bidirectionally (Fig. 4F, see also table 1).\n\nWe conclude that mCherry-SERT displays highly dynamic vesicular trafficking in hippocampal neurons and that these mCherry-SERT vesicles do not have a preference for a direction of movement.\n\n**Table 1: Overview of mCherry-SERT vesicle dynamics**\n\n| | Velocity (µm/s) | Percentage of total | Percentage pausing time |\n|------------------|-----------------|---------------------|-------------------------|\n| Anterograde | $1.31 \\pm 0.05$ | 13.83 | 42.13 |\n| Retrograde | $1.34 \\pm 0.06$ | 12.06 | 39.21 |\n| Bidirectional | $1.12 \\pm 0.06$ | 8.63 | 41.04 |\n| Stationary | - | 65.48 | - |\n\nVelocity data are shown as mean ± standard error of the mean (SEM). Data of 927 mCherry-SERT puncta in 6 neurons.\n\nDiscussion\n\nHere we studied the trafficking of SERT in hippocampal neurons. We used human SERT cDNA tagged at its N-terminus with the red fluorescent protein mCherry. Overexpression of mCherry-SERT in hippocampal neurons resulted in a distinct pattern of expression between axons and dendrites; in dendrites SERT displayed a uniform distribution, whereas in axons SERT displayed a punctate distribution. Using electron microscopy, it has been shown that in 5-HT neurons endogenous SERT predominantly localized to varicosities and the membrane in axons, and in soma and dendrites SERT localized predominantly in the cytoplasm (Zhou et al., 1998; Tao-Cheng and Zhou, 1999). Thus, although we are using hippocampal neurons, mCherry-SERT distribution seems to mimic the distribution of endogenous SERT in 5-HT neurons.\n\nUltrastructural studies revealed that SERT labeling was associated with vesicular organelles, which possibly are large dense cored vesicles (LDCVs) (Pickel and Chan, 1999). Here, we co-transfected mCherry-SERT with tPA-GFP for which it has been shown that these are transported in LDCVs (also called secretory vesicles) (Silverman et al., 2005). The majority of mCherry-SERT puncta co-localized with tPA-GFP, strongly suggesting that SERT is transported in secretory vesicles. Several mCherry-SERT vesicles are localized in close proximity (~1 µm), and some co-localized, with presynaptic terminals. This suggests that the majority of SERT is not localized in the presynaptic terminal itself, but rather is localized close to a synapse. Indeed, previous research found that presynaptic membranes lacked SERT, but SERT was present on extrasynaptic domains, either in close proximity or at some distance away from synapses (Zhou et al., 1998).\n\nLive cell imaging revealed that mCherry-SERT vesicles display highly dynamic transport, i.e. there are vesicles which move anterogradely or retrogradely without interruptions, stationary vesicles or vesicles which move bidirectional. Analysis of average velocity revealed that both anterograde and retrograde vesicles moved with an average velocity of ~1.3 µm/s. The average velocity is in analogy with other transport or transport associated proteins which are transported in secretory vesicles, like the p75 neurotrophin receptor and Gephyrin (Maas et al., 2006; Formaggio et al., 2008). Surprisingly, we found that mCherry-SERT vesicles did not display a preference for direction of movement. For other releasable fluorescent cargos that are transported in secretory vesicles, such as Sema3A, BDNF and CCL21 it has been shown that transport is predominantly anterogradely (Adachi et al., 2005; de Wit et al., 2006; de Jong et al., 2008). In contrast, the p75 neurotrophin receptor, which is transported in vesicular structures, does not display a preference for direction of movement (Formaggio et al., 2008). Moreover, the scaffold protein Gephyrin,\n\nwhich is associated and co-transported with the glycine receptor also does not display a preference for anterograde or retrograde movement (Maas et al., 2006). Therefore, it appears that proteins which can be secreted and are transported in secretory vesicles predominantly move in the anterograde direction, whereas receptor/transporter proteins move both in the anterograde and retrograde direction.\n\nAn explanation for this could be that cargos which can be secreted are transported towards synaptic release sites distant from the cell soma. After a vesicle has been released, the secreted compounds either diffuse in the extracellular space (as is the case for Sema3A) or are taken up again and degraded in the presynaptic terminal. Hence, releasable cargos are hardly transported back towards the cell soma. On the other hand, the level of membrane expression for receptor and transporter protein is tightly regulated, and these proteins can be rapidly internalized (Loder and Melikian, 2003; Melikian, 2004; Zhu et al., 2004). Upon internalization, transporters are packaged in vesicles again and are recycled. Finally, receptors and transporters can maintain functioning without a need for intracellular transport, whereas secreted components are dependent on a new supply of vesicles. Therefore, vesicles containing transporters or receptors can be transported both in anterograde and retrograde directions.\n\nIn conclusion, expression of mCherry-SERT in hippocampal neurons differs between axons and dendrites. In axons mCherry-SERT is distributed in puncta, which co-localize with tPA-GFP and therefore presumably are secretory vesicles. Several of these mCherry-SERT vesicles are localized in close proximity of presynaptic terminals, but only few mCherry-SERT vesicles co-localized with presynaptic terminals. Live cell imaging revealed that mCherry-SERT displays highly dynamic trafficking behaviour with no preference for direction of movement.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Plasmids*\n\nConstruction of the human SERT cDNA with ECFP at the 5’ end (ECFP-SERT in the pECFP-C1 backbone (Clontech)) has been described before ((Schmid et al., 2001); a kind gift of Dr. H. Sitte). To construct mCherry-SERT, ECFP was excised with AseI and BsrGI and replaced for mCherry.\n\n*Neuronal culturing and transfection*\n\nHippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with\n\n0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium supplemented with B27 supplement, 18 mM HEPES, 0.5 mM Glutamax and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine 2000 was added to 25 µl of serum free glutamax medium (Invitrogen) and incubated for 5 minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free glutamax containing Lipofectamine 2000. This mixture was incubated for 30 minutes at room temperature before adding to the cells. After 4-6 hours of incubation on the cells at 37°C, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2-7 and imaged at DIV9-12.\n\n**Live cell imaging**\n\nCells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Grafelfing, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.\n\n**Immunocytochemistry and confocal microscopy**\n\nFor immunocytochemistry, neurons were fixed by incubation in 4% paraformaldehyde in phosphate buffered saline (PBS) for 20 minutes. Subsequently, to block aspecific binding and for permeabilization, the neurons were incubated in PBS containing 0.2% Triton X-100 and 4% fetal calf serum for 20 minutes. Neurons were incubated in primary antibody diluted in PBS containing 0.2% Triton X-100 for one hour. After washing three times in PBS, neurons were incubated in secondary antibodies in PBS for one hour. Neurons were washed again three times in PBS and mounted in Dabco-Mowiol. All reactions were carried out at room temperature. As primary antibodies, monoclonal MAP2 (Chemicon) and polyclonal Synapsin (E028) were used. As secondary antibodies Alexa fluor-conjugated antibodies were used (Invitrogen).\n\nFor confocal analysis, neurons were examined on a confocal LSM510 microscope (Zeiss, B.V. The Netherlands)\n\n**Trafficking analysis**\n\nIn order to analyze the trafficking dynamics of SERT, one minute movie image stacks with 1 s interval between images, were analyzed in the custom written software program Fluotrack (Broeke et al., 2008). To this end, metamorph files (.stk) were converted to .avi files using ImageJ, and analyzed using Fluotrack. Data analysis was performed using the SPSS statistical package. Data shown are mean values ± SEM.\n\n**References**\n\nAdachi N, Kohara K, Tsumoto T (2005) Difference in trafficking of brain-derived neurotrophic factor between axons and dendrites of cortical neurons, revealed by live-cell imaging. BMC Neurosci 6:42.\n\nAnsorge MS, Morelli E, Gingrich JA (2008) Inhibition of serotonin but not norepinephrine transport during development produces delayed, persistent perturbations of emotional behaviours in mice. J Neurosci 28:199-207.\n\nAnsorge MS, Zhou M, Lira A, Hen R, Gingrich JA (2004) Early-life blockade of the 5-HT transporter alters emotional behaviour in adult mice. Science 306:879-881.\n\nBroeke JH, Ge H, Dijkstra IM, Cemgil AT, Riedl JA, Niels Cornelisse L, Toonen RF, Verhage M, Fitzgerald WJ (2008) Automated quantification of cellular traffic in living cells. J Neurosci Methods.\n\nChen NH, Reith ME, Quick MW (2004) Synaptic uptake and beyond: the sodium- and chloride-dependent neurotransmitter transporter family SLC6. Pflugers Arch 447:519-531.\n\nde Jong EK, Vinet J, Stanulovici VS, Meijer M, Wesseling E, Sjollema K, Boddeke HW, Biber K (2008) Expression, transport, and axonal sorting of neuronal CCL21 in large dense-core vesicles. Faseb J 22:4136-4145.\n\nde Wit J, Toonen RF, Verhaagen J, Verhage M (2006) Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites. Traffic 7:1060-1077.\n\nFabre V, Beaufour C, Evrard A, Rioux A, Hanoun N, Lesch KP, Murphy DL, Lanfumey L, Hamon M, Martres MP (2000) Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter. Eur J Neurosci 12:2299-2310.\n\nFjorback AW, Pla P, Muller HK, Wiborg O, Saudou F, Nyengaard JR (2009) Serotonin transporter oligomerization documented in RN46A cells and neurons by sensitized acceptor emission FRET and fluorescence lifetime imaging microscopy. Biochem Biophys Res Commun 380:724-728.\n\nFormaggio E, Cantu C, Chiamulera C, Fumagalli GF (2008) p75 neurotrophin receptor distribution and transport in cultured neurons. Neurosci Res 62:32-42.\n\nHolmes A, Murphy DL, Crawley JN (2003a) Abnormal behavioural phenotypes of serotonin transporter knockout mice: parallels with human anxiety and depression. Biol Psychiatry 54:953-959.\n\nHolmes A, Yang RJ, Lesch KP, Crawley JN, Murphy DL (2003b) Mice lacking the serotonin transporter exhibit 5-HT(1A) receptor-mediated abnormalities in tests for anxiety-like behaviour. Neuropsychopharmacology 28:2077-2088.\n\nJust H, Sitte HH, Schmid JA, Freissmuth M, Kudlacek O (2004) Identification of an additional interaction domain in transmembrane domains 11 and 12 that supports oligomer formation in the human serotonin transporter. J Biol Chem 279:6650-6657.\n\nLau T, Horschitz S, Bartsch D, Schloss P (2008a) Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy. Neurochem Int.\n\nLau T, Horschitz S, Berger S, Bartsch D, Schloss P (2008b) Antidepressant-induced internalization of the serotonin transporter in serotonergic neurons. Faseb J 22:1702-1714.\n\nLebrand C, Cases O, Adelbrecht C, Doye A, Alvarez C, El Mestikawy S, Seif I, Gaspar P (1996) Transient uptake and storage of serotonin in developing thalamic neurons. Neuron 17:823-835.\n\nLochner JE, Kingma M, Kuhn S, Meliza CD, Cutler B, Sealettar BA (1998) Real-time imaging of the axonal transport of granules containing a tissue plasminogen activator/green fluorescent protein hybrid. Mol Biol Cell 9:2463-2476.\n\nLoder MK, Melikian HE (2003) The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines. J Biol Chem 278:22168-22174.\n\nMaas C, Tagnaouti N, Loebrich S, Behrend B, Lappe-Sieke C, Kneussel M (2006) Neuronal cotransport of glycine receptor and the scaffold protein gephyrin. J Cell Biol 172:441-451.\n\nMathews TA, Fedele DE, Coppelli FM, Avila AM, Murphy DL, Andrews AM (2004) Gene dose-dependent alterations in extraneuronal serotonin but not dopamine in mice with reduced serotonin transporter expression. J Neurosci Methods 140:169-181.\n\nMelikian HE (2004) Neurotransmitter transporter trafficking: endocytosis, recycling, and regulation. Pharmacol Ther 104:17-27.\n\nNarboux-Neme N, Pavone LM, Avallone L, Zhuang X, Gaspar P (2008) Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs). Neuropharmacology 55:994-1005.\n\nPickel VM, Chan J (1999) Ultrastructural localization of the serotonin transporter in limbic and motor compartments of the nucleus accumbens. J Neurosci 19:7356-7366.\n\nSchmid JA, Schölze P, Kudlacek O, Freissmuth M, Singer EA, Sitte HH (2001) Oligomerization of the human serotonin transporter and of the rat GABA transporter 1 visualized by fluorescence resonance energy transfer microscopy in living cells. J Biol Chem 276:3805-3810.\n\nShaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins\n\nderived from *Discosoma* sp. red fluorescent protein. Nat Biotechnol 22:1567-1572.\n\nSilverman MA, Johnson S, Gurkins D, Farmer M, Lochner JE, Rosa P, Scalettar BA (2005) Mechanisms of transport and exocytosis of dense-core granules containing tissue plasminogen activator in developing hippocampal neurons. J Neurosci 25:3095-3106.\n\nTao-Cheng JH, Zhou FC (1999) Differential polarization of serotonin transporters in axons versus soma-dendrites: an immunogold electron microscopy study. Neuroscience 94:821-830.\n\nZhou FC, Tao-Cheng JH, Segu L, Patel T, Wang Y (1998) Serotonin transporters are located on the axons beyond the synaptic junctions: anatomical and functional evidence. Brain Res 805:241-254.\n\nZhu CB, Hewlett WA, Feoktistov I, Biaggioni I, Blakely RD (2004) Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation. Mol Pharmacol 65:1462-1474.\n\nZhuang X, Masson J, Gingrich JA, Rayport S, Hen R (2005) Targeted gene expression in dopamine and serotonin neurons of the mouse brain. J Neurosci Methods 143:27-32.\n\nChapter 4\n\nPresynaptic localization of tryptophan hydroxylase 2 in mature hippocampal neurons\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1} and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\n\\textit{In preparation}\n\nAbstract\n\nTryptophan hydroxylase (Tph) is the rate limiting enzyme in serotonin (5-HT) synthesis. Two genes named Tph1 and Tph2 synthesize 5-HT in peripheral tissues and the central nervous system, respectively. When expressed in neurons, Tph1 displays a cytoplasmic distribution in the cell body and is associated with microtubules in the axon and dendrites. Axonal Tph1 is associated with 5-HT varicosities. Here, we studied the subcellular distribution of Tph2. To this end we expressed Tph2-EGFP in hippocampal neurons. In young neurons, before synaptogenesis has occurred, Tph2-EGFP displayed a cytoplasmic distribution throughout the neurites. Live cell imaging revealed that Tph2-EGFP is not transported in vesicles. In mature neurons, Tph2-EGFP accumulated in presynaptic terminals of axons, whereas in dendrites it was distributed throughout the cytoplasm, also in spines. In live cell imaging, Tph2-EGFP hardly displayed any movement. These results suggest that Tph2 is freely diffusible in the dendrites and soma, but accumulates at the synaptic terminal by a mechanism that deserves further study.\n\nIntroduction\n\nThe neurons of the 5-HT system are among the first neurons in the brain synthesizing a specific neurotransmitter. Two enzymes are necessary to synthesize 5-HT from its precursor L-tryptophan. Of these, the rate limiting enzyme is Tph which converts L-tryptophan to 5-hydroxy-L-tryptophan. For decades only one Tph isoform was known in the mammalian brain. However, deletion of this gene did not result in a significant decrease in brain 5-HT levels, which led to the discovery of a second Tph gene called Tph2 (Walther et al., 2003). Tph2 is exclusively expressed in the raphe nuclei, whereas Tph1 is expressed in the pineal gland and in the peripheral 5-HT system (Patel et al., 2004; Gutknecht et al., 2009).\n\nSince 5-HT projections are distributed throughout the brain, it is unlikely that Tph2 is only present in the cell body to synthesize 5-HT. Indeed, previous research showed that Tph was present in the cytoplasm of the cell body, and in axons and dendrites where it was associated with microtubules (Joh et al., 1975). Additionally, Tph was associated with 5-HT varicosities, suggesting local synthesis of 5-HT (Pickel et al., 1976; Pickel et al., 1977).\n\nAlthough the subcellular distribution of Tph1 is known, it is currently unknown how Tph is transported to synaptic terminals and whether Tph2 also\n\nlocalizes to presynaptic terminals. Therefore we tagged Tph2 with EGFP, and expressed this chimaeric protein in hippocampal neurons. We show here that in young neurons (before synaptogenesis), Tph2-EGFP is homogenously distributed throughout the neurites. However, in mature neurons, Tph2-EGFP accumulated at presynaptic terminals in the axon. In the dendrites of mature neurons, Tph2-EGFP displayed a homogenous distribution, and also localized to spines. Live cell imaging suggested that Tph2 is a soluble protein and is not transported in vesicles to presynaptic terminals. We conclude that Tph2-EGFP accumulates at presynaptic terminals in mature hippocampal neurons.\n\n**Results**\n\n*Cytoplasmic distribution of Tph2-EGFP in young neurons*\n\nTo study the distribution of Tph2 in neurons, we inserted EGFP at the N-terminus of Tph2 and expressed Tph2-EGFP in hippocampal neurons. First, we started with imaging Tph2-EGFP in neurons at 9 days in vitro (DIV), which are still immature neurons in terms of synapse development (Basarsky et al., 1994) Transfected neurons showed a uniform distribution of Tph2-EGFP through the whole neuron, indicating that Tph2-EGFP is cytoplasmic (Fig. 1A). We did not observe Tph2-EGFP puncta, suggesting that Tph2-EGFP is not transported in\n\n\n\nTph2-EGFP was expressed in young hippocampal neurons before synaptogenesis and imaged at DIV9. (A) Tph2-EGFP displays a cytoplasmic distribution throughout all the neurites. (B) Live cell imaging revealed that Tph2-EGFP is not transported in vesicles, and only little movement is detected. Images are taken 10 seconds apart, first image is green and image 10 seconds later is in red. (C) Zooming in on the axon (arrow in A), as identified by its smaller diameter compared to dendrites shows that already puncta are observed which might be immature synapses or varicosities (arrows). (D) In the dendrites, Tph2-EGFP distribution is cytoplasmic, and Tph2-EGFP also localized to dendritic filopodia. Scale bars: 10 µm in B, 5 µm in C and D.\n\nA\n\nTph2-EGFP\n\nB\n\nSynaptophysin-mCherry\n\nC\n\nMerge\n\nD\n\n*\n\n***\n\nE\n\nF\n\nG\n\nH\n\nI\n\nJ\n\n*\n\n*\n\nK\n\nL\n\nM\n\n1s 30s\n\nN\n\n1s 30s\n\nScale bars: A, B, C = 5 μm; D, E, F = 10 μm; G, H, I = 20 μm; J, K, L = 20 μm; M, N = 10 μm.\n\nNeurons expressing Tph2-EGFP and the presynaptic marker Synaptophysin-mCherry were imaged at DIV23. (A) Tph2-EGFP was expressed in the cytoplasm of dendrites. (B) Expression of Synaptophysin-mCherry in the same neuron. (C) Merged image. (D) Blow up of the white box in C shows that some Tph2-EGFP appears to accumulate in the tip of dendritic spines (asterisks). (E) Synaptophysin-mCherry expression. (F) Merged image. (G) Axon of a neuron expressing Tph2-EGFP. Tph2-EGFP displays a punctate expression, indicative of presynaptic localization. (H) Indeed, Tph2-EGFP co-localizes with Synaptophysin-mCherry. (I) Merged image. (J) A EGFP expressing neuron as a control shows that EGFP is homogenously distributed in the dendrites (asterisks) and in the axon as identified by the absence of MAP2 staining (arrows). (K) MAP2 staining. (L) Merged image. (M) Live cell imaging revealed that Tph2-EGFP is static. Images are taken 30 seconds apart, with the green image after 1 second, and the red image after 30 seconds. (N) Blow up of the white box in J shows that some dendrites which contain Tph2-EGFP displayed some movement (arrows). Scale bars: 10 µm in C, 5 µm in F, 10 µm in I, 10 µm in L, 10 µm in M and 5 µm in N.\n\nvesicles, but instead is a soluble protein. Live cell imaging revealed that Tph2-EGFP is rather static, and no Tph2-EGFP puncta or other movements were observed (Fig. 1B). The axon, as identified by its smaller diameter and larger length as compared to dendrites, also had a uniform distribution of Tph2-EGFP, although some Tph2-EGFP appeared to accumulate in puncta along the axon (arrows in Fig. 1C). As in axons, in dendrites Tph2-EGFP displayed a uniform distribution and also filopodia were labelled (Fig. 1D).\n\n**Tph2-EGFP localizes to presynaptic terminals in mature neurons**\n\nNext, we investigated the distribution of Tph2-EGFP in mature neurons after synaptogenesis had occurred. To this end we transfected hippocampal neurons with Tph2-EGFP at DIV18 and imaged Tph2-EGFP at DIV23. Tph2-EGFP was uniformly distributed in the cell body, dendrites and dendritic spines (Fig. 2A-C). We expressed EGFP in neurons as a control to compare the distribution of EGFP with Tph2-EGFP (Fig. 2J-L). In contrast to EGFP, which labels the whole dendritic spine, it appeared that in neurons transfected with Tph2-EGFP in some spines Tph2-EGFP accumulated in the tip of the spine, suggesting that Tph2-EGFP was enriched in postsynaptic structures (Fig. 2D-F). We co-transfected Tph2-EGFP with Synaptophysin-mCherry, which is a marker for synaptic vesicles and presynaptic terminals (Jahn et al., 1985; Granseth et al., 2006). In the axon, identified by the punctate labelling of Synaptophysin-mCherry, Tph2-EGFP displayed a punctate distribution (Fig. 2G). The Tph2-EGFP puncta co-localized with Synaptophysin-mCherry puncta, strongly suggesting that Tph2-EGFP accumulated at presynaptic terminals (Fig. 2H,I). In neurons transfected\n\nwith EGFP as a control, EGFP displayed a uniform distribution throughout the axon (Fig. 2J-L). Next, we imaged the neurons expressing Tph2-EGFP during 1 minute. Hardly any Tph2-EGFP movement could be observed (Fig. 2M). The only movement which could be observed was some motility in postsynaptic spines (Fig. 2N).\n\nTo further test if Tph2-EGFP is localized to presynaptic terminals in mature neurons, we fixed neurons expressing Tph2-EGFP at DIV23 and performed immunocytochemistry for the synaptic marker Synapsin and the dendritic marker MAP2 to discriminate dendrites and axons (Fig. 3A-C). In the axon, as identified by the absence of MAP2 labelling, Tph2-EGFP displayed a punctate distribution (Fig. 3D-F). Immunocytochemistry for Synapsin showed that in dendrites of neurons expressing Tph2-EGFP, spines were labelled, and some spine heads were localized in close proximity with Synapsin puncta, suggesting that this is a synapse (Fig. 3G-I). Finally, we focussed on the axon of a neuron expressing Tph2-EGFP which we stained for Synapsin. The boutons with Tph2-EGFP co-localized with Synapsin, strongly suggesting that Tph2-EGFP localized to presynaptic terminals (Fig. 3J-L).\n\n**Discussion**\n\nWe have shown here that Tph2, the rate limiting enzyme in 5-HT synthesis localized to presynaptic terminals in mature hippocampal neurons. Moreover, in dendrites and in the cell body Tph2-EGFP had a cytoplasmic distribution and appeared to accumulate in the tip of some dendritic spines. Thus, it appears that Tph2-EGFP displayed a polarized distribution in mature hippocampal neurons. Live cell imaging revealed that hardly any Tph2-EGFP movement could be observed, and no Tph2-EGFP vesicles were observed, indicating that Tph2 is a soluble protein. Accordingly, Tyrosine hydroxylase, an enzyme related to Tph2, also was shown to be a soluble protein (Mockus et al., 1997). Electron microscope studies on Tph and TH localization revealed that these proteins were enriched on presynaptic terminals, and were associated with microtubules in somatodendritic compartments (Pickel et al., 1976). Therefore, Tph2-EGFP seems to mimic the endogenous distribution of Tph in 5-HT neurons.\n\nTph2-EGFP displayed a polarized distribution in mature hippocampal neurons, i.e. in dendrites the protein is distributed throughout the cytoplasm, whereas in the axon the protein accumulated in presynaptic terminals. This suggests that in presynaptic terminals and in 5-HT varicosities, there is local synthesis of 5-HT. In dendrites, Tph2-EGFP localized to spines, which prompts the question whether 5-HT could be synthesized in dendrites.\n\nFigure 3. Immunocytochemistry on a mature neuron expressing Tph2-EGFP\n\nNeurons (DIV23) expressing Tph2-EGFP were fixed and immunocytochemistry was performed for MAP2 and Synapsin. (A) Tph2-EGFP expression. (B) MAP2 immunostaining to distinguish between dendrites and the axon. (C) Merged image. (D) Blow up of the white box in C shows a dendrite (asterisk) which has Tph2-EGFP through the whole cytoplasm, and the axon (arrows) in which Tph2-EGFP displays a punctate distribution. (E) MAP2 staining. (F) Merged image. (G) Tph2-EGFP expressing neurons were stained for the presynaptic marker Synapsin. (H) Synapsin staining. (I) Merged image, which shows that in the close proximity of some dendritic spines there is a Synapsin puncta, suggesting that these are true synapses (asterisks). (J) Distribution of Tph2-EGFP in an axon. (K) Synapsin staining. (L) Tph2-EGFP puncta were positive for Synapsin (arrows), showing that these are presynaptic terminals. Scale bars: 20 µm in C, 10 µm in F, 5 µm in I and L.\n\nTph is localized in dendrites, where it associated with microtubules (Joh et al., 1975). Correspondingly, 5-HT vesicles are detected in dendrites, either associated with a synaptic specialization or with the membrane (Chazal and Ralston, 1987). After stimulation of Retzius neurons in the leech, which contain large dense core vesicles (LDCVs) filled with 5-HT, several vesicles are localized close to the cell membrane as studied by electron microscopy (Trueta et al.,\n\nTherefore, this strongly suggests that 5-HT release can occur from the somatodendritic compartment. Our results indicate that 5-HT can also be synthesized in the somatodendritic compartment.\n\nTph2-EGFP was not associated with vesicles and the protein interactions for its presynaptic accumulation remain unclear. Tph is associated with microtubules, suggesting it could interact with molecular motors of the dynein and kinesin family which move along microtubules (Hirokawa and Takemura, 2005). For Tph2, several polymorphisms are known, some of which associate with neuropsychiatric conditions (Zhang et al., 2006). Both in humans and in mice a polymorphism in the Tph2 gene is found which results in a marked reduction in Tph2 activity (Zhang et al., 2004; Zhang et al., 2005). Finally, it would be interesting to investigate whether these polymorphisms not only affect Tph2 activity, but could also affect Tph2 transport and localization.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Plasmids*\n\nTph2 cDNA in pCR4-TOPO vector was obtained from Geneservice. To clone Tph2 in an expression vector, EGFP was digested using PstI and cloned in the PstI site of pBiotag-EGFP-C1. Synaptophysin-mCherry was a kind gift from Dr. A. Jeromin (Allen Brain Institute).\n\n*Neuronal culturing and transfection*\n\nHippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks’ Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with 0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium supplemented with B27 supplement, 18 mM HEPES, 0.5 mM Glutamax and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine was added to 25 µl of serum free glutamax medium and incubated for five minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free\n\nglutamax containing Lipofectamine. This mixture was incubated for 30 minutes before adding to the cells. After 4-6 hours of incubation on the cells, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2-7 and DIV18 and imaged at DIV10-14 and DIV23.\n\n**Live cell imaging**\n\nCells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Grafelfing, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.\n\n**Immunocytochemistry**\n\nFor immunocytochemistry, primary antibodies against MAP2 (monoclonal, Chemicon) and synapsin (polyclonal Eo28) were used in a 1:1000 dilution. As secondary antibodies, goat-anti-mouse Alexa488 and goat-anti-rabbit Alexa 543 were used (Invitrogen). To perform immunocytochemistry, cells were incubated in 4% paraformaldehyde dissolved in phosphate buffered saline (PBS, pH 7.4), for 20 minutes. Subsequently, cells were washed two times with PBS and incubated in PBS containing 0.2% Triton X-100 and 4% fetal calf serum for 20 minutes. Cells were incubated in primary antibody in PBS containing 0.2% Triton X-100 for one hour, washed three times with PBS and incubated in the secondary antibody in PBS for one hour. After washing the cells three times with PBS, coverslips were mounted using Dabco-Mowiol. All reactions were performed at room temperature. Cells were analyzed on a confocal LSM510 microscope (Zeiss, B.V. The Netherlands) using a 63x oil immersion lens and appropriate lasers and filters to visualize EGFP fluorescence and secondary antibodies.\n\n**References**\n\nBasarsky TA, Parpura V, Haydon PG (1994) Hippocampal synaptogenesis in cell culture: developmental time course of synapse formation, calcium influx, and synaptic protein distribution. J Neurosci 14:6402–6411.\n\nChazal G, Ralston HJ, 3rd (1987) Serotonin-containing structures in the nucleus raphe dorsalis of the cat: an ultrastructural analysis of dendrites, presynaptic dendrites, and axon terminals. J Comp Neurol 259:317–329.\n\nGranseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51:773–786.\n\nGutknecht L, Kriegebaum C, Waider J, Schmitt A, Lesch KP (2009) Spatio-temporal expression of tryptophan hydroxylase isoforms in murine and human brain: convergent data from Tph2 knockout mice. Eur Neuropsychopharmacol 19:266–282.\n\nHirokawa N, Takemura R (2005) Molecular motors and mechanisms of directional transport in neurons. Nat Rev Neurosci 6:201-214.\n\nJahn R, Schiebler W, Ouimet C, Greengard P (1985) A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci U S A 82:4137-4141.\n\nJoh TH, Shikimi T, Pickel VM, Reis DJ (1975) Brain tryptophan hydroxylase: purification of, production of antibodies to, and cellular and ultrastructural localization in serotonergic neurons of rat midbrain. Proc Natl Acad Sci U S A 72:3575-3579.\n\nMockus SM, Kumer SC, Vrana KE (1997) A chimeric tyrosine/tryptophan hydroxylase. The tyrosine hydroxylase regulatory domain serves to stabilize enzyme activity. J Mol Neurosci 9:35-48.\n\nPatel PD, Pontrello C, Burke S (2004) Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiatry 55:428-433.\n\nPickel VM, Joh TH, Reis DJ (1976) Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons. Immunocytochemical localization by light and electron microscopy. J Histochem Cytochem 24:792-306.\n\nPickel VM, Joh TH, Reis DJ (1977) A serotonergic innervation of noradrenergic neurons in nucleus locus coeruleus: demonstration by immunocytochemical localization of the transmitter specific enzymes tyrosine and tryptophan hydroxylase. Brain Res 131:197-214.\n\nTrueta C, Sanchez-Armass S, Morales MA, De-Miguel FF (2004) Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons. J Neurobiol 61:309-316.\n\nWalther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, Bader M (2003) Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76.\n\nZhang X, Beaulieu JM, Gainetdinov RR, Caron MG (2006) Functional polymorphisms of the brain serotonin synthesizing enzyme tryptophan hydroxylase-2. Cell Mol Life Sci 63:6-11.\n\nZhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG (2004) Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217.\n\nZhang X, Gainetdinov RR, Beaulieu JM, Sotnikova TD, Burch LH, Williams RB, Schwartz DA, Krishnan KR, Caron MG (2005) Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45:11-16.\n\nChapter 5\n\nThe effect of a polymorphism in Piccolo on localization and trafficking dynamics of the serotonin reuptake transporter\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1} and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\nAbstract\n\nThe active zone protein Piccolo is involved in formation of synapses and stabilization of existing synapses. Recently, a short nucleotide polymorphism (SNP) in the C2A domain of Piccolo was found in association with depression. In this study we addressed whether knockdown of Piccolo affected the localization and trafficking of the serotonin reuptake transporter (SERT) in hippocampal neurons. We co-expressed a short nucleotide RNA which resulted in an efficient knockdown of Piccolo with mCherry-SERT to evaluate the effect of knockdown of Piccolo on SERT localization and trafficking in hippocampal neurons. We found that knockdown of Piccolo did not alter SERT trafficking dynamics. Subsequently, we expressed the two variants of the Piccolo C2A domain together with SERT in heterologous cells. SERT displayed a uniform distribution at the membrane upon co-expression of the wildtype C2A variant. However, upon co-expression of the mutant C2A variant, SERT displayed a more patchy distribution at the membrane, indicating that membrane levels of SERT might be decreased. These data suggest that knockdown of Piccolo does not alter SERT trafficking dynamics, but the mutant variant of the Piccolo C2A domain might alter SERT membrane localization.\n\nIntroduction\n\nMajor depression is a neuropsychiatric disorder affecting millions of people world wide each year. The serotonin (5-HT) system is implicated in depression. An important protein in the 5-HT system is the SERT which regulates the transport of 5-HT back into the synapse after its release, thus regulating the extracellular 5-HT concentration. Therefore, SERT is an important factor in determining efficacy of 5-HT neurotransmission. Several polymorphisms have been described in SERT which associate with psychopathological processes such as depression, anxiety, aggression and autism (Murphy et al., 2004; Serretti et al., 2006).\n\nSeveral decades ago it was hypothesized that psychopathological processes such as depression and anxiety were the result of an alteration in extracellular 5-HT content. This hypothesis, the so-called monoamine hypothesis, was based on the fact that blockade of the SERT by selective 5-HT reuptake inhibitors (SSRIs) resulted in an alleviation in depressive symptoms, possibly due to the increase in extracellular 5-HT levels. However, between starting SSRI treatment and alleviation of depressive symptoms was a time period of 3-4 weeks, whereas 5-\n\nHT levels increased immediately after SSRI intake. Therefore the monoamine hypothesis was abandoned and shifted towards the so-called network hypothesis, which stated that psychopathological processes such as anxiety and depression could be the result of an altered 5-HT network connectivity (Castren, 2005). The evidence for this hypothesis was based on human imaging studies which showed that humans with depression had a reduced grey matter volume in cortex and hippocampus. However, evidence for this hypothesis on a cellular level is still lacking. Recently, a SNP in the presynaptic gene Piccolo was found which might be associated with major depressive disorder (Sullivan et al., 2008). Therefore, this revitalizes the network hypothesis for depression and opens up new opportunities to study the relation between the presynaptic terminal, proper formation of networks, and the relation between 5-HT network (formation) and psychopathological processes such as depression.\n\nPiccolo is a 420 kDa protein which is enriched in presynaptic terminals where it is a structural component of the presynaptic cytoskeletal cytomatrix (PCM) (Cases-Langhoff et al., 1996). Piccolo is a multidomain zinc finger protein which is structurally related to Bassoon, another component of the PCM (tom Dieck et al., 1998; Fenster et al., 2000). Piccolo contains several coiled coil domains, a PDZ domain and two C2 domains (Fenster and Garner, 2002). The C2A domain functions as a low-affinity calcium sensor in presynaptic terminals (Gerber et al., 2001). Piccolo and Bassoon, along with other presynaptic constituents, are transported in 80 nm large dense core vesicles (LDCVs) to nascent synapses, and 2 or 3 of these vesicles are sufficient to form an active synapse (Zhai et al., 2001; Shapira et al., 2003; Tao-Cheng, 2007). The recent described nonsynonymous SNP rs2522833 in Piccolo encodes a common variation in the C2A domain, where a serine is substituted for an alanine (Sullivan et al., 2008).\n\nPiccolo is involved in the regulation of the internalization of the dopamine transporter (DAT) in heterologous cells. Specifically, expression of the C2A domain of Piccolo reduces methamphetamine-induced DAT internalization. (Cen et al., 2008). DAT and SERT are structurally related proteins, which belong to the solute carrier superfamily. It could be likely that membrane incorporation and internalization of these proteins is regulated in the same manner. Additionally, Piccolo influences synaptic function by negatively regulating synaptic vesicle exocytosis (Leal-Ortiz et al., 2008). These data led us to hypothesize that possibly, Piccolo might contribute to the overall risk of depression by affecting the membrane incorporation of SERT.\n\nTo test this hypothesis we expressed mCherry-SERT in hippocampal neurons and investigated whether knockdown of Piccolo or the variation rs2522833 affected the localization and trafficking of SERT. We show here that\n\nknockdown of Piccolo in neurons did not affect SERT trafficking. However, expression of the Piccolo C2A domain variation rs2522833 in heterologous cells might affect membrane localization of SERT.\n\nResults\n\n*Piccolo localized to neurotransmitter release sites in hippocampal and 5-HT neurons*\n\nFirst, we analyzed the cellular localization of Piccolo in hippocampal neurons and 5-HT neurons. Hippocampal neurons (14 days in vitro (DIV)) were fixed and immunocytochemistry was performed for Piccolo and for the presynaptic terminal marker Synaptobrevin-2 (VAMP2). Piccolo immunoreactivity was distributed in a punctate pattern, indicative for labeling of presynaptic terminals (Fig. 1A). Indeed, the majority of Piccolo puncta co-localized with VAMP2 puncta, showing that these represented presynaptic terminals (Fig. 1B,C). Zooming in on part of a neurite showed that >90% of Piccolo puncta co-localized with VAMP2 (Fig. 1D-F). Thus, Piccolo was present in presynaptic terminals.\n\nNext, we investigated the localization of Piccolo in 5-HT neurons. We used a coronal brain slice from an adult mouse through the dorsal raphe nucleus (DRN), the region in the brain where the majority of 5-HT neurons is localized. We performed immunocytochemistry for tryptophan hydroxylase (Tph), the rate limiting enzyme for 5-HT synthesis and a marker for the 5-HT system. In a DRN slice, several 5-HT neurons were present (Fig. 1G). Immunocytochemistry for Piccolo resulted in a punctate labeling in the DRN slice (Fig. 1H). Piccolo staining was barely detectable in the 5-HT cell bodies (Fig. 1I). However, Piccolo puncta co-localized with Tph puncta. Tph is present in varicosities and presynaptic terminals (chapter 4 of this thesis) (Pickel et al., 1976).\n\n**Figure 1 next page. Piccolo localizes to presynaptic terminals in neurons**\n\n(A-C) Hippocampal neurons were fixed at DIV14, and immunocytochemistry was performed for Piccolo (A) and the presynaptic marker VAMP2 (B). (C) Merged image shows that several Piccolo puncta co-localized with VAMP2 puncta. (D-F) Blow up of the white box in C shows that almost every Piccolo puncta co-localizes with a VAMP2 puncta. (D) Piccolo staining. (E) VAMP2 staining. (F) Merged image. (G) A coronal brain slice through the DRN from an adult mouse was stained against the 5-HT marker Tph. This shows that 5-HT cell bodies and fibers are present. (H) Piccolo staining on this slice shows that Piccolo expression is low in 5-HT cell bodies. (I) Merged image. (J) Blow up of the white box in I shows a 5-HT cell body and several Tph puncta, which might be 5-HT varicosities. (K) Piccolo staining shows that in several of the Tph puncta Piccolo is present. (L) Merged image. Scale bars: 20 µm in C and I, 10 µm in F and L.\n\nThese Tph puncta might therefore represent 5-HT varicosities, axonal sites where 5-HT release occurs, often without a post-synaptic specialization. Therefore, it seems that also in 5-HT neurons Piccolo is localized in release sites (Fig 1J-L).\n\n**Efficient knockdown of Piccolo in hippocampal neurons**\n\nNext, we investigated whether knockdown of Piccolo affected trafficking of mCherry-SERT. In hippocampal neurons, mCherry-SERT is transported in secretory vesicles which move through the axon with no preference for direction of movement (Chapter 3, this thesis). To achieve efficient knockdown of Piccolo,\n\nwe designed six short hairpin RNA (shRNA) nucleotides, targeted towards the N-terminus of Piccolo. These short hairpins were inserted in a vector containing also an internal ribosomal entry site (IRES) and EGFP, as a control for transfected neurons. We tested the shRNAs for their Piccolo knockdown efficiency. To this end we transfected the shRNA constructs in hippocampal neurons, and four days later fixed and processed the neurons for immunocytochemistry. Several constructs tested did not show any knockdown of Piccolo. We decided to use shRNA#5 as a negative control. Analysis of Piccolo expression in MAP2 negative axons of neurons transfected with shRNA#5 was not reduced compared to untransfected cells (Fig. 2A-D). The axon had a punctate labeling of Piccolo, which was indistinguishable from untransfected neurons. On the other hand, transfection of shRNA#6 caused a reduction in levels of Piccolo in axons (Fig. 2E-H, 2I-L).\n\nQuantification of the number of Piccolo puncta per μm neurite revealed that in control condition there was on average one Piccolo puncta per two μm neurite, whereas in shRNA#6 mediated knockdown there was on average one Piccolo puncta per ten μm neurite (control $0.49 \\pm 0.11$ Piccolo puncta per μm axon; knockdown $0.11 \\pm 0.02$ Piccolo puncta per μm axon, $p=0.002$) (Fig. 2M). Thus, shRNA#6 resulted in an efficient, approximately 80%, reduction in number of Piccolo puncta. Therefore, we used this construct to test the effect of Piccolo knockdown on mCherry-SERT localization and trafficking.\n\n**Knockdown of Piccolo does not affect SERT trafficking**\n\nWe used shRNA#6 to investigate the effect of Piccolo knockdown on mCherry-SERT localization and trafficking. SERT tagged with mCherry is transported in secretory vesicles, which move in anterograde and retrograde direction, with no preference for direction of movement (chapter 3 of this thesis). We transfected mCherry-SERT and shRNA#6 in hippocampal neurons, and used EGFP as a control. One minute movies of mCherry-SERT trafficking were made, and the movies were analyzed using the custom written Fluotrack software program (Broeke et al., 2008). The velocity of moving puncta was quantified, as well as the direction of movement. For direction we discerned anterograde direction, retrograde direction and bidirectional, vesicles which changed direction during imaging. Both in neurons expressing shRNA#6 and EGFP, we observed several puncta of mCherry-SERT, which displayed rapid movements in both the anterograde and retrograde direction. Additionally, also stationary vesicles were observed (Fig. 3A-D). We quantified the average velocity of mCherry-SERT moving puncta in control neurons expressing EGFP, and neurons expressing shRNA#6. A velocity histogram shows the distribution of the average velocity and direction of movement between control and knockdown (Fig. 3E).\n\nFigure 2. Efficient shRNA-mediated knockdown of Piccolo\n\nSeveral shRNA constructs against Piccolo were tested for their efficacy to reduce Piccolo expression in cultured hippocampal neurons. ShRNA#5 did not result in Piccolo knockdown. (A) EGFP expression, showing that the shRNA construct is expressed. (B) Piccolo staining. (C) Neurite is MAP2 negative, showing that this is an axon. (D) Merged image. (E) EGFP expression in a neuron transfected with shRNA#6-EGFP. (F) Piccolo staining. (G) MAP2 staining. (H) Merged image, showing that shRNA#6 results in efficient knockdown of Piccolo in the axon. (I-L) Blow up of the white box in H, with EGFP, Piccolo staining, MAP2 staining and merged image respectively. (M) Quantification of the level of Piccolo knockdown shows that shRNA#6 resulted in a ~80% reduction in Piccolo puncta (control $0.49 \\pm 0.11$ Piccolo puncta per $\\mu m$ axon ($n=7$); knockdown $0.11 \\pm 0.02$ Piccolo puncta per $\\mu m$ axon ($n=8$)). Scale bars: 5 $\\mu m$ in D and L, 10 $\\mu m$ in H.\n\nTrafficking of mCherry-SERT did not differ between control and knockdown (average velocity control 1.30 ± 0.042 µm/s (n=8 neurons); knockdown 1.36 ± 0.039 µm/s, (n=18 neurons) (Fig. 3F). Also, percentage of moving vesicles was not different between control and knockdown (control 46.69 ± 5.03%; knockdown 39.88 ± 3.39%) (Fig. 3G). Additionally, percentage of vesicles moving anterogradely, retrogradely or bidirectionally did not differ between control and knockdown (data not shown).\n\nFigure 3 previous page. Piccolo knockdown does not affect SERT trafficking dynamics\n\n(A) Neurons transfected with EGFP and mCherry-SERT. Images are taken 5 seconds apart. Several mCherry-SERT puncta are observed which are either stationary (yellow), or are moving anterogradely or retrogradely. (B) A neuron transfected with shRNA#6EGFP and mCherry-SERT. Images taken 5 seconds apart show that after Piccolo knockdown also several mCherry-SERT puncta are observed, some of which are stationary, and other which display anterograde or retrograde movements. (C) and (D) are blow ups of the white boxes in A and B, respectively. (E) Velocity histogram of mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. Negative values are retrograde movements, positive values are anterograde movements. (F) Average velocity of mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. (G) Percentage moving mCherry-SERT puncta in control neurons and Piccolo knockdown neurons. Scale bars: 10 µm in A and B, 5 µm in C and D.\n\nExpression of C2A variant rs2522833 alters SERT distribution at the membrane in heterologous cells\n\nFinally, we were interested whether the SNP in the C2A domain of Piccolo which was found to associate with major depression, affected the membrane localization of SERT. Therefore, we constructed the C2A\\textsubscript{wt} domain of Piccolo in an IRES2-EGFP vector and a C2A\\textsubscript{mut} domain with the rs2522833 SNP which results in a serine to alanine substitution at amino acid 4684. We transfected these constructs, together with mCherry-SERT, in heterologous Neuro2A cells, which do not express endogenous Piccolo. In Neuro2A cells transfected with mCherry-SERT together with Piccolo C2A\\textsubscript{wt}, SERT was localized predominantly at the cell membrane. The majority of cells displayed this SERT distribution, although also in some cells mCherry-SERT was homogenously distributed. This may be due to the level of expression, since in several cells which showed this mCherry-SERT distribution, also EGFP fluorescence intensity was increased compared to other cells (Fig. 4A-C). Zooming in on a few cells expressing EGFP and mCherry-SERT showed that SERT is localized at the membrane, as mCherry fluorescence appeared like a ring at the membrane. Moreover, mCherry-SERT also seemed to associate with intracellular organelles, as some mCherry-SERT appeared as puncta in the cytoplasm. (Fig. 4D-F). Expressing mCherry-SERT together with Piccolo C2A\\textsubscript{mut} resulted in cells in which mCherry-SERT was localized at the membrane, but also some cells in which mCherry-SERT displayed a more uniform distribution in the cytoplasm which might again be due to the level of expression (Fig. 4G-I). A typical example of three cells expressing both constructs showed that in one cell mCherry-SERT was localized at the membrane, and in the other cells mCherry-SERT displayed a more uniform distribution in the cytoplasm (Fig. 4J-L). Strikingly, membrane bound mCherry-SERT in Piccolo C2A\\textsubscript{mut} expressing cells displayed a more patchy\n\ndistribution than in Piccolo C2Awt expressing cells (Fig. 4M-O). A line scan profile of a typical cell co-expressing mCherry-SERT with Piccolo C2Awt showed that the highest level of fluorescence was at the membrane (Fig. 4P). In contrast, a line scan profile of a cell co-expressing mCherry-SERT with Piccolo C2Amut showed that fluorescence levels in the cytoplasm were comparable with fluorescence levels at the membrane (Fig. 4Q).\n\nThus, it appeared that co-expression of the Piccolo C2Amut with the rs2522833 SNP resulted in an altered distribution of mCherry-SERT at the membrane. These data suggest that Piccolo is not involved in the trafficking of SERT, but might be involved in regulating SERT membrane levels.\n\n**Discussion**\n\nPiccolo is a large protein, which is part of the active zone in the presynaptic terminal. There, Piccolo probably has a scaffolding role, but Piccolo also contains a PDZ domain and two C2 domains. Piccolo can be alternatively spliced, resulting in absence or presence of the C2B domain (Wang et al., 1999). Additionally, in the C2A domain a nine-residue sequence has been found which is subject of alternative splicing, resulting in variants of the C2A domain with different calcium affinities (Garcia et al., 2004). In this study we focused exclusively on the long C2A variant. The gene region in which Piccolo is localized has been linked to autism (Nabi et al., 2003). The SNP rs2522833 is localized in the C2A domain (Sullivan et al., 2008). The C2A domain has a low calcium affinity, suggesting an involvement in presynaptic plasticity processes (Gerber et al., 2001). It is currently unknown whether the SNP affects PIP$_2$ or Ca$^{2+}$ binding to the C2A domain.\n\n*Figure 4 next page. Expression of Piccolo C2Awt and C2Amut with mCherry-SERT in heterologous cells*\n\n(A-C) Expression of C2Awt–IRES-EGFP and mCherry-SERT in Neuro2A cells. (D-F) Blow up of white box in C shows that in the majority of cells mCherry-SERT is localized at the membrane. In the cytoplasm, mCherry-SERT displays a punctate distribution, suggesting association with intracellular organelles. (G-I) Expression of C2Amut–IRES-EGFP and mCherry-SERT in heterologous cells. (J-L) Blow up of white box #1 in I shows that in several Neuro2A cells expressing C2Amut–IRES-EGFP, mCherry-SERT seems to have a more uniform cytoplasmic distribution. (M-O) Blow up of white box #2 in I shows that in cells in which mCherry-SERT is localized at the membrane, mCherry-SERT is not uniformly distributed at the membrane, but instead is distributed in patches along the membrane. (P,Q) Line scan profiles of a typical cell expressing mCherry-SERT with C2Awt–IRES-EGFP or C2Amut–IRES-EGFP (lines in figures E and K respectively). Arrows indicate cell membrane. Scale bars: 20 µm in C and I, 10 µm in F and L and 5 µm in O.\n\n**EGFP**\n\n- **A**: EGFP expression in cells.\n- **B**: mCherry-SERT expression in cells.\n- **C**: Merge of EGFP and mCherry-SERT expressions.\n\n**C2Awt-IRESGFP**\n\n- **D**: EGFP expression in cells.\n- **E**: mCherry-SERT expression in cells.\n- **F**: Merge of EGFP and mCherry-SERT expressions.\n\n**C2Amut-IRESGFP**\n\n- **G**: EGFP expression in cells.\n- **H**: mCherry-SERT expression in cells.\n- **I**: Merge of EGFP and mCherry-SERT expressions.\n\n**P**: Fluorescence intensity profile across a cell with a nucleus.\n\n**Q**: Fluorescence intensity profile across a cell without a nucleus.\n\nThe regulation of SERT membrane localization and internalization is important in determining efficacy of 5-HT transmission. Increases in membrane localization of SERT increase 5-HT reuptake and thereby reduce extracellular levels of 5-HT and binding of 5-HT to postsynaptic 5-HT receptors. In contrast, internalization of SERT results in increased extracellular levels of 5-HT and increased postsynaptic activation of 5-HT receptors.\n\nProtein kinase C (PKC) reduces the expression level of SERT, presumably by altering the membrane localization of SERT (Qian et al., 1997). Indeed, application of phorbol esters results in increased internalization of SERT. In contrast, substrates of SERT, such as 5-HT, reduce PKC dependent SERT internalization, whereas SERT antagonists, such as antidepressants, block this 5-HT mediated reduction in SERT internalization (Ramamoorthy and Blakely, 1999). On the other hand, protein kinase G (PKG) activity increases the membrane localization of SERT (Zhu et al., 2004). Therefore, there is an extensive level of regulation of membrane expression of SERT. There is an indirect link between Piccolo and SERT; Syntaxin1A is known to interact with SERT, and to regulate SERT activity (Quick, 2003). The C2A domain of Synaptotagmin, which shares similarity with the C2A domain of Piccolo, interacts with Syntaxin1A (Shao et al., 1997). Therefore this makes it likely that also the C2A domain of Piccolo is able to interact with Syntaxin1A, and thus possibly with SERT.\n\nWe used heterologous Neuro2A cells, which do not express Piccolo endogenously, to study the effect of expression of both C2A variants on SERT localization. We showed that distribution of SERT at the membrane might be altered after co-expression with the mutant C2A variant. Previously it has been shown that DAT trafficking and internalization is not altered in heterologous cells upon Piccolo knockdown. However, upon treatment of cells with 3,4-methylenedioxy-N-methylamphetamine (MDMA), there is a significant increase in DAT internalization. Thus, Piccolo seems to regulate the MDMA dependent internalization of DAT. For SERT, it has been shown that application of antidepressants and the PKC activator β-phorbol 12-myristate 13-acetate (β-PMA) results in an increased internalization of SERT (Lau et al., 2008a; Lau et al., 2008b). In view of this, it would be interesting to challenge SERT with an antidepressant after expression of both C2A variants, to investigate whether this results in an altered distribution of SERT between the two C2A variants.\n\nSeveral studies show that the expression level, membrane level, or transport activity of SERT might be affected in psychopathological processes. Levels of SERT expression in DRN are reduced in brains of depressed suicide victims (Arango et al., 2001). In contrast, long-term administration of antidepressants seemed to also reduce SERT surface expression (Hirano et al., 2005). Recent\n\nstudies showed that a SNP in SERT which associated with obsessive compulsive disorder resulted in increased transport activity of SERT (Kilic et al., 2003). Thus, further studies are required to investigate the relation between the SNP in Piccolo, SERT expression and activity, and major depression disorder.\n\nEXPERIMENTAL PROCEDURES\n\nPlasmids\n\nHuman SERT (hSERT) cDNA tagged with ECFP at the 5' end in the Clontech ECFP-C1 vector was a kind gift from Dr. H. Sitte. To clone a red fluorescent version of hSERT we replaced ECFP for the red fluorescent protein mCherry. To this end mCherry was digested out of the vector using AseI and BsrGI and cloned sticky in the ECFP-hSERT vector from which ECFP had been excised. For construction of pSuper-CMV-EGFP-shRNA-Piccolo, pSuper was digested using EcoRI and PstI and CMV-EGFP was inserted after isolation of this fragment from pEGFP-C3 (Clontech) using BsrGI and NsiI. The shRNA against Piccolo was constructed using the following primers: forward 5'-GATCCCCACCCCAAAATATAAAGCTTTCAAGAGAAGCCTTATATTITGTGGGTTTTTTTGGAAGA-3' and reverse 5'-AGCTTTTCTCAAAGCTTTATATTGTGGGGTGGG-3'. These primers were annealed by heating to 100°C, cooling down to room temperature and inserted in pSuper-CMV-EGFP by digesting this vector with BglII and HindIII. For construction of C2Awt, this fragment was isolated from a Yeast-2-Hybrid mouse cDNA bank using the following primers: forward 5'-GGCGTGGATCCCAAGCAGCTG-3' and reverse 5'-GTTTTGACTGGAGTGGAGACTG-3' and subsequently a PCR was performed using the following primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3' to add BamHI and NotI sites. For C2Amut (S468A), the C2A domain was isolated using two PCR reactions using the following primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-CGAGGAGTGTTGTCCAGATGAGCAGTACTAGATAAAATCAATCAATAC-3' and forward 5'-GTATGATGTGATTATCTAGTACTGCTCATCTGGACAACACTCCTCG-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3'. These two PCR products were annealed and a PCR was performed on this product using primers: forward 5'-AAGGATCCGCCTCTCACCCAATTACAGGA-3' and reverse 5'-TTTCGGGCCGATGCTTTCACTGCTGTITTTTCAG-3' to add BamHI and NotI sites and C2Awt and C2Amut fragments were ligated in pGEX. To construct pCMV-C2A-IRES2EGFP, both fragments were isolated from pGEX by\n\nPCR using the following primers: forward 5'-AAAGGATCCACATGGCCTCTCACCCCAATTACAGG-3' and reverse 5'-TTTGAATTCTCAGCTTTTCAGTCTGTCTTTTCAG-3' to add BamHI and EcoRI sites and a Kozak and stop sequence. Fragments were inserted in pIRES2EGFP by ligating this vector with BglII and EcoRI.\n\n**Cell culture and transfections**\n\nHippocampi from embryonic day 18 C57/Bl6 mouse embryos were dissected free of meninges and collected in Hanks’ Balanced Salt Solution (HBSS; Invitrogen) supplemented with 7 mM HEPES (Invitrogen). Hippocampi were digested with 0.25% trypsin (Invitrogen) in HBSS supplemented with 7 mM HEPES for 15 minutes at 37°C. After digestion tissue was washed three times with HBSS supplemented with 7 mM Hepes and subsequently triturated with a fire-polished glass pipette. Dissociated neurons were plated at a density of 25,000 cell/well on rat glial cells on 18 mm glass coverslips (Menzel Glaser, Braunschweig, Germany) in 12 well plates. Cells were cultured in neurobasal medium (Invitrogen) supplemented with B27 supplement (Invitrogen), 18 mM HEPES, 0.5 mM Glutamax (Invitrogen) and penicillin/streptomycin (Invitrogen). Once a week half of the medium was replaced. The hippocampal cultures were transfected using Lipofectamine 2000 (Invitrogen). Before transfection, half of the neurobasal medium was collected from each well and replaced with new neurobasal medium. For one well, 1 µl of Lipofectamine was added to 25 µl of serum free glutamax (Invitrogen) medium and incubated for 5 minutes. Subsequently, from each construct 1 µg of DNA was added to 25 µl of serum free glutamax and immediately mixed with the 25 µl of serum free glutamax containing Lipofectamine. This mixture was incubated for 30 minutes before adding to the cells. After 4–6 hours of incubation on the cells, half of the neurobasal medium was replaced again with the earlier collected neurobasal medium. Cells were routinely transfected at DIV2–7 and imaged at DIV10–14.\n\n**Immunocytochemistry**\n\nFor immunocytochemistry, neuronal cells were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) (pH 7.4) for 20 minutes at room temperature. Cells were washed in PBS and blocked in PBS containing 4% fetal calf serum and 0.2% Triton X-100 for 20 minutes at room temperature. Cells were incubated overnight with primary antibodies at 4°C in PBS containing 0.2% Triton X-100. As primary antibodies monoclonal MAP2 (Chemicon), and polyclonal Piccolo (Synaptic Systems) and Synapsin (Eo28) were used. After incubation in primary antibodies, cells were washed three times using PBS. Subsequently, cells were incubated in Alexa Fluor-conjugated secondary antibodies (Invitrogen) in PBS\n\nfor one hour at room temperature. All antibodies were used in a 1:1000 dilution. Finally, cells were washed three times in PBS and mounted using Dabco-Mowiol.\n\nFor immunohistochemistry on mouse brain slices, mice were anaesthetized and transcardially perfused with 4% paraformaldehyde (PFA) in PBS (pH 7.4). Brains were post fixed in PFA for one day, and subsequently cryoprotected in increasing concentrations of sucrose. Thirty μm coronal adult brain slices were made using a Cryostat (Leica). Midbrain slices containing the dorsal and medial raphe nucleus were blocked for two hours in PBS containing 10% normal goat serum and 0.5% Triton X-100. Subsequently, the slices were incubated overnight in primary antibodies in PBS containing 0.5% Triton X-100 at 4°C. Brain slices were washed three times two hours in PBS and incubated in secondary antibodies in PBS overnight at 4°C. After washing three times two hours, slices were mounted using Dabco-Mowiol. As primary antibodies monoclonal Tph (Sigma, recognizing both Tph2 and Tph2) and polyclonal Piccolo (Synaptic systems) were used in a 1:1000 dilution. Alexa Fluor-conjugated secondary antibodies (Invitrogen) were used in a 1:1000 dilution. Cells and slices were examined on a Zeiss LSM 510 confocal laser scanning microscope (Zeiss, BV The Netherlands).\n\n**Live cell imaging**\n\nCells were imaged in a chamber perfused with Tyrode’s solution (119 mM NaCl, 2.5 mM KCl, 2 mM MgCl$_2$, 2 mM CaCl$_2$, 30 mM glucose, 25 mM HEPES, pH 7.4) on an Axiovert II Microscope (Zeiss, Oberkochen, Germany) equipped with a Coolsnap HQ camera (Photometrics, Tucson, Arizona, USA) and a Polychrome IV illumination unit (TILL photonics, Graefling, Germany). Images were acquired in MetaMorph 6.2 software (Universal Imaging, Downingtown, Philadelphia, USA) using a 100x oil objective.\n\n**Image analysis**\n\nTo analyze the trafficking of mCherry-hSERT, image stacks were converted to .avi movie files using ImageJ software. Movies were analyzed using the Fluotrack software program (Broeke et al., 2008) to quantify the average number of moving puncta, the average velocity of moving puncta, the average pausing time of moving puncta and the direction of moving puncta.\n\n**References**\n\nArango V, Underwood MD, Boldrini M, Tamir H, Kassir SA, Hsiung S, Chen JJ, Mann JJ (2001) Serotonin 1A receptors, serotonin transporter binding and\n\nserotonin transporter mRNA expression in the brainstem of depressed suicide victims. *Neuropsychopharmacology* 25:892-903.\n\nBroeke JH, Ge H, Dijkstra IM, Cemgil AT, Riedl JA, Niels Cornelisse L, Toonen RF, Verhage M, Fitzgerald WJ (2008) Automated quantification of cellular traffic in living cells. *J Neurosci Methods*.\n\nCases-Langhoff C, Voss B, Garner AM, Appeltaner U, Takei K, Kindler S, Veh RW, De Camilli P, Gundelfinger ED, Garner CC (1996) Piccolo, a novel 420 kDa protein associated with the presynaptic cytomatrix. *Eur J Cell Biol* 69:214-223.\n\nCastrén E (2005) Is mood chemistry? *Nat Rev Neurosci* 6:241-246.\n\nCen X, Nitta A, Ibi D, Zhao Y, Niwa M, Taguchi K, Hamada M, Ito Y, Ito Y, Wang L, Nabeshima T (2008) Identification of Piccolo as a regulator of behavioural plasticity and dopamine transporter internalization. *Mol Psychiatry* 13:349, 451-363.\n\nFenster SD, Garner CC (2002) Gene structure and genetic localization of the PCLO gene encoding the presynaptic active zone protein Piccolo. *Int J Dev Neurosci* 20:161-171.\n\nFenster SD, Chung WJ, Zhai R, Cases-Langhoff C, Voss B, Garner AM, Kaempf U, Kindler S, Gundelfinger ED, Garner CC (2000) Piccolo, a presynaptic zinc finger protein structurally related to bassoon. *Neuron* 25:203-214.\n\nGarcia J, Gerber SH, Sugita S, Sudhof TC, Rizo J (2004) A conformational switch in the Piccolo C2A domain regulated by alternative splicing. *Nat Struct Mol Biol* 11:45-53.\n\nGerber SH, Garcia J, Rizo J, Sudhof TC (2001) An unusual C(2)-domain in the active-zone protein piccolo: implications for Ca(2+) regulation of neurotransmitter release. *Embo J* 20:1605-1619.\n\nHirano K, Seki T, Sakai N, Kato Y, Hashimoto H, Uchida S, Yamada S (2005) Effects of continuous administration of paroxetine on ligand binding site and expression of serotonin transporter protein in mouse brain. *Brain Res* 1053:154-161.\n\nKilic F, Murphy DL, Rudnick G (2003) A human serotonin transporter mutation causes constitutive activation of transport activity. *Mol Pharmacol* 64:440-446.\n\nLau T, Horschitz S, Bartsch D, Schloss P (2008a) Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy. *Neurochem Int*.\n\nLau T, Horschitz S, Berger S, Bartsch D, Schloss P (2008b) Antidepressant-induced internalization of the serotonin transporter in serotonergic neurons. *Faseb J* 22:1702-1714.\n\nLeal-Ortiz S, Waites CL, Terry-Lorenzo R, Zamorano P, Gundelfinger ED, Garner CC (2008) Piccolo modulation of SynapsinIa dynamics regulates synaptic vesicle exocytosis. *J Cell Biol* 181:831-846.\n\nMurphy DL, Lerner A, Rudnick G, Lesch KP (2004) Serotonin transporter: gene, genetic disorders, and pharmacogenetics. *Mol Interv* 4:109-123.\n\nNabi R, Zhong H, Serajee RJ, Huq AH (2003) No association between single nucleotide polymorphisms in DLX6 and Piccolo genes at 7q21-q22 and autism. *Am J Med Genet B Neuropsychiatr Genet* 119B:98-101.\n\nPickel VM, Joh TH, Reis DJ (1976) Monoamine-synthesizing enzymes in central dopaminergic, noradrenergic and serotonergic neurons.\n\nImmunocytochemical localization by light and electron microscopy. J Histochem Cytochem 24:792-306.\n\nQian Y, Galli A, Ramamoorthy S, Risso S, DeFelice LJ, Blakely RD (1997) Protein kinase C activation regulates human serotonin transporters in HEK-293 cells via altered cell surface expression. J Neurosci 17:45-57.\n\nQuick MW (2003) Regulating the conducting states of a mammalian serotonin transporter. Neuron 40:537-549.\n\nRamamoorthy S, Blakely RD (1999) Phosphorylation and sequestration of serotonin transporters differentially modulated by psychostimulants. Science 285:763-766.\n\nSerretti A, Calati R, Mandelli L, De Ronchi D (2006) Serotonin transporter gene variants and behaviour: a comprehensive review. Curr Drug Targets 7:1659-1669.\n\nShao X, Li C, Fernandez I, Zhang X, Sudhof TC, Rizo J (1997) Synaptotagmin-synaptaxin interaction: the C2 domain as a Ca2+-dependent electrostatic switch. Neuron 18:133-142.\n\nShapira M, Zhai RG, Dresbach T, Bresler T, Torres VI, Gundelfinger ED, Ziv NE, Garner CC (2003) Unitary assembly of presynaptic active zones from Piccolo-Bassoon transport vesicles. Neuron 38:237-252.\n\nSullivan PF, de Geus EJ, Willemsen G, James MR, Smit JH, Zandbelt T, Arolt V, Baune BT, Blackwood D, Cichon S, Coventry WL, Domschke K, Farmer A, Fava M, Gordon SD, He Q, Heath AC, Heutink P, Holstboer F, Hoogendijk WJ, Hottenga JJ, Hu Y, Kohli M, Lin D, Lucae S, Macintyre DJ, Maier W, McGhee KA, McGuffin P, Montgomery GW, Muir WJ, Nolen WA, Nothen MM, Perlis RH, Pirlo K, Posthuma D, Rietschel M, Rizzu P, Schosser A, Smit AB, Smoller JW, Tzeng JY, van Dyck R, Verhage M, Zitman FG, Martin NG, Wray NR, Boomsma DI, Penninx BW (2008) Genome-wide association for major depressive disorder: a possible role for the presynaptic protein piccolo. Mol Psychiatry.\n\nTao-Cheng JH (2007) Ultrastructural localization of active zone and synaptic vesicle proteins in a preassembled multi-vesicle transport aggregate. Neuroscience 150:575-584.\n\ntom Dieck S, Sammarti-Vila L, Langnaese K, Richter K, Kindler S, Soyke A, Wex H, Smalla KH, Kampf U, Franzer JT, Stumm M, Garner CC, Gundelfinger ED (1998) Bassoon, a novel zinc-finger CAG/glutamine-repeat protein selectively localized at the active zone of presynaptic nerve terminals. J Cell Biol 142:499-509.\n\nWang X, Kibschull M, Laue MM, Lichte B, Petrasch-Parwez E, Kilimann MW (1999) Acczonin, a 550-kD putative scaffolding protein of presynaptic active zones, shares homology regions with Rim and Bassoon and binds profilin. J Cell Biol 147:151-162.\n\nZhai RG, Vardinon-Friedman H, Cases-Langhoff C, Becker B, Gundelfinger ED, Ziv NE, Garner CC (2001) Assembling the presynaptic active zone: a characterization of an active one precursor vesicle. Neuron 29:131-143.\n\nZhu CB, Hewlett WA, Feoktistov I, Biaggioni I, Blakely RD (2004) Adenosine receptor, protein kinase G, and p38 mitogen-activated protein kinase-dependent up-regulation of serotonin transporters involves both transporter trafficking and activation. Mol Pharmacol 65:1462-1474.\n\nChapter 6\n\nDeletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and early postnatal lethality\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1}, R.F.T. Toonen\\textsuperscript{1}, Neuro-BSIK Mouse Phenomics consortium and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\nSubmitted\n\nAbstract\n\nThe serotonin (5-HT) system densely innervates many brain areas and is important for proper brain development. To specifically ablate the 5-HT system we generated mutant mice carrying a floxed Munc18-1 gene and Cre recombinase driven by the 5-HT-specific serotonin reuptake transporter (SERT) promoter. Mutant mice were smaller than their control littermates and the majority died within a few days after birth. Immunohistochemical analysis of brains of these mice showed that initially 5-HT neurons are formed and the cortex is innervated with 5-HT projections. From embryonic day (E) 16 onwards, however, 5-HT neurons started to degenerate and at postnatal day (P) 2 hardly any 5-HT projections were present in the cortex. The 5-HT system of mice heterozygous for the floxed Munc18-1 allele was indistinguishable from control mice. Behavioural analysis of these heterozygous mice showed that there is no difference in basal behaviour, conditioning and anxiety-related behaviour compared to control mice. These data show that deletion of Munc18-1 in 5-HT neurons results in rapid degeneration of the 5-HT system and suggests that the 5-HT system is important for postnatal survival.\n\nIntroduction\n\nThe 5-HT system consists of clusters of cell bodies in the midbrain raphe nuclei, with the largest clusters in the median raphe nucleus (MRN) and the dorsal raphe nucleus (DRN). Several brain areas receive dense 5-HT innervation and 5-HT is released both synaptically and as volume transmission (Ridet et al., 1993; Bunin and Wightman, 1998). Due to this and to the several 5-HT receptor subtypes which are present in the brain, 5-HT has many roles and influences many processes in the brain (Jacobs and Azmitia, 1992).\n\nNeurogenesis of 5-HT neurons in the mouse brain occurs in the ventral rhombencephalon around E10 (Wallace and Lauder, 1983). One day later, 5-HT neurons begin to synthesize and secrete 5-HT and start growing out axons. Around birth, target areas such as the forebrain and the hippocampus are densely innervated with 5-HT projections. Only after birth, the maturation of the 5-HT network is completed.\n\nSeveral studies have addressed the role of 5-HT on the development of the 5-HT system and brain development. In a conditional Lmx1b knockout (KO) mouse, almost all 5-HT neurons fail to survive, resulting in a significant decrease in brain tissue 5-HT levels (Zhao et al., 2006). However, these mice do not show\n\nan overt phenotype and survive to adulthood (Zhao et al., 2006). In contrast, it was shown that maternal 5-HT is required for embryonic development (Cote et al., 2007). Furthermore, in tryptophan hydroxylase 2 (Tph2) KO mice 5-HT neurons are completely devoid of 5-HT, but the morphology and neurite distribution of the 5-HT system is not affected, and these mice do not show any behavioural phenotype (Gutknecht et al., 2008). Neonatal depletion of 5-HT by the neurotoxin 5,7-Dihydroxytryptamine results in rather subtle changes in behavioural response and brain development (Hohmann et al., 2007; Bennett-Clarke et al., 1995).\n\nIn this study we silenced the 5-HT system by conditional deletion of Munc18-1 in 5-HT neurons. Neurons that lack the presynaptic protein Munc18-1 have a complete absence of neurotransmitter secretion and these mice are born paralyzed and die immediately after birth (Verhage et al., 2000). In these mice, initially synapses are formed and the assembly of the brain is normal. However, in later stages of brain development there is massive neuronal cell death and brain degeneration (Verhage et al., 2000).\n\nIn SERT-Cre\\textsuperscript{wt} Munc18-1\\textsuperscript{lox/lox} mice 5-HT neurons were initially generated and 5-HT projections innervated the midbrain and cortex, later followed by degeneration and loss of 5-HT projections in the cortex. The majority of these mice died within a few days after birth. These data suggest that the 5-HT system contributes importantly to postnatal brain development.\n\n**Results**\n\n*Deletion of Munc18-1 in SERT expressing neurons results in postnatal lethality*\n\nWe have shown previously that Munc18-1 mutant mice lack regulated secretion and have a lethal phenotype (Verhage et al., 2000). Therefore, we have generated Munc18-1\\textsuperscript{lox/lox} mutant mice in order to conditionally delete Munc18-1. Crossing these mice with a L7-Cre line, with Cre expressed in Purkinje neurons in the cerebellum, resulted in mice which developed severe ataxia, suggesting a cerebellar phenotype (Heeroma et al., 2004). Here we crossed Munc18-1\\textsuperscript{lox/lox} mice with SERT-Cre mice, which express Cre in SERT expressing neurons. These are the 5-HT neurons in the raphe nuclei, but also some hippocampal neurons and thalamocortical neurons which express SERT transiently during development (Zhuang et al., 2005; Narboux-Neme et al., 2008). Crossing SERT-Cre mice with Munc18-1\\textsuperscript{lox/lox} mice results in mice in which Munc18-1 is specifically removed in SERT expressing neurons (Fig. 1A). We crossed SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} mice with SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt} mice which should result in 12.5% of offspring which have SERT-Cre\\textsuperscript{cre/wt} and Munc18-1\\textsuperscript{lox/lox}\n\ngenotypes. Genotyping mice sacrificed at E16 or E18 revealed that there were more SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} and SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/lox} mice and less SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} mice than expected (p = 0.007) (Fig. 1B). Genotyping 101 mice three weeks after birth revealed that genotype frequencies were not distributed at Mendelian ratio (p = 0.027). Only three SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice were found, whereas based on Mendelian ratio the expected number of SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice should be 13, giving a 77% mortality rate in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice (Fig. 1C). Also, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice were smaller compared to littermates (data not shown). These three remaining SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice all died in the fourth postnatal week. This shows that Munc18-1 deletion in SERT expressing neurons results in postnatal lethality.\n\nFigure 1. Postnatal lethality in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice\n\n(A) Overview of breeding approach. Crossing SERT-Cre with Munc18-1\\textsuperscript{lox/lox} mice results in deletion of Munc18-1 only and specifically in SERT expressing neurons, whereas all other neurons still express Munc18-1.\n\n(B) Genotypes of mice sacrificed at E16 or E18 differed from the expected Mendelian ratio. (C) Genotyping mice after weaning at 3 weeks of age revealed that only few SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice survived up to three weeks.\n\n5-HT neurons are initially generated but then quickly degenerate\n\nTo assess the effect of Munc18-1 deletion in 5-HT neurons on the development of the 5-HT system, we made coronal sections of paraformaldehyde (PFA) fixed brains at different developmental stadia and performed immunohistochemistry for 5-HT. As developmental time points we chose E16, E18 and P2. Expression of SERT, and thus of Cre recombinase, starts at E11 and the earliest recombination observed in the DRN in SERT-Cre mice was at E12.5 (Narboux-Neme et al., 2008). Since it will take some days before all remaining Munc18-1 mRNA and protein is degraded, we chose E16 as the first time point. We first focused on the 5-HT cell bodies in the DRN. Immunohistochemistry for 5-HT on brain slices from E16 SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} and control mice showed that in the DRN 5-HT neurons are present and these showed the characteristic DRN topology (Fig. 2A,B). The number of 5-HT neurons did not differ from control (control 312 ± 17.67, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 305 ± 25.43; Fig. 2G,H,O). However, morphological analysis of the neurons showed that these were already degenerating, as assessed by the round morphology and reduced number of primary neurites. At E18, there was a ~80% decrease in the number of 5-HT neurons in SERT-Cre\\textsuperscript{cre/wt} M18\\textsuperscript{lox/lox} mice compared to control (control 358 ± 10.82, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 59 ± 4.84, Fig. 2C,D,O). At P2 there were only few 5-HT neurons left in the DRN (control 364 ± 19.68, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 25 ± 2.89, Fig. 2E,F,O). At E18 and P2 the remaining 5-HT neurons in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} brain sections displayed an altered morphology compared to control 5-HT neurons (Fig. 2I,J,K,L). 5-HT neurons in control brains had a characteristic fusiform or ovoid shape and grew out several primary neurites (Fig. 2M). In contrast, the mutant 5-HT neurons had a rounded morphology and the majority did not contain primary neurites, indicative of degenerating neurons (Fig. 2N). This showed that between E16 and P2 there is a massive degeneration of 5-HT neurons in the DRN.\n\n5-HT projections innervate the DRN and cortex but are degenerated at P2\n\nNext, we focused on the innervation of the midbrain and cortex with 5-HT projections. Midbrain and cortex are the first regions which are densely innervated with 5-HT projections during development. We quantified 5-HT neurite density first in DRN midbrain sections at E16, E18 and P2. At E16 the sections from both control and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice contained several 5-HT neurites and there was no significant difference in 5-HT innervation density (1.31 ± 0.51% and 0.63 ± 0.24% respectively, Fig. 3A,B). At E18, in control sections the 5-HT innervation density was slightly increased compared to E16 (Fig. 3C). However, in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections the 5-HT innervation density was decreased compared to control (control 2.32 ± 0.86%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 0.31 ± 0.27%, Fig. 3D).\n\nFigure 2. Rapid degeneration of 5-HT neurons in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice\n\nWe compared the number of 5-HT cell bodies in the DRN in control and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice at E16, E18 and P2. (A-F) At E16, in both control mice (A) and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} (lox/lox) mice (B) the 5-HT cell bodies are distributed in the DRN topology. At E18 and P2, however, in midbrain sections from SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} brains (D, F respectively) only few 5-HT neurons are present compared to control sections (C, E respectively). (G-L) Zooming in on 5-HT cell bodies shows that in control sections at E16 (G), E18 (I) and P2 (K) the cell bodies grow out several neurites and have a fusiform or ovoid morphology. However, remaining 5-HT cell bodies in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections at E16 (H), E18 (J) and P2 (L) have a round morphology with hardly any neurites. (M,N) Blow up of some 5-HT cell bodies in control and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice sections shows the differences in 5-HT cell body morphology. (O) Analysis of the number of 5-HT cell bodies in the sections showed that there is no difference at E16, but at E18 and P2 there is a decrease of 70% to 80% in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections. Scale bars: 200 µm in F, 50 µm in L and 20 µm in N. Data shown are mean ± standard error of the mean (SEM). * p < 0.05.\n\nAt P2 only few 5-HT neurites are left in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections (control 1.6 ± 0.41%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 0.33 ± 0.14%, Fig. 3E,F). Next, we focused on the 5-HT innervation density in the cortex. This revealed that already at E16 there is a reduction in 5-HT innervation density in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} sections (control 3.27 ± 1.14%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 0.65 ± 0.19%, Fig. 3G,H). At E18 and P2 the reduction in 5-HT innervation density is augmented, with only very few 5-HT neurites left at P2 in the cortex (E18 control 1.73 ± 0.45%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} 0.36 ± 0.18%, P2 control\n\n1.78 ± 0.52%, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} 0.19 ± 0.04%, Fig. 3I,J,K,L). Quantification showed that in the midbrain sections the 5-HT innervation density was reduced to ~20% and ~10% at E18 and P2 respectively (Fig. 3M). In the cortex, the 5-HT innervation density was reduced to ~20%, ~10% and ~5% at E16, E18 and P2 respectively (Fig. 3N).\n\nIn summary, deletion of Munc18-1 in 5-HT neurons resulted in rapid degeneration of 5-HT cell bodies and the absence of 5-HT projections in the postnatal brain.\n\n**Figure 3. Degeneration of 5-HT neurites in DRN and cortex**\n\n(A-F) At E16 in the midbrain containing the DRN, there was no difference in 5-HT neurite density between control (A) and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} (B) mice. In control sections at E18 (C) and P2 (E) several 5-HT neurites were present. However, in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} sections at E18 (D) and P2 (F) only very few 5-HT neurites are left. (G-L) In the cortex already at E16 there is a reduction in 5-HT neurite density in sections from SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} (H) mice compared to control (G). At E18 (J) and P2 (L), in cortical sections from SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsubscript{lox/lox} mice only very few remaining 5-HT neurites are present, in contrast to E18 (I) and P2 (K) control cortical sections. (M) Quantification of the 5-HT neurite density in the DRN revealed that at E16 there is no difference, but at E18 and P2 5-HT neurite density is reduced to ~20% and ~10% respectively. (N) In cortical sections, at E16 the 5-HT neurite density is reduced to ~20% and at E18 and P2 the 5-HT neurite density is reduced to ~10% and ~5% respectively. Scale bar 50 µm in L. Data shown are mean ± SEM * p < 0.05.\n\nBasal behaviour is not affected in SERT-Cre\\textit{er/cut} Munc18-1\\textit{fl/ut} mice\n\nFinally, we were interested whether deletion of one allele of Munc18-1 in SERT expressing cells affected mouse behaviour. Previously, we have shown that Munc18-1 expression levels regulate the readily releasable pool (RRP) size. In Munc18-1 heterozygous mice there is a faster synaptic rundown during episodes of high activity, a decreased RRP size and a decrease in docked vesicles at the synapse (Toonen et al., 2006). Therefore it is likely that deletion of one functional Munc18-1 allele in SERT expressing neurons affects the efficacy of 5-HT neurotransmission.\n\nFigure 4. Deletion of one functional allele of Munc18-1 in SERT expressing neurons does not affect number of 5-HT cell bodies and 5-HT innervation density\n\n(A-D) Midbrain sections containing the DRN from control (A) and hz (B) mice shows that 5-HT neurons are distributed in the characteristic DRN topology. Zooming in on 5-HT cell bodies show that both in control (C) and hz sections (D) these neurons have a fusiform or ovoid morphology and grow out several neurites. (E-H) 5-HT neurite density is not different between control and hz mice in the midbrain containing the DRN (E and F respectively) or in cortical sections from control and hz mice (G and H respectively). (I) Number of 5-HT cell bodies per section does not differ between control and hz. (J) 5-HT neurite density in midbrain containing DRN and cortical sections is not different between control and hz. Scale bars: 200 µm in B, 50 µm in D and H.\n\nFirst, we investigated whether deletion of one allele of Munc18-1 in SERT expressing neurons affected the number of 5-HT cell bodies in the DRN, or the 5-HT fiber density in the DRN and cortex. Analysis of midbrain sections from control and SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} (hz) mice showed that 5-HT neurons are distributed in the characteristic DRN topology, and control and hz are indistinguishable (Fig. 4A,B). 5-HT cell bodies in both control and hz had a fusiform or ovoid shape (Fig. 4C,D). Quantification of the number of 5-HT cell bodies in the DRN revealed no difference between control and hz (control 364 ± 14.78, hz 361 ± 15.01, Fig. 4I). Likewise, the 5-HT neurite density in the midbrain and cortex were similar (midbrain control 4.38 ± 0.93%, hz 4.67 ± 0.55%; cortex control 2.18 ± 0.35%, hz 2.87 ± 0.99%, Fig. 4E-H,I,J). Thus, deletion of one allele of Munc18-1 does not affect 5-HT neuronal survival, 5-HT neurite outgrowth or 5-HT projection density.\n\nWe tested several behavioural characteristics of hz mice by monitoring them for six days in home cages equipped with a video camera on top to visualize and analyze mouse behaviour. We analyzed the behaviour of eleven hz mice and compared several behavioural parameters with eleven control mice. First, we analyzed whether there was a difference in total distance moved in the open space and time spent under the shelter. Control and hz mice moved a similar total distance in the open space (control dark phase 2231.78 ± 139.39 cm/hour, light phase 483.91 ± 32.30 cm/hour; hz dark phase 2150.61 ± 112.70 cm/hour, light phase 433.69 ± 26.15 cm/hour; Fig. 5A,B), spent a similar proportion of time in a sheltered compartment (control dark phase 58.99 ± 2.03%, light phase 86.37 ± 2.36%; hz dark phase 60.04 ± 1.11%, light phase 90.38 ± 0.45%; Fig. 5C) and spent a similar time drinking (control dark phase 33.92 ± 1.99 s/hour, light phase 7.50 ± 0.52 s/hour; hz dark phase 41.06 ± 4.72 s/hour, light phase 8.06 ± 0.79 s/hour; Fig. 5D). Also, time spent in the feeder was indistinguishable between control and hz (control dark phase 476.77 ± 15.17 s/hour, light phase 160.55 ± 6.58 s/hour; hz dark phase 497.73 ± 18.41 s/ hour, light phase 165.33 ± 6.99 s/hour; Fig. 5E). Thus, basal behavioural parameters were indistinguishable between control and hz.\n\n\\textit{Conditioning and anxiety related behaviour is not affected in SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} mice}\n\nTo investigate whether hz mice display altered conditioning behaviour, a free operant conditioning task was performed, where a jump on the shelter resulted in the drop of a sucrose pellet. The number of directed short movement bouts (SMBs) increased during day four and five compared to day 1-3, but this conditioning behaviour did not differ between control and hz.\n\nFigure 5. Hz mice do not display altered basal behaviour\n\n(A) Six day plot of average distance moved (cm / 12 hours) during dark and light phase. Note the increase in distance moved during the dark phase at day 4 due to the free operant conditioning test. (B) No difference in average distance moved (cm/hour) between control and Hz mice during the dark and light phase. (C) Proportion of time spent under the shelter does not differ between control and Hz mice. (D) There is no difference in time spent drinking (s/hour) between control and Hz mice. (E) Also, there is no difference in time spent in the feeder (s/hour). Data shown are mean ± SEM.\n\n(day 1-3 control $17.07 \\pm 2.12$, hz $16.33 \\pm 2.17$; day 4-5 control $45.3 \\pm 5.27$, hz $46.32 \\pm 5.78$; Fig. 6A). Number of pellets dropped during pre-training phase (session 1-10, day four) did not differ between control and hz mice (control $4.88 \\pm 0.43$, hz $5.99 \\pm 0.45$; Fig. 6B). Also during the sessions at day five (session 11-12), there was no difference in the number of pellets dropped (control $20.36 \\pm 2.81$, hz $19.95 \\pm 2.34$; Fig. 6B).\n\nTo assess whether hz mice displayed altered anxiety related behaviour, a one trial anxiety task was performed at day six. To this end a lightspot was created at the feeder place, and the duration of time the animal spent in the lightspot and the feeder was recorded and compared with the time the animal spent at the feeder without a lightspot on. At day five (baseline) and day six (test) there was no difference between control and hz, but both groups spent less time in the lightspot at day six (day five control $1798.72 \\pm 208.1$ s, hz $2108.01 \\pm 284.63$ s; day six control $668.76 \\pm 61.28$ s, hz $682.19 \\pm 83.98$ s; Fig. 6C). Next, we investigated whether the frequency of entering the feeder zone at day five (baseline) and day six (test) was different. Frequency of entering the feeder zone did not differ between control and hz mice, but both groups displayed a decreased frequency during the lightspot test during day six (day five control $16.64 \\pm 2.51$, hz $23.09 \\pm 3.51$; day six control $7.15 \\pm 1.40$, hz $8.12 \\pm 1.56$; Fig. 6D). Also, time spent in the feeder zone during day five and day six did not differ between control and hz (day five control $528.15 \\pm 50.21$ s, hz $621.91 \\pm 65.99$ s; day six control $191.01 \\pm 30.36$ s, hz $198.46 \\pm 33.53$ s; Fig. 6E). Finally, analysis of latency to enter the lightspot zone after lightspot off revealed that this did not differ between control and hz (control $1567.71 \\pm 377.86$ s; hz $1293.87 \\pm 329.17$ s; Fig. 6F). Thus, mice in which one allele of Munc18-1 is deleted in SERT expressing neurons do not display altered basal, conditioning or anxiety-related behaviour.\n\n**Discussion**\n\nIn this study, we showed that SERT-Cre$^{cre/cre}$wt Munc18-1$^{lox/lox}$ mice display a postnatal lethality phenotype, accompanied by a rapid degeneration of the 5-HT system. Inactivation of one allele of Munc18-1 in SERT expressing neurons does not affect 5-HT cell number or 5-HT innervation density, and does not affect anxiety related behaviour or conditioning related behaviour.\n\nCrossing Munc18-1$^{lox/lox}$ mice with SERT-Cre mice results in deletion of Munc18-1 from SERT expressing cells. It was shown previously that crossing SERT-Cre mice with loxSTOPlox-EYFP mice results in >99% of 5-HT neurons in the DRN which are EYFP positively labeled (Zhuang et al., 2005).\n\nFigure 6. Hz behaviour on free operant conditioning test and one trial anxiety test does not differ from control\n\n(A) The number of directional SMB’s is increased at day 4 and 5 compared to day 1-3, but does not differ between control and hz. (B) Number of sucrose pellets acquired is increased at session 11 and 12 (day 5), compared to session 1-10 (day 4, pre-training day), but does not differ between control and hz. (C) Time spent in the lightspot (s) is decreased at day 6 (lightspot on) compared to day 5 (baseline, without lightspot on), but does not differ between control and hz. (D) Frequency of entering the feeder zone is decreased at day 6 compared to day 5, but there is no difference between control and hz mice. (E) During lightspot on on day 6 mice spent less time in the feeder (s) which does not differ between control and hz. (F) No difference in latency (s) to enter the lightspot zone after lightspot off. Data shown are mean ± SEM.\n\nHowever, SERT is also transiently expressed in some other brain regions during development, such as the thalamocortical neurons (Lebrand et al., 1996). In SERT-Cre ROSA-loxstoplox-LacZ double positive mice, strong LacZ expression was not only observed in the DRN, but also in the thalamus, cingulate cortex and\n\nin the CA3 region of the hippocampus (Zhuang et al., 2005; Narboux-Neme et al., 2008). Therefore, in SERT-Cre Munc18-1\\textsuperscript{lox/lox} mice also in some non-5-HT neurons Munc18-1 will be deleted. Genotyping sacrificed mice at E16 or E18 revealed that there were more SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice than expected, showing that these mice do not die during embryonic development. However, genotyping mice three weeks after birth showed that the majority of SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice had died, and the remaining SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice died within the fourth postnatal week. SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice were smaller compared to control mice which is in analogy with the VMAT2 KO mice which are hypoactive, smaller compared to control littermates, do not feed and die within a few days after birth (Fon et al., 1997; Wang et al., 1997).\n\nPreviously we showed that in Munc18-1 KO mice, the neuromuscular junction initially develops, but at later stages most of the motor neuronal cell bodies in the spinal cord degenerate (Heeroma et al., 2003). In analogy with this, in the absence of regulated secretion the hypothalamo-neurohypophysial system is initially normally formed, but in later stages of development degenerates (Korteweg et al., 2004). This is consistent with our observation that Munc18-1 is dispensable for initial generation and outgrowth of 5-HT neurons, but required for their survival.\n\nSince also in some other neurons Munc18-1 is deleted, it is unclear whether the observed phenotypes can be attributed to the degeneration of the 5-HT system. In Tphz2 KO mice, 5-HT neurons are completely devoid of 5-HT, yet there are no alterations in 5-HT neuron morphology and 5-HT neurite distribution (Gutknecht et al., 2008). Although these mice can survive into adulthood, approximately 50% does not survive the first 4 weeks and surviving mice have growth retardation (Alenina et al., 2009). Additionally, in a conditional Lmx1b KO mouse, 5-HT neurons are initially formed but almost all central 5-HT neurons fail to survive and brain 5-HT levels are reduced to ~10% (Zhao et al., 2006). Remarkably, these mice display normal locomotor behaviour and show no abnormalities in gross brain morphology. Recently, it was shown that these mice have severe apnea and have a ~20% mortality rate during the neonatal period, although the majority of these mice survive beyond P28 (Hodges et al., 2008; Hodges et al., 2009).\n\nApparently, even the absence of central 5-HT synthesis or an almost complete removal of 5-HT neurons does not result in a severe (postnatal) lethality phenotype. Thus, although several of these mice display high (postnatal) mortality, the phenotypes observed are not as severe as in the SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice.\n\nOn the other hand, mice which lack GAP43, display disrupted barrel cortex formation and thalamocortical connections fail to form. These mice display a\n\npostnatal lethality phenotype, with 50% of homozygotes which die between P0 and P2, and >95% of homozygotes which die before P21 (Maier et al., 1999). Although these mutant mice display a reduced 5-HT innervation in the cortex and hippocampus, they have no significant difference in the number of 5-HT neurons, and other areas such as the piriform cortex and amygdale receive normal 5-HT innervation (Donovan et al., 2002). Thus, based on these data it is unclear whether the postnatal lethality phenotype observed in this study is attributable to a degeneration of thalamocortical or 5-HT neurons. It would be very interesting to delete the Munc18-1 gene specifically in 5-HT neurons using the Pet-Cre transgenic line, in which expression of Cre is restricted to 5-HT neurons (Scott et al., 2005). This approach could be used to investigate whether the phenotype we observed could indeed be attributed to degeneration of the 5-HT system rather than degeneration of thalamocortical neurons.\n\nWe have shown that mice in which one allele of Munc18-1 is deleted in SERT expressing neurons did not display altered basal, conditioning or anxiety-related behaviour. In these mice number of 5-HT neurons and 5-HT neurite density was indistinguishable from control mice. However, we have shown previously in neurons cultured from Munc18-1 heterozygous mice that there is a subtle secretion phenotype (Toonen et al., 2006). Thus, one could argue that possibly deletion of one functional allele of Munc18-1 affects 5-HT release and thus efficacy of 5-HT neurotransmission.\n\nThere could be a few explanations why reduced 5-HT neurotransmission in the heterozygous mice used in this study does not affect behaviour. First of all, the reduction in 5-HT neurotransmission might just be too subtle to affect mouse behaviour. In Munc18-1\\(^{-/-}\\) neuronal cultures, there is a ~25% reduction in synaptic vesicle release (Toonen et al., 2006). In contrast, expression of tetanus toxin light chain in 5-HT neurons in vivo results in a robust (>90%) inhibition in 5-HT neurotransmission and these mice display decreased anxiety-related behaviour and enhanced associative learning (Kim et al., 2009). This suggests that a substantial reduction in 5-HT release is required to affect behaviour and that deletion of only one allele of Munc18-1 is not sufficient to achieve this reduction in 5-HT release.\n\nSecondly, in Munc18-1 null mice synaptic vesicle release is completely abolished. However, at least in chromaffin cells lacking Munc18-1, there is still some large dense core vesicle (LDCV) release, although this is reduced to ~10% (Voets et al., 2001). In Munc18-1 heterozygous mice LDCV release is presumably largely unaffected. In the 5-HT system, the bulk amount of 5-HT is released from LDCVs, which contain up to ~17 times as much 5-HT molecules as a single synaptic vesicle (Bruns and Jahn, 1995). Thus, if 5-HT released from synaptic vesicles is more severely affected than 5-HT released from LDCVs, there is only a\n\nminor reduction in release of 5-HT and thus in efficacy of 5-HT neurotransmission. Finally, (minor) reductions in extracellular 5-HT levels might be compensated for, such as by reduced SERT levels at the synaptic membrane and/or increased expression of postsynaptic 5-HT receptors, to guarantee proper levels of 5-HT neurotransmission.\n\nIn conclusion, deletion of one allele of Munc18-1 in 5-HT neurons does not affect mouse behaviour presumably due to the fact that there is a too subtle reduction in 5-HT neurotransmission in these mice. However, deletion of both alleles of Munc18-1 in SERT expressing neurons results in a rapid degeneration of the 5-HT system and postnatal lethality.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Laboratory animals*\n\nGeneration and characterization of SERT-Cre mice has been described (Zhuang et al., 2005). Briefly, immediately upstream of the SERT translational start codon a cassette containing Cre and a FRT-flanked neomycin cassette was inserted. To generate conditional Munc18-1 mice (Heeroma et al., 2004), floxed Munc18-1 mice were generated by insertion of LoxP sites flanking the second exon using homologous recombination. To obtain SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/wt} mice were crossed with SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt} mice. Pregnant females were sacrificed by cervical dislocation. Mouse embryos were obtained by caesarean section of pregnant females from timed matings. To investigate the effect of deletion of Munc18-1 in the 5-HT system on the development of the 5-HT system, SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{lox/lox} mice were used. SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/lox}, SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt}, or SERT-Cre\\textsuperscript{cre/wt} Munc18-1\\textsuperscript{wt/wt} littermates were not different from SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{wt/wt} mice and were used as controls. SERT-Cre mice were genotyped as has been previously described (Zhuang et al., 2005). For genotyping of Munc18\\textsuperscript{lox/lox} mice the following primers were used: 5’-ttgtggtcgaatggccagtag-3’, 5’-cctgtatgggtactgttcgttcaactaaataa-3’ and 5’-tctgaaccttgaggccagtcgagacacag-3’. Animals were housed and bred according to institutional and Dutch guidelines.\n\n*Immunohistochemistry*\n\nFor immunohistochemical analysis of 5-HT, embryonic mouse brains were dissected and immediately fixed by immersion in 4% PFA in phosphate buffered saline (PBS, pH 7.4). For analysis of 5-HT neurons and projections in adult brains, mice were anaesthetized and transcardially perfused with 4% PFA in PBS. For post-fixation, brains were incubated in 4% PFA in PBS overnight. For cryoprotection, brains were incubated in increasing concentrations of sucrose.\n\nSubsequently, coronal slices of 40 µm were made. For immunohistochemistry, brain slices were first incubated in blocking buffer containing PBS supplemented with 0.5% Triton X-100 and 10% normal goat serum for two hours. Subsequently, slices were incubated overnight with primary antibodies in PBS containing 0.5% Triton X-100 at 4°C in under gentle agitation. The next day, the slices were washed three times two hours in PBS and incubated with secondary antibodies in PBS for one hour under gentle agitation. Finally, slices were washed again three times two hours in PBS and mounted in Dabco-Mowiol for analysis. All procedures were performed at room temperature unless otherwise stated. Polyclonal anti-5-HT (1:1000) (Immunostar/Diasorin) was used as the primary antibody, and as a secondary antibody GAR-546 (1:1000) was used (Invitrogen). To analyze number of 5-HT cell bodies in DRN sections, at least three sections per experimental group were imaged using a CLSM 510 microscope. Number of 5-HT cell bodies was manually counted in the slices. For analysis of 5-HT fiber density in DRN and cortex a Z-stack of at least three positions per group was made. To analyze the 5-HT fiber density, the Z-stacks were projected in a single image and binarized. The 5-HT fiber density was quantified as the area in the image occupied by 5-HT neurites compared to the total area.\n\nMouse behaviour\n\nBehavioural analysis of mice was tested in the Phenotyper® (Noldus Information Technology, Wageningen, The Netherlands). For the home cage test, one animal per cage was tested for six days. Eleven SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt} mice were used in the experimental group and eleven SERT-Cre\\textsuperscript{wt/wt} Munc18-1\\textsuperscript{lox/wt} mice were used as a control group. Each cage contains a top unit equipped with built-in hardware for videotracking, containing a digital infrared sensitive video camera and infrared lights. Using this method the animal behaviour can be continuously assessed, both in the light and in the dark phase. Ethovision 3.0 software was used as videotracking software. The phenotyper cages (30x30x35 cm) were made of transparent Perspex walls with an aluminium floor, and a feeding station and a water bottle attached to the outside. To the waterspout a lickometer was attached to determine the total duration the animal is touching the waterspout. A shelter unit (diameter: 9 cm, height: 10 cm, non-transparent material) was placed in the cage, which was defined as a hidden zone in Ethovision, and the software program could distinguish between in the shelter and on the shelter. Videotracking was performed at a rate of 12.5 samples per second and the following parameters were determined: average velocity of movement, total distance moved in the open space, proportion of time spent in the feeder, proportion of time spent in the shelter, and time spent drinking.\n\nThese parameters were calculated in two hour bins for both the dark and light phase. For the analysis of movement and velocity, only movement was scored when the mouse moved at a velocity of 3.5 cm/s and higher, averaged over twelve samples. Furthermore, total proportion of time spent in a zone of the home cage (on shelter, in shelter, feeder, pellet or waterspout), frequency of zone visits and frequency on the shelter during the dark period was quantified.\n\nOn day four and five a free operant conditioning task was performed as follows. The number of SMBs was quantified, which is a directed movement of the animal from one point in the cage to another. A distinction is made between raw and directional SMBs; When the animal moves with a higher velocity than the threshold value, which is determined by dividing the total distance moved by the total duration of movement, the SMB is considered to be a directional SMB. When the animal is moving at a lower velocity, it is considered as a raw SMB. Thus, in a directional SMB the animal is either moving with a greater velocity, or is taking a shorter route. At day four and five, when the mice gets on its shelter, this will result in the drop of a sucrose pellet. Thus, the mouse ‘learns’ that it has to jump onto its shelter in order to receive a new sucrose pellet.\n\nOne day six a one trial anxiety test was performed as follows. Fifteen minutes after the change from light to dark phase, a lightspot is on the feeder, thus creating a lightspot zone. The lightspot lasts for three hours, and the total amount of time the animal is in the lightspot zone in this period is recorded and compared with a baseline reference. The baseline reference is acquired by recording the amount of time the animal is in the feeder zone at day five at the corresponding hours, without a lightspot on.\n\n**Statistical analysis**\n\nData were analyzed using SPSS 17.0. For analysis of number of 5-HT cell bodies in DRN and 5-HT neurite density in DRN and cortex, data was analyzed using the independent samples t-test. To analyze whether observed genotype frequencies differed significantly from a Mendelian ratio, a one-way chi-square test was performed. Mouse behaviour parameters were analyzed using the non-parametric Wilcoxon test after checking for normal distribution. Data shown are mean ± SEM. Significance level was set at p <0.05.\n\n**References**\n\nAlenina N, Kikic D, Todiras M, Mosienko V, Qadri F, Plehm R, Boye P, Vilianovitch L, Sohr R, Tenner K, Hortnagl H, Bader M (2009) Growth retardation and altered autonomic control in mice lacking brain serotonin. Proc Natl Acad Sci U S A 106:10332–10337.\n\nBennett-Clarke CA, Lane RD, Rhoades RW (1995) Fenfluramine depletes serotonin from the developing cortex and alters thalamocortical organization. Brain Res 702:255-260.\n\nBruns D, Jahn R (1995) Real-time measurement of transmitter release from single synaptic vesicles. Nature 377:62-65.\n\nBunin MA, Wightman RM (1998) Quantitative evaluation of 5-hydroxytryptamine (serotonin) neuronal release and uptake: an investigation of extrasynaptic transmission. J Neurosci 18:4854-4860.\n\nCote F, Fligny C, Bayard E, Launay JM, Gershon MD, Mallet J, Vodjdani G (2007) Maternal serotonin is crucial for murine embryonic development. Proc Natl Acad Sci U S A 104:329-334.\n\nDonovan SL, Mamounas LA, Andrews AM, Blue ME, McCasland JS (2002) GAP-43 is critical for normal development of the serotonergic innervation in forebrain. J Neurosci 22:3543-3552.\n\nFon EA, Pothos EN, Sun BC, Killeen N, Sulzer D, Edwards RH (1997) Vesicular transport regulates monoamine storage and release but is not essential for amphetamine action. Neuron 19:1271-1283.\n\nGutknecht L, Waider J, Kraft S, Kriegebaum C, Holtmann B, Reif A, Schmitt A, Lesch KP (2008) Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2 knockout mice. J Neural Transm 115:1127-1132.\n\nHeeroma JH, Plomp JJ, Roubos EW, Verhage M (2003) Development of the mouse neuromuscular junction in the absence of regulated secretion. Neuroscience 120:733-744.\n\nHeeroma JH, Roelandse M, Wierda K, van Aerde KI, Toonen RF, Hensbroek RA, Brussaard A, Matus A, Verhage M (2004) Trophic support delays but does not prevent cell-intrinsic degeneration of neurons deficient for munc18-1. Eur J Neurosci 20:623-634.\n\nHodges MR, Wehner M, Aungst J, Smith JC, Richerson GB (2009) Transgenic mice lacking serotonin neurons have severe apnea and high mortality during development. J Neurosci 29:10341-10349.\n\nHodges MR, Tattersall GJ, Harris MB, McEvoy SD, Richerson DN, Denneris ES, Johnson RL, Chen ZF, Richerson GB (2008) Defects in breathing and thermoregulation in mice with near-complete absence of central serotonin neurons. J Neurosci 28:2495-2505.\n\nHohmann CF, Walker EM, Boylan CB, Blue ME (2007) Neonatal serotonin depletion alters behavioural responses to spatial change and novelty. Brain Res 1139:163-177.\n\nJacobs BL, Azmitia EC (1992) Structure and function of the brain serotonin system. Physiol Rev 72:165-229.\n\nKim JC, Cook MN, Carey MR, Shen C, Regehr WG, Dyamecki SM (2009) Linking genetically defined neurons to behaviour through a broadly applicable silencing allele. Neuron 63:305-315.\n\nKorteweg N, Maia AS, Verhage M, Burbach JP (2004) Development of the mouse hypothalamo-neurohypophysial system in the munc18-1 null mutant that lacks regulated secretion. Eur J Neurosci 19:2944-2952.\n\nLebrand C, Cases O, Adelbrecht C, Doye A, Alvarez C, El Mestikawy S, Seif I, Gaspar P (1996) Transient uptake and storage of serotonin in developing thalamic neurons. Neuron 17:823-835.\n\nMaier DL, Mani S, Donovan SL, Soppet D, Tessarollo L, McCasland JS, Meiri KF (1999) Disrupted cortical map and absence of cortical barrels in growth-associated protein (GAP)-43 knockout mice. Proc Natl Acad Sci U S A 96:9397-9402.\n\nNarboux-Neme N, Pavone LM, Avallone L, Zhuang X, Gaspar P (2008) Serotonin transporter transgenic (SERTcre) mouse line reveals developmental targets of serotonin specific reuptake inhibitors (SSRIs). Neuropharmacology 55:994-1005.\n\nRidet JL, Rajaofetra N, Teilhac JR, Geffard M, Privat A (1993) Evidence for nonsynaptic serotonergic and noradrenergic innervation of the rat dorsal horn and possible involvement of neuron-glia interactions. Neuroscience 52:143-157.\n\nScott MM, Wylie CJ, Lerch JK, Murphy R, Lobur K, Herlitze S, Jiang W, Conlon RA, Strowbridge BW, Deneris ES (2005) A genetic approach to access serotonin neurons for in vivo and in vitro studies. Proc Natl Acad Sci U S A 102:16472-16477.\n\nToonen RF, Wierda K, Sons MS, de Wit H, Cornelisse LN, Brussaard A, Plomp JJ, Verhage M (2006) Munc18-1 expression levels control synapse recovery by regulating readily releasable pool size. Proc Natl Acad Sci U S A 103:18332-18337.\n\nVerhage M, Maia AS, Plomp JJ, Brussaard AB, Heeroma JH, Vermeer H, Toonen RF, Hammer RE, van den Berg TK, Missler M, Geuze HJ, Sudhof TC (2000) Synaptic assembly of the brain in the absence of neurotransmitter secretion. Science 287:864-869.\n\nVoets T, Toonen RF, Brian EC, de Wit H, Moser T, Rettig J, Sudhof TC, Neher E, Verhage M (2001) Munc18-1 promotes large dense-core vesicle docking. Neuron 31:581-591.\n\nWallace JA, Lauder JM (1989) Development of the serotonergic system in the rat embryo: an immunocytochemical study. Brain Res Bull 10:459-479.\n\nWang YM, Gainetdinov RR, Fumagalli F, Xu F, Jones SR, Bock CB, Miller GW, Wightman RM, Caron MG (1997) Knockout of the vesicular monoamine transporter 2 gene results in neonatal death and supersensitivity to cocaine and amphetamine. Neuron 19:1285-1296.\n\nZhao ZQ, Scott M, Chiechio S, Wang JS, Renner KJ, Gereau RWt, Johnson RL, Deneris ES, Chen ZF (2006) Lmx1b is required for maintenance of central serotonergic neurons and mice lacking central serotonergic system exhibit normal locomotor activity. J Neurosci 26:12781-12788.\n\nZhuang X, Masson J, Gingrich JA, Rayport S, Hen R (2005) Targeted gene expression in dopamine and serotonin neurons of the mouse brain. J Neurosci Methods 143:27-32.\n\nChapter 7\n\nTowards a genetic approach to study serotonergic outgrowth and connectivity in vivo\n\nJ.J. Dudok\\textsuperscript{1}, A.J.A. Groffen\\textsuperscript{1}, J. Wortel\\textsuperscript{1} and M. Verhage\\textsuperscript{1}\n\n\\textsuperscript{1}Department of Functional Genomics, Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, the Netherlands\n\nAbstract\n\nAltered connectivity of the serotonin (5-HT) system might underlie psychopathological processes such as anxiety and depression. Therefore, it would be very interesting to be able to visualize all 5-HT fibers, axons or presynaptic terminals/varicosities in living mouse brain. To specifically label 5-HT neurons and fibers, our aim is to express Cre recombinase in the 5-HT system. We describe here the generation of a tryptophan hydroxylase 2 (Tph2)-iCre targeting construct, the targeting of mouse embryonic stem (ES) cells and the blastocyst injections of positively targeted clones. Moreover, to study 5-HT connectivity in vivo, we generated several transgenic lines using the loxSTOPlox (LSL) methodology.\n\nEventually, we hope to use these techniques to create mice in which specifically in the 5-HT system reporter genes are expressed, so that these can be used to study the 5-HT outgrowth and connectivity in vivo.\n\nIntroduction\n\nThe 5-HT system has its cell bodies in the midbrain raphe nuclei. Although only few 5-HT neurons are present, virtually every brain area receives dense 5-HT innervation which triggers signaling pathways through several different 5-HT receptors. For these reasons the 5-HT system influences a wide variety of processes. Moreover, the 5-HT system is implicated in major depressive disorder and anxiety. One of the current hypotheses for psychopathological processes such as anxiety and depression is that 5-HT connectivity might be affected. Therefore, it would be very interesting to study the connectivity of the 5-HT system in vivo.\n\nTo study 5-HT outgrowth and connectivity in vivo, our aim is to generate a mouse line with expression of improved Cre recombinase (iCre) specifically in 5-HT neurons. As locus for the iCre knockin, we selected the Tph2 gene, which is selectively expressed in the 5-HT neurons in the raphe nuclei (Patel et al., 2004; Zhang et al., 2005; Clark et al., 2006). To be able to specifically express transgenes in the 5-HT system, we relied on the Cre-loxP technique (Tsien et al., 1996; Nagy, 2000). We selected to use iCre instead of Cre, since iCre gives improved Cre expression due to the application of mammalian codon usage (Shimshek et al., 2002).\n\nIn addition we generated several transgenic lines that carry a ubiquitous promoter, a LSL sequence and synaptically or axonally targeted fluorescent fusion proteins. Our ultimate goal is to combine the Tph2-iCre knockin allele with these transgenes, which would constitute an ideal model to study 5-HT\n\nconnectivity and dynamics in the living mouse brain. Here we describe the generation of the Tph2-iCre targeting construct, and the generation of Tph2-iCre chimaeric mice. Additionally, we describe the generation of several LSL transgenic lines.\n\nResults\n\nConstruction and design of the Tph2-iCre targeting construct\nIn order to specifically express iCre in 5-HT neurons, we selected the locus for Tph2 which is localized at chromosome 10 D2. Tph2 is the rate limiting enzyme in 5-HT synthesis and is specifically expressed in 5-HT neurons in the brain. To generate mice with expression of iCre specifically in 5-HT neurons, our approach was to generate a targeting construct in which iCre cDNA is inserted at the endogenous Tph2 start codon. We designed the targeting construct in a way that the 1.2 kb short arm at the 5’ side of the construct stops just upstream of the endogenous Tph2 start codon. The 5.5 kb long arm was designed to start 53 bp downstream of the short arm, so that only 53 bp of endogenous DNA, including the endogenous Tph2 start codon, was deleted in case of homologous recombination (Fig. 1A). In between the short and long arm the iCreVenus cDNA was inserted and a neomycin phosphotransferase cassette (neo) for positive selection, flanked by FRT sites. These can be used to later on remove the neo cassette using the flippase/FRT system (O’Gorman et al., 1991).\n\nIn case of homologous recombination of the targeting construct in ES cells and subsequent generation of Tph2-iCre mice our approach was to use heterozygous Tph2-iCre mice. These contain one functional allele with Tph2 expression, whereas in the other allele, part of the first exon of Tph2 including the start codon is replaced for iCreVenus. In this case these mice will still have a functional Tph2 allele and expression of iCreVenus from the Tph2 promoter (Fig. 1B).\n\nGeneration of Tph2-iCre chimaeric mice\nAfter designing and constructing the Tph2-iCre targeting construct, the construct was sequence verified. This showed that the construct had been correctly made and that the iCreVenus cDNA contained no mutations. The linearized targeting construct was used for ES cell transfection. The antibiotic G418 was used for positive selection of the neomycin resistance marker (Gossler et al., 1986). Eventually five clones were found in which homologous recombination had occurred. Blastocyst injection of clone 1C11 resulted in several chimaeric mice. Although we performed several rounds of chimaeric breeding, we have unfortunately not achieved germline transmission of the Tph2-iCre allele until now.\n\n**Figure 1. Tph2-Cre knockin approach**\n\n(A) The targeting construct was created to replace the start codon of the first exon of Tph2 with a iCre cassette and a neomycin resistance cassette flanked with FRT sites. The targeting construct consisted of a short arm of 1.2 kb, a long arm of 5.5 kb and a iCreVenus and neo cassette. In case of homologous recombination only 53 bp of endogenous DNA is deleted and replaced for the iCreVenus cassette and the neomycin resistance cassette. To identify ES cell clones in which homologous recombination occurred, a PCR screening approach was used. A forward primer (arrow) outside the short arm of the targeting construct and a reverse primer (arrow) in the iCre cassette were used to amplify a 1.5 kb fragment of DNA in case of homologous recombination (dotted line). (B) Approach for Southern blot screening of ES cell clones. An inside and an outside probe were used (grey boxes). For the inside probe, genomic DNA is digested with BsrGI. This will result in a 5.3 kb fragment in WT DNA, but in a 3.3 kb fragment in clones in which homologous recombination occurred due to the presence of an additional BsrGI site in the targeting construct. For the outside probe, genomic DNA is digested with SwaI, which will result in a 14.2 kb fragment in WT DNA, but in a 9.8 kb fragment in clones in which homologous recombination occurred, due to the presence of an additional SwaI site in the targeting construct.\n\n**Generation of LSL transgene lines**\n\nFor construction of LSL transgene lines we used several marker genes tagged with either EGFP or mCherry, which is an improved variant of mRFP that matures more completely and is more photostable (Shaner et al., 2004). As marker genes we used SynaptophysinpHluorin (SypHy), TMSyxEGFP and TMSyxmCherry, which all specifically label axons and presynaptic terminals, and VenusKras4b which is a general membrane marker.\n\n**Figure 2. Validation of transgene constructs**\n\nTo investigate whether the LSL approach worked, we expressed LSL transgene construct with or without iCre cDNA in HEK293 cells. (A,B) No Expression of SypHy or TMSyxEGFP in HEK293 cells after transfection of LSL-SypHy or LSL-TMSyxEGFP without iCre. (C,D) After transfection of LSL-SypHy or LSL-TMSyxEGFP with iCre, SypHy and TMSyxEGFP were expressed in HEK293 cells respectively. (E,F) In neurons, VenusKras4b or TMSyxEGFP are not expressed when transfected without iCre. (G) Upon transfection of LSL-VenusKras4b with iCre, VenusKras4b is expressed in neurons, and the membrane is labeled, also in growth cones (inset). (H) Upon transfection of LSL-TMSyxEGFP with iCre, TMSyxEGFP is expressed in axons, as shown by the absence of MAP2 staining. Immunocytochemistry for the synaptic marker Synapsin shows that there is colocalization between TMSyxEGFP and Synapsin, showing that TMSyxEGFP is targeted to synapses. Scale bars: A-G 10 µm, inset in G 2 µm, H 5 µm.\n\nSypHy is the improved variant of Synaptophysin and detects secretion events due to the increase in pH in synaptic vesicles after exposure to the extracellular medium (Miesenbock et al., 1998; Granseth et al., 2006). TMSyxEGFP contains part of the transmembrane domain of SyntaxinA coupled to EGFP; TMSyxmCherry is the same but with a red fluorophore. VenusKras4b is the EYFP derived Venus fused with the 20 amino acid C-terminal plasma membrane targeting domain of Kras4b (Welman et al., 2000). As ubiquitous promoter either the chicken β-actin promoter or the Thy1 promoter was used (Aigner et al., 1995; Ludin et al., 1996). In between the promoter and the transgene, we inserted a floxed stop sequence in order to acquire iCre mediated transgene expression (Lakso et al., 1992). Some of the constructs were expressed in heterologous cells or neurons to test whether the floxed stop sequence prevented expression and whether co-transfection of iCre resulted in expression of the transgene. Transfection of β-actin-LSL-SypHy or Thy1-LSL-TMSyxEGFP alone in heterologous cells did not result in expression of the transgene (Fig. 2A,B). In contrast, after co-transfection with iCre, there was strong expression of\n\nboth transgenes (Fig. 2C,D). Also in neurons, expression of β-actin-LSL-VenusKras4b or Thy1-LSL-TMSyxEGFP alone did not result in detectable transgene expression (Fig. 2E,F). However, after co-expression with iCre, VenusKras4b expression was detected (Fig. 2G). After co-expression of iCre and Thy1-LSL-TMSyxEGFP neurons were found which expressed TMSyxEGFP and this localized exclusively to axons and synapses as shown by the absence of staining for the dendritic marker MAP2 and co-localization with the synaptic marker Synapsin (Fig. 2H).\n\nThese transgenic constructs were linearized and injected in oocytes to generate seven founder lines of β-actin-LSL-SypHy, five founder lines of β-actin-LSL-VenusKras4b, five founder lines of Thy1-LSL-TMSyxEGFP and eleven founder lines of Thy1-LSL-TMSyxmCherry.\n\n**Screening of expression of transgenes in founder lines**\n\nIn order to screen the expression of the transgenes per founder line, and to find out whether there are lines with expression of the transgene in the raphe nuclei, we first removed the LSL cassette by crossing the founder lines with the CMV-Cre mouse line, a line in which Cre is ubiquitously expressed (Schwenk et al., 1995).\n\nThis breeding strategy resulted in mice in which the LSL cassette was removed in all cells (Fig. 3A). This in contrast to crossing with Tph2-iCre mice, which is expected to result in removal of the LSL cassette only and specifically in 5-HT neurons. Thus, in 5-HT neurons the transgene will be expressed, whereas in all other cells the LSL is still present and the transgene is not expressed (Fig. 3B).\n\nAfter screening all transgenic founder lines, we found several lines in which the transgene was very low and mosaically expressed. For β-actin-LSL-SypHy we found one founder line with low expression in the Purkinje cell layer of the cerebellum, and two founder lines with expression in the hippocampal CA1-CA3 region and dentate gyrus (data not shown). For β-actin-LSL-VenusKras4b we only found very low and mosaic expression of the transgene (data not shown). For Thy1-LSL-TMSyxEGFP and TMSyxmCherry, we found one line with TMSyxEGFP ubiquitously expressed in neocortex, and one line with TMSyxmCherry mosaically expressed in cortex and hippocampus (data not shown). Unfortunately, not one founder line did show expression of the transgene in midbrain or raphe nuclei areas.\n\n**Discussion**\n\nHere we have described the generation of Tph2-iCre chimaeric mice and several LSL transgenic mouse lines. We would like to eventually create mice with expression of a marker gene specifically in 5-HT neurons. This would allow us to\n\nFigure 3. Approach for screening expression pattern of transgenic mice\n\n(A) To screen the expression pattern of LSL transgenic mice, these mice were crossed with a CMV-Cre mouse, which ubiquitously expressed Cre recombinase. This results in mice in which the LSL is removed in all cells, thus allowing an analysis of transgene expression pattern. (B) Once LSL mice are identified with expression in the 5-HT system, they can be used to cross with Tph2-Cre mice to drive transgene expression specifically in the 5-HT system.\n\nWe choose to target iCreVenus to the first exon of Tph2, so that the endogenous start codon and small part of the first exon of Tph2 is replaced for iCreVenus. In this way we anticipated that Tph2 expression relies on endogenous Tph2 promoter activity and is specifically expressed in 5-HT neurons in which Tph2 is expressed. In contrast to this approach we could also have chosen to target iCreVenus to the 3’ side of the Tph2 open reading frame using an internal ribosomal entry site (IRES). We did, however, not use this approach because the expression of a reporter gene behind an IRES is often rather low. Although using our approach we sacrifice one functional Tph2 allele, we expect that this will not significantly affect brain development and behaviour in Tph2 heterozygotes. Deletion of both Tyrosine hydroxylase alleles results in mid-gestational lethality.\n\nHowever, TH heterozygotes showed no obvious anatomical or behavioural defects (Zhou et al., 1995). Therefore, we reason that our approach will not significantly affect brain development or behaviour.\n\nUsing a transgenic approach with a LSL cassette to allow transgene expression only in cells in which iCre is also present, we aimed to express these marker genes specifically in 5-HT neurons. However, none of the founder lines we screened showed expression of the transgene construct in the midbrain or raphe nuclei. It has been shown for both the chicken β-actin and mouse Thy1 promoter that these are able to induce expression of transgenes in the cortex and hippocampus (Feng et al., 2000; Roelandse et al., 2003). However, it appears more difficult to also induce expression in midbrain and raphe nuclei for these promoters, since none of our transgenic lines expressed the transgene in these regions. Recently, it was shown that a 1.8 kb region immediately upstream the Pet-1 gene is sufficient to acquire 5-HT neuron specific expression of a transgene (Scott et al., 2005a; Scott et al., 2005b). Therefore, in new experiments we could try to rely on the Pet-1 promoter to express transgenes in the 5-HT neurons. Possibly, future experiments with novel blastocyst injections of Tph2-iCre targeted ES clones and transgene injections using the Pet-1 promoter will result in a Tph2-iCre mouse line and transgenic mouse lines with expression of reporter genes in 5-HT neurons. These mouse lines can then be used to create mouse lines with expression of reporter genes specifically in 5-HT neurons to possibly resolve the longstanding question whether psychopathological processes are a result of altered 5-HT network connectivity.\n\n**EXPERIMENTAL PROCEDURES**\n\n*Generation of Tph2-iCre targeting construct*\n\nThe Tph2-iCre targeting construct was designed in such a way that in the wildtype Tph2 locus, 53 bp in exon 1 are deleted, including the start codon, and replaced for a iCreVenus cassette. The Tph2-iCre targeting construct was generated as follows. The 1.2 kb short arm upstream the endogenous start codon of Tph2 was generated with PCR primers forward 5’CATGCATAGAAAGTGTCCAG3’ reverse 5’TTCTITCTCAGCAGCAGGGG3’. The 5.5 kb long arm starting 34 bp behind the Tph2 start codon was generated with PCR primers forward 5’GGGCCAGGAGAGGGTTGTCTCTTGGAITICTG3’ and reverse 5’CCGTACATGAGGACTCGGTGAGAGCATCTG3’. Both arms were subcloned in the pGEM-T easy vector (Promega). In the piCreVenus vector the NotI site behind Venus was removed and a linker containing SwaI, BsiWI, MluI and NotI sites was placed in the AflII site behind the polyA tail. The short arm was cloned sticky using EcoRI in front of iCreVenus in the modified iCreVenus vector. A neomycin resistance cassette flanked by FRT sites was removed from\n\nthe ploxPFRTNEOFRTloxP vector (a kind gift from Jeroen Pasterkamp, Utrecht) using partial digestion with BamHI and cloned blunt in the unique SpeI site behind the long arm in the pGEM-T easy vector. Finally, the FRTNEOFRT long arm Tph2 was cloned sticky behind the short arm iCreVenus using NotI. The construct was verified by sequence analysis. The construct was linearized with KspI for targeting.\n\n**Targeting ES cells**\n\nIn order to target ES cells with the Tph2-iCre targeting construct, approximately $1 \\times 10^6$ ES cells (line E14.1) were electroporated with 100 µg of linearized Tph2-Cre targeting construct and plated on murine embryonic feeders. The subsequent day, neomycin selection was started by adding 400 µg/ml G418. After 10 days, non targeted clones had died and 700 surviving clones were picked and individually grown. DNA of all these clones was screened to distinguish between clones in which the targeting construct was randomly inserted and clones in which homologous recombination had occurred.\n\nTo select for clones in which homologous recombination occurred, the clones were screened with a nested PCR approach using the following primers: 1st PCR forward 5’ CACTTTTCTTCACACCCITCTC3’ and reverse 5’ CCTGACTTTCATCAGAGGTGGCATC3’ nested PCR forward 5’ TTATTTGTGTGAGACACGTGA3’ and reverse 5’ TTTTGGTGCAACAGTCAGCAG3’ which results in a 1.6 kb PCR fragment in positively targeted clones. From the 700 targeted clones, eventually 5 clones were selected in which homologous recombination occurred designated 2F11, 1A1, 1C11, 4E1 and 2E11. Homologous recombination was verified using a 5’ primer outside the forbidden construct and sequence analysis of this region. ES cell clones 1C11 and 4E1 were injected in blastocysts to generate chimaeric mice.\n\n**Generation of transgenic mice**\n\nIn order to generate transgenic mice for conditional expression of reporter genes, the LSL cassette derived from the Pbs302 vector was used (Sauer, 1993). As promoter, the chicken β-actin and regulatory elements of the mouse Thy1 gene were used. As reporter genes, SypHy (a kind gift from Andreas Jeromin), VenusKras4b, TMSyx (transmembrane Syntaxin1A) EGFP and TMSyxmCherry were used. The constructs were cloned as follows. For β-actin-LSL-SypHy, the LSL cassette was excised from the Pbs302 vector using EcoRI and SpeI and cloned blunt in the unique XbaI site in the pβ-actin-SypHy vector. For β-actin-LSL-VenusKras4b, the LSL cassette was excised from the Pbs302 vector using EcoRI and SpeI and cloned blunt in the BglII site in the pVenusKras4b vector. Subsequently, LSL-VenusKras4b was excised using NheI and XbaI and cloned blunt in BlnI and XbaI in pβ-actin-EGFP to replace EGFP. Both β-actin-LSLSypHy and β-actin–LSL-VenusKras4b were linearized with AatII for transgene injection. For Thy1-LSL-TMSyxEGFP, TMSyxEGFP was excised with NheI and SspI and cloned blunt in the unique NotI site in pThy1-LSL (a kind gift from Christiaan Levelt, Amsterdam). To clone TMSyxmCherry, TMSyx was excised from pTMSyxEGFP using NheI and EcoRI and cloned blunt in pmCherry-N3 in the EcoXI site. Then TMSyxmCherry was excised using NheI and SspI and cloned blunt in the NotI site the in pThy1-LSL vector. Both Thy1-LSL-TMSyxEGFP and Thy1-LSL-TMSyxmCherry were linearized with AatII for transgene injection. To create transgenic mice, linearized DNA constructs were injected in the pronucleus of fertilized C57BL/6 oocytes.\n\n**Transfection of transgene plasmids in heterologous cells and neurons**\n\nTransgene plasmids were transfected in HEK293 cells or neurons using Lipofectamine 2000 reagent according to the manufacturer’s protocol. HEK293 cells and neurons were cultured as has been published before (Groffen et al., 2004; de Wit et al., 2006). In HEK293 cells, one day post-transfection expression of transgene constructs was visualized using an inverted microscope fitted with fluorescence filters. In neurons and HEK293 cells, expression of transgene constructs was analyzed using a LSM510 confocal laser scanning microscope (Zeiss, B.V. The Netherlands). Immunocytochemical analysis of neurons was performed as has been described before using monoclonal MAP2 (Chemicon) and polyclonal Synapsin I (E028) as primary antibodies and goat-anti-mouse Alexa 543/644 and goat-anti-rabbit Alexa 543 (Molecular Probes) (de Wit et al., 2006).\n\n**Screening of expression pattern**\n\nIn order to investigate the expression pattern of the transgene construct per transgenic line, the founder lines were crossed with the 129-Cre mouse line, which has ubiquitous expression of Cre (Schwenk et al., 1995). Pups positive for Cre and the transgene were used for in vivo paraformaldehyde (PFA) perfusion between two and four months age. For in vivo PFA fixation, mice were anaesthetized and transcardially perfused with 4% PFA dissolved in phosphate buffered saline (PBS) (pH 7.4). After fixation, brains were removed and postfixed in 4% PFA for 24 hours at 4°C. Subsequently, brains were cryoprotected in increasing concentrations of sucrose dissolved in PBS at 4°C. For screening of expression pattern of the transgenes, the fixed and cryoprotected brains were sagitally cut in 30 µm slices using a microtome. In case the EGFP fluorescence was too low, Immunohistochemistry was performed using the free floating method. To this end, slices were first blocked using blocking solution which contained 0.1% Triton X-100 and 10% normal goat serum for two hours under gentle agitation. Slices were incubated overnight at 4°C using primary antibody,\n\nwashed three times two hours the next day with PBS 0.1% Triton X-100 and incubated with the secondary antibody for two hours. After washing for three times two hours in PBS, slices were positioned on a glass slide, allowed to dry and overlaid with a glass coverslip using Dabco Mowiol. All reactions were carried out at room temperature unless otherwise stated.\n\n**Analysis of transgene expression**\n\nTo analyze the expression pattern of the transgene constructs, brain slices were analyzed using an inverted microscope equipped with fluorescence filters and a Sony camera. A LSM510 confocal laser scanning microscope was used to investigate the expression of the transgene constructs in individual neurons.\n\n**References**\n\nAigner L, Arber S, Kapfhammer JP, Laux T, Schneider C, Botteri F, Brenner HR, Caroni P (1995) Overexpression of the neural growth-associated protein GAP-43 induces nerve sprouting in the adult nervous system of transgenic mice. Cell 83:269–278.\n\nClark MS, McDevitt RA, Neumaier JF (2006) Quantitative mapping of tryptophan hydroxylase-2, 5-HT1A, 5-HT1B, and serotonin transporter expression across the anteroposterior axis of the rat dorsal and median raphe nuclei. J Comp Neurol 498:611–623.\n\nde Wit J, Toonen RF, Verhaagen J, Verhage M (2006) Vesicular trafficking of semaphorin 3A is activity-dependent and differs between axons and dendrites. Traffic 7:1060–1077.\n\nFeng G, Mellor RH, Bernstein M, Keller-Peck C, Nguyen QT, Wallace M, Nerbonne JM, Lichtman JW, Sanes JR (2000) Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41–51.\n\nGossler A, Doetschman T, Korn R, Serfling E, Kemler R (1986) Transgenesis by means of blastocyst-derived embryonic stem cell lines. Proc Natl Acad Sci U S A 83:9065–9069.\n\nGranseth B, Odermatt B, Royle SJ, Lagnado L (2006) Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron 51:773–786.\n\nGroffen AJ, Brian EC, Dudok JJ, Kampmeijer J, Toonen RF, Verhage M (2004) Ca(2+)-induced recruitment of the secretory vesicle protein DOC2B to the target membrane. J Biol Chem 279:23740–23747.\n\nKerr JN, Denk W (2008) Imaging in vivo: watching the brain in action. Nat Rev Neurosci 9:195–205.\n\nLakso M, Sauer B, Mosinger B, Jr., Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H (1992) Targeted oncogene activation by site-specific recombination in transgenic mice. Proc Natl Acad Sci U S A 89:6232–6236.\n\nLudin B, Doll T, Meili R, Kaech S, Matus A (1996) Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins. Gene 173:107–111.\n\nMiesenbock G, De Angelis DA, Rothman JE (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394:192–195.\n\nNagy A (2000) Cre recombinase: the universal reagent for genome tailoring. Genesis 26:99–109.\n\nO’Gorman S, Fox DT, Wahl GM (1991) Recombinase-mediated gene activation and site-specific integration in mammalian cells. Science 251:1351-1355.\n\nPatel PD, Pontrullo C, Burke S (2004) Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiatry 55:428-433.\n\nRoelandse M, Welman A, Wagner U, Hagmann J, Matus A (2003) Focal motility determines the geometry of dendritic spines. Neuroscience 121:39-49.\n\nSauer B (1993) Manipulation of transgenes by site-specific recombination: use of Cre recombinase. Methods Enzymol 225:890-900.\n\nSchwenk F, Baron U, Rajewsky K (1995) A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells. Nucleic Acids Res 23:5080–5081.\n\nScott MM, Krueger KC, Deneris ES (2005a) A differentially autoregulated Pet-1 enhancer region is a critical target of the transcriptional cascade that governs serotonin neuron development. J Neurosci 25:2628–2636.\n\nScott MM, Wylie CJ, Lerch JK, Murphy R, Lobur K, Herlitze S, Jiang W, Conlon RA, Strowbridge BW, Deneris ES (2005b) A genetic approach to access serotonin neurons for in vivo and in vitro studies. Proc Natl Acad Sci U S A 102:16472–16477.\n\nShaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22:1567–1572.\n\nShimshick DR, Kim J, Hubner MR, Spergel DJ, Buchholz F, Casanova E, Stewart AF, Seeburg PH, Sprengel R (2002) Codon-improved Cre recombinase (iCre) expression in the mouse. Genesis 32:19–26.\n\nTsien JZ, Chen DF, Gerber D, Tom C, Mercer EH, Anderson DJ, Mayford M, Kandel ER, Tonegawa S (1996) Subregion- and cell type-restricted gene knockout in mouse brain. Cell 87:1317–1326.\n\nWelman A, Burger MM, Hagmann J (2000) Structure and function of the C-terminal hypervariable region of K-Ras4B in plasma membrane targeting and transformation. Oncogene 19:4582–4591.\n\nZhang X, Gainetdinov RR, Beaulieu JM, Sotnikova TD, Burch LH, Williams RB, Schwartz DA, Krishnan KR, Caron MG (2005) Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45:11-16.\n\nZhou QY, Quaife CJ, Palmiter RD (1995) Targeted disruption of the tyrosine hydroxylase gene reveals that catecholamines are required for mouse fetal development. Nature 374:640-643.\n\nChapter 8\n\nGeneral discussion\n\nGeneral discussion\n\nThe aim of the studies described in this thesis was to investigate different cellular aspects of the outgrowth and connectivity of the brain 5-HT system. The 5-HT system has the remarkable feature that although there are only few 5-HT neurons, localized in the midbrain raphe nuclei, virtually every brain area receives dense 5-HT innervations. These innervations contain several 5-HT varicosities, where 5-HT is released both synaptically and as volume transmission. Due to several 5-HT receptors which are present in the brain, 5-HT has many functions and influences a wide variety of processes. Moreover, the 5-HT system is implicated in several psychopathological processes such as anxiety and depression. Despite the high social and economical importance of these conditions, there is a lot of uncertainty about the involved mechanisms. Therefore, a further cellular characterization of the outgrowth and connectivity of the 5-HT system is of high interest. We used several experimental approaches to study different cellular aspects of the outgrowth and connectivity of the 5-HT system.\n\nIn chapter 2 we used organotypic slice cultures to study 5-HT outgrowth and connectivity and the effect of pharmacological manipulations in vitro. This revealed that the 5-HT neurite density was not affected by blocking the SERT or application of a 5-HT$_{1A}$ agonist, but the density was reduced by application of a 5-HT$_2$ agonist. This was presumably due to inhibited outgrowth of the 5-HT system.\n\nIn chapter 3, we investigated the trafficking of SERT. We expressed SERT tagged with the fluorescent protein mCherry in hippocampal neurons. We showed that mCherry-SERT is transported in vesicles which displayed a dynamic trafficking.\n\nIn chapter 4 we used the same approach as in chapter 3, but now we studied the localization and trafficking of Tph2, the rate limiting enzyme in 5-HT synthesis. We tagged Tph2 with EGFP and studied the localization of Tph2-EGFP in hippocampal neurons. In axons Tph2-EGFP displayed a punctate distribution and co-localized with Synaptophysin-mCherry, a marker for synaptic terminals, suggesting that Tph2-EGFP localized to synaptic terminals.\n\nIn chapter 5 we studied the effect of a SNP in the presynaptic gene Piccolo on the trafficking and localization of SERT. Knockdown of Piccolo in neurons did not affect mCherry-SERT trafficking. The SNP in Piccolo possibly affects the localization of SERT, although future research is needed to further resolve this issue.\n\nIn chapter 6 we crossed SERT-Cre mice with floxed Munc18-1 mice to specifically abolish Munc18-1 in SERT expressing neurons. This resulted in early postnatal lethality and a rapid degeneration of the 5-HT system. We used heterozygous mice to study the effect of removal of one Munc18-1 allele in SERT expressing neurons on behaviour using a phenotyping experiment. Basal behaviour, conditioning and anxiety related behaviour were not affected.\n\nIn chapter 7 we describe our approach to create mice which have Cre expression specifically in 5-HT neurons. Moreover, we show that we have generated transgenic lines with a floxed stop cassette which can be used for spatial regulation of transgene expression upon Cre mediated recombination. In the future, these mice can be used to specifically label 5-HT neurons, or to specifically remove a gene in 5-HT neurons.\n\nRoles of 5-HT in outgrowth, connectivity and behaviour\n\nOne of the current hypotheses for psychopathological processes such as depression is that altered 5-HT network connectivity might be involved in these processes. Furthermore, it is hypothesized that antidepressants such as fluoxetine might restore aberrant 5-HT network connectivity (Castren, 2005). We used organotypic slices to study these processes in an in vitro model system. Our data showed that fluoxetine did not affect 5-HT innervation density.\n\nIn adult rats, chronic fluoxetine application did result in an increased density of 5-HT projections in the forebrain (Zhou et al., 2006). Interestingly, an in vivo study using chronic fluoxetine treatment either during development or during adulthood, showed that when applied during adulthood, fluoxetine did not affect behaviour. In contrast, fluoxetine treatment during development resulted in increased anxiety-related behaviour during adulthood (Ansorge et al., 2004; Ansorge et al., 2008). Our data suggests, although it is in vitro, that this effect of fluoxetine on behaviour does not result from an altered innervation of 5-HT projections. However, in our model system we were unable to investigate whether the number of 5-HT varicosities was affected by fluoxetine application. Thus, although 5-HT projection density is unaffected, connectivity of the 5-HT system might be affected due to an altered number of 5-HT varicosities or an altered rate of 5-HT release.\n\nAt least in the snail Helisoma, 5-HT acts as an autotrophic factor on its own outgrowth; 5-HT applied at µM concentrations to the growth cones of 5-HT neurons resulted in an abrupt cessation of growth cone motility and outgrowth (Haydon et al., 1984; Haydon et al., 1987).\n\nThe 5-HT\\textsubscript{IA} and 5-HT\\textsubscript{3C} receptor might mediate this effect of 5-HT, as these receptors are expressed in the DRN (Wright et al., 1995; Clemett et al., 2000). Our data showed that 5-HT\\textsubscript{IA} receptor activation slightly increased 5-HT neurite\n\ndensity whereas chronic activation of the 5-HT\\textsubscript{2} receptor decreased 5-HT neurite density. It is tempting to speculate that these receptors act in concert during outgrowth to regulate a proper development of 5-HT projections towards target areas mediated by 5-HT. Interestingly, 5-HT\\textsubscript{1A} and 5-HT\\textsubscript{2} receptors have different affinities for 5-HT; 5-HT\\textsubscript{1A} receptors are high affinity 5-HT receptors which have an EC\\textsubscript{50} value in the nM range, whereas 5-HT\\textsubscript{2} receptors are low affinity 5-HT receptors, which have an EC\\textsubscript{50} value in the \\(\\mu\\)M range (Fargin et al., 1989; Marek and Aghajanian, 1994; Azmitia, 2001). The 5-HT\\textsubscript{3C} receptor is present on GABA-ergic cells in the DRN, and it is unknown whether this receptor is present on 5-HT neurons (Serrats et al., 2005). This suggests that the inhibitory effect of 5-HT\\textsubscript{3C} receptor activation is not a direct effect on 5-HT neurons, but might be an indirect effect via GABA-ergic neurons. Indeed, in vivo data supports that 5-HT\\textsubscript{3C} receptor activation inhibits neuronal firing of 5-HT neurons via GABA-ergic neurons (Boothman et al., 2006). Thus, in the DRN \\(\\mu\\)M levels of 5-HT might inhibit outgrowth, whereas nM levels might stimulate outgrowth of 5-HT innervations.\n\nMice in which the 5-HT\\textsubscript{1A} receptor is deleted have increased anxiety-related behaviour, as they spend less time in the center in the open field test and avoid the open arms in the elevated plus maze (Parks et al., 1998; Ramboz et al., 1998). Overexpression of the 5-HT\\textsubscript{1A} receptor, on the other hand, results in reduced anxiety-related behaviour (Deng et al., 2007). A very elegant study was performed by Gross and colleagues, who showed that restoring 5-HT\\textsubscript{1A} receptor expression in the forebrain during development, but not during adulthood rescued the behavioural phenotype of 5-HT\\textsubscript{1A} KO mice. (Gross et al., 2002). Clearly, the 5-HT\\textsubscript{1A} receptor is important during development to establish normal anxiety-like behaviour. Unfortunately, no studies were performed to resolve whether 5-HT connectivity is affected in these mice.\n\nDeletion of the 5-HT\\textsubscript{3C} receptor, on the other hand, results in compulsive-like behaviour, and these mice are obese and suffer from seizures (Tecott et al., 1995; Chou-Green et al., 2003). Unfortunately, also for these mice it is not known whether 5-HT connectivity is affected.\n\n**Trafficking of SERT and Tph2 in neurons**\n\nAs the 5-HT system has long projections to several brain areas, proteins involved in 5-HT synthesis and reuptake are transported over large distances towards axonal growth cones and varicosities. In chapter 3 and 4 we focused on SERT and Tph2 transport in hippocampal neurons and observed that SERT and Tph2 displayed a distinct distribution. SERT was predominantly localized extrasynaptically and some SERT puncta localized in the vicinity of presynaptic terminals. In contrast, Tph2 accumulated in puncta which colocalized with\n\nsynaptic markers. Obviously, Tph2 is required at presynaptic terminals and varicosities for local synthesis of 5-HT. Indeed in 5-HT neurons Tph is enriched in presynaptic terminals (Pickel et al., 1976; Pickel et al., 1977). On the other hand, SERT is localized extra-synaptically to transport extracellular 5-HT which has diffused away from its release sites back into the neuron. An electron microscopy study showed that the majority of SERT in 5-HT neurons was located outside synapses (Zhou et al., 1998). Thus, the distribution of Tph2 and SERT which we found in hippocampal neurons seems to mimic the distribution of endogenous SERT and Tph2 in 5-HT neurons.\n\nWe also showed that SERT and Tph2 are transported in distinct ways through the neuron. In contrast to Tph2, SERT displayed a vesicular trafficking. Apparently, SERT contains a signal peptides which mediates the transport in vesicles. Alternatively, SERT contains transmembrane domains, which requires that SERT is transported in vesicles as these lipophilic domains are insoluble.\n\nAs SERT is transported in secretory vesicles, there should be one or more motor proteins responsible for this transport. There are several families of motor proteins which are involved in the transport of several different proteins through neurites (for a review see (Schlager and Hoogenraad, 2009). The average velocity of SERT transport vesicles and the fact that these do not display a preference for direction of movement is in accordance with the average velocity of transport vesicles containing glycine (Maas et al., 2006). Also in this study, it was shown that dynein motors are responsible for the retrograde transport of glycine (Maas et al., 2006). Dynein motors are involved in transporting other cargo molecules through neurons, such as the Trkb receptor and Bassoon (Yano et al., 2001; Fejtova et al., 2009). The trafficking dynamics of Trkb and Bassoon are comparable with the trafficking dynamics of SERT, strongly suggesting that the dynein motor protein is responsible for SERT transport (Ha et al., 2008; Fejtova et al., 2009).\n\n**A SNP in Piccolo associates with depression**\n\nRecently, a SNP in Piccolo was found which was associated with depression (Sullivan et al., 2008; Bochdanovits et al., 2009). Piccolo is a structural component of the presynaptic cytoskeletal cytomatrix (PCM) and structurally related to Bassoon, another component of the PCM (Cases-Langhoff et al., 1996; tom Dieck et al., 1998; Fenster et al., 2000). Piccolo is a multidomain zinc finger protein which contains several coiled coil domains and a PDZ domain (Fenster and Garner, 2002). Piccolo also contains two C2 domains, C2A and C2B, of which C2A functions as a low-affinity calcium sensor in presynaptic terminals (Gerber et al., 2001). The SNP in Piccolo was found to be closely localized to the C2A domain (Sullivan et al., 2008).\n\nIn the presynaptic terminal, Piccolo is involved in different processes. First of all, Piccolo, together with Bassoon, is involved in presynaptic terminal formation. Piccolo and Bassoon are transported in Piccolo-Bassoon transport vesicles together with other constituents of presynaptic terminals, and two or three of these vesicles are sufficient to form an active synapse (Zhai et al., 2001; Shapira et al., 2003; Tao-Cheng, 2007). In chapter 5 we have shown that 5-HT neurons contain Piccolo. Thus, in 5-HT varicosities Piccolo might be involved in formation of varicosities, regulating 5-HT secretion and regulation of SERT internalization. We have shown that knockdown of Piccolo did not affect SERT trafficking dynamics, suggesting that Piccolo is not involved in SERT transport. Secondly, since Piccolo is present in the presynaptic terminal, Piccolo might be involved in the modulation of synaptic vesicle exocytosis. Indeed, Piccolo influences synaptic function by negatively regulating synaptic vesicle exocytosis, possibly via a calmodulin kinase II-dependent mechanism, mediated through the modulation of Synapsin1A dynamics (Leal-Ortiz et al., 2008). Thus, Piccolo might be involved in modulating 5-HT release from synaptic vesicles. However, the majority of 5-HT is secreted as bulk release from large dense core vesicles (LDCVs) (Bruns and Jahn, 1995). Therefore, although it might be that a SNP in Piccolo could modulate 5-HT release from synaptic vesicles, this would probably only have minor effects on extracellular 5-HT levels.\n\nThirdly, it was shown in heterologous cells that Piccolo is involved in the regulation of DAT internalization. Specifically, the C2A domain was involved in this process (Cen et al., 2008). As DAT and SERT belong to the same gene family and are structurally related it is conceivable that Piccolo could be involved in SERT internalization. In chapter 5, we have investigated in heterologous cells whether the SNP in the C2A domain of Piccolo alters the localization of mCherry-SERT. This showed that expression of the C2A domain of Piccolo containing the SNP seemed to alter the membrane distribution of SERT compared to the control C2A variant. It would be very interesting to further investigate whether the SNP in the C2A domain affects varicosity density of 5-HT projections or regulation of SERT membrane localization. If so, this would be the first cellular evidence that altered 5-HT network connectivity might be involved in the etiology of depression.\n\n**Deletion of Munc18-1 in SERT expressing neurons results in early postnatal lethality**\n\nOur data in chapter 6 showed that deletion of Munc18-1 in SERT expressing neurons resulted in early postnatal lethality. Although neurons are initially formed and there is normal brain development, deletion of Munc18-1 eventually results in degeneration (Verhage et al., 2000; Heeroma et al., 2003; Korteweg et\n\nOur data showed that also 5-HT neurons in which Munc18-1 is deleted initially develop and grow out projections which innervated the cortex. However, after the initial generation, the 5-HT neurons undergo rapid degeneration, and at P2 only very few 5-HT neurons were left. This is the first mouse model which has a complete removal of the 5-HT system. This in contrast to the Tph2 KO mouse, which has no 5-HT synthesis in the central nervous system, but still 5-HT neurons and projections are unaffected.\n\nDeletion of Tph2 resulted in ~50% lethality in the first four weeks (Alenina et al., 2009). Surprisingly, however, another research group did not find any increased lethality in Tph2−/− mice ((Savelieva et al., 2008) and personal communication). Deletion of Pet1 and Lmx1b results in mice in which ~70% and >99% of 5-HT neurons fail to survive respectively. Concerning the marked reduction in 5-HT neurons and projections in these mice, it is surprising that these mice only display subtle phenotypes (Ding et al., 2003; Hendricks et al., 2003; Zhao et al., 2006)\n\nUnfortunately, as also thalamocortical neurons and some hippocampal neurons briefly express SERT, and thus Cre recombinase, during development, it is difficult to assess whether the observed phenotype is attributable to a degenerating 5-HT system. Therefore, crossing floxed Munc18-1 mice with Pet-Cre or Tph2-Cre mice which express Cre exclusively in 5-HT neurons should finally resolve whether complete removal of the 5-HT system affects brain development and survival.\n\n**Altered connectivity of the 5-HT system in human psychopathological processes?**\n\nDescriptive studies on the effect of psychopathological processes on human 5-HT connectivity rely on postmortem brain tissue. The last decade, however, more and more human imaging studies are performed using functional magnetic resonance imaging (fMRI) and positron emission tomographic (PET) imaging to resolve whether brain circuitry is affected in psychopathological processes associated with the 5-HT system.\n\nResearch on postmortem brain material from depressive people who committed suicide predominantly focused on mRNA expression levels or binding of radiolabeled 5-HT to 5-HT receptors. Several studies showed that there is an elevated 5-HT2A and 5-HT2C receptor abundance in the frontal cortex of depressed suicide victims (Arango et al., 1990; Xu and Pandey, 2000; Prasad et al., 2005). Other studies reported either reduced or increased binding or availability of 5-HT1A receptors, depending on the brain region studied (Drevets et al., 2007). From these data, however, it cannot be deduced whether the altered\n\nlevels of mRNA expression or altered levels of receptor binding are the result of an alteration in expression levels or of an altered 5-HT projection density.\n\nPET imaging is used to measure cerebral blood flow and rate of glucose metabolism as a measure for brain activity. Functional MRI is used to measure blood oxygen level dependent (BOLD) fluctuations which are a measure for activity and connectivity of brain regions. PET and fMRI studies have reported a decreased activation of cortical areas such as the dorsolateral prefrontal cortex and the anterior cingulate cortex in depressed subjects (Drevets et al., 1997; Mayberg et al., 1999). On the other hand, other studies reported an increased activation of limbic areas such as the amygdala in depression (Sheline et al., 2001; Siegle et al., 2002). Several studies have explored whether antidepressants restore aberrant activation patterns observed in depression. Antidepressant treatment increases glucose metabolism in prefrontal cortex and reduces activity in limbic areas (Sheline et al., 2001; Harvey et al., 2004b).\n\nAlthough it is difficult to conclude from these imaging studies if altered brain network connectivity is a cause or consequence of the depression, one could at least speculate that connectivity changes occur at some stage. Clearly, more research is needed to further elucidate whether network connectivity alterations underlie psychopathological processes associated with the 5-HT system.\n\n**Future perspectives**\n\nThere are a number of approaches which could be taken to further elucidate the connectivity of the 5-HT system, the relation between the 5-HT system and behaviour, and the involvement of the 5-HT system in psychopathology. First of all, it would be of great interest to label all 5-HT neurons and neurites with fluorescent markers. This could be achieved by creating knockin mice using the Tph2 promoter to drive expression of Cre and to cross these mice with floxed stop fluorescent reporter mice. Additionally, it has already been shown that Pet-Cre mice express Cre specifically in 5-HT neurons, and crossing with reporter mice showed that >99% of 5-HT neurons were labeled (Scott et al., 2005). These mice could then be used to reconstruct the connectivity of the 5-HT system. Secondly, there are several subpopulations of 5-HT neurons that can be distinguished based upon their precursor genes (Jensen et al., 2008). If Cre mouse lines could be made for these specific subpopulations, these could be crossed with reporter mice or with the so-called brainbow mice (Livet et al., 2007). It would be very interesting to find out whether different subpopulations of 5-HT neurons innervate different brain regions, and are involved in different aspects of behaviour.\n\nAdditionally, these mouse lines could be used to specifically inhibit neurotransmission from these populations of 5-HT neurons, or to inhibit neurotransmission from all 5-HT neurons using Tph2-Cre mice. Expressing tetanus toxin in neurons blocks synaptic activity since tetanus toxin cleaves VAMP2 which is a protein necessary for vesicle fusion. It has been shown that this approach works in vivo, e.g. in the retina, but also for 5-HT neurons (Kerschensteiner et al., 2009; Kim et al., 2009). The work of Kim et al showed that silencing a subset of 5-HT neurons resulted in mice with behavioural alterations (Kim et al., 2009). These mice could be used to further explore the role of the 5-HT system and subsets of the 5-HT system on behaviour and to what extend these contribute to pathophysiological processes such as anxiety and depression. The other way around, these mice could be used to study whether silencing of 5-HT neurons affects 5-HT connectivity, i.e. 5-HT projection and varicosity density.\n\nA third interesting future direction is to use mice which express Cre specifically in 5-HT neurons to inactivate 5-HT autoreceptors only in 5-HT neurons. A lot of research has been conducted in elucidating the role of 5-HT autoreceptors in determining 5-HT transmission and behaviour. The 5-HT\\textsubscript{IA} and 5-HT\\textsubscript{IB} knockout mice display altered anxiety-related and aggression-related behaviours. However, in these mice it is difficult to assess whether these phenotypes observed are due to deletion of 5-HT autoreceptors, or deletion of these receptors from non-5-HT neurons. A far more elegant approach, however, would be to generate floxed 5-HT\\textsubscript{IA} and 5-HT\\textsubscript{IB} mice and cross these with Pet-Cre or Tph2-Cre mice to selectively inactivate these receptors only in 5-HT neurons. This will allow to distinguish between the role of 5-HT autoreceptors and heteroreceptors, and to investigate to what extend the deletion of 5-HT autoreceptors affects mouse behaviour.\n\nRecently, also a tamoxifen inducible Tph2-Cre mouse line has been generated, which allows to temporally regulate Cre expression in 5-HT neurons (Weber et al., 2009). This mouse line allows to investigate whether deleting a gene in the 5-HT system either during development or during adulthood affects 5-HT outgrowth, connectivity or mouse behaviour. Finally, it would be interesting to further investigate whether connectivity is affected in anxiety and depression in human subjects. 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J Neurochem 96:396-406.\n\nNederlandse samenvatting\n\nCellulaire karakterisatie van de uitgroei en connectiviteit van het serotonine systeem in het brein\n\nNederlandse samenvatting\n\nDe menselijke hersenen bestaan uit ongeveer 100 miljard zenuwcellen (neuronen). Neuronen zijn de basis elementen die bijvoorbeeld gedrag en geheugen mogelijk maken. Neuronen hebben een aantal uitlopers waarmee ze met elkaar kunnen communiceren. Een neuron heeft een axon, waarmee het signalen naar een ander neuron kan versturen, en meerdere dendrieten, waarmee een neuron signalen van andere neuronen kan ontvangen. Neuronen communiceren met elkaar door het uitscheiden van signaalstoffen, neurotransmitters genaamd. Deze neurotransmitters kunnen binden aan bepaalde receptoren in een ontvangende cel, waardoor een signaal doorgegeven kan worden. De punten waar twee cellen met elkaar communiceren worden synapsen genoemd. Hier wordt door de zendende cel een presynaptische terminal gevormd op de ontvangende cel. Vanuit deze presynaptische terminal worden de neurotransmitters uitgescheiden, en deze worden opgevangen door receptoren in de postsynaptische terminal in de ontvangende cel.\n\nEr zijn veel verschillende soorten neuronen, deze worden onder andere in groepen ingedeeld door de neurotransmitter die ze uitscheiden. Een van de groepen neuronen zijn de zogenaamde serotonerge neuronen, neuronen die de neurotransmitter serotonine uitscheiden. Deze neuronen zijn in een aantal clusters gelokaliseerd in de hersenstam. In tegenstelling tot vele andere groepen neuronen, hebben de serotonerge neuronen een aantal opvallende kenmerken. Ten eerste is het een erg kleine groep neuronen, in de menselijke hersenen gaat het ongeveer om een half miljoen cellen. Ten tweede zijn ondanks dit kleine aantal in bijna alle andere hersengedeelten uitlopers van deze serotonerge neuronen aanwezig. Ten derde wordt deze neurotransmitter in deze hersengebieden in grote hoeveelheden uitgescheiden, waardoor grote groepen andere neuronen door deze serotonerge neuronen beïnvloed kunnen worden. Ten vierde zijn deze serotonerge neuronen, het zogenaamde serotonerge systeem, betrokken bij het beïnvloeden van verschillende soorten gedrag. Als laatste is het serotonerge systeem betrokken bij aandoeningen zoals depressiviteit, angststoornissen, autisme en obsessief compulsief gedrag. Een welbekend antidepressivum zoals Prozac grijpt bijvoorbeeld aan op de serotonerge neuronen, door de heropname van uitgescheiden serotonine terug het neuron in te blokkeren.\n\nHet idee achter aandoeningen zoals depressie is dat er een verandering ontstaat in de uitlopers van deze serotonerge neuronen naar andere hersengebieden en dat antidepressiva de uitlopers weer in de oude, gezonde staat terugbrengen. In dit proefschrift hebben we op een aantal verschillende\n\nmanieren geprobeerd te kijken naar de dynamiek van de uitgroei van deze serotonerge neuronen.\n\nIn hoofdstuk 2 hebben we uit muizenhersenen, die we als modelsysteem hebben gebruikt, de serotonerge neuronen gesneden in een hersenplakje, een zogenaamde organotypische slice. Dit plakje hebben we naast een plakje van een ander hersengebied, de hippocampus, gelegd om te kijken of in dit kweeksysteem de serotonerge neuronen uitlopers vormden het hippocampale plakje in. Inderdaad bleken de uitlopers van de serotonerge neuronen binnen vier dagen het andere plakje in te groeien. Vervolgens hebben we onderzocht of door toediening van stoffen zoals het hierboven genoemde Prozac de uitgroei van de serotonerge neuronen geremd of juist gestimuleerd werd. Voor de stof Prozac bleek dat het toedienen geen effect had op de uitgroei van de serotonerge neuronen. Echter, toediening van een andere stof die aan een bepaalde serotonerge receptor bindt, bleek een effect te hebben op de serotonerge uitlopers. Na zeven dagen waren er namelijk minder serotonerge uitlopers in het hippocampale plakje dan zonder de toediening van deze stof. Hieruit blijkt dat chronische activatie van deze serotonerge receptor leidt tot een remming in de uitgroei van de serotonerge neuronen.\n\nIn hoofdstuk 3 hebben we een belangrijk eiwit in serotonerge neuronen, het eiwit dat zorgt voor heropname van serotoninine in het neuron na afgifte (de serotoninine heropname transporter, SERT) voorzien van een rood fluorescerend eiwit (mCherry). Dit zogenaamde fusie-eiwit, mCherry-SERT genaamd, hebben we tot expressie gebracht in neuronen om te onderzoeken hoe de verdeling van dit eiwit er in neuronen uitziet. Het bleek dat dit eiwit in het neuron aanwezig was in vesicles. Dit zijn pakketjes waarmee eiwitten in een neuron getransporteerd worden. Vervolgens hebben we door middel van microscopie ‘live’ naar de beweging van pakketjes mCherry-SERT gekeken. Uit de analyse van de snelheid en de richting van de beweging bleek dat de pakketjes zich voortbewogen met een gemiddelde snelheid van 1.2 µm/s (=4.32 mm/uur) en dat de pakketjes geen voorkeur hadden voor de richting waarin ze zich voortbewogen in de uitlopers.\n\nIn hoofdstuk 4 hebben we een soortgelijke aanpak gekozen als in hoofdstuk 3. Echter, nu hebben we gekeken naar het eiwit dat belangrijk is voor de aanmaak van serotoninine, tryptophan hydroxylase 2 (Tph2). Dit eiwit hebben we voorzien van een groen fluorescerend eiwit, (Enhanced Green Fluorescent Protein, EGFP). Dit fusie-eiwit hebben we tot expressie gebracht in neuronen om de verdeling van dit eiwit, ook in vergelijking met mCherry-SERT, te onderzoeken. Hieruit bleek dat, in tegenstelling tot mCherry-SERT, Tph2-EGFP zich door de hele cel bevond, en niet in vesicles zat. In jonge neuronen had Tph2-EGFP geen voorkeur voor een bepaald deel van de cel. In oudere neuronen echter, bleek dat Tph2-EGFP voornamelijk aanwezig was in presynaptische\n\nterminals, maar tegelijkertijd ook in de dendrieten. Ook in deze cellen hebben we door middel van microscopie live naar de beweging van Tph2-EGFP gekeken. Hieruit bleek dat Tph2-EGFP, in tegenstelling tot mCherry-SERT, zich niet in vesicles bevond en daardoor ook geen beweging liet zien.\n\nIn hoofdstuk 5 hebben we onderzocht of een variatie in een gen dat codeert voor het eiwit Piccolo, effect heeft op de verdeling en dynamiek van het eiwit SERT. Deze variatie in het gen wordt geassocieerd met depressie, waardoor het erg interessant is om te onderzoeken of de variatie in dit gen verantwoordelijk is voor een verandering in de dynamiek van het serotonine systeem.\n\nHiertoe hebben we eerst onderzocht of het eiwit Piccolo voorkwam in de serotonerge neuronen en uitlopers, en dit bleek inderdaad het geval. Vervolgens hebben we een zogenaamde knockdown construct gemaakt waarmee de hoeveelheid Piccolo in een cel grotendeels gereduceerd kan worden. Na het tot expressie brengen van dit construct in neuronen bleek inderdaad dat de hoeveelheid Piccolo in een cel met meer dan 80% gereduceerd was. Vervolgens hebben we onderzocht of een vermindering in de hoeveelheid Piccolo van invloed is op de beweging en dynamiek van het eiwit SERT. Om dit te onderzoeken hebben we mCherry-SERT tot expressie gebracht in neuronen waarin de hoeveelheid Piccolo gereduceerd was en hebben we door middel van live microscopie naar de dynamiek van het eiwit mCherry-SERT gekeken. Dit hebben we vergeleken met neuronen waarin de hoeveelheid Piccolo niet gereduceerd was. We concludeerden uit deze vergelijking dat een vermindering in de hoeveelheid Piccolo geen effect heeft op de beweging en snelheid van bewegen van mCherry-SERT. Als laatste hebben we gekeken, in een ander cel type, of de mutante variant van het eiwit Piccolo, in vergelijking met de normale variant de verdeling van het eiwit SERT beïnvloedt. Om dit te onderzoeken hebben we de mutante variant van Piccolo en mCherry-SERT in cellen tot expressie gebracht en gekeken of de verdeling van mCherry-SERT veranderd was. In cellen waarin mCherry-SERT tot expressie gebracht was met de normale variant van Piccolo was mCherry-SERT, zoals verwacht, gelocaliseerd aan het membraan. Na expressie van mCherry-SERT met de mutante variant van Piccolo bleek echter dat de hoeveelheid mCherry-SERT aan het membraan verminderd was. Hieruit concludeerden wij dat mogelijk in serotonerge neuronen de mutante variant van het eiwit Piccolo ervoor zorgt dat er een verandering is in de hoeveelheid SERT aan het membraan. Hierdoor zou de signaaldoorgifte van serotonerge neuronen naar andere neuronen verstoord kunnen zijn.\n\nIn hoofdstuk 6 hebben we onderzocht wat het effect is van het uitschakelen van het serotonine systeem in de muis. Om dit te onderzoeken hebben we door muizen te kruisen een muis verkregen waarbij een gen in het serotonine systeem uitgeschakeld is. Uit onderzoek naar de embryonale ontwikkeling van het serotonerge systeem in deze muizen bleek dat het serotonerge systeem in eerste\n\ninstantie tot ontwikkeling kwam. De serotonerge neuronen waren aanwezig, en serotonerge vezels groeiden uit naar andere hersengebieden. We hebben het serotonerge systeem ook bestudeerd iets later in de hersenontwikkeling van deze muizen. Dit toonde aan dat de serotonerge neuronen een veranderde morfologie vertoonden, waaruit afgeleid kan worden dat deze neuronen degenereren. Uiteindelijk zijn bijna al de serotonerge neuronen verdwenen. Ook de projecties van deze neuronen verdwenen later tijdens de hersenontwikkeling. Hieruit concludeerden wij dat het uitschakelen van dit gen uiteindelijk leidt tot een verdwijning van de serotonerge neuronen. Vervolgens hebben we gekeken wat het effect hiervan is op de ontwikkeling en het overleven van deze muizen. Dit toonde aan dat de meeste muizen vlak na de geboorte doodgingen. Enkele muizen die wel overleefden waren kleiner dan controle muizen die dit gen nog wel hadden, en gingen uiteindelijk ook binnen drie weken dood. We waren ook geïnteresseerd of het gedrag van de muizen veranderd was. Echter, aangezien de meeste muizen al snel na de geboorte dood gingen, was dit niet mogelijk. Daarom hebben we het gedrag geanalyseerd van muizen die nog een kopie van dit gen hadden, en dit vergeleken met controle muizen. Om het gedrag van deze muizen te analyseren hebben we deze muizen zeven dagen in een speciale kooi gehouden. Deze kooien bevatten een videocamera zodat later een groot aantal parameters geanalyseerd kan worden, zoals de hoeveelheid beweging, hoe vaak deze muizen drinken of hoeveel deze muizen eten. Verder konden we ook nog het angst gerelateerde gedrag en het leergedrag van deze muizen analyseren. Uit analyses van muizen met een kopie van het gen bleek dat deze geen veranderd gedrag vertoonden ten opzichte van controle muizen.\n\nHet doel in hoofdstuk 7 was om een muis te maken waarbij alle serotonerge neuronen en uitlopers naar andere hersengebieden gelabeld waren. Om dit doel te bereiken hebben we een gen gebruikt dat specifiek tot expressie komt in serotonerge neuronen, Tph2. Een stuk DNA voor een gen, een promoter genoemd, is verantwoordelijk voor waar en wanneer het gen afgeschreven wordt. Achter de promoter van Tph2 hebben een eiwit genaamd Cre recombinase gezet, zodat de expressie van Cre recombinase wordt gereguleerd door de Tph2 promoter en zodoende alleen tot expressie komt in serotonerge neuronen. In dit hoofdstuk hebben we beschreven hoe we embryonale stamcellen gemanipuleerd hebben en hoe we deze hebben geïnjecteerd in muizen blastocysten. Helaas is het nog niet gelukt om deze muis te genereren. Om specifiek serotonerge neuronen te labelen hebben we nog een aantal andere muizen gemaakt, waarbij het eiwit alleen aangemaakt wordt als in de cell Cre recombinase tot expressie komt. Door deze muizen met elkaar te kruisen zou Cre recombinase alleen in serotonerge neuronen ‘aan’ staan, en zou alleen in deze neuronen een zogenaamd reporter eiwit aangezet worden. Op deze manier hopen we uiteindelijk muizen te hebben waarbij specifiek in serotonerge neuronen reporter genen tot expressie komen,\n\nwaardoor we deze uiteindelijk kunnen gebruiken om in levende muizen naar de dynamische uitgroei en connectiviteit van het serotonerge systeem te kijken. Verder kunnen we dan onderzoeken of manipulatie aan dit systeem leidt tot een verandering in uitgroei van serotonerge neuronen en tot muizen die bepaalde aspecten van humane depressiviteit nabootsen. Aan de andere kant kunnen we symptomen van humane depressiviteit in muizen induceren, en onderzoeken of dit leidt tot veranderingen in het serotonerge systeem. Uiteindelijk zouden we op deze manier ook kunnen onderzoeken of medicatie tegen depressiviteit leidt tot een verandering in de connectiviteit van het serotonerge systeem en deze weer terugbrengt in de oude staat.\n\n| Abbreviation | Description |\n|--------------|-------------|\n| 5-HIAA | 5-hydroxyindolacetic acid |\n| 5-HT | serotonin |\n| 5-HT | 5-hydroxytryptamine |\n| 8-OH-DPAT | 8-hydroxy-2-(di-n-propylamino)tetraline |\n| BOLD | blood oxygen level dependent |\n| cAMP | cyclic adenosine monophosphate |\n| CSF | cerebrospinal fluid |\n| DAT | dopamine transporter |\n| dGBSS | dissection Gey’s balanced salt solution |\n| DIV | days in vitro |\n| DOI hydrochloride | (+)-2,5-dimethoxy-4-iodoamphetamine hydrochloride |\n| DRN | dorsal raphe nucleus |\n| E | embryonic day |\n| EGFP | enhanced green fluorescent protein |\n| ES | embryonic stem |\n| fMRI | functional magnetic resonance imaging |\n| GIRK | G protein-activated inwardly rectifying K⁺ |\n| HBSS | Hanks Balanced Salt Solution |\n| iCre | improved Cre recombinase |\n| IP₃ | inositol 3-phosphate |\n| IRES | internal ribosomal entry site |\n| KO | knockout |\n| l | long |\n| LAT₁ | large neutral amino acid transporter |\n| LDCV | large dense core vesicle |\n| Lmx1 | LIM homeobox transcription factor 1 |\n| LSD | lysergic acid diethylamide |\n| LSL | loxSTOPlox |\n| MAO-A | monoamine Oxidase-A |\n| MAP2 | microtubule associated protein 2 |\n| MDMA | 3,4-methylenedioxy-N-methylamphetamine |\n| MHO | midbrain-hindbrain organizing centre |\n| MRN | median raphe nucleus |\n| neo | neomycin phosphotransferase cassette |\n| OCD | obsessive-compulsive disorder |\n| p | postnatal day |\n| PBS | phosphate buffered saline |\n| PCM | presynaptic cytoskeletal cytomatrix |\n\n| Abbreviation | Description |\n|--------------|-------------|\n| PCPA | p-chlorophenylalanine |\n| PET | positron emission tomography |\n| Pet1 | PC12 Ets factor |\n| PFA | paraformaldehyde |\n| PKA | protein kinase A |\n| PKC | Protein kinase C |\n| PKG | protein kinase G |\n| β-PMA | β-phorbol 12-myristate 13-acetate |\n| RRP | readily releasable pool |\n| s | short |\n| SEM | standard error of mean |\n| SERT | 5-HT reuptake transporter |\n| SMB | short movement bouts |\n| SNP | short nucleotide polymorphism |\n| SSRI | selective serotonin reuptake inhibitor |\n| SSV | small synaptic vesicle |\n| Tetrahydrobiopterin BH4 (6R)-L-erythro-5,6,7,8-Tetrahydrobiopterin | |\n| TH | tyrosine hydroxylase |\n| tPA-GFP | tissue plasminogen activator-GFP |\n| Tph | tryptophan hydroxylase |\n| Tph1 | tryptophan hydroxylase 1 |\n| Tph2 | tryptophan hydroxylase 2 |\n| VAMP2 | synaptobrevin-2 |\n| VMAT2 | Vesicular Monoamine Transporter 2 |\n\nDankwoord\n\nNu ik aan het einde van mijn promotietraject ben gekomen en het boekje af is, besef ik dat ik een ontzettend leuke en leerzame tijd heb gehad, en daar wil ik graag een aantal mensen voor bedanken.\n\nIn de eerste plaats mijn promotor Matthijs. Beste Matthijs, ik wil je bedanken voor het feit dat ik dit project op je lab heb mogen uitvoeren, de begeleiding en de dingen die ik van je geleerd heb. Verder ben ik je dankbaar dat ik ook de ruimte kreeg om mijn eigen invulling aan dit project te geven.\n\nIn de tweede plaats mijn copromotor Sander: Beste Sander, door een stage die ik bij jou gelopen heb ben ik op dit lab terecht gekomen, ik ben je daar nog steeds dankbaar voor. Tijdens mijn promotieproject dank ik je voor de begeleiding en het nakijken van mijn manuscripten.\n\nDe leescommissie dank ik voor de aandacht die ze aan mijn proefschrift hebben willen besteden.\n\nVerder heb ik veel gehad aan de suggesties en input van Ruud en Joris tijdens onze meetings, mijn dank daarvoor. Oliver, ik zou je graag willen bedanken voor de suggesties met betrekking tot gedragsproeven in muizen en de hulp met de perfusies. Ik dank Pieter Voorn en Menno Witter voor het meedenken met het slice hoofdstuk en Pieter en Allert tevens voor alle hulp op de MCID microscoop.\n\nMijn dank gaat uit naar mijn beide paranimfen. Beste Joost, bedankt voor al die keren dat je me op het lab een hoetje(s) liet schrikken 😂 Ik heb altijd met heel veel plezier met jou samengewerkt op het lab, maar we hebben ook heel veel lol gehad buiten het werk om.\n\nBeste Arthur, ondanks dat je pas in mijn laatste jaar mijn kamergenoot werd, bleek al snel dat we het qua humor goed met elkaar konden vinden. Behalve onze liefde voor flauwekul bleek ook onze liefde voor (speciale) bier(en) overeen te komen (zou daar een relatie tussen zitten?). Ik wens je alle succes met jouw verdere afronding van je promotietraject en met de voortzetting van het door ons opgezette ‘bureau voor flauwekul’.\n\nBeste Ineke, ik wil je danken voor alle gezelligheid, maar ook voor alle goede gesprekken over de wetenschap en aanverwante zaken. Ik wens je heel veel succes met de verdere afronding van je opleiding tot klinisch chemicus.\n\nBuiten het feit dat ik veel geleerd heb, heb ik ook de sfeer op de afdeling en het lab altijd als zeer positief ervaren. Op het DNA lab, het kloppend hart van de afdeling, heb ik altijd met heel veel plezier gewerkt. Niet in de laatste plaats door\n\nalle gezelligheid die daar altijd te vinden was. Voor al deze gezelligheid, maar zeker ook voor alle hulp ben ik de analisten grote dank verschuldigd.\n\nBeste Robbie, Mr. Salmon, ik ben je dankbaar voor de stage die ik bij jou gelopen heb waarin je me hebt ingewijd in de geheimen van het kloneren. Hoewel ik nog niet eens jouw pipetten mag vasthouden denk ik dat ik inmiddels aardig kan kloneren.\n\nBeste Ingrid, bedankt voor je humor op het lab en voor de hulp met de kloneringen. N.B. Ik heb in de wandelgangen vernomen dat zelfs *jij* nu getrouwd bent :-)\n\nIk ben ook veel dank verschuldigd aan Desiree en later Boukje voor alle hulp met de neuronen kweek. Bastijn, jou dank ik voor het uitwerken van de Phenotyper data.\n\nBeste Joke, bedankt voor alle hulp met de muizen, maar zeker ook voor je altijd vriendelijke houding.\n\nDit proefschrift was ook zeker niet mogelijk geweest zonder alle hulp vanuit de muizenstallen. Dus Chris, Bert en Lieselotte, ook jullie hartelijk dank voor alle hulp met de muizen.\n\nMijn mede aio’s Jurjen, Juliane, Sabine, Marieke, Asiya, Rhea, Jiun, Tony en Gregoire dank ik voor alle discussies en ik wens jullie allen veel succes met de verdere voortzetting van jullie project en de afronding van jullie proefschrift.\n\nTina en Els jullie dank ik voor alle administratieve ondersteuning. Els, als ik een vraag had wist je die altijd binnen no time te beantwoorden.\n\nAlle andere nog niet genoemde collega’s van het lab dank ik voor alle discussies tijdens labmeetings, gezelligheid tijdens de dagelijkse lablunch en het jaarlijkse labuitje.\n\nRoel, ik dank je voor alle gezelligheid tijdens de treinreizen vanuit Hoofddorp, en ik vind het erg leuk dat we elkaar nog weleens tegenkomen.\n\nMijn nieuwe lab Neuromedische Genetica op het NIN dank ik voor de gastvrijheid waarmee ik in hun lab ben opgenomen en de plezierige werksfeer.\n\nGijs, Ray en Robert, we hebben altijd ontzettend veel lol gehad tijdens onze studieperiode. Ik ben blij dat we nog weleens afspreken. Wanneer gaan we weer poolen (of was het nou snooker...?).\n\nAls laatste zou ik graag een aantal mensen willen bedanken uit mijn persoonlijke omgeving.\n\nDe mannen uit Renesse: Alex, Bart, Kenneth, Michael; bedankt voor alle nachtelijk stapuurtjes en (wintersport) vakanties.\n\nBeste familie Visser, ik dank jullie voor alle gezelligheid en alle borreltjes die we al met elkaar gedronken hebben. Wat is het bij jullie toch altijd lekker rustig... :-)\n\nIk dank mijn schoonouders, Jan en Christien, mijn schoonzussen en zwagers voor de interesse in mijn proefschrift en alle feesten, familiedagen en andere gezellige activiteiten. Is die Eiffeltoren nou al gevonden?\n\nMijn broer(tje) en zus(je), ik dank jullie voor de interesse in mijn proefschrift, de gezelligheid tijdens het meerijden naar Renesse en ik wens jullie alle succes met de afronding van jullie studie.\n\nMijn ouders, ‘promoveren zit dat erin dan?’, Frans en Lia, ik ben heel trots op jullie en ik ben blij jullie zoon te zijn. Ik realiseer me maar al te vaak wat een rijkdom het was om zo te mogen opgroeien. Ik dank jullie voor alle steun en vertrouwen, maar ook voor alle gezelligheid. Als we elkaar zien is het altijd erg gezellig en vaak ineens midden in de nacht als we zitten te kletsen onder het genot van een wijntje.\n\nAls laatste dank ik natuurlijk mijn lieve vrouw Judith. Sinds die kapotte printer zijn we alweer bijna 7 jaar bij elkaar, wonen we al ruim 3 jaar in Hoofddorp en zijn we al ruim 1 jaar getrouwd. Time flies when having fun, wil ik daar alleen maar even mee zeggen. Ik wens jou ook alle succes met de verdere afronding van jouw promotieproject en boekje, en heb zin om samen de toekomst tegemoet te gaan!\n\nJeroen\n\nHoofddorp, Juli 2010\n\nCurriculum vitae\n\nJacobus Johannis Dudok werd geboren op 13 juni 1981 te Zierikzee. In 1993 begon hij aan zijn VWO opleiding aan het Buys Ballot College te Goes, waar hij in 1999 zijn Gymnasium Beta diploma haalde. In augustus 1999 begon hij aan de Bachelor Biomedische Wetenschappen aan de Vrije Universiteit te Amsterdam. In deze opleiding liep hij zijn Bachelor stage van februari 2002-juni 2002 aan de vakgroep pathologie in het VU medisch centrum. Hier bestudeerde hij, begeleidt door Dr. R. Veerhuis en Ing. E. van Elk, de verschillende aggregatie vormen van Aβ1-42, het eiwit betrokken bij de ziekte van Alzheimer. In september 2002 begon hij aan de Master of Neurosciences aan de Vrije Universiteit te Amsterdam. Tijdens deze master deed hij twee stages bij de afdeling Functionele Genoomanalyse. Tijdens de eerste masterstage deed hij onderzoek naar de calcium afhankelijke translocatie van het presynaptische eiwit Doc2B, begeleidt door Dr. S. Groffen. Tijdens zijn tweede masterstage, begeleidt door Ing. R. Zalm, cloneerde hij een targeting construct en gebruikte deze om muis embryonale stamcellen mee te targetten. Zijn masterscriptie schreef hij onder leiding van Dr. J. Pronk en ging over de in silico clonering en karakterisatie van het eiwit Tph2. In augustus 2004 studeerde hij af aan de Vrije Universiteit te Amsterdam. Van oktober 2004 tot april 2009 deed hij zijn promotieonderzoek naar de uitgroei en connectiviteit van het serotonerge systeem. Dit onderzoek deed hij onder begeleiding van professor Verhage aan de vakgroep Functionele Genoomanalyse aan de Vrije Universiteit te Amsterdam. De resultaten van dit promotieonderzoek zijn in dit proefschrift beschreven. Vanaf april 2009 werkt hij als postdoctoraal onderzoeker op het Nederlands Instituut voor Neurowetenschappen (NIN) bij de vakgroep Neuromedische Genetica onder leiding van Dr. J. Wijnholds. Hier doet hij onderzoek naar de functionele rol van het eiwit MPP3 in het Crumbs complex en in neuronale cellen.\n\nJacobus Johannis Dudok was born on the 13th of June 1981 in Zierikzee, the Netherlands. In 1993 he started his secondary education at the Buys Ballot College in Goes, where he graduated in 1999. In August 1999 he started the Bachelor Medical Biology at the Vrije Universiteit Amsterdam. During this Bachelor he carried out a research project from February 2002 to June 2002 at the research department of pathology at the VU medical center. Here he studied the different aggregation states of Aβ1-42, the peptide involved in Alzheimer’s disease, supervised by Dr. R. Veerhuis and Ing. E. van Elk. In September 2002 he started the Master of Neuroscience at the Vrije Universiteit Amsterdam. During this master he carried out two research projects at the department of Functional Genomics. During his first research project he studied the calcium dependent translocation of the presynaptic protein Doc2B, supervised by Dr. S. Groffen. During his second research project he constructed a targeting construct and used this to target mouse embryonic stem cells, supervised by Ing. R. Zalm. He wrote his Master Thesis under supervision of Dr. J. Pronk about the in silico cloning and characterization of the protein Tph2. In August 2004 he graduated at the Vrije Universiteit Amsterdam. From October 2004 until April 2009 he did his PhD project on the outgrowth and connectivity of the serotonin system. This PhD project was supervised by professor Verhage and carried out at the department of Functional Genomics at the Vrije Universiteit Amsterdam. The results of that study are described in this thesis. He is currently working as a postdoctoral fellow at the Netherlands Institute for Neuroscience (NIN) in the department of Neuromedical Genetics headed by Dr. J. Wijnholds. Here he studies the functional role of the protein MPP3 in the Crumbs complex and in neuronal cells."}}}],"truncated":false,"partial":true},"paginationData":{"pageIndex":2046,"numItemsPerPage":100,"numTotalItems":208496,"offset":204600,"length":100}},"jwt":"eyJhbGciOiJFZERTQSJ9.eyJyZWFkIjp0cnVlLCJwZXJtaXNzaW9ucyI6eyJyZXBvLmNvbnRlbnQucmVhZCI6dHJ1ZX0sImlhdCI6MTc3ODk1NjA4MSwic3ViIjoiL2RhdGFzZXRzL1RlbGVBSS1BSS1GbG93L0luZm9ybWF0aW9uQ2FwYWNpdHkiLCJleHAiOjE3Nzg5NTk2ODEsImlzcyI6Imh0dHBzOi8vaHVnZ2luZ2ZhY2UuY28ifQ.w5l6coOB7e6wCfp1mPSeEp33lAnHXmF2DLomo2kiXpqBcJUXr20IBzHDF3F_SZmd16K9ow-rM_oCs5GWNTZkDw","displayUrls":true,"splitSizeSummaries":[{"config":"default","split":"train","numRows":208496,"numBytesParquet":2762384337}]},"discussionsStats":{"closed":1,"open":0,"total":1},"fullWidth":true,"hasGatedAccess":true,"hasFullAccess":true,"isEmbedded":false,"savedQueries":{"community":[],"user":[]}}">
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Reckless thrust by Quality
Quality recording act Reckless are on release with Heart Of Steel, an LP produced by Ross Munro at Metalworks in Mississauga, Ont. At press time, the label had not chosen a single, although a video is imminent for the Hot 'n Ready track. Following a couple of lineup changes, Reckless are cur... |
IN THE DISTRICT COURT OF CLEVELAND COUNTY
STATE OF OKLAHOMA
STATE OF OKLAHOMA, ex rel.,
MIKE HUNTER,
ATTORNEY GENERAL OF OKLAHOMA,
Plaintiff,
vs.
(1) PURDUE PHARMA L.P.;
(2) PURDUE PHARMA, INC.;
(3) THE PURDUE FREDERICK COMPANY;
(4) TEVA PHARMACEUTICALS USA, INC.;
(5) CEPHALON, INC.;
(6) JOHNSON & JOHNSON;
(7) JANS... |
PHOTODYNAMICS STUDIES OF LIGAND-PROTECTED GOLD NANOCENTERS BY USING ULTRAFAST TRANSIENT INFRARED SPECTROSCOPY
BY
SATU MUSTALAHTI
Academic Dissertation for the Degree of Doctor of Philosophy
To be presented, by permission of the Faculty of Mathematics and Science of the University of Jyväskylä, for public examinatio... |
Ambient Occlusion and Shadows for Molecular Graphics
Robert Easdon
A thesis submitted for the degree of
Master of Science
at the University of East Anglia
September 2013
Ambient Occlusion and Shadows for Molecular Graphics
Robert Easdon
© This copy of the thesis has been supplied on condition that anyone who consu... |
THE FAMILY OF
Hans and Margaret Zum (Bach) Biettschi
THE FAMILY OF
Hans and
Margaret Zum (Bach)
Biettschi
Compiled by Raymond and Darla Beachy
1626 - 2004
© 2004 Darla Kizer
All rights reserved. No portion of this book may be reproduced by any means, electronic or mechanical, including photocopying, recording, or ... |
COMPTOMETER
medley
A COPY of each edition is sent free to every operator on Felt & Tarrant's mailing list. The intention is to interest and encourage Comptometer Operators for the benefit of themselves and their employers.
May We Remind You—
the name "Comptometer" belongs to Felt & Tarrant and cannot rightly be used... |
(54) USE OF MONOCYTES DERIVED CELLS, ANTIGENS AND ANTIBODIES FOR OPTIMAL INDUCTION OF IMMUNOTHERAPEUTIC EFFICIENCY
ANWENDUNG VON MONOCYTE-ABSTAMMENDE ZELEN, ANTIGENE UND ANTIKÖRPER ZUR OPTIMALEN INDUZIERUNG EINER IMMUNTHERAPEUTISCHEN LEISTUNGSFÄHIGKEIT
UTILISATION DE CELLULES DERIVEES DE MONOCYTES, D'ANTIGENES ET D'A... |
AN ORDINANCE ADOPTING AND ENACTING A NEW CODE OF ORDINANCES OF THE CITY OF ANDERSON, COUNTY OF MCDONALD, STATE OF MISSOURI; ESTABLISHING THE SAME; PROVIDING FOR THE REPEAL OF CERTAIN ORDINANCES NOT INCLUDED THEREIN, EXCEPT AS HEREIN EXPRESSLY PROVIDED; PROVIDING FOR THE MANNER OF AMENDING SUCH CODE OF ORDINANCES; PROVI... |
KILLER KUTS
(See Consensus Cuts on P. 2)
Crosby, Stills, Nash - "Dark Star"
"Shadow Captain"
"Cathedral"
"Just a Song Before I Go"
Roger Daltrey - "Avenging Annie"
"Say It Ain't So Joe"
Alan Parsons - "I Wouldn't Wanna Be..."
"I Robot"
Steve Winwood - "Time is Running Out"
"Hold On"
"Vacant Chair"
CONCERT RAVES
... |
CONTENTS
Editorial_______________________________________________________ 1
The Rose Cross and Holy Grail, Costello ___________________________ 2
Reasons for Bacon’s Participation, Brameld ________________________ 4
Alchemy and The Holy Grail, Bayley _______________________________ 26
Francis Bacon: A Forerunner, Srig... |
Protective Effect of Phloroglucinol on Oxidative Stress-Induced DNA Damage and Apoptosis through Activation of the Nrf2/HO-1 Signaling Pathway in HaCaT Human Keratinocytes
Cheol Park 1,†, Hee-Jae Cha 2,†, Su Hyun Hong 3,4, Gi-Young Kim 5, Suhkmann Kim 6, Heui-Soo Kim 7, Byung Woo Kim 8, You-Jin Jeon 5* and Yung Hyun C... |
HOT HITS
WHAT BECOMES OF THE BROKENHEARTED
Paul Young
MISSING YOU NOW
Michael Bolton
I LOVE YOUR SMILE
Shanice
THOUGHT I'D DIED AND GONE TO HEAVEN
Bryan Adams
HEAD OVER HEELS
Frozen Ghost
UNTIL YOUR LOVE COMES AROUND
RTZ
YOUR SONG
Rod Stewart
CHURCH OF YOUR HEART
Roxette
SAVE THE BEST FOR LAST
Vanessa Williams... |
BPM
Optimism marks first quarter after X-MAS sell-through
by Sean LaRose
As reported last week in RPM, 1996 for the most part had the Canadian music industry on edge with soft sales at retail. Ironically, Soundscan and CRIA statistics for '96 didn't fair too badly in regards to numbers and certifications. Some recor... |
Large deflections of a clamped-slider-pinned rod with uniform intrinsic curvature
V.G.A. Goss*, P. Singh, R. Chaouki
School of Engineering, London South Bank University, London SE10 0AA, United Kingdom
ARTICLE INFO
Article history:
Received 4 May 2017
Revised 20 July 2017
Available online 12 September 2017
Keyword... |
Crime Breaking Open
INSIDE
Banking Sector
NRNs: Making Differences
Social Inclusion
SOUND. STRONG. STABLE.
Your bank stands at the top when it comes to setting benchmarks.
• No. 1 lender in Nepal with total loans and advances of NRs 36.20 billion.
• No. 1 Private sector bank in Deposits with NRs 46.70 billion.
• ... |
OUR MISSION STATEMENT:
"Saint Francis Xavier Parish is a Christian Family dedicated to the mission of Jesus and the teachings of the Catholic Church. We are committed to fostering spiritual growth and to responding to the human needs of both our faith community and the larger community by sharing our gifts and resource... |
RADIO AT WAR
IN THIS ISSUE
Serving Uncle Sam
War Communications
Walkie-Talkies
The Army Hour
Stars in Uniform
Women's Uniforms
Entertainment in Camps
Army, Navy, Marine, Coast Guard and Civilian Insignias
How to Display the Flag
V-Mail
Honor Roll
TOPEKA WIBW KANSAS
COLUMBIA BROADCASTING SYSTEM
Dear Folks:
This book... |
Protecting Life and Liberty: The Constitutionality and Necessity of Civil Commitment of Sexual Predators
Jennifer Ann Smulin
Follow this and additional works at: https://via.library.depaul.edu/law-review
Recommended Citation
Jennifer A. Smulin, Protecting Life and Liberty: The Constitutionality and Necessity of Civi... |
OUR MISSION STATEMENT:
"Saint Francis Xavier Parish is a Christian Family dedicated to the mission of Jesus and the teachings of the Catholic Church. We are committed to fostering spiritual growth and to responding to the human needs of both our faith community and the larger community by sharing our gifts and resource... |
Northside Middle School
JETS
Title I Handbook
2024-2025
Dr. Alma Mundy, Principal
500 Johnson Road
Warner Robins, GA 31088
Ms. Tiffany Davis, Family Engagement Liaison
Revised: July 30, 2024
A Parent’s Guide to Title I
What is Title I?
Title I, Part A is a federally funded program under the Every Student Succee... |
IMPACT OF THE COVID-19 PANDEMIC ON SMEs IN CHINA, CROATIA, NORTH MACEDONIA AND SLOVENIA
Recommendations for improvement of policy measures
ISBN 978-953-7520-15-1 (e-book)
CEPOR – SMEs & Entrepreneurship Policy Centre (Croatia)
The Centre for Polish and Central and Eastern European Studies, School of International St... |
Implications of anomalous relative sea-level rise for the peopling of Remote Oceania
Juliet P. Sefton\textsuperscript{1,2*}, Andrew C. Kemp\textsuperscript{1}, Simon E. Engelhart\textsuperscript{3}, Joanna C. Ellison\textsuperscript{4}, Makan A. Karegar\textsuperscript{5}, Blair Charley\textsuperscript{6}, and Mark D.... |
OUR MISSION STATEMENT:
"Saint Francis Xavier Parish is a Christian Family dedicated to the mission of Jesus and the teachings of the Catholic Church. We are committed to fostering spiritual growth and to responding to the human needs of both our faith community and the larger community by sharing our gifts and resource... |
ATTENDEES:
Mayor L. Kelly Jones
Council Member Phillip Poole
Council Member Brian Libbey
Council Member Michael Dingman
Council Member Robert Fitzgerald
Council Member Halden Griffith
City Administrator/Sec Brandy Barrett
Police Chief Kevin Reaves
Deputy City Secretary Elisa Hickey
Interim City Attorney Tracie Kenan
A... |
La mer
うみ
1990年2月
日仏海洋学会
La Société franco-japonaise d'océanographie
Tokyo, Japon
SOCIETE FRANCO-JAPONAISE D'OCEANOGRAPHIE
Comité de Rédaction
(de l'exercice des années de 1988 et 1989)
Directeur et rédacteur: Y. ARUGA
Comité de lecture: S. AOKI, M. HANZAWA, M. HORIKOSHI, A. KAMATANI, M. MAEDA,
M. MURANO, M. OGU... |
OUR MISSION STATEMENT:
"Saint Francis Xavier Parish is a Christian Family dedicated to the mission of Jesus and the teachings of the Catholic Church. We are committed to fostering spiritual growth and to responding to the human needs of both our faith community and the larger community by sharing our gifts and resource... |
Edumilestones Platform
Setup Your own Advanced Career Counselling Technology Platform
12 Hours
Get your own Branded Career Assessment & Counselling Platform
Industry Leading Platform
Career Assessment tools and platform training
Module 1: Career Assessment tools, Counselling platform and Reports
Module 2: Career... |
Set Your Child’s Biological Clock for Better Sleep
**DIM THE LIGHTS AT NIGHT**
Darkness causes a release of melatonin, the body's natural sleep hormone. Help your child feel tired by dimming the lights the hour before bedtime, and keeping the room dark all night.
**HAVE BRIGHT DAYS**
Light signals the brain to be ale... |
AUGSA Minutes
Date: July 14, 6:00pm MDT
Place: Web conference
Attendance:
Executive Committee: Mary-Anne Parker (President), Ashley Ravenscroft (VP Operations & Finance), Judd Asoyuf (VP Academic), Crys Vincent (VP External)
Faculty of Business Representatives: Margaret Clappison, David Newman, Brandon Simmons
Facu... |
Deglaciation of the Eurasian ice sheet complex
Henry Patton\textsuperscript{a, *}, Alun Hubbard\textsuperscript{a}, Karin Andreassen\textsuperscript{a}, Amandine Auriac\textsuperscript{b}, Pippa L. Whitehouse\textsuperscript{b}, Arjen P. Stroeven\textsuperscript{c, d}, Calvin Shackleton\textsuperscript{a}, Monica Wins... |
A method, system, and computer program product in a data processing system are disclosed for dynamically selecting software buffers for aggregation in order to optimize system performance. Data to be transferred to a device is received. The data is stored in a chain of software buffers. Current characteristics of the s... |
A genome wide transcriptional model of the complex response to pre-TCR signalling during thymocyte differentiation
Hemant Sahni¹, Susan Ross¹, Alessandro Barbarulo¹, Anisha Solanki¹, Ching-In Lau¹, Anna Furmanski¹, José Ignacio Saldaña¹, Masahiro Ono¹, Mike Hubank¹, Martino Barencro¹,* and Tessa Crompton¹,*
¹ Institu... |
A Home-made Automatic Noise-Figure Measuring System
A Publication for the Radio-Amateur Especially Covering VHF, UHF and Microwaves
VHF communications
Volume No. 15 · Spring · 1/1983 · DM 6.50
A Publication for the Radio Amateur
Especially Covering VHF, UHF, and Microwaves
Volume No. 15 · Spring · Edition 1/1983
... |
Negative regulation of transcription of the *Saccharomyces cerevisiae* catalase T (*CTT1*) gene by cAMP is mediated by a positive control element
T.Belazzi, A.Wagner, R.Wieser, M.Schanz, G.Adam, A.Hartig and H.Ruis
Institut für Allgemeine Biochemie der Universität Wien and Ludwig Boltzmann-Forschungsstelle für Bioche... |
Diamond Firetail Finch
by Matthew M. Vriends, Ph.D.
The Netherlands
The Diamond Fire-tailed Finch comes from many parts of Australia. It is commonly known to U.S. aviculturists as the Diamond Sparrow. It is not, however, related to sparrow-like birds.
Scientific names:
Emblema guttata, Stagonopleura guttata, and Zon... |
A STUDY OF FUEL INJECTION AND MIXTURE FORMATION FOR A GASOLINE DIRECT INJECTION ENGINE
2005.
SCHOOL OF ENGINEERING
PhD Thesis
A STUDY OF FUEL INJECTION AND MIXTURE FORMATION FOR A GASOLINE DIRECT INJECTION ENGINE
Supervisor: Professor D A Greenhalgh
September 2005
This thesis is submitted in partial fulfilment o... |
Rational Design of a GFP-Based Fluorogenic Caspase Reporter for Imaging Apoptosis In Vivo
Highlights
- Split GFP is “zipped” or redesigned to sense protease activity
- ZipGFP-based caspase reporter detects apoptosis in human cells
- ZipGFP caspase reporter enables imaging physiological apoptosis in zebrafish
Authors... |
Katariina Heikkilä
SUSTAINABILITY STUDIES IN UNIVERSITIES
– Review on Study Modules of Sustainable Development and Responsible Business in Finnish and Some European Universities
FINLAND FUTURES RESEARCH CENTRE
FFRC eBook 13/2015
Katariina Heikkilä
Project Manager
Finland Futures Research Centre, University of Turku
... |
STUDIES OF THE DROSOPHILA BRAIN USING P[GAL4] ENHANCER TRAP LINES
A Thesis Submitted for the Degree of Doctor of Philosophy at the University of Glasgow
by
Ming Yao Yang
Division of Molecular Genetics, University of Glasgow
Glasgow G11 5JS, Scotland, UK.
January 1996
All rights reserved
INFORMATION TO ALL USERS
... |
**HOT HITS**
- SENZA UNA DONNA (Without A Woman)
Zucchero w/Paul Young
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PM Dawn
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Sarah McLachlan
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Cher
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Mariah Carey
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James Taylor
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Dan Hill
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Tomahawk, April 12, 1932
College of the Holy Cross
Follow this and additional works at: https://crossworks.holycross.edu/crusader
Part of the Higher Education Commons, and the Social History Commons
Recommended Citation
College of the Holy Cross, "Tomahawk, April 12, 1932" (1932). Student Newspapers. 214.
https://c... |
June 20, 2024
Ms. MARIA IRISH ALOHA CUBILLAN
PIO, Navotas City
Mr. RENATO REYES
ICTO, Navotas City
Dear Sir / Madam,
Respectfully forwarded herewith are copies of Invitation to Bid for the procurement proceeding of the City Government of Navotas, to wit;
1. PROCUREMENT OF SECURITY SERVICES FOR THE USE OF NAVOT... |
CHAPTER 6
Disaster in the Mediterranean
It was in September, 1943, while I was on the second day of a course at Brighton where German was being taught that I was hastily called back to Goodwood because my request for an overseas posting had been granted. I was sent to a tropical medicine course in London, being bille... |
This is Exhibit 5 referred to in the affidavit of Samuel Berg sworn before me, this 18th day of February, 2015.
A witness, after taking affidavits
Hotels and Accomodations
COURTYARD Marriott
(http://www.nfcourtyard.com/)
HEART OF NIAGARA
MEET EAT SLEEP
(http://www.heartofniagarahotels.com/)
DOUBLE TREE BY HILTON
(... |
Rational and Constitutional Approaches to Airline Safety in the Face of Terrorist Threats
Jamie L. Rhee
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Recommended Citation
Jamie L. Rhee, Rational and Constitutional Approaches to Airline Safety in the Face of Terrorist Threats, 49 DePa... |
Diffusion of Ultra-Cold Neutrons in Randomly Rough Channels
Sarah Elizabeth Brent
University of Rhode Island, firstname.lastname@example.org
Follow this and additional works at: https://digitalcommons.uri.edu/oa_diss
Terms of Use
All rights reserved under copyright.
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Brent, Sarah Elizabeth, "Diff... |
High Temperature Oxidation Kinetics of Dysprosium Particles
Brian J. Jaques
*Boise State University*
Darryl P. Butt
*Center for Advanced Energy Studies*
**Publication Information**
Jaques, Brian J. and Butt, Darryl P.. (2014). "High Temperature Oxidation Kinetics of Dysprosium Particles". *Journal of Alloys and... |
Animal sexual signals:
Do they maximise or optimise information content?
Submitted by Iker Vaquero-Alba to the University of Exeter
as a thesis for the degree of
Doctor of Philosophy in Biological Sciences
In December 2011
This thesis is available for Library use on the understanding that it is copyright material and... |
Virtual Chinese Literature: A Comparative Case Study of Online Poetry Communities*
Michel Hockx
ABSTRACT This article looks at the practices of communities that employ internet technologies in order to produce, distribute, consume and value Chinese poetry. The article is in three parts. The first part provides a bri... |
MARY KAY SEMINAR 2024
This is the final month to register. Plus, skin care is on sale!
NOW'S THE TIME FOR SKY-HIGH SUCCESS!
Sherell McDonald Love, an Independent Sales Director and former military gunnery sergeant, shares how trusting herself and learning the importance of work-life balance helped her achieve her goa... |
NOW, MORE THAN EVER,
ALL RADIO DEPENDS ON
THE RADIO SERVICEMAN!
"Keep 'em listening!"
Despite restricted production and material shortages, as a radio service engineer, we urge you to "keep 'em listening!"
We know how essential a home front job you perform. And, you know you can depend on Mallory for practical help ... |
As Big Country enters its 18th year and presents its awards for the 16th time, RPM reflects on the history of the event and its role in helping develop the Canadian country music industry.
**HIT PICK**
- CAN'T GET ENOUGH OF YOUR LOVE - Taylor Dayne
Arista
**ALBUM PICK**
- ROD STEWART
Unplugged And Seated
Warn... |
DISPOSITION: STIPULATIONS ADOPTED
In this order, we adopt two stipulations filed by the parties in this case. Adopting the terms of the stipulations will reduce the size of the Oregon Universal Service Fund (OUSF), provide clarity for the next several years, and strengthen reporting and information requirements for no... |
This is a repository copy of *A critical analysis of calcium carbonate mesocrystals*.
White Rose Research Online URL for this paper:
http://eprints.whiterose.ac.uk/83533/
Version: Published Version
**Article:**
Kim, YY orcid.org/0000-0002-8503-4554, Schenk, AS, Ihli, J et al. (7 more authors) (2014) A critical analy... |
This is Exhibit B referred to in the affidavit of Lukas Walter sworn before me, this 11th day of March, 2015.
A commissioner for taking affidavits
Western Hockey League
Standard Player Agreement
Terms and Conditions Schedule
INTRODUCTION - POLICIES AND PROCEDURES
1) The WHL must approve and register all agreements... |
Effects of disparity on visual discomfort caused by short-term stereoscopic viewing based on electroencephalograph analysis
Xiao Wang, Liuye Yao, Yuemei Zhao, Lidong Xing, Zhiyu Qian*ID, Weitao Li and Yamin Yang
*Correspondence:
email@example.com
College of Automation Engineering, Nanjing University of Aeronautics an... |
Commencement Program, December (2013)
Minnesota State University Moorhead
Follow this and additional works at: https://red.mnstate.edu/commencement
Researchers wishing to request an accessible version of this PDF may complete this form.
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Minnesota State University Moorhead, "Commencement Program... |
Lanthorn, vol. 49, no. 45, February 19, 2015
Grand Valley State University
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Part of the Archival Science Commons, Education Commons, and the History Commons
Recommended Citation
Grand Valley State University, "Lanthorn, vol. 49, no. 45... |
BEFORE THE PUBLIC UTILITY COMMISSION
OF OREGON
UG 360
In the Matter of
AVISTA CORPORATION, dba AVISTA UTILITIES,
Purchased Gas Cost Adjustment, Schedule 461 and 462.
ORDER
DISPOSITION: STAFF’S RECOMMENDATION ADOPTED
At its Special Public Meeting on October 11, 2018, the Public Utility Commission of Oregon adopt... |
Creep crack initiation and growth in 2.25% chromium - 1% molybdenum alloy steel
COLLINGTON, Rachel A.
Available from the Sheffield Hallam University Research Archive (SHURA) at:
http://shura.shu.ac.uk/3172/
A Sheffield Hallam University thesis
This thesis is protected by copyright which belongs to the author.
The... |
APRIL 1999
APRIL CHAIRMAN: DICK GOLZE
2 Speaker: Charles Forbes, Forbes Management, Inc.
Subject: “Moving the Gem Theatre”
Applicants invited S - Z
9 Speaker: Thomas G. Cutler, Brigadier General, Michigan Air National Guard
Subject: “127th Wing Role in National Defense”
Applicants invited A- E
16 Speaker: Honorable... |
Berlin cop serves town and country
By Josh Davis
Associate Editor
(Oct. 20, 2016) Cpl. Merle Bragg was less than 24 hours away from another lengthy deployment with the U.S. Army National Guard. It would have been a familiar story for the 50-year-old veteran, before the state granted a hardship approval memorandum to ... |
NEW YORK STATE COUNCIL ON THE ARTS 1962
NEW YORK STATE
Library of Congress Catalogue Card Number 62-62582
| Section | Page |
|----------------------------------------------|------|
| FOREWORD | 3 |
| Nelson A. Rockefeller, Governor ... |
Searching for Worlds beyond the Canon: Narrative, Rhetoric, and Legal Change
Susan Bandes
DePaul College of Law
Follow this and additional works at: https://via.library.depaul.edu/lawfacpubs
Part of the Law Commons
Recommended Citation
Susan Bandes, Searching for Worlds beyond the Canon: Narrative, Rhetoric, and Le... |
HPC-GAP: engineering a 21st-century high-performance computer algebra system
BEHREND, Reimer, HAMMOND, Kevin, JANJIC, Vladimir, KONOVALOV, Alexander, LINTON, Steve, LOIDL, Hans-Wolfgang, MAIER, Patrick <http://orcid.org/0000-0002-7051-8169> and TRINDER, Phil
Available from Sheffield Hallam University Research Archive... |
Community in Postmodern Philosophy with an emphasis on the work of Jean-Luc Nancy
M.Phil. thesis
Gender Studies
Candidate:
Oleg Domanov
Supervisors:
Professor Linda Fisher
Professor Gabriele Griffin
Budapest, Novosibirsk 2004
Submission date: 1 December 2004
Award date: 9 March 2005
All rights reserved
INFOR... |
RADIO AT WAR
UNITED STATES
GREAT BRITAIN
CHINA
RUSSIA
AUSTRALIA
CANADA
WLAC
50,000 WATTS - NASHVILLE, TENNESSEE
A MESSAGE TO OUR LISTENERS
Dear Friends:
On September 1st, 1942, the power of WLAC was increased to 50,000 watts, making this station as powerful as any standard broadcast station in the United States.
... |
Climate forcings in Goddard Institute for Space Studies SI2000 simulations
Citation
Hansen, J., M. Sato, L. Nazarenko, R. Ruedy, A. Lacis, D. Koch, I. Tegen, T. Hall, D. Shindell, B. Santer, P. Stone, T. Novakov, L. Thomason, R. Wang, Y. Wang, D. Jacob, S. Hollandsworth, L. Bishop, J. Logan, A. Thompson, R. Stolarski,... |
Analyzing the Diverse Impacts of Conventional Distributed Energy Resources on Distribution System
Muhammad Aamir Aman\textsuperscript{1}, Sanaullah Ahmad\textsuperscript{1}, Azzam ul Asar\textsuperscript{2}, Babar Noor\textsuperscript{1}
\textsuperscript{1}Electrical Engineering Department, IQRA National University, ... |
Computers can make it easier, quicker, and cheaper to print formal publications; individuals can do what was once practical only for organizations. ... As the computer increases the freedom of writers, so does it increase the responsibility of readers.
John Shore
*The Sachertorte Algorithm and other antidotes to compu... |
Brown & Root/NORTHROP
The image shows an aerial view of a large industrial or commercial facility, likely from the mid-20th century based on the style of vehicles and architecture. The building is rectangular with a flat roof and appears to be surrounded by open land. There are several parking lots filled with cars, i... |
CYTOLOGICAL AND GENETICAL STUDIES ON
PUCCINIA STRIIFORMIS WESTEND.
ROBERT GEOFFREY WRIGHT, B.Sc. (Wales)
Doctor of Philosophy
Edinburgh University
1977
DECLARATION
This thesis has been composed by myself and describes experimental work which I carried out between August 1974 and August 1977.
| CONTENTS ... |
TREVOR HAS A HIT AND A BIG WEEK
Toronto: When Date recording artist, Van Trevor signed into the Edison Hotel, he was already batting a thousand. Columbia's promotion man Charlie Cannilieri had laid a carpet of promotion that only required a fast moving car and 26 hours a day.
It would appear that Van Trevor is off an... |
Multi-Dimensional Regression Host Utilization algorithm (MDRHU) for Host Overload Detection in Cloud Computing
Ali A. El-Moursy\textsuperscript{1}*, Amany Abdelsamea\textsuperscript{2}, Rukshanda Kamran\textsuperscript{2} and Mohamed Saad\textsuperscript{1}
Abstract
The use of cloud computing data centers is growing ... |
Devices, methods and computer-readable media for providing control of switching of an interactive session in a communications network. Content may be switched between media presentation screens. Collected data is analyzed and a location of a user within the communications network is monitored. A presentation of a first... |
Theses Digitisation:
https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/
This is a digitised version of the original print thesis.
Copyright and moral rights for this work are retained by the author
A copy can be downloaded for personal non-commercial research or study, without prior permission ... |
INTERACT HANDBOOK
A Guide for Interactors
This handbook is for youth interested in forming or participating in an Interact club. You will learn how to:
- Start an Interact club
- Build a dynamic club that offers all members meaningful service and leadership opportunities
- Promote your club’s work to your community a... |
Microtubule-associated protein tau in oligodendrocytes following acute brain injury
Elaine Alison Irving BSc (Hons)
A thesis submitted for the Degree of Doctor of Philosophy to the Faculty of Medicine, University of Glasgow.
Wellcome Surgical Institute and Hugh Fraser Neuroscience Laboratories,
Garscube Estate,
Bear... |
Theses Digitisation:
https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/
This is a digitised version of the original print thesis.
Copyright and moral rights for this work are retained by the author
A copy can be downloaded for personal non-commercial research or study, without prior permission ... |
Electric Dockyard Crane
A No.10 Set model built & described by B.N. LOVE
Because of the size and complexity of this outstanding, advanced model we are unable to provide the full building instructions in this one feature. They would take up most of the magazine! We are therefore splitting the instructions into two ins... |
SINGLES TO WATCH
HARD SUN
Indio
HARRY HOUDINI
Kon Kan
WILDFIRE
The Nylons
KEEP EACH OTHER WARM
Barry Manilow
DON'T SAY YOU LOVE ME
Billy Squier
WE CAN LAST FOREVER
Chicago
TOY SOLDIERS
Martika
HOT ALBUMS
MADONNA
Like A Prayer
ROXETTE
Look Sharp
GUNS 'N' ROSES
G'N'R Lies
SIMPLY RED
A New Flame
ROD STEWART
O... |
EDITOR'S NOTE:
This issue of the FLY PAPER really is a flying paper! Thru an error, Boss Riddle boarded a northbound Eastern Air Lines ship with the FLY PAPER copy in his brief case. We caught him by telegraph at Jacksonville airport, and he shipped the copy back by the next returning plane. Apologies for being a day ... |
REINFORCED CONCRETE STRUCTURES UNDER IMPACT LOADS
Abdul Jabbar Sangi BE, M.Engg
Submitted for the degree of Doctor of Philosophy
Heriot-Watt University
School of the Built Environment
April 2011
The copyright in this thesis is owned by the author. Any quotation from the thesis or use of any of the information con... |
Theses Digitisation:
https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/
This is a digitised version of the original print thesis.
Copyright and moral rights for this work are retained by the author
A copy can be downloaded for personal non-commercial research or study, without prior permission ... |
October 23, 2009
Via Electronic Filing and U.S. Mail
Public Utility Commission of Oregon
Attention: Filing Center
PO Box 2148
Salem, OR 97308-2148
Re: INTERNATIONAL PAPER COMPANY, Complainant, vs.
PACIFICORP dba PACIFC POWER, Respondent
OPUC Docket No. UM 1449
Attention Filing Center:
Enclosed for filing ... |
NATIONAL ADVISORY COMMITTEE FOR AERONAUTICS
WARTIME REPORT
ORIGINALLY ISSUED
March 1946 as Advance Confidential Report 5K27
THEORY AND APPLICATION OF HOT-WIRE INSTRUMENTS IN THE INVESTIGATION OF TURBULENT BOUNDARY LAYERS
By G. B. Schubauer and P. S. Klebanoff
National Bureau of Standards
WASHINGTON
NACA WARTIME ... |
SINISTER WISDOM
Inside the Archives
Founders and publishers: Harriet Desmoines & Catherine Nicholson
Editors: C. Colette, Harriet Desmoines, Catherine Nicholson, Carolyn Shafer
Poetry Editor: Susan Leigh Star
Review Editor: Julia Penelope
Contributing Editors: Sarah Hoagland, Beth Hodges, Mab Segrest
Cover Desig... |
WIN A VIDEO RECORDER!
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STUBBLE, BUBBLE, toll and trouble: as a million small girls recoil in horror at Georgie Michael's recently acquired beard, RM presents its exclusive guide to pop's flirtation with facial hair... |
Gregory L. Lippetz (State Bar No. 154228)
firstname.lastname@example.org
Laurie M. Charrington (State Bar No. 229679)
email@example.com
JONES DAY
Silicon Valley Office
1755 Embarcadero Road
Palo Alto, CA 94303
Telephone: 650-739-3939
Facsimile: 650-739-3900
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INTERSIL CORPORATION AND ELANTEC SEM... |
Development of diagnostic tools and analysis of the density functional theory zoo
Pierpaolo Morgante
Follow this and additional works at: https://repository.fit.edu/etd
Part of the Chemistry Commons
Development of diagnostic tools and analysis of the density functional theory zoo.
by
Pierpaolo Morgante
A dissert... |
Avoidance in Nonpayment Situations and Fundamental Breach Under the 1980 U.N. Convention on Contracts for the International Sale of Goods
Olof Clausson
Follow this and additional works at: https://digitalcommons.nyls.edu/journal_of_international_and_comparative_law
Part of the Law Commons
Recommended Citation
Claus... |
CUSTOMS MEMORANDUM CIRCULAR
NO. 150-2019
SUBJECT: Intellectual Property Recordation of JUUL LABS INC. Trademarks
Attached are copies of Certificates of Registration No. 4-2018-011466, 4-2018-022134 & 4-2018-022133 certified as true copies by the Intellectual Property Office on 26 April 2019, issued by the Bureau of T... |
**CULTURAL DATA BANK**
**BARTLETT**
**KAOS PIRATE TV LIBRARY**
**POLITICAL:**
- ABC CBS NBC
- Kenya, Laos, Cambodia May 1970 2 hrs
- Demetrius & The Gladiators 2 hrs
- Samson & Delilah 2 hrs
- % Paths of Glory 1 hr
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**ARTS/CULTURE:**
- How Life Begins 1 hr
**MISCELLANEOUS:**
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Loudspeakers in Vented Boxes: Part II*
A. N. THIELE
Australian Broadcasting Commission, Sydney, N.S.W. 2001, Australia
Editor's Note: Part I of *Loudspeakers in Vented Boxes* was published in the May, 1971 issue of the Journal.
VIII. LOUDSPEAKER EFFICIENCY
In Eq. (12) an expression was derived for the efficiency o... |
Causal Inference
Nancy Cartwright
1. Problems for Causation
Causation has long been a vexed notion across the sciences, so much so that there have been repeated cries that it must be banished forever from both the natural and the social sciences. Yet, whatever is the case with the natural sciences, causation remains... |
NEUROENDOCRINE REGULATION OF GASTRIC ENDOCRINE CELL FUNCTION
Thesis submitted in accordance with the requirements of The University of Liverpool for the degree of Doctor of Philosophy by Andrew Damian Higham B.Sc., M.B. Ch.B., M.R.C.P.
March 1998
LIVERPOOL UNIVERSITY LIBRARY
LATIVX
The last decade has seen major a... |
AN ORDINANCE APPROVING, ADOPTING AND ENACTING AMERICAN LEGAL PUBLISHING'S OHIO BASIC CODE, 2017 EDITION, AS THE CODE OF ORDINANCES FOR THE MUNICIPALITY OF Amanda Village, OHIO, AND DECLARING AN EMERGENCY.
WHEREAS, the present general and permanent ordinances of the municipality are inadequately arranged and classified... |
Project Overview
Whether traveling to school, running errands, or enjoying a stroll through the park, many people in Pekin choose to walk and bike, and many more would choose to do so if conditions were better.
A safe and connected system of sidewalks, trails, and bikeways not only supports people to traveling by bik... |
Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy
Pascal J. Lafontant
*DePauw University*, email@example.com
Follow this and additional works at: [https://scholarship.depauw.edu/bio_facpubs](... |
Cellular characterization of the outgrowth and connectivity of the brain serotonin system
Jeroen Dudok
The research described in this thesis was conducted at the Center for Neurogenomics and Cognitive Research (CNCR), Vrije Universiteit Amsterdam, The Netherlands, and was supported by a grant from the Dutch Organizat... |
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