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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We next calculated the subtype composition of vmPFC neurons projecting to each downstream brain region.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Interestingly, we found that these target specific projection neurons were transcriptionally heterogeneous, which were composed of different neuronal subtypes (Figure 2F).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Neurons projecting to LH or MD were mainly L6-Syt6 subtype, whereas neurons projecting to AI, DMS, or BLA were mainly composed of L5-S100b, and to a lesser extent L6-Npy and L2/3-Rorb subtypes (Figure 2F).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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It is worth noting, based on the observed ratios, that the cellular composition of target-specific projection neurons from FAC-sorted or unsorted groups is similar (Figure 2—figure supplement 1E).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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As the layer distribution of each neuron subtype can be inferred by their layer specific marker gene expression, these results also implied the layer distribution of neurons projecting to each target (Figure 2E, Figure 2—figure supplement 1B).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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By calculating the projection properties of each vmPFC neuron subtype, we found that each transcriptome-defined neuron subtype can project to specific but multiple targets.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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For instance, L5-S100b, L6-Npy and L2/3-Rorb mainly projected to AI, DMS and BLA, while L6-Syt6 mainly projected to MD and LH (Figure 2G).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Interestingly, we also found that different neuron subtypes localized in the same layer could project to distinct targets.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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For instance, L6-Npy neurons projecting to AI, DMS, and BLA, while L6-Syt6 neurons projecting to MD, DMS, and LH (Figure 2G).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Similar phenotypes were observed for L5-S100b and L5-Bcl6 subtypes (Figure 2G), suggesting transcriptomic and projection/function diversities in the spatially close neurons within the same cortical layer.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Together, by MERGE-seq analysis, we have revealed the heterogeneity and cellular composition of vmPFC neurons projecting to different target.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Our results demonstrate that vmPFC neurons projecting to a certain target are composed of different transcriptome-defined neuronal subtypes, and individual transcriptome-defined subtypes of vmPFC neuron project to multiple targets.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Interestingly, we found that a portion of barcoded vmPFC neurons had more than one type of barcode, suggesting collateral projection of these neurons.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We therefore analyzed the projection pattern of each barcoded vmPFC neuron by calculating the number of valid barcode types (see Materials and methods).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We defined the dedicated projection neuron as a neuron containing only one type of barcode, the collateral projection neuron as a neuron containing more than one type of barcode.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We found 64.88% of 2050 viral-barcoded neurons belonged to dedicated projection and the remaining belonged to collateral projection.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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A total of 23.37% had dual targets (bifurcated projection), 8.15% had triple targets, and 3.61%, if any, projected to more than three targets (Figure 3A).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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It is worth mentioning that the definition of ‘dedicated’ and ‘collateral’ projections relies solely on the analysis of MERGE-seq data.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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The quantitative resolution of dedicated and collateral projections of vmPFC neurons will depend on the comprehensiveness of retrograde labeling from all postsynaptic targets and labeling efficiency.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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By calculating the conditional probability that the same neuron projects to two targets (see Materials and methods), we found that vmPFC neurons projecting to AI or BLA were more likely to have collateral projection to DMS (Figure 3B).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We also observed a relatively high conditional probability of collateral projection between MD and LH, or DMS and LH, or DMS and MD (Figure 3B), suggesting bifurcated projections to these paired targets for single vmPFC neuron. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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A) Pie chart indicating the number of projection targets for barcoded vmPFC neurons recovered by MERGE-seq. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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B) Heatmap showing the probability that a neuron projecting to area A also projects to area B. (C) Bar graph illustrating the percentage of neuronal projection pattern of all projection patterns given five projection targets inferred by MERGE-seq (red bars) versus the 1155 fMOST-based single-neuron projectome data (blue bars) (Gao et al., 2022). (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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D) Boxplot comparison of percentage of neurons with different projection targets identified by MERGE-seq and fMOST. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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E) Heatmap showing normalized projection strength.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Rows represent the projection targets and columns represent the cells labeled by the top 10 binary projection patterns or labeled by transcriptional neuron subtypes. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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F) Alluvial plot showing the 10 most frequent projection patterns distribution into neuronal subtypes. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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G) Pie charts describing the projection patterns from (E) partitioned by neuronal subtype.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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In (A, B), 2050 barcoded neurons were represented.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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In (C, D), 2050 barcoded neurons from MERGE-seq data were represented, 1155 cells with fMOST data were represented (Gao et al., 2022).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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In (E–G), 1853 barcoded neurons (top 10 frequent projection patterns) were represented.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Figure 3—figure supplement 1.Immunostaining of dual-color, retrogradely labeled neurons and quantification, PCA plot of projection clusters.(A–L) Immunostaining of dual-color traced retrograde labeled neurons of selected targets DMS (GFPnls) /LH (tdTomato), AI (GFP) /DMS (tdTomato), DMS (GFPnls) /BLA (tdTomato), and BLA (GFP) /LH (tdTomato). (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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A, D, G, J).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Dotted line depicts layers 2/3, 5, and 6 of the vmPFC.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Scale bars, 500 µm. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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B, E, H, K) Enlarged view of the dotted box in (A, D, G, J).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Scale bars, 100 µm. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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C, F, I, L) Histogram shows quantitative data for single- (red, green) and double- (yellow) labeled neurons as mean percentages of total rAAV2-retro labeled neurons (n=3 mice).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Data are presented as mean ± SD.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Pie chart showing layer distribution of double (yellow) labeled neurons. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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M) Normalized projection index barcode expression on PC1 and PC2 embeddings and binary projection annotation labeled on PC1 and PC2 embeddings.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Note that only the 10 most frequent binary projection patterns were included.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Data are the mean ± SD. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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A–L) Immunostaining of dual-color traced retrograde labeled neurons of selected targets DMS (GFPnls) /LH (tdTomato), AI (GFP) /DMS (tdTomato), DMS (GFPnls) /BLA (tdTomato), and BLA (GFP) /LH (tdTomato). (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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A, D, G, J).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Dotted line depicts layers 2/3, 5, and 6 of the vmPFC.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Scale bars, 500 µm. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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B, E, H, K) Enlarged view of the dotted box in (A, D, G, J).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
|
Scale bars, 100 µm. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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C, F, I, L) Histogram shows quantitative data for single- (red, green) and double- (yellow) labeled neurons as mean percentages of total rAAV2-retro labeled neurons (n=3 mice).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Data are presented as mean ± SD.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Pie chart showing layer distribution of double (yellow) labeled neurons. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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M) Normalized projection index barcode expression on PC1 and PC2 embeddings and binary projection annotation labeled on PC1 and PC2 embeddings.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Note that only the 10 most frequent binary projection patterns were included.
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PMC10914349
|
High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Data are the mean ± SD.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We first validate the bifurcated projection patterns (2 targets) inferred from the digital projectome.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We injected retrograde AAV2 encoding different fluorescent proteins (EGFP or tdTomato) into different combinations of projection targets (dual-color rAAV2-retro labeling assay), and analyzed the projection patterns by immunohistochemistry.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Consistent with MERGE-seq identifying DMS + LH bifurcated projections (Figure 3B), dual-color labeling revealed 17.8% ± 0.11% of vmPFC neurons collateralize to DMS and LH (Figure 3—figure supplement 1A–C).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Of these, 73.28% ± 7.60% localized to layer 5 (Figure 3—figure supplement 1A–C).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Other bifurcated projection patterns inferred by MERGE-seq was also verified by our dual-color retro-AAV labeling assay.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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These patterns included DMS + AI (23.1% ± 2.03% of all dual-color neurons) and DMS + BLA (6.59% ± 1.55%) (Figure 3—figure supplement 1D–I).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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In contrast, we only observed 1.66% ± 0.92% of dual-color labeled neurons in BLA + LH group (Figure 3—figure supplement 1J–L).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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This result is consistent with our MERGE-seq analysis, in which BLA + LH was not inferred as bifurcated projection targets (Figure 3B), further supporting the accuracy of the digital projectome based on MERGE-seq analysis.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Since dual-color labeling can only validate two targets, we additionally validated inferred projections by quantifying MERGE-seq patterns as percentages of totals and comparing to published single-neuron PFC projectome data (Gao et al., 2022).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We found that DMS, AI + DMS, MD, and LH projection pattern appear as the most frequent projection patterns in both studies, with a relatively higher percentage of DMS dedicated projection pattern in MERGE-seq data (Figure 3C).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We further categorized projection patterns by number of targets and found no significant differences versus imaging-based reconstruction (Figure 3D), indicating MERGE-seq faithfully identifies projection patterns.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Next, we focused our analysis on the 5 dedicated projections (DMS, AI, MD, LH, and BLA) and most frequent five collateral projections (DMS + AI, DMS + MD, DMS + LH, DMS + AI + MD, and DMS + AI + MD + LH).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We conducted a principal component analysis (PCA) of the projection matrix and mapped binary projection labels on PC embeddings.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Results from binary projection clustering aligned well with clusters at PC1- and PC2-defined embeddings (Figure 3—figure supplement 1M).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We further clustered cells according to projection strength (defined as normalized projection barcode UMI counts; Figure 3E).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We found that cells exhibited collateral projections to DMS + AI, or DMS + MD, or DMS + LH, or DMS + AI + MD, or DMS + AI + MD + LH (Figure 3E), a pattern very similar to that we observed in binary projection model, indicating that projection strength-based clustering is comparable to binary projection pattern model (Figure 3B).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We next explored the cell type composition of the top 10 dedicated or collateral projection neurons.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We mapped transcriptomic clusters to projection patterns (Figure 3F).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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While dedicated and collateral projection neurons were largely transcriptionally diverse (≥3 subtypes, Figure 3G), certain projections like MD-projecting and DMS + MD-projecting were highly homogeneous, composed of >90% L6-Syt6 cells (Figure 3G).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Overall, MERGE-seq elucidated dedicated and collateral vmPFC neuron projections at the single-neuron level, demonstrating diversity in projection patterns within individual vmPFC neurons.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Furthermore, projection-defined (collateral or bifurcated) neurons have specific cell type composition and layer distributions.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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It is worth noting that as a proof of concept, we only acquired the vmPFC projectome from five downstream targets.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Definitions of dedicated or collateral projections are thus limited to these five targets and some collateral projections may be underestimated.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Next, we sought to determine the molecular features of neurons projecting to different downstream targets.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We calculated DEGs for each target-specific projection neurons (Figure 4A, Figure 4—figure supplement 1).
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We found that some of projection-specific DEGs are marker genes of typical neuronal types.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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For example, Syt6, Foxp2, and Cyr61 are both MD-projecting DEGs and marker genes of L6-Syt6 neurons; Rorb and Slc24a3 are both DMS-projecting DEGs and marker genes of layer 2/3 neurons (neuronal subtype L2/3-Rorb; Figure 2C, Figure 4A, Figure 4—figure supplement 1). (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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A) Volcano plots DEGs of MD-projecting versus non-MD-projecting vmPFC neurons.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Assigned DEGs (red dots) were determined using threshold: Log2 fold change = 0.5, p value cutoff=10. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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B) Immunostaining of EGFP (MD) and tdTomato (LH), and RNA FISH of Syt6. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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i, ii) Enlarged view of dotted box in (B). (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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i) represents typical view at layer 6 and (ii) represents typical view at layer 5.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Arrow head indicates Syt6EGFP neurons. (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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C) Quantifications of (B). (
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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B) Scale bars, 200 μm.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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i, ii in (B) Scale bars, 50 μm.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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N=3 mice.
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PMC10914349
|
High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Data are presented as mean ± SD.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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In (A), 8210 cells were represented.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Figure 4—figure supplement 1.Transcriptional profiling of projection target-specific vmPFC neurons.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Volcano plots DEGs of DMS-projecting versus non-DMS-projecting vmPFC neurons, AI-projecting versus non-AI-projecting vmPFC neurons, BLA-projecting versus non-BLA-projecting vmPFC neurons, and LH-projecting versus non-LH-projecting vmPFC neurons.
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PMC10914349
|
High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Assigned DEGs (red dots) were determined using threshold: Log2 fold change = 0.5, p value cutoff=10.
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PMC10914349
|
High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
|
Volcano plots DEGs of DMS-projecting versus non-DMS-projecting vmPFC neurons, AI-projecting versus non-AI-projecting vmPFC neurons, BLA-projecting versus non-BLA-projecting vmPFC neurons, and LH-projecting versus non-LH-projecting vmPFC neurons.
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PMC10914349
|
High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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Assigned DEGs (red dots) were determined using threshold: Log2 fold change = 0.5, p value cutoff=10.
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PMC10914349
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High-throughput mapping of single-neuron projection and molecular features by retrograde barcoded labeling.
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We further validated the molecular features of neurons associated with their specific projections by combining RNA fluorescence in situ hybridization (FISH) and retrograde labeling.
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