Single-cell transcriptomic evidence for dense intracortical neuropeptide networks
- Allen Institute for Brain Science, United States
- Neuroscience Paris Seine, Sorbonne Université, France
Figures
Figure 1
Single-cell NPP gene expression and co-expression statistics for distant neocortical areas VISp and ALM show that expression patterns for 18 NPP genes are highly differential within both neocortical areas but highly conserved between areas.
(A) Different NPP genes show very different expression level distributions across the 22,439 VISp+ALM neurons sampled. Color-coded solid curves plot single-cell CPM values for the specified …
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Figure 1—source data 1
Sorted NPP CPM distributions for all neurons.
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig1-data1-v2.csv
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Figure 1—source data 2
Single-cell NPP CPM expression table.
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig1-data2-v2.csv
Figure 2 with 1 supplement
Single-cell NP-GPCR gene expression and co-expression statistics for distant neocortical areas VISp and ALM show that expression patterns for 29 NP-GPCR genes are highly differential within neocortical areas but conserved between areas.
(A) Different NP-GPCR genes show very different expression level distributions across the 22,439 VISp+ALM neurons sampled. Color-coded solid curves plot single-cell CPM values for the specified …
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Figure 2—source data 1
Sorted NP-GPCR CPM distributions for all neurons.
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig2-data1-v2.csv
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Figure 2—source data 2
Single-cell NP-GPCR CPM expression table.
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig2-data2-v2.csv
Figure 2—figure supplement 1
Co-expression of cognate NPP/NP-GPCR pairs.
A large fraction of cortical neurons co-express cognate NPP/NP-GPCR pairs, and conservation of these fractions between cortical areas VISp and ALM argues in favor of some important underlying …
Figure 3 with 3 supplements
Neurotaxonomic heatmaps reveal highly neuron-type-specific expression of (A) 18 NPP and (B) 29 NP-GPCR genes in 22,439 individual neurons harvested from areas VISp and ALM.
Trimmed-mean (5% trim) CPM expression values for each of the 115 VISp+ALM glutamatergic and GABAergic neuron types (see Figure 3—figure supplement 1) are normalized per gene to maximum value …
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Figure 3—source data 1
Cell types, cluster ids and color codes for ALM and VISp regions.
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig3-data1-v2.csv
Figure 3—figure supplement 1
Tasic 2018 neurotaxonomy.
Tasic and co-workers (Tasic et al., 2018, Nature 563:72–78) generated the hierarchical taxonomy of glutamatergic and GABAergic neurons represented here by dimensionality reduction and iterative …
Figure 3—figure supplement 2
NPP expression variation within subclasses.
Coefficient of variation (c.v. = standard deviation/mean) of expression for each NPP gene for Tasic et al., 2018 cell types pooled to 12 major types, glutamatergic types (IT, PT, NP, CT, L6b) and …
Figure 3—figure supplement 3
NP-GPCR expression variation within subclasses.
Coefficient of variation (c.v. = standard deviation/mean) of expression for each NP-GPCR gene for Tasic et al., 2018 cell types pooled to 12 major types, glutamatergic types (IT, PT, NP, CT, L6b) …
Figure 4 with 2 supplements
Neurons can be clustered effectively based on just 47 NP genes (18 NPP and 29 NP-GPCR).
(A) A two-dimensional latent space representation of 22,439 cells based on 6083 highly expressed (HE) genes obtained by an autoencoding neural network. Dots represent individual cells, colored …
Figure 4—figure supplement 1
Autoencoder expression clustering performance by neuron type.
Results here compare classification based on 6,083 HE genes (dashed line) with results based on 47 NP genes (colored vertical bars). The resolution index (RI), as described in connection with Figure …
Figure 4—figure supplement 2
1```Determination of optimal autoencoder latent space dimensionality.
To determine the latent space dimensionality for optimal HE gene representation, we performed hyperparameter tuning by varying the latent space dimensionality between 2 and 20 dimensions. The …
Figure 5
Neurotaxonomic heatmaps show strong conservation of NPP and NP-GPCR expression patterns between two distant neocortical areas.
(A) Expression heatmap for 18 NPP and 29 NP-GPCR genes in 13,491 single VISp neurons classified by type. (B) Similar heatmap for 8948 single neurons harvested from ALM. Heatmaps generated and …
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Figure 5—source data 1
Cell types, cluster ids and color codes for VISp region.
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig5-data1-v2.csv
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Figure 5—source data 2
Cell types, cluster ids and color codes for ALM region.
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig5-data2-v2.csv
Figure 6 with 10 supplements
Neurotaxonomic expression profiles of 37 cognate NPP/NP-GPCR pairs predict 37 peptidergic networks.
Weighted adjacency matrix plots predicting local peptidergic coupling amongst neuron types (A–E) and subclasses (F). Matrices were computed as outer products (CPM*CPM units) of row and column factor …
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Figure 6—source data 1
NP coupling predictions by cognate pair and type for area VISp (archive containing 37 CSV files, one for each of the 37 cognate pairs listed in Table 3 and represented in Figure 6—figure supplements 1–8).
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig6-data1-v2.zip
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Figure 6—source data 2
NP coupling predictions by cognate pair and type for area ALM (archive containing 37 CSV files, one for each of the 37 cognate pairs listed in Table 3 and represented in Figure 6—figure supplements 1–8).
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig6-data2-v2.zip
Figure 6—figure supplement 1
Neurotaxonomic coupling predictions for cognate NPP/NP-GPCR pairs #1-#4.
Predictions for all 37 cognate pairs in both VISp and ALM cortical areas are grouped and color-coded according to Gα signal transduction family. All predictions were generated as described in …
Figure 6—figure supplement 2
Neurotaxonomic coupling predictions for cognate NPP/NP-GPCR pairs #5-#9.
Predictions for all 37 cognate pairs in both VISp and ALM cortical areas are grouped and color-coded according to Gα signal transduction family. All predictions were generated as described in …
Figure 6—figure supplement 3
Neurotaxonomic coupling predictions for cognate NPP/NP-GPCR pairs #10-#14.
Predictions for all 37 cognate pairs in both VISp and ALM cortical areas are grouped and color-coded according to Gα signal transduction family. All predictions were generated as described in …
Figure 6—figure supplement 4
Neurotaxonomic coupling predictions for cognate NPP/NP-GPCR pairs #15-#18.
Predictions for all 37 cognate pairs in both VISp and ALM cortical areas are grouped and color-coded according to Gα signal transduction family. All predictions were generated as described in …
Figure 6—figure supplement 5
Neurotaxonomic coupling predictions for cognate NPP/NP-GPCR pairs #19-#23.
Predictions for all 37 cognate pairs in both VISp and ALM cortical areas are grouped and color-coded according to Gα signal transduction family. All predictions were generated as described in …
Figure 6—figure supplement 6
Neurotaxonomic coupling predictions for cognate NPP/NP-GPCR pairs #24-#27.
Predictions for all 37 cognate pairs in both VISp and ALM cortical areas are grouped and color-coded according to Gα signal transduction family. All predictions were generated as described in …
Figure 6—figure supplement 7
Neurotaxonomic coupling predictions for cognate NPP/NP-GPCR pairs #28-#32.
Predictions for all 37 cognate pairs in both VISp and ALM cortical areas are grouped and color-coded according to Gα signal transduction family. All predictions were generated as described in …
Figure 6—figure supplement 8
Neurotaxonomic coupling predictions for cognate NPP/NP-GPCR pairs #33-#37.
Predictions for all 37 cognate pairs in both VISp and ALM cortical areas are grouped and color-coded according to Gα signal transduction family. All predictions were generated as described in …
Figure 6—figure supplement 9
Hierarchical relationships of coupling matrices.
For area VISp (left) and ALM (right), the NPP, NP-GPCR coupling matrices C are linearized to vectors and then clustered using hierarchical clustering (average linkage) of the Pearson correlation …
Figure 6—figure supplement 10
Coupling matrix localization and correlation metrics for VISp and ALM areas.
To measure the extent to which each (NPP, NP-GPCR) pair p demonstrates localized signal in its coupling matrix in a region , we extract the top 10% percentile of , then reduce the remaining …
Figure 7
Pooling NP-GPCRs by primary Gα family enables neurotaxonomic prediction of primary NP-GPCR signaling impacts across 37 cognate NP pairs (see text) for each of areas VISp and ALM.
(A) ISQ color maps representing coupling matrix predictions at the Tasic 2018 neurotaxonomy type level, merging Gi/o (red), Gs (green) and Gq/11 (blue) primary Gα family components. (B) Component …
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Figure 7—source data 1
VISp coupling predictions by Gα family and type.
(archive containing three CSV files, one for each of Gi/o, Gs and Gq/11 families).
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig7-data1-v2.zip
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Figure 7—source data 2
ALM coupling predictions by Gα family and type.
(archive containing three CSV files, one for each of Gi/o, Gs and Gq/11 families).
- https://cdn.elifesciences.org/articles/47889/elife-47889-fig7-data2-v2.zip
Figure 8
Neurotaxonomy offers a framework for integrating statistical descriptions of multiple modulatory and synaptic networks, as schematized here for purely fictitious neuron types, transcriptomic and connectomic data.
Multiple directed network graphs are predicted here for peptidergic networks from transcriptomic data (A–C) and for synaptic networks from connectomic data (D,E) to predict a modulated synaptic …
Author response image 1
Tables
Table 1
Single-cell RNA-seq expression statistics for 18 highly expressed neuropeptide precursor protein (NPP) genes cognate to locally expressed NP-GPCR genes (see Table 2).
NPP genes are tabulated here along with peak single-cell expression levels as pFPKM (Peak FPKM, see Materials and methods), percentile and absolute ranking of these pFPKM values across pFPKMs for …
| NPP Gene | Peak FPKM | pFPKM Percentile | pFPKM Rank | % Cells | Predicted Neuropeptides | Cognate NP-GPCR Genes | |
|---|---|---|---|---|---|---|---|
| Npy | 108,865 | 100.00 | 1 | 42 | Neuropeptide Y | Npy1r, Npy2r, Npy5 | |
| Sst | 70,274 | 99.99 | 2 | 26 | Somatostatins | Sstr1, Sstr2, Sstr3, Sstr4 | |
| Vip | 48,747 | 99.99 | 3 | 33 | Vasoactive Intestinal Peptide | Vipr1, Vipr2 | |
| Tac2 | 18,284 | 99.98 | 4 | 15 | Neurokinin B | Tacr3 | |
| Cck | 16,396 | 99.97 | 6 | 69 | Cholecystokinins | Cckbr | |
| Penk | 11,160 | 99.96 | 8 | 26 | Enkephalins | Oprd1, Oprm1 | |
| Crh | 9,118 | 99.95 | 10 | 17 | Corticotropin-Releasing Hormone | Crhr1, Crhr2 | |
| Cort | 7,477 | 99.93 | 15 | 32 | Cortistatin | Sstr1, Sstr2, Sstr3, Sstr4 | |
| Tac1 | 5,728 | 99.92 | 18 | 11 | Substance P, Neurokinin A | Tacr1 | |
| Pdyn | 2,813 | 99.69 | 68 | 8 | Dynorphins | Oprd1, Oprk1, Oprm1 | |
| Pthlh | 1,656 | 99.29 | 156 | 18 | Parathyroid-Hormone-Like Hormone | Pth1r | |
| Pnoc | 698 | 97.68 | 509 | 23 | Nociceptins | Oprl1 | |
| Trh | 510 | 96.51 | 766 | 3 | Thyrotropin-Releasing Hormone | Trhr, Trhr2 | |
| Grp | 435 | 95.59 | 968 | 12 | Gastrin-Releasing Peptide | Grpr | |
| Rln1 | 258 | 91.99 | 1757 | 7 | Relaxin 1 | Rxfp1, Rxfp2 | Rxfp3 |
| Adcyap1 | 165 | 87.29 | 2788 | 26 | Adenylate Cyclase-Activating Polypeptides | Adcyap1r1, Vipr1, Vipr2 | |
| Nts | 121 | 82.14 | 3917 | 1 | Neurotensin | Ntsr1, Ntsr2 | |
| Nmb | 112 | 80.53 | 4270 | 14 | Neuromedin B | Nmbr | |
Table 2
Single-cell RNA-seq expression statistics for 29 neuropeptide-selective, G-protein-coupled receptor (NP-GPCR) genes cognate to locally expressed NPP genes (see Table 1).
NP-GPCR gene peak FPPM values, percentile ranking, and percentage sampled as for NPP genes in Table 1. The table names encoded NP-GPCR proteins, A-F class of NP-GPCR, primary Gα signal transduction …
| NP-GPCR Gene | Peak FPKM | pFPKM Percentile | % Cells | Neuropeptide Receptor | GPCR Class | Primary Gα Family | Cognate NPP Genes |
|---|---|---|---|---|---|---|---|
| Sstr2 | 413 | 95.3 | 42 | Somatostatin Receptor 2 | A4 | Gi/o | Sst, Cort |
| Npy2r | 291 | 93.1 | 10 | Neuropeptide Y Receptor Y2 | A9 | Gi/o | Npy |
| Npy1r | 272 | 92.4 | 50 | Neuropeptide Y Receptor Y1 | A9 | Gi/o | Npy |
| Grpr | 231 | 91 | 10 | GRP Receptor | A7 | Gq/11 | Grp |
| Cckbr | 210 | 90 | 52 | Cholecystokinin B Receptor | A6 | Gq/11 | Cck |
| Ntsr2 | 161 | 86.9 | 17 | Neurotensin Receptor 2 | A7 | Gq/11 | Nts |
| Npy5r | 152 | 86.1 | 28 | Neurpeptide Y Receptor Y5 | A9 | Gi/o | Npy |
| Nmbr | 123 | 82.4 | 8 | Neuromedin B Receptor | A7 | Gq/11 | Nmb |
| Rxfp1 | 121 | 82 | 22 | Relaxin Family Receptor 1 | A5 | Gs | Rln1 |
| Sstr4 | 106 | 79.5 | 28 | Somatostatin Receptor 4 | A4 | Gi/o | Sst, Cort |
| Trhr | 101 | 78.4 | 3 | TRH Receptor | A7 | Gq/11 | Trh |
| Sstr1 | 90 | 76 | 38 | Somatostatin Receptor 1 | A4 | Gi/o | Sst, Cort |
| Adcyap1r1 | 89 | 75.8 | 71 | ADCYAP1 Receptor 1 | B1 | Gs | Adcyap1, Vip |
| Crhr1 | 86 | 74.9 | 28 | CRH Receptor 1 | B1 | Gs | Crh |
| Rxfp3 | 85 | 74.7 | 5 | Relaxin Family Receptor 3 | A5 | Gi/o | Rln1 |
| Oprl1 | 82 | 73.8 | 48 | Opioid Receptor-Like 1 | A4 | Gi/o | Pnoc |
| Crhr2 | 72 | 70.7 | 3 | CRH Receptor 2 | B1 | Gs | Crh |
| Tacr3 | 65 | 68 | 3 | Tachykinin Receptor 3 | A9 | Gq/11 | Tac2 |
| Oprk1 | 64 | 67.4 | 3 | Kappa-Opioid Receptor | A4 | Gi/o | Pdyn |
| Tacr1 | 56 | 64.2 | 3 | Tachykinin Receptor 1 | A9 | Gq/11 | Tac1 |
| Pth1r | 51 | 61.6 | 15 | PTH 1 Receptor | B1 | Gq/11 | Pthlh |
| Vipr1 | 41 | 56.1 | 28 | VIP Receptor 1 | B1 | Gs | Vip, Adcyap1 |
| Oprm1 | 35 | 52.1 | 43 | Mu-Opioid Receptor | A4 | Gi/o | Penk, Pdyn |
| Trhr2 | 30 | 48.9 | 10 | TRH Receptor 2 | A7 | Gq/11 | Trh |
| Vipr2 | 30 | 48.4 | 0.5 | VIP Receptor 2 | B1 | Gs | Vip, Adcyap1 |
| Rxfp2 | 28 | 47.3 | 4 | Relaxin Family Receptor 2 | A5 | Gs | Rln1 |
| Oprd1 | 26 | 45.8 | 13 | Delta-Opioid Receptor | A4 | Gi/o | Penk, Pdyn |
| Ntsr1 | 24 | 44.3 | 10 | Neurotensin Receptor 1 | A7 | Gq/11 | Nts |
| Sstr3 | 17 | 39.5 | 21 | Somatostatin Receptor 3 | A4 | Gi/o | Sst, Cort |
Table 3
The 18 NPP and 29 NP-GPCR genes of Tables 1 and 2 constitute 37 cognate NPP/NP-GPCR pairs and predict at least 37 potentially distinct peptidergic modulatory networks.
The 37 pairs are enumerated here along with indications of the expected primary GPCR signal transduction class for each NP-GPCR (Alexander et al., 2017) and a fraction denoting frequency with which …
| # | Cognate Pair Symbol | NPP Gene | NP-GPCR Gene | Primary Gα Family | Fraction of Type Pairs | # | Cognate Pair Symbol | NPP Gene | NP-GPCR Gene | Primary Gα Family | Fraction of Type Pairs |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | Npy→Npy1r | Npy | Npy1r | Gi/o | 0.7805 | 19 | Vip→Vipr1 | Vip | Vipr1 | Gs | 0.496 |
| 2 | Npy→Npy2r | Npy | Npy2r | Gi/o | 0.341 | 20 | Vip→Vipr2 | Vip | Vipr2 | Gs | 0.052 |
| 3 | Npy→Npy5r | Npy | Npy5r | Gi/o | 0.8095 | 21 | Crh→Crhr1 | Crh | Crhr1 | Gs | 0.3925 |
| 4 | Sst→Sstr1 | Sst | Sstr1 | Gi/o | 0.751 | 22 | Crh→Crhr2 | Crh | Crhr2 | Gs | 0.2035 |
| 5 | Sst→Sstr2 | Sst | Sstr2 | Gi/o | 0.836 | 23 | Rln1→Rxfp1 | Rln1 | Rxfp1 | Gs | 0.2465 |
| 6 | Sst→Sstr3 | Sst | Sstr3 | Gi/o | 0.405 | 24 | Rln1→Rxfp2 | Rln1 | Rxfp2 | Gs | 0.07 |
| 7 | Sst→Sstr4 | Sst | Sstr4 | Gi/o | 0.806 | 25 | Adcyap1→Adcyap1r1 | Adcyap1 | Adcyap1r1 | Gs | 0.284 |
| 8 | Penk→Oprd1 | Penk | Oprd1 | Gi/o | 0.4955 | 26 | Adcyap1→Vipr1 | Adcyap1 | Vipr1 | Gs | 0.1465 |
| 9 | Penk→Oprm1 | Penk | Oprm1 | Gi/o | 0.9 | 27 | Adcyap1→Vipr2 | Adcyap1 | Vipr2 | Gs | 0.0155 |
| 10 | Cort→Sstr1 | Cort | Sstr1 | Gi/o | 0.6265 | 28 | Tac2→Tacr3 | Tac2 | Tacr3 | Gq/11 | 0.0955 |
| 11 | Cort→Sstr2 | Cort | Sstr2 | Gi/o | 0.6965 | 29 | Cck→Cckbr | Cck | Cckbr | Gq/11 | 0.6635 |
| 12 | Cort→Sstr3 | Cort | Sstr3 | Gi/o | 0.338 | 30 | Tac1→Tacr1 | Tac1 | Tacr1 | Gq/11 | 0.119 |
| 13 | Cort→Sstr4 | Cort | Sstr4 | Gi/o | 0.672 | 31 | Pthlh→Pth1r | Pthlh | Pth1r | Gq/11 | 0.392 |
| 14 | Pdyn→Oprd1 | Pdyn | Oprd1 | Gi/o | 0.2115 | 32 | Trh→Trhr | Trh | Trhr | Gq/11 | 0.016 |
| 15 | Pdyn→Oprk1 | Pdyn | Oprk1 | Gi/o | 0.0745 | 33 | Trh→Trhr2 | Trh | Trhr2 | Gq/11 | 0.055 |
| 16 | Pdyn→Oprm1 | Pdyn | Oprm1 | Gi/o | 0.4 | 34 | Grp→Grpr | Grp | Grpr | Gq/11 | 0.113 |
| 17 | Pnoc→Oprl1 | Pnoc | Oprl1 | Gi/o | 0.654 | 35 | Nts→Ntsr1 | Nts | Ntsr1 | Gq/11 | 0.0225 |
| 18 | Rln1→Rxfp3 | Rln1 | Rxfp3 | Gi/o | 0.106 | 36 | Nts→Ntsr2 | Nts | Ntsr2 | Gq/11 | 0.054 |
| 37 | Nmb→Nmbr | Nmb | Nmbr | Gq/11 | 0.5655 |
Additional files
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Supplementary file 1
Gene ontology results for 18 NPP genes.
- https://cdn.elifesciences.org/articles/47889/elife-47889-supp1-v2.csv
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Supplementary file 2
Gene ontology results for 29 NP-GPCR genes.
- https://cdn.elifesciences.org/articles/47889/elife-47889-supp2-v2.csv
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Supplementary file 3
pFPKM expression data for 94 putative mouse NPP genes.
- https://cdn.elifesciences.org/articles/47889/elife-47889-supp3-v2.csv
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Supplementary file 4
Criteria for selecting 18 genes from a set of 39 cortically expressed NPP genes.
- https://cdn.elifesciences.org/articles/47889/elife-47889-supp4-v2.csv
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Supplementary file 5
pFPKM expression data for 84 putative mouse NP-GPCR genes.
- https://cdn.elifesciences.org/articles/47889/elife-47889-supp5-v2.csv
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Transparent reporting form
- https://cdn.elifesciences.org/articles/47889/elife-47889-transrepform-v2.docx
