Circuit and molecular architecture of a ventral hippocampal network

Abstract

The ventral hippocampus (vHPC) is a critical hub in networks that process emotional information. While recent studies have indicated that ventral CA1 (vCA1) projection neurons are functionally dissociable, the basic principles of how the inputs and outputs of vCA1 are organized remain unclear. Here, we used viral and sequencing approaches to define the logic of the extended vCA1 circuit. Using high-throughput sequencing of genetically barcoded neurons (MAPseq) to map the axonal projections of thousands of vCA1 neurons, we identify a population of neurons that simultaneously broadcast information to multiple areas known to regulate the stress axis and approach–avoidance behavior. Through molecular profiling and viral input–output tracing of vCA1 projection neurons, we show how neurons with distinct projection targets may differ in their inputs and transcriptional signatures. These studies reveal new organizational principles of vCA1 that may underlie its functional heterogeneity.

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Fig. 1: High-throughput mapping of vCA1 projections using MAPseq.
Fig. 2: vCA1 projection motifs.
Fig. 3: Viral input–output tracing of vCA1.
Fig. 4: RV-labeled inputs to vCA1 output neurons.
Fig. 5: Transcriptional profiling of vCA1 neurons defined by connectivity.

Data availability

RNA-seq data are available in the NCBI’s Gene Expression Omnibus under accession number GSE150869, and MAPseq data are available at NLM Sequence Read Archive BioProject under accession number PRJNA633836. RNA-seq differential expression data is provided in Supplementary Table 4. All MAPseq source data can be downloaded at https://github.com/mkheirbek/. MAPseq motif counts are provided in Supplementary Table 1. All counts for input–output rabies tracing (n, mean and s.e.m.) are provided in Supplementary Table 2.

Code availability

Code for analysis is posted on the Kheirbek Lab GitHub site (https://github.com/mkheirbek/).

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Acknowledgements

We thank J. Jimenez and L. Drew for feedback on the manuscript, A. Zador for initial guidance on MAPseq, H. Zhan and the CSHL MAPseq core facility, and Z. Knight, C. Zimmerman and D. Lieb for advice on TRAP experiments. A.V.M. is supported by the Pew Charitable Trusts, the National Institute of Mental Health (NIMH; R01MH119349 and DP2MH116507), the Brain and Behavior Research Foundation and the Burroughs Welcome Fund. M.A.K. is supported by NIMH (R01 MH108623, R01 MH111754 and R01 MH117961), a One Mind Rising Star Award, the Human Frontier Science Program, the Pew Charitable Trusts, the Esther A. and Joseph Klingenstein Fund, the McKnight Memory and Cognitive Disorders Award and The Ray and Dagmar Dolby Family Fund.

Author information

Affiliations

Authors

Contributions

Conceptualization: M.A.K.; methodology: M.A.K.; investigation: M.M.G., K.J.H., K.J.C., H.S.C. and V.S.T.; formal analysis: M.A.K., M.M.G., K.J.H., H.S.C., B.B., I.D.V. and A.V.M.; writing: M.A.K. (original draft) and all authors (review, editing and methods); resources: M.A.K.; and supervision: M.A.K.

Corresponding author

Correspondence to Mazen A. Kheirbek.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Neuroscience thanks Lucas Pozzo-Miller and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Clustering MAPseq data and projection strengths of individual vCA1 neurons.

a. k-means clustering of MAPseq data. Clusters are plotted as a grid-structured scatter plot where each row is a cluster and the color of the dots along each column correspond to the normalized projection strength to a region b. Plots of normalized projection strength of all multiple projection barcode motifs from MAPseq data. As in Fig. 2 each line (different color) is an individual neuron’s projection strength to each of the 7 target regions, normalized to the maximal value in that row, resulting in a projection strength scale from 0 to 1 (see Methods). Black line is mean projection strength for all neurons in that motif, and grey is SEM. Exact cell number for each motif is provided in the inset (observed and expected from null model, see Methods), and in Supplementary Data Table 1.

Extended Data Fig. 2 Brainwide extra-hippocampal input to vCA1 neurons that project to LH, BA, NAc, adBNST, LS and mPFC.

a–d. Fraction of extrahippocampal input from nuclei in the (a) thalamus, (b) amygdala, (c) contralateral CA3, (d) midbrain/hindbrain and (e) basal forebrain/septum. See Methods for all abbreviations. n= 6 vCA1-mPFC, 8 vCA1-NAc, 9 vCA1-LH, 9 vCA1-BA, 4 vCA1-adBNST and 3 vCA1-LS mice . Error bars represent standard error of the mean. See Extended data table 2 for proportion counts (mean ± SEM) for all assayed input regions and all statistical values. f. Controls for non-specific rabies infection. No long-range inputs were labeled in mice injected with Cre-dependent rabies helper virus and EnvA-G deleted rabies mCherry virus in the vCA1 subregion of vHPC in the absence of AAV2retro Cre injection (n=2 mice, no long-range input cells detected). right, image of red channel from thalamus, basal forebrain and amygdala of control mice. g. Number of extrahippocampal inputs correlates with the number of starter cells in RV samples (Pearson’s r=0.4035, two-tailed, p=0.037). AA-anterior amygdala, AD-anterodorsal nucleus, AM-anteromedial nucleus, BLA-basolateral amygdala, BMA-basomedial amygdala, CeA-central amygdala, CL-central lateral nucleus of the thalamus, CLI-central linear nucleus raphe, CM-central medial nucleus of the thalamus, CoA-cortical amygdala, DR-dorsal raphe nucleus, DTN-dorsal tegmental nucleus, IAD-interanterodorsal nucleus, IF-interfascicular nucleus raphe, IMD-Intermediodorsal nucleus of the thalamus, IPN-interpeduncular nucleus, LA-lateral amygdala, LC-locus coeruleus, LDT-laterodorsal tegmental nucleus, LHA-lateral hypothalamic area, LP-lateral posterior nucleus of the thalamus, LS-lateral septum, MR-median raphe, MA-magnocellular nucleus, MD-intermediodorsal nucleus of the thalamus, mPOA-medial preoptic area, MS-medial septum, MY-medulla, NDB-diagonal band nucleus, NI-nucleus incertus, NLoT-nucleus of lateral olfactory tract, P-pons, PAA-piriform-amygdalar area, PAG-periaqueductal gray, PB-parabrachial nucleus, PCG-pontine central gray, PF-parafascicular nucleus, PPN-pedunculopontine nucleus, PR-perireunensis nucleus, PRNr-pontine reticular nucleus, PT-parataenial nucleus, PVH-paraventricular nucleus of hypothalamus, PVT, paraventricular nucleus of the thalamus, RE-nucleus of reuniens, RPO-nucleus raphe pontis, RT- reticular nucleus of the thalamus, SC- superior colliculus, SG-supragenual nucleus, SI- substantia innominate, SUT-supratrigeminal nucleus, TR-piriform transition area, VAL-ventral anterior-lateral complex of the thalamus, VII-facial motor nucleus, VM-ventral medial nucleus of the thalamus, VTA-ventral tegmental, Xi-xiphoid thalamic nucleus.

Extended Data Fig. 3 Transcriptional profiling of vCA1 projection neurons.

a. Volcano plots of differentially expressed genes for each set of pairwise comparisons. Red dots indicate differentially expressed genes that passed p<0.05 cutoff using Wald test, two-sided, and after correction for multiple comparisons with Benjamini Hochberg. All exact p-values provided in Extended Data Table 4. b. Normalized relative expression of the all 24 genes that passed the significance threshold for differential expression in pairwise comparisons . n=3 vCA1 to mPFC replicates, 2 vCA1 to LH replicates, 3 vCA1 to NAc replicates and 3 vCA1 to BA replicates. Error bars indicate SEM. c. RNAscope of CDR1 transcript in vCA1 neurons defined by their projection to the mPFC. Arrowheads indicate co-labeled neurons. Scale bar 40 μm. Right. Quantification of overlap in CTB labeled and CDR1 labeled cells, n=9 FOVs from 2 mice, error bars indicate SEM. d. Heatmap correlations of total log2 normalized read counts from individual sample sets used for profiling experiments. e. Principal components plots of 500 most variable genes in the dataset, showing first 5 PCs.

Supplementary information

Reporting Summary

Supplementary Table 1

MAPseq motif counts. Sheet 1: co-projection motif counts and percentages (within a motif) from MAPseq dataset. Sheet 2: observed and expected motif counts from MAPseq dataset with effect size and P values.

Supplementary Table 2

Input–output rabies tracing data. Sheet 1: proportion of inputs to mPFC-, NAc-, LH-, BA-, adBNST- and LS-projecting vCA1 neurons. Sheet 2: statistical results for TRIO dataset.

Supplementary Table 3

RNA-seq mapping, alignment and raw counts data. Sheet 1: table containing summary information regarding the mapping of each RNA-seq sample. Sheet 2: table containing summary information regarding counts of alignment types from each RNA-seq sample. Sheet 3: table of all RNA-seq samples and their raw (unnormalized) read counts per gene.

Supplementary Table 4

RNA-seq differential expression data. Sheet 1: all genes from TRAP dataset with counts and fold changes. Sheet 2: differentially expressed genes for vCA1–mPFC versus vCA1–subcortical targets.

Supplementary Table 5

Metascape GO Metabolism table.

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Gergues, M.M., Han, K.J., Choi, H.S. et al. Circuit and molecular architecture of a ventral hippocampal network. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-0705-8

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