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Single-cell transcriptomic analysis of the lateral hypothalamic area reveals molecularly distinct populations of inhibitory and excitatory neurons

Nature Neuroscience volume 22, pages 642–656 (2019)Cite this article

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Abstract

The lateral hypothalamic area (LHA) coordinates an array of fundamental behaviors, including sleeping, waking, feeding, stress and motivated behavior. The wide spectrum of functions ascribed to the LHA may be explained by a heterogeneous population of neurons, the full diversity of which is poorly understood. We employed a droplet-based single-cell RNA-sequencing approach to develop a comprehensive census of molecularly distinct cell types in the mouse LHA. Neuronal populations were classified based on fast neurotransmitter phenotype and expression of neuropeptides, transcription factors and synaptic proteins, among other gene categories. We define 15 distinct populations of glutamatergic neurons and 15 of GABAergic neurons, including known and novel cell types. We further characterize a novel population of somatostatin-expressing neurons through anatomical and behavioral approaches, identifying a role for these neurons in specific forms of innate locomotor behavior. This study lays the groundwork for better understanding the circuit-level underpinnings of LHA function.

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Fig. 1: scRNA-seq of LHA cell populations.
Fig. 2: Classification of LHAGlut and LHAGABA neuronal cell types in the LHA.
Fig. 3: Validation of discriminatory marker expression in LHAGlut Hcrt-expressing neurons.
Fig. 4: Validation of discriminatory marker expression in LHAGlut Pmch-expressing neurons and subpopulations.
Fig. 5: Validation of discriminatory marker expression in LHAGlut Trh-expressing neurons and subpopulations.
Fig. 6: Validation of discriminatory marker expression in LHAGABA Nts-expressing neurons and subpopulations.
Fig. 7: Defining four molecularly distinct populations of Sst-expressing neurons in the LHA:
Fig. 8: Functional and anatomical interrogation of Sst-expressing neurons in the LHA:

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Code availability

No specific custom code was developed for this analysis. The analysis routine for the single-cell data is defined in the scRNA-seq analysis section and further details, if needed, provided upon request. Interactive analysis was done using the CellView RShiny web application65.

Data availability

All data that support the findings of this study are available as raw data in GEO at GSE125065.

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Acknowledgements

The authors gratefully acknowledge A. Lucido, R. Kanadia, M. Andermann, A. Chesler, B. Jones, A. Nishiyama and A. Tzingounis for valuable discussions and comments on the manuscript. They also thank H. Fitch, J. Salamone, C. Cain and L. Ostroff for advice on behavioral experiments. The authors also gratefully acknowledge T. Sakurai (University of Tsukuba) and M. Myers (University of Michigan) for the use of mutant mouse lines, W. He for flow cytometry support, J. Nasuta for genotyping support, C. O’Connell for imaging support and W. Flynn for assistance with sequencing data. The project was supported by National Institutes of Health grants R00MH097792 and R01MH112739 (to A.C.J.), a Connecticut Institute for the Brain and Cognitive Sciences Seed Grant (to A.C.J.) and student fellowships (to L.E.M., A.F. and J.R.N.), a Connecticut Innovations Regenerative Medicine Research Fund grant 15-RMD-UCHC-01 (to P.R.), the Beckman Scholars Program (to B.R.C. and E.B.), and a NIH Shared Instrumentation Grant S10OD016435 (to A. Nishiyama) for imaging support.

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Author notes

  1. Mohan Bolisetty

    Present address: Bristol-Myers Squibb, Pennington, NJ, USA

  2. These authors contributed equally: Laura E. Mickelsen and Mohan Bolisetty.

Authors and Affiliations

  1. Department of Physiology and Neurobiology, University of Connecticut, Storrs, CT, USA

    Laura E. Mickelsen, Brock R. Chimileski, Akie Fujita, Eric J. Beltrami, James T. Costanzo, Jacob R. Naparstek & Alexander C. Jackson

  2. Connecticut Institute for the Brain and Cognitive Sciences, Storrs, CT, USA

    Laura E. Mickelsen, Akie Fujita & Alexander C. Jackson

  3. The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA

    Mohan Bolisetty & Paul Robson

  4. Department of Biomedical Engineering, University of Connecticut, Storrs, CT, USA

    Akie Fujita

  5. Department of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington, CT, USA

    Paul Robson

  6. Institute for Systems Genomics, University of Connecticut, Farmington, CT, USA

    Paul Robson & Alexander C. Jackson

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Contributions

L.E.M. designed experiments, performed microdissections, single-cell isolation, sc-qPCR experiments and analyses, FISH experiments and analyses, stereotactic injections, neuroanatomical tracing and imaging, behavioral experiments and analyses and edited the manuscript. M.B. performed single-cell capture, scRNA-seq library preparation and sequencing, developed bioinformatic pipelines, performed bioinformatic analyses and edited the manuscript. B.R.C. performed FISH experiments and analyses. A.F. performed slice electrophysiology and analysis. E.B. performed behavioral experiments and analyses. J.T.C. and J.R.N. performed neuroanatomical tracing and imaging. P.R. designed experiments, provided intellectual guidance on bioinformatics and edited the manuscript. A.C.J. conceived and supervised the study, designed experiments and wrote the manuscript.

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Correspondence to Paul Robson or Alexander C. Jackson.

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Journal peer review information: Nature Neuroscience thanks Arpiar Saunders and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Integrated supplementary information

Supplementary Figure 1 Comparison of female and male single-cell LHA samples.

(a) Comparison of male (green) and female (blue) LHA cells (total n = 6944 cells; from 3 male and 2 female mice). LHA single cells shown on t-SNE plots after the first iteration of unsupervised clustering show near-identical cell type assignments. (b) Proportion of male vs. female cells that comprise the 13 clusters of non-neuronal cells; (c) 15 clusters of LHAGlut neurons; and (d) 15 clusters of LHAGABA neurons.

Supplementary Figure 2 Classification of major non-neuronal cell types in the LHA.

(a) Unsupervised clustering of non-neuronal cell types in the LHA represented as a t-SNE plot (n = 2536 cells). Different cell type clusters are color coded below. (b) Heatmap showing scaled expression of discriminative marker gene sets across all 13 non-neuronal clusters, with cells as columns and genes as rows (n = 2536 cells). Color scheme represents Z-score distribution with −3 (blue) to 3 (dark orange). (c) Violin plot, representing the distribution of log transformed normalized gene expression (gene UMIs/total cell UMIs) in each cluster, of selected markers for non-neuronal populations (n = 2536 cells). These include three populations of oligodendrocyte lineage cells: a large population of mature oligodendrocytes (cluster 1: Olig1+, Mag+, Opalin+); proliferating oligodendrocyte progenitors (cluster 3: Olig1+, Cspg4+, Pdgfra+); and immature oligodendrocytes (cluster 13: Olig1+, Mag+, Pdgfra-, Fyn+). Two clusters of astrocytes (clusters 6 and 8) exhibited robust expression of Gja1+, Aqp4+ and Agt+, but could be distinguished on the basis of Slc7a10 (high in 6, low in 8) and Gfap (low in 6, high in 8) expression. Other clusters were identified as endothelial cells (cluster 2: Flt1+); red blood cells (cluster 4: Hba-a1+); fibroblasts (cluster 5: Pdgfra+, Col1a2+, Gja1+); macrophages (cluster 7: C1qa+, Cx3cr1+, Mrc1+); pericytes (cluster 9: Cspg4+, Acta2+); and microglia (cluster 10: C1qa+, Cx3cr1+, Tmem119+). All neurons are represented as N. Number of cells, mean genes/cell and mean UMIs/cell is provided in Supplementary Table 1. (d) Distribution of genes and transcripts (UMIs) detected in each non-neuronal cell type in the data and all neurons (N).

Supplementary Figure 3 Genes and transcripts detected in LHAGlut and LHAGABA neuronal cell types in the LHA.

(a) Violin plots representing the distribution of log transformed normalized gene expression (gene UMIs/total cell UMIs) in each LHAGlut neuronal cluster in the data (n = 1537 cells). (b) Heatmap showing scaled expression (log CPM) of discriminative marker gene sets across all 15 LHAGlut clusters (n = 1537 cells), with cells as columns and genes as rows. Color scheme represents Z-score distribution with −3 (blue) to 3 (dark orange). (c) Violin plot showing distribution of genes and transcripts detected in each LHAGABA neuronal cluster in the data (n = 1900 cells). (d) Heatmap showing scaled expression (log CPM) of discriminative marker gene sets across all 15 LHAGABA clusters (n = 1900 cells), with cells as columns and genes as rows. Color scheme represents Z-score distribution with −3 (blue) to 3 (dark orange). Total number of cells, mean genes/cell and mean UMIs/cell is provided in Supplementary Table 1.

Supplementary Figure 4 Comparison of Allen Brain Atlas ISH expression for single discriminatory markers for each LHAGABA and LHAGlut cluster.

Representative Allen Mouse Brian Atlas (Lein et al, 2007) micrographs of ISH expression of single discriminatory markers for (a) LHAGlut clusters, and (b) LHAGABA clusters, in coronal sections containing the LHA (corresponding to middle/posterior LHA). Image credit for all ISH images: Allen Institute. Note that some identical ISH figures, for discriminatory markers that define more than one cluster, appear multiple times (ex. Sst, Trh), and one ISH is unavailable (Atp1a2).

Supplementary Figure 5 Control for FISH experiments using RNAScope.

(a) Left: Representative Allen Brian Atlas (Lein et al, 2007) micrograph of Hcrt ISH expression in a coronal section containing the LHA. Middle: Representative Allen Brian Atlas micrograph of Slc2a13 ISH expression in a coronal section containing the LHA. Image credit: Allen Institute. Right: Schematic of a coronal section (modified from58) containing the LHA, where boxes (red) indicate localization of LHA, cortex (Ctx) and hippocampus (Hipp). (b) Confocal (40X) micrographs of coronal sections of wild type mice showing expression of mRNA for Hcrt and Slc2a13 in the LHA (left), Ctx (middle) and Hipp (right). All counterstained with DAPI (blue). Note the absence of Hcrt signal in the Ctx and Hipp relative to the robust signal in the LHA. Experiments were repeated in n = 3 mice.

Supplementary Figure 6 Additional discriminatory-marker expression in LHAGlut Hcrt-expressing neurons and possible subpopulations.

(a) Confocal micrographs (40X) of coronal sections of wild-type mice and corresponding pie charts representing co-expression of mRNA for Hcrt and Scg2 (n = 203 cells; 3 mice) (top), Hcrt and Slc2a13 (n = 254 cells; 3 mice) (bottom); all counterstained with DAPI (blue). White arrowheads indicate co-localization, scale bar 50 μm. (b) Heatmap showing scaled expression (log CPM) of discriminative genes across the two Hcrt+ subclusters (n = 162 cells). (c) Unsupervised clustering of Hcrt+ neurons of the LHA represented as a t-SNE plot (n = 162 cells), showing subcluster 1 (blue) and subcluster 2 (green). (d) Normalized expression of discriminatory markers for both subclusters 1 and 2, shown as t-SNE plots (Ddx3y, Fos, Pde10a, Xist, Tox2 and Gm10076) (n = 162 cells).

Supplementary Figure 7 Anatomical distribution of Trh-expressing subpopulations throughout the LHA.

(a) Left: Anatomical schematic of the LHA corresponding to anterior (bregma −1.34 mm), middle (bregma −1.58 mm) and posterior (bregma −1.82 mm). Boxes indicate localization of individual confocal micrographs across LHA sections (red). Right: Confocal micrographs (40X) of coronal sections of wild type mice and corresponding pie charts representing co-expression of mRNA for Trh, Syt2 and Cbln2 in anterior (n = 458 cells, 3 mice) (top), middle (n = 371 cells, 3 mice) (middle), and posterior (n = 258 cells, 3 mice) (bottom); all counterstained with DAPI (blue). White arrowheads indicate co-localization of Trh/Cbln2 and white asterisks indicate co-localization of Trh/Syt2, scale bar 50 μm.

Supplementary Figure 8 Gal-expressing LHAGABA neurons and anatomical analysis of Nts–Cartpt co-expression throughout the LHA.

(a) Violin plot, representing the distribution of log transformed normalized gene expression (gene UMIs/total cell UMIs), of Slc32a1, Slc17a6, and discriminatory markers for LHAGlut cluster 1 (Gal/Dlk1) neurons (n = 450 cells). Total number of cells, mean genes/cell and mean UMIs/cell provided in Supplementary Table 1. (b) Confocal micrographs (40X) of coronal sections of wild type mice and corresponding pie charts representing co-expression of mRNA for Slc32a1, Dlk1, and Gal (n = 728 cells, 3 mice). (c) Table illustrating proportion of cells from scRNA-seq data co-expressing Slc32a1, Slc17a6, Nts and Nts/Cartpt. (d) Left: Pie chart representing proportion of all Nts+ neurons in the scRNAseq dataset that are either Slc32a1+ (blue) or Slc17a6+ (pink) (total n = 380 cells) Right: Pie chart representing proportion of all Nts+ neurons (n = 380) that are assigned to LHAGABA cluster 3 (Nts/Cartpt) (n = 74). (e) Left: Anatomical schematic of the LHA corresponding to anterior (bregma −1.34 mm), middle (bregma −1.58 mm) and posterior (bregma −1.82 mm). Boxes indicate localization of individual confocal micrographs across LHA sections (red). Right: Confocal micrographs (40X) of coronal sections of wild type mice and corresponding pie charts representing co-expression of mRNA for Nts and Cartpt in anterior (n = 254 cells, 4 mice) (top), middle (n = 559 cells, 4 mice) (middle), and posterior (n = 523 cells, 4 mice) (bottom) LHA; all counterstained with DAPI (blue). White arrowheads indicate co-localization, scale bar 50 μm.

Supplementary Figure 9 Expression of Lepr and Mc4r relative to LHAGABA Nts+ and Gal+ neurons.

(a) Normalized expression of Nts, Gal, Lepr and Mc4r expression among all LHAGABA clusters shown in t-SNE plots (n = 1900 cells). Dashed circles denote the location of LHAGABA clusters 1 (Gal/Dlk1) and 3 (Nts/Cartpt). Note low/sparse expression of Lepr and Mc4r in these or any LHAGABA cluster. (b) Schematic of LHA tissue microdissected from Lepr-Cre;EYFP mice for sc-qPCR. (c) Heatmap of 53 single Lepr+ neurons (from 2 mice) and their expression of 12 genes by qPCR. Heatmap colors depict expression levels on a log2 scale from low (blue) to high (red). Note the high proportion of Lepr+ neurons that exhibit a GABAergic phenotype (Slc32a1+/Gad1+/Gad2+) and an enrichment in neurons that co-express Nts and Gal.

Supplementary Figure 10 Anatomical distribution of GABAergic vs glutamatergic Sst-expressing neurons across the rostrocaudal axis.

(a) Confocal micrographs (40X) of coronal sections of wild-type mice containing the perifornical LHA and corresponding pie chart representing co-expression of mRNA for Sst, Slc32a1, and Slc17a6 in anterior (n = n = 419 cells, 4 mice) (top), middle (n = 243 cells, 4 mice) (middle) and posterior (n = 190 cells, 4 mice) (bottom). (b) Confocal micrographs (40X) of coronal sections of wild-type mice from the tuberal region and corresponding pie chart representing co-expression of mRNA for Sst, Slc32a1, and Slc17a6 in middle (n = 243 cells, 4 mice) (top) and posterior (n = 570 cells, 4 mice) (bottom). White arrowheads indicate co-localization of Sst/Slc32a1 and white asterisks indicate co-localization of Sst/Slc17a6. All counterstained with DAPI (blue), scale bar 50 μm.

Supplementary Figure 11 Additional discriminatory markers that define LHAGABA Sst-expressing neurons distributed among the perifornical LHA and tuberal region.

(a) Confocal micrographs (40X) of coronal sections of wild-type mice and corresponding pie chart representing co-expression of mRNA within the perifornical LHA for Sst, Slc32a1, and Meis2 (n = 117 cells, 2 mice), Sst, Otp, and Slc32a1 (n = 57 cells, 2 mice), Sst, Slc32a1, and Ptk2b (n = 179 cells, 3 mice), Sst, Nrgn, and Slc32a1 (n = 129 cells, 2 mice). (b) Confocal micrographs (40X) of coronal sections of wild-type mice and corresponding pie charts representing co-expression of mRNA within the tuberal region for Sst, Slc32a1, and Meis2 (n = 140 cells, 2 mice), Sst, Otp, and Slc32a1 (n = 153 cells, 2 mice), Sst, Slc32a1, and Ptk2b (n = 247 cells, 3 mice), Sst, Nrgn, and Slc32a1 (n = 89 cells, 2 mice). All counterstained with DAPI (blue). White arrowheads indicate co-localization, scale bar 50 μm.

Supplementary Figure 12 Additional discriminatory markers that define LHAGlut Sst-expressing neurons in the perifornical LHA.

(a) Confocal micrographs (40X) of coronal sections of wild type mice and corresponding pie chart representing co-expression of mRNA within the perifornical LHA for Sst, Slc17a6, and Nkx2.1 (n = 90 cells, 3 mice), Sst, Npy, and Slc17a6 (n = 103, cells, 3 mice), Sst, Slc17a6, and Calcr (n = 238 cells, 3 mice). All counterstained with DAPI (blue). White arrowheads indicate co-localization, scale bar 50 μm.

Supplementary Figure 13 Electrophysiological signatures distinguish Sst-expressing neurons in the perifornical LHA and tuberal region.

(a) Schematic illustrating anatomical location of 59 recorded EYFP+ neurons found in the perifornical (PeF) LHA (n = 40 cells; red) and tuberal (Tub) region (n = 19 cells; grey) (approximate distance from bregma, −1.58 mm) in 10 Sst-Cre;EYFP mice (4 males and 6 females). (b) Representative confocal images of a biocytin-filled (immunostained with streptavidin-conjugated Alexa Fluor 594), EYFP+ PeF neuron (left) and Tub neuron (right). Scale bars represent 30 μm. (c) Representative single action potential (AP) waveforms of EYFP + PeF and Tub neurons. Traces are overlaid and aligned at the voltage and time of peak for visual comparison. (d) Bar plots showing significant differences in AP half-width (p = 0.019) (left), decay time (p = 0.002) (middle) and afterhyperpolarization (AHP) amplitude (p = 0.002) (right) for PeF (n = 40) and Tub (n = 19) neurons. (e) Representative responses to a 1 s hyperpolarizing step of −60 pA in representative EYFP+ PeF and Tub neurons. (f) Plot of average repolarization latency in response to 1 s hyperpolarizing steps of 0 to −60 pA with steps of −20 pA (left) and bar plot of spike ratio at −60 pA (p = 0.003) (right). (g) Plot of average firing frequency in response to 1 s depolarizing steps of 0 to 200 pA with steps of + 20 pA (left) and bar plot of maximum firing rate (p = 0.003) (right). Asterisks indicate statistical significance *p < 0.05, **p < 0.01 using an unpaired two-sample Wilcoxon rank sum test. Center values indicate mean, error bars represent ± s.e.m.

Supplementary Figure 14 Chemogenetic viral injections, cFos staining and behavioral analysis.

(a) Left: Schematic of injection sites (red) for control animals injected with AAV-DIO-mCherry (n = 6 mice) in anterior, middle and posterior LHA sections. Right: Representative fluorescence micrographs (10X) of control animal. Counterstained with DAPI, scale bar represents 500 μm (applies to all images). (b) Schematic of injection sites (red) for hM3Dq animals injected with AAV-DIO-hM3Dq-mCherry (n = 4 mice) in anterior, middle and posterior sections. Right: Representative fluorescence micrographs (10X) of hM3Dq animal. Counterstained with DAPI. (c) Fluorescence micrographs (10X) of representative LHA sections from control (n = 2 mice) (top) and hM3Dq (n = 2 mice) (bottom) mice IP injected with CNO 90 min prior to sacrifice, counterstained with anti-cFos and DAPI. Scale bar represents 500 μm. (d) Bar graphs showing average time spent (%) for control (gray) and hM3Dq (blue) mice for the 10 manually scored behaviors across a 1 h pre-injection period. (e) Bar graphs showing average time spent (%) for control (gray) and hM3Dq (blue) mice for 10 manually scored behaviors across a 1 h post-injection period. P values are as follows: gnawing p = 0.011, digging p = 0.026, resting p = 0.014, eating p = 0.005, rearing p = 0.023. Open circles represent individual data points, asterisks indicate statistical significance *p < 0.05, **p < 0.01 using an unpaired two-sample Wilcoxon rank sum test. Center values indicate mean, error bars represent ± s.e.m.

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Mickelsen, L.E., Bolisetty, M., Chimileski, B.R. et al. Single-cell transcriptomic analysis of the lateral hypothalamic area reveals molecularly distinct populations of inhibitory and excitatory neurons. Nat Neurosci 22, 642–656 (2019). https://doi.org/10.1038/s41593-019-0349-8

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