Molecularly defined subpopulations of leptin receptor neurons dissociate the control of food intake from blood pressure

Figures

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  Figure 1.. Roles for LeprGlp1r neurons in the control of cardiovascular function by leptin. (a) UMAP plot showing MBH neuronal cell types identified during snRNA-seq analysis of tissue from mice treated with saline or exogenous leptin [18]. Lepr-expressing population are denoted by labels and colors. (b) Lepr expression for the 5 identified MBH Lepr cell types, along with all other cell types. (c) Correlation of systolic BP-associated GWAS genes with the transcriptional response to leptin (left panels) or saline (right panels) for LeprGlp1r neurons from WT mice. (d) Schematic showing generation of LeprFlox/Flox control (CT) and Glp1rCre;LeprFlox/Flox (LeprGlp1rKO; KO) mice. (e) Body weight for CT and KO animals (n=11 CT and 12 KO). (f, g) Mean arterial pressure (MAP; f) and HR (g) over 24 hours (left panel) and averaged over the first 30 minutes of the light cycle (right panel) for CT (n=3) and KO (n=3) females. *P < 0.05 by Welche’s t test.

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  Figure 2.. Hypothalamic Lepr neuron-specific snRNA-seq identifies novel populations of Lepr neurons, including distinct subpopulations of LeprGlp1r neurons. (a) UMAP projection of hypothalamic neuron clusters for GFP-expressing cells from LeprSun1-sfGFP mice. (b) Heatmap of the top five genes per cluster with a log2 fold change > 2. (c) Average scaled Lepr expression for each neuron cluster. (d) Heatmap of gene expression the top 10 genes differentially expressed between LeprGlp1r−1 and LeprGlp1r−2 populations shown for all major GABAergic (Slc32a1-expressing) Lepr neuron clusters. Genes examined were all detected in >25% of the relevant cell cluster. (e-h) UMAP feature plots showing cells expressing Glp1r (e), Trh (f), Ebf1 (g), and Bnc2 (h); expression scales for the relevant gene shown to the right of each plot.

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  Figure 3.. Distribution of LeprGlp1r neurons and their subtypes throughout the hypothalamus. (a) Schematic of LeprGFP;Glp1rtdT mouse model. (b-m) Representative images showing GFP (green) and tdTomato (red) (c, e, g, I, k, m) neurons or neurons containing colocalized GFP plus tdTomato (white) (d, f, h, j, l, n) throughout the LeprGFP;Glp1rtdT mouse hypothamalus (representative of n= 4 female and 6 male mice), running caudal (bregma −2.30; b,c) to rostral. Scale bars=100nm. (n) Mapping of LeprGlp1r−1 (Lepr+Glp1r+Trh; blue) and LeprGlp1r−2 (Lepr+Glp1r+Ebf1; red and Lepr+Glp1r+Trh+Ebf1; yellow) neurons onto a sagittal mouse section brain at the indicated rostral-caudal position in the hypothalamus. Distribution of Sim1 (purple) and Nr5a1 (green) signals is also shown.

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  Figure 4.. RAbies Dual Recominase Retrograde (RADRR) system-mediated tracing from ARC and DMH LeprGlp1r neurons. (a) Schematic of RADRR tracing system. Adeno-associated viruses (AAVs) containing rabies glycoprotein (G) AAV8-hSyn1-DIO-TVA-mCherry (purple plasmid) expresses receptor for EnvA (TVA) in LeprCre cells; AAV8-hSyn-fDIO-Rabies-G (grey plasmid) expresses rabies-G in Glp1rFlp cells. Pseudotyped Rabies-ΔG-GFP (green plasmid) infects all LeprCre cells and can propagate one synapse retrograde from LeprCre;Glp1rFlp cells. (b, c) Representative images showing Rabies-ΔG-GFP-infected cells (green) at the viral hit site (*) in the ARC (b, representative of n=1 female and n=3 males) and DMH (c, representative of n=2 females and n=2 males) in LeprCre;Glp1rFlp mice. (d-m) Representative images showing Rabies-ΔG-GFP-infected cells (green) in regions afferent to ARC (d-h) or DMH (i-m) LeprGlp1r cells. Shown are ventral tegmental area (VTA) (d, i), DMH (e, j), PVH, anterior hypothalamic nucleus (AHA) (f, k), periventricular thalamus (PVT), medial preoptic (MPO), and bed nucleus of the stria terminalis (BST) (g-m). Main images, scale bars= 200 um. Insets show digital zooms of relevant areas in main…

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  Figure 5.. Downstream projections from ARC and DMH LeprGlp1r neurons. (a) Schematic of the tTA-driven Recombinase-Guided Intersectional Targeting (tTARGIT) viral system consisting of AAV8-SYN1-TETOX2-FLEX-FRT-tTA (grey plasmid) used to express tetracycline transactivator (tTA) in Glp1rFlp cells; tTA drives the expression of channelrhodopsin-2 (ChR2-eYFP) from rAAV8-TRE-DIO-hChR2-eYFP LeprCreGlp1rFlp cells. (b-k) Representative images showing GFP-immunoreactivity (green) in GFP-IR containing regions from LeprCreGlp1rFlp mice injected in the ARC (b-f, representative of n=2 males and 2 females) and DMH (g-k, representative of n=3 males). Viral hit site indicated by *. Scale bars in main images= 200 um. Scale bars in digital zoom inlay images= 100um.

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  Figure 6.. Modulation of food intake and energy expenditure by ARC and DMH LeprGlp1r neurons in female mice. (a) Schematic of the tTARGIT viral system used to express hM3Dq in LeprGlp1r neurons. AAV8-SYN1-TETOX2-FLEX-FRT-tTA (grey plasmid) mediates the expression of tTA in Glp1rFlp cells, which drives the expression of hM3Dq-mCherry from AAV8-TRE-DIO-hM3Dq-mCherry in LeprCreGlp1rFlp cells. (b, c) IP (core body) temperature (b, c) and intrascapular (BAT) temperature (d, e) over the 360 minutes following the activation of ARC (b, d, n=5) or DMH (c, e, n=4) LeprGlp1r neurons. Shown are averages +/− SEM; *P < 0.05 by Paired t test. (f-p) Continuous measurement of energy expenditure (f-i), cumulative locomotor activity (j-m), and cumulative food intake (n-q) following the injection of saline or CNO in LeprGlp1r-ARC-Dq (yellow; n=5) or LeprGlp1r-DMH-Dq mice (blue; n=4). Measurements were taken automatically using the SABLE metabolic cage system with 12 hour light and dark cycle, during which the animals were acclimated for 2 days, and then treated BID (0900 and 1600) with saline for 3 days and then with CNO (1 mg/kg, IP) for 3 days. Comparisons were made between the third day of saline injections…

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  Figure 7.. Modulation of cardiovascular function by ARC and DMH LeprGlp1r neurons. Shown are mean arterial pressure (MAP; a, b), systolic BP (c, d), diastolic BP (e, f), and HR (g, h) prior to and following the injection of saline (grey lines) or CNO in male LeprGlp1r-ARC-Dq (yellow; n=3) or LeprGlp1r-DMH-Dq mice (blue; n=3) mice. Left panels show continuous measurements; injection (t=40 minutes) is indicated by the arrowhead. Right panels show mean values (−/+ SEM) for 60–120 minutes (60 minutes, starting 20 minutes after the injection). *p< 0.05 and **p< 0.01 by Paired t test.