Molecular profile and response to energy deficit of leptin-receptor neurons in the lateral hypothalamus

Abstract

Leptin exerts its effects on energy balance by inhibiting food intake and increasing energy expenditure via leptin receptors in the hypothalamus. While LepR neurons in the arcuate nucleus of the hypothalamus, the primary target of leptin, have been extensively studied, LepR neurons in other hypothalamic nuclei remain understudied. LepR neurons in the lateral hypothalamus contribute to leptin's effects on food intake and reward, but due to the low abundance of this population it has been difficult to study their molecular profile and responses to energy deficit. We here explore the transcriptome of LepR neurons in the LH and their response to energy deficit. Male LepR-Cre mice were injected in the LH with an AAV carrying Cre-dependent L10:GFP. Few weeks later the hypothalami from fed and food-restricted (24-h) mice were dissected and the TRAP protocol was performed, for the isolation of translating mRNAs from LepR cells in the LH, followed by RNA sequencing. After mapping and normalization, differential expression analysis was performed with DESeq2. We confirm that the isolated mRNA is enriched in LepR transcripts and other known neuropeptide markers of LepR^LH neurons, of which we investigate the localization patterns in the LH. We identified novel markers of LepR^LH neurons with association to energy balance and metabolic disease, such as Acvr1c, Npy1r, Itgb1, and genes that are differentially regulated by food deprivation, such as Fam46a and Rrad. Our dataset provides a reliable and extensive resource of the molecular makeup of LH LepR neurons and their response to food deprivation.

Figure 1

Targeting and RNA enrichment from LepR^LH cells (A) LepR-Cre mice and wild type mice were injected in the LH with an AAV carrying Flex-L10:GFP (graphic of coronal section does not represent exact coordinates). In wild-type littermates (wt/wt) the GFP expression was very low (right image) compared to LepR-cre mice. In homozygous Cre (cre/cre) mice, 83.5% of the cells with immunohistochemically-detected GFP were in the LH (mean, n = 12 hypothalami). (B) Scatterplot of RNAseq results showing normalized counts from LepR^LH fraction (IP) in x axis plotted against normalized counts from Hypothalamus fraction (IN) in y axis. Violet-colored dots represent genes that are enriched in IP (with log2FoldChange > 1.5 and adjusted p < 0.01) and dark grey-colored dots represent genes that are enriched in IN (with log2FoldChange < − 1.5 and adjusted p < 0.01). GFP, LepR and known marker genes of LepR^LH cells are represented as dots colored in dark violet. (C) Scatterplot of RNAseq results showing genes de-enriched in IP fraction (enriched in IN fraction) as log2 of normalized counts from IN fraction (x-axis) plotted against Fold-enrichment (log2 of IP/IN normalized counts) (y-axis). Markers known to not be present in LepR^LHcells (Agrp, Npy) and non-neuronal markers are represented as dark violet dots. (D) Barplot showing Fold-enrichment (log2 of IP/IN normalized counts) of GABAergic markers Slc32a1, Gad1 and Gad2 and glutamatergic marker Slc17a6 (mean ± SEM). IP: immunoprecipitated samples, IN: input samples.

Figure 2

Expression of neuropeptides in LepR^LH cells and co-localization patterns in the LH. (A) Fixed coronal sections from LepR-Cre x L-tdTomato mice underwent fluorescent in situ hybridization (FISH) with anti-sense RNA probes for Nts, Gal, Cartpt, Tac1 and Crh ("Neuropeptide"—red channel) followed by immunohistochemistry (IHC) for tdTomato ("LepR"—green channel) and imaging. The percentage of Neuropeptide⁺LepR⁺ cells out of the total LepR⁺ in the LH was quantified, as represented in the barplots (mean, n = 3 hypothalami). (B) Localization of Neuropeptide⁺LepR⁺ cells was observed under the microscope and drawn on representative graphic images of coronal sections from the mouse Brain Atlas, which were adapted for this figure. Arc: Arcuate nucleus of the Hypothalamus, 3 V: 3rd Ventricle, DMH: Dorsomedial Hypothalamus, VMH: Ventromedial Hypothalamus, LH: Lateral Hypothalamus, PeF: Perifornical area, f: Fornix, MTu: Medial Tuberal nucleus.

Figure 3

Genes significantly enriched in IP over IN fraction categorized by molecular function. Barplots represent RNAseq results of the Fold-enrichment (IP/IN of normalized counts) in x-axis of the top 10 most significantly enriched genes between the IP and IN fractions (mean ± SEM) per type of molecular function (sorted by adjusted p value).

Figure 4

Genes differentially regulated by fasting (A) Heatmap of ten (10) genes that were differentially regulated by 24-h fasting (adjusted p < 0.05, n = 3 samples/condition). (B) Representative images of in situ hybridization (ISH) stainings for Fam46a and Rrad in the mouse hypothalamus (bregma points − 1.4 and − 1.8 mm) from the Allen Brain Atlas database.