Figure 5.
Intersectional regulation of hypothalamic OT neurons by CCKAR and κ-opioid receptors is dependent on dietary context
(A) Confocal micrographs of coronal brain section from an adult male OT:Sun1-sfGFP mouse, depicting the nuclear localization of sfGFP (green) in hypothalamic OT neurons (gray) of the PVN and SON. III, third ventricle; ot, optic tract. Scale bars, 200 μm and 25 μm (inset).
(B) Schematic of the workflow used to sort individual sfGFP+ nuclei into 384-well plates using FANS (top left) with a representative FANS plot displayed (top right); isolation was followed by low-volume pipetting robot-assisted single nucleus lysis, cDNA synthesis, and library preparation for snRNA-seq2 (bottom).
(C) Volcano plot highlighting differential gene expression changes across the sum of individual OT nuclei from adult male OT:Sun1-sfGFP mice chronically fed an HFHS diet relative to SC diet-fed littermate controls. n = 2 mice.
(D) Upset plot visualization of intersectional expression of select receptors in individual OT nuclei from SC diet-fed (top) and HFHS diet-fed (bottom) OT:Sun1-sfGFP mice. Transcripts for Cckar and Oprk1 are highlighted (red dash boxes), as well as their intersections (blue bars). Percentages of Cckar+ OT nuclei also expressing Oprk1 are visualized as pie charts for each panel.
(E) UMAP plot visualization of individual OT nuclei from SC diet-fed (top) and HFHS diet-fed (bottom) OT:Sun1-sfGFP mice, colored according to their expression of either Cckar (magenta), Oprk1 (cyan), their combination (blue), or none (gray). N = 2; 614 cells (SC diet) and 588 cells (HFHS diet).
(F) Representative traces of action potential frequency of magnOT neurons derived from adult male OT:Ai14-tdTomato reporter mice fed either an SC diet or HFHS diet in response to bath-applied A-71623 (25 nM) with or without pre-treatment with nor-BNI (200 nM). Scale bar, 25 μm.
(G) Summary of changes in action potential frequency visualized in (F). Data are presented before and after application of A-71623 as mean ± SEM. ∗p < 0.05. n = 2–3 mice/5–8 neurons per mouse (paired Student’s t test).
(H) Cumulative food intake of HFHS diet-fed male C57BL/6J wild-type mice upon injection of a CCK + nor-BNI combination (20 μg/kg BW and 10 mg/kg BW, respectively; i.p.), nor-BNI alone (10 mg/kg BW, i.p.), or vehicle. Data are presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01. n = 6–7 mice (two-way ANOVA).