Figure 3.

PVN^OT neurons are activated by CCK via a direct, CCK_AR-dependent mechanism in lean but not obese mice

(A) 2-photon micrograph of an acute brain slice expressing the Ca²⁺ indicator GCaMP6f (green, top left) in PVN^OT neurons conditionally tagged by Ai14-tdTomato (red, bottom left) in a cell-type-specific manner (merge, center). Representative images were taken during the time-lapse recording before (top right) and after bath application of 50 nM CCK (bottom right) in the presence of synaptic blockers. Scale bars, 25 μm.

(B) Heatmap representation of cytosolic Ca²⁺ transients of individual PVN^OT neurons upon bath application of CCK (50 nM) in the presence of synaptic blockers. n = 1 mouse, 49 neurons.

(C) Pie chart illustrating the percentages of PVN^OT neurons that increase (yellow) or decrease their activity (purple) upon CCK application or that do not exhibit any change (gray).

(D) Quantification of Ca²⁺ events over time, displayed in 1-min bins. n = 1 mouse, 49 neurons.

(E) 3D-rendered, high-power confocal micrograph depicting Ot mRNA (green), Cckar mRNA (magenta), and DAPI (gray) upon FISH (fluorescence in situ hybridization; RNAscope). Individual nuclei are outlined for demarcation. Scale bar, 20 μm.

(F) Corresponding quantification of FISH using background-corrected mean fluorescence intensity (M.F.I.) of Cckar mRNA per Ot⁺ soma in the rostromedial and caudal PVN of adult male C57BL/6J mice fed either an SC or HFHS diet. Data are presented as mean of all somata analyzed ± SEM. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001. n = 3 mice, 4–8 hemisections per mouse (unpaired Student’s t test).

(G) Representative traces of action potential frequency of putative magnOT neurons derived from SC diet-fed versus HFHS diet-fed mice in response to increasing concentrations of bath-applied A-71623 (5, 25, and 50 nM), followed by superfusion with native CCK (50 nM).

(H) Summary of changes in action potential frequency visualized in (G). Data are presented as mean superimposed with individual data points. ^∗p < 0.05, n = 2–3 mice/6 neurons per mouse (two-way ANOVA).