Figure 4.

CCK^LPBN Neurons Engage SF1^VMH Neurons to Mediate CR-Responses

(A) Unilateral stereotaxic injection of Cre-dependent synpatophysin-mCherry virus into the LPBN of CCK-ires-Cre mice facilitated genetically defined tract tracing of CCK^LPBN neuron projections.

(B and C) CCK^LPBN neurons send ascending projections to the ipsilateral hypothalamus, including the lateral hypothalamus (LH), dorsomedial nucleus (DMH), and VMH.

(D) CNO-mediated activation of unilateral CCK-ires-Cre::hM3Dq-mCherry^LPBN neurons evokes cFOS-IR (magenta) within ipsilateral SF1^VMH neurons (green) in CCK-ires-Cre::SF1-Cre::R26-loxSTOPlox-L10-GFP mice.

(E and F) A total of 55% (5/9) of synaptically isolated SF1^VMH neurons were activated by CCK (CCK-8S, 100 nM) in ex vivo slice preparations maintained under hypoglycemic conditions (0.5 mM glucose).

(E) Representative electrophysiological trace of a SF1^VMH neuron demonstrating CCK-induced activation.

(F) CCK-responsive SF1^VMH neurons exhibited a 2.5-fold increase in firing frequency over baseline upon CCK-8S administration (n = 6, paired t test, t₍₄₎ = 4.1, p = 0.01).

(G and H) Functional occlusion of CCK^LPBN neuron glucoregulation through concomitant silencing of downstream SF1^VMH neurons.

(H) SF1-Cre::hM4D_i-mCherry^VMH silencing prevents the CCK-ires-Cre::hM3D_q-mCherry^LPBN-mediated CR-response in CNO-treated double transduced mice, as compared to CCK-ires-Cre::hM3D_q-mCherry^LPBN only transduced mice (n = 5 per group; two way ANOVA, main effect of treatment [F_(1,56) = 188.2, p < 0.0001], main effect of time [F_(6,56) = 11.9, p < 0.0001], and interaction [F_(6,56) = 4.5, p = 0.0009]; Sidak’s post hoc test for individual time point analysis).

(I) SF1^VMH neuron silencing does not influence blood glucose concentrations compared to saline (n = 4; repeated-measures ANOVA; main effects of treatment, time, and interaction not significant).

(J) The CR-response to 2DG was significantly attenuated by pretreatment with the selective CCK_B-receptor antagonist CI988 (n = 5 per group; two-way ANOVA; main effect of treatment [F_(3,112) = 369.1, p < 0.0001], main effect of time [F_(6,112) = 121.7, p < 0.0001], and interaction [F_(18,112) = 36.2, p < 0.001]; post hoc comparisons determined by Tukey’s post hoc test for individual time point analysis).

Abbreviations: ARC, arcuate nucleus of the hypothalamus; DMH, dorsomedial nucleus of the hypothalamus; LHA, lateral hypothalamic area; MeP, medial amygdaloid nucleus posterior part; PVT, paraventricular nucleus of the thalamus; SAL, saline; VMH, ventromedial nucleus of the hypothalamus; c, central; vl, ventrolateral; dm, dorsomedial. All data are presented as mean ± SEM; ^∗p < 0.05; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.