A parabrachial-hypothalamic cholecystokinin neurocircuit controls counterregulatory responses to hypoglycemia

Abstract

Hypoglycemia engenders an autonomically mediated counterregulatory (CR)-response that stimulates endogenous glucose production to maintain concentrations within an appropriate physiological range. Although the involvement of the brain in preserving normoglycemia has been established, the neurocircuitry underlying centrally mediated CR-responses remains unclear. Here we demonstrate that lateral parabrachial nucleus cholecystokinin (CCK(LPBN)) neurons are a population of glucose-sensing cells (glucose inhibited) with counterregulatory capacity. Furthermore, we reveal that steroidogenic-factor 1 (SF1)-expressing neurons of the ventromedial nucleus of the hypothalamus (SF1(VMH)) are the specific target of CCK(LPBN) glucoregulatory neurons. This discrete CCK(LPBN)→SF1(VMH) neurocircuit is both necessary and sufficient for the induction of CR-responses. Together, these data identify CCK(LPBN) neurons, and specifically CCK neuropeptide, as glucoregulatory and provide significant insight into the homeostatic mechanisms controlling CR-responses to hypoglycemia.

Graphical abstract

Figure 1

CCK^LPBN Neurons Are Activated by Glucoprivation (A–D) 2DG- and INS-induced glucoprivation promoted cFOS-IR (red) within the LPBN, as compared to saline controls. Both glucoprivic stimuli elicited cFOS-IR within the sLPBN (B,C), with 2DG also increasing neural activity within the external compartment of the LPBN (B). (D) Quantification of cFOS-IR across the rostral-to-caudal extent of the LPBN (see Figure S1) revealed a significant elevation of cFOS-IR in 2DG- and INS-treated mice above saline controls (n = 3–5 per group; one-way ANOVA, F_(2,8) = 31.1, p = 0.0002 with Tukey’s post hoc comparison). (E–G) The sLPBN is located at the rostral and dorsal extreme of the LPBN and defined by the expression of CCK. (F and G) Transgenic labeling of CCK neurons (green) in a CCK-ires-Cre::R26-loxSTOPlox-L10-GFP mouse line recapitulated the known endogenous expression profile. (H–K) 2DG- and INS-induced glucoprivation increased cFOS-IR (red) within CCK^LPBN neurons (green) compared to saline controls (white arrows denote colocalized neurons) (n = 3–5 per group; one-way ANOVA, F_(2,9) = 23.2, p = 0.0003 with Tukey’s post hoc comparison). (L–N) A subset of CCK^LPBN neurons were inhibited by glucose. (L and M) 5/14 CCK^LPBN neurons exhibited reversible membrane depolarization in response to a downward glucose step form 5 mM to 0.5 mM (n = 5, repeated-measures one-way ANOVA, F_(2,4) = 77.3, p = 0.0003 with Tukey’s post hoc comparison). (N) Representative electrophysiological trace from a spontaneously active glucose-inhibited CCK^LPBN neuron. 2DG, 2-deoxyglucose; e, external LPBN; INS, insulin; SAL, saline; scp, superior cerebellar peduncle; s, superior LPBN. All data are presented as mean ± SEM. ^∗∗p < 0.01; ^∗∗∗p < 0.001.

Figure 2

CCK^LPBN Neurons Promote SNS-Mediated CR-Like Responses (A and B) Bilateral stereotaxic injection of Cre-dependent excitatory hM3D_q-mCherry virus into the LPBN of male CCK-ires-Cre mice facilitated real-time activation of CCK^LPBN neurons. (A) Representative image of Cre-dependent expression of hM3D_q-mCherry specifically within the LPBN of a CCK-ires-Cre mouse. (B) Membrane potential and firing rate of CCK-ires-Cre::hM3D_q-mCherry^LPBN neurons increased upon 5 μM CNO application. (C) CCK-ires-Cre::hM3D_q-mCherry^LPBN mice exhibited a significant hyperglycemic response to CNO, compared to saline, administration (n = 7; repeated-measures ANOVA, main effect of treatment [F_(1,36) = 39.6, p < 0.0001], main effect of time [F_(5,36) = 6.6, p = 0.0002], and interaction [F_(5,36) = 4.3, p = 0.003]; post hoc comparisons determined by Sidak’s post hoc test for individual time point analysis). (D–F) CNO treatment evoked an increase in serum glucagon (D; n = 3 per group; t test, t₍₄₎ = 26.0, p < 0.0001), corticosterone (E; n = 3 to 4 per group; t test, t₍₆₎ = 4.4, p = 0.004), and epinephrine concentrations (F; n = 6–8 per group; t test, t₍₁₂₎ = 2.3, p = 0.04). (G) CCK^LPBN neuron activation was associated with increased hepatic G6pc mRNA expression (n = 3 to 4 per group; t test, t₍₄₎ = 2.7, p = 0.05) compared to saline controls. (H) CNO-induced hyperglycemia was abolished by pretreatment with pan-specific CCK-receptor antagonist (20 mg/kg proglumide: PROG) (data shown at 60 min after CNO/SAL administration; n = 5; repeated-measures ANOVA, F_(4,12) = 5.0, p = 0.04 with Tukey’s post hoc comparison). (I and J) CNO treatment did not influence feeding behavior compared to saline. (I) Three hour dark-cycle food intake (n = 10; paired t test, t₍₉₎ = 0.4, p = 0.7) and (J) 3 hr light-cycle food intake (n = 5; paired t test, t₍₄₎ = 1.8, p = 0.1) in ad-libitum-fed mice. CNO, clozapine-N-oxide; G6pc, glucose-6-phosphatase; scp, superior cerebellar peduncle; SAL, saline. All data are presented as mean ± SEM; ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 3

CCK^LPBN Neurons Are Necessary for CR-Response to Glucoprivation (A and B) Bilateral stereotaxic injection of Cre-dependent inhibitory hM4D_i-mCherry virus into the LPBN of male CCK-ires-Cre mice facilitated the real-time inhibition of CCK^LPBN neurons. (A) Representative image of Cre-dependent expression of hM4D_i-mCherry specifically within the LPBN of a CCK-ires-Cre mouse. (B) Membrane potential and firing rate of CCK-ires-Cre::hM4Di-mCherry^LPBN neurons decreased upon 5 μM CNO application. (C) CNO-induced CCK^LPBN neuron silencing in normoglycemic CCK-ires-Cre::hM4D_i-mCherry^LPBN mice had no effect on blood glucose levels, compared to saline controls (n = 11; repeated-measures ANOVA, main effects of treatment and time, and interaction, not significant). (D and E) CNO-induced CCK^LPBN neuron silencing under glucoprivic conditions attenuated the CR-response. (D) 2DG-induced CR was significantly diminished by CNO pretreatment compared to saline (n = 8; repeated-measures ANOVA, main effect of treatment [F_(1,49) = 14.3, p < 0.0004], main effect of time [F_(6,49) = 64.7, p < 0.0001], and interaction [F_(6,49) = 2.8, p = 0.02]; post hoc comparisons determined by Sidak’s post hoc test for individual time point analysis). (E) Likewise, INS-induced CR was significantly diminished by CNO pretreatment compared to saline (n = 5; repeated-measures ANOVA, main effect of treatment [F_(1,32) = 4.6, p = 0.04], main effect of time [F_(7,32) = 22.3, p < 0.0001], and interaction [F_(7,32) = 2.8, p = 0.02]; post hoc comparisons determined by Sidak’s post hoc test for individual time point analysis). Abbreviations: 2DG, 2-deoxyglucose; INS, insulin; scp, superior cerebellar peduncle; SAL, saline. All data are presented as mean ± SEM.; ^∗∗p < 0.01; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001.

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.