Functional identification of a neurocircuit regulating blood glucose

Abstract

Previous studies implicate the hypothalamic ventromedial nucleus (VMN) in glycemic control. Here, we report that selective inhibition of the subset of VMN neurons that express the transcription factor steroidogenic-factor 1 (VMN(SF1) neurons) blocks recovery from insulin-induced hypoglycemia whereas, conversely, activation of VMN(SF1) neurons causes diabetes-range hyperglycemia. Moreover, this hyperglycemic response is reproduced by selective activation of VMN(SF1) fibers projecting to the anterior bed nucleus of the stria terminalis (aBNST), but not to other brain areas innervated by VMN(SF1) neurons. We also report that neurons in the lateral parabrachial nucleus (LPBN), a brain area that is also implicated in the response to hypoglycemia, make synaptic connections with the specific subset of glucoregulatory VMN(SF1) neurons that project to the aBNST. These results collectively establish a physiological role in glucose homeostasis for VMN(SF1) neurons and suggest that these neurons are part of an ascending glucoregulatory LPBN→VMN(SF1)→aBNST neurocircuit.

Keywords: bed nucleus of the stria terminalis; counter regulation; glucoregulatory circuit; hyperglycemia; ventromedial nucleus.

Fig. 1.

Photoinhibition of VMN^SF1 neurons disrupts recovery from insulin-induced hypoglycemia. (A) Scheme demonstrating bilateral stereotaxic microinjection of the Cre-dependent inhibitory channelrhodopsin-EYFP (SwiChR_CA) virus into the VMN of SF1-Cre⁺ mice and (B) implantation of the optic fiber dorsal to the injection site. (C) Representative image of EYFP fluorescence showing bilateral infection and expression specifically within the VMN of SF1-Cre⁺ mice. 3V, third cerebral ventricle; VMN, ventromedial hypothalamic nucleus. (D) Blood glucose and plasma (E) glucagon and (F) corticosterone levels in SF1-Cre⁺ mice during photoinhibition of VMN^SF1 neurons (Laser On) relative to the Laser Off during insulin-induced hypoglycemia (n = 7–8 per group). *P < 0.05 vs. Laser off. All data are expressed as mean ± SEM. (G) Blood glucose levels before (pre), during (inhib), and 1 h after (post) bilateral photoinhibition of VMN^SF1 neurons in SF1-Cre⁺ mice in the basal state (n = 8) analyzed by one-way ANOVA with Sidak’s post hoc test. All comparisons are nonsignificant.

Fig. 2.

Photoactivation of VMN^SF1 neurons induces hyperglycemia and causes impaired glucose and pyruvate tolerance in nondiabetic mice. (A) Scheme depicting unilateral stereotaxic microinjection of the Cre-dependent light-activated channelrhodopsin (ChR2-EYFP) virus into the VMN of SF1-Cre⁺ mice and (B) implantation of the optic fiber dorsal to the injection site. (C) Representative image indicating unilateral infection and expression of EYFP fluorescence within the VMN of SF1-Cre⁺ mice and c-Fos within both the VMN and surrounding area 1 h after photostimulation. 3V, third cerebral ventricle; VMN, ventromedial hypothalamic nucleus. Blood glucose levels before (Pre), during (Stim) and 1 h after (Post) unilateral stimulation of VMN^SF1 neurons in (D) SF1-Cre⁺ mice and (E) VMN^Control animals (Cre-negative) (n = 5–8 per group). *P < 0.05 vs. Pre. Blood glucose levels during an i.p. (F) glucose- (GTT) and (G) pyruvate-tolerance test (PTT) in both the presence and absence of unilateral photoactivation of VMN^SF1 neurons in SF1-Cre⁺ mice. *P < 0.05 vs. Laser Off. All data are expressed as mean ± SEM.

Fig. 3.

Photoactivation of VMN^LepRb neurons fails to raise blood glucose levels. (A) Scheme demonstrating unilateral stereotaxic microinjection of the Cre-dependent light-activated channelrhodopsin (ChR2-EYFP) virus into the VMN of Lepr-IRES-Cre mice and (B) implantation of the optic fiber dorsal to the injection site. (C) Representative image depicting unilateral infection and expression of EYFP fluorescence within the VMN of Lepr-IRES-Cre mice and c-Fos within both the VMN and surrounding area 1 h after photostimulation. 3V, third cerebral ventricle; VMN, ventromedial hypothalamic nucleus. (D) Blood glucose levels before (Pre), during (Stim), and 1 h after (Post) unilateral stimulation of VMN^LepRb neurons in Lepr-IRES-Cre mice. All comparisons are nonsignificant (n = 11 per group). All data are expressed as mean ± SEM.

Fig. 4.

Photoactivation of VMN^SF1 neurons mediates CRR responses. (A) Blood glucose, (B) plasma insulin, and (C) plasma corticosterone either prior (Pre), or 1 h after Cre-dependent channelrhodopsin stimulation (Stim) and (D) plasma glucagon and hepatic expression of (E) Pepck and (F) G6Pase as measured by real-time PCR 1 h after Cre-dependent channelrhodopsin activation of VMN^SF1, VMN^Control, and VMN^LepRb cell types, respectively (n = 5–8 per group). *P < 0.05 vs. VMN^Control; ^#P < 0.05 vs. VMN^LepRb. All data are expressed as mean ± SEM.

Fig. 5.

Photoactivation of VMN^SF1→aBNST projections selectively promote hyperglycemia in nondiabetic mice. (A) Schematic showing unilateral stereotaxic microinjection of the Cre-dependent light-activated channelrhodopsin (ChR2-EYFP) virus into the VMN of SF1-Cre⁺ mice (i.e., VMN^SF1 neurons) and implantation of the optic fiber above each of four projection sites in separate cohorts of animals. Image adapted from connectivity.brain-map.org/; experiment ID 182337561. aBNST, anterior bed nucleus of the stria terminalis; CeA, central nucleus of the amygdala; PAG, periaqueductal gray; PVN, paraventricular nucleus; VMN, ventromedial nucleus. Fluorescently labeled projections of channelrhodopsin in VMN^SF1 neurons detected in terminal targets including the (B) aBNST, (C) PVN, (D) CeA, and (E) PAG. (Magnification: 4×.) 3V, third cerebral ventricle; CA, cerebral aqueduct. (F–I) Blood glucose, (J–M) plasma insulin, and (N–Q) plasma corticosterone (CORT) levels before (Pre), during (Stim), and 1 h after (Post) photoactivation of VMN^SF1 neuron axon projection fields. (aBNST, n = 7; PVN, n = 6; CeA, n = 5; and PAG, n = 6 per group). *P < 0.05 vs. Pre. (R) Plasma glucagon 1 h after photoactivation of each site. *P < 0.05 vs. CeA and PAG; no significant difference from PVN. All data are expressed as mean ± SEM.

Fig. S1.

Activation of VMN projections selectively to the aBNST induces hyperglycemia. (A) Diagram showing a coronal section of the mouse brain at the level of the aBNST. (B) Representative image (magnified 4×) depicting scar tissue after an intraparenchymal injection at the coordinates used for aBNST fiber placement. (C, Left) Expression of fluorescently labeled neuronal tracer cholera toxin subunit B (CTB) in green at the injection site and along the needle track (indicated by white arrows). CTB infects all neurons (not cre-dependent) at the site of injection and is transported in the retrograde direction. (Middle) Red autofluorescence in the same tissue section, confirming the tissue damage seen under light microscopy. (Right) A merge of green and red filters. AC, anterior commissure; LV, lateral cerebral ventricle. Blood glucose before (Pre), during (Stim), and 1 h after (Post) unilateral (D) anterior BNST and (E) medial BNST stimulation of ChR2-EYFP 6 wk after viral injection into the VMN of SF1-Cre⁺ mice (n = 4 per group). *P < 0.05 vs. Pre. All data are expressed as mean ± SEM.

Fig. 6.

Cell bodies of VMN^SF1 neurons projecting to the aBNST are located primarily in central and dorsomedial VMN. (A) Diagram showing unilateral stereotaxic microinjection of fluorescently labeled retrograde neuronal tracer cholera toxin subunit B (CTB) into the aBNST of WT mice and (B) expression of the virus at injection site and associated coronal illustration for orientation. AC, anterior commissure; LV, lateral cerebral ventricle. Expression of the fluorescently labeled retrograde neuronal tracer CTB throughout the mediobasal hypothalamus (MBH) at either magnification (C) 4× or (D) 10×. (E) Diagram showing unilateral microinjection of ChR2-EYFP into the VMN of SF1-Cre⁺ mice followed by 1 h photoactivation of the aBNST projection field. (F) Representative 10× magnification of VMN^SF1 projections within the aBNST (green-labeled fibers), with (G) c-Fos (red stain) and the (H) merged image after aBNST photoactivation of VMN^SF1projections. (I) Control staining for c-Fos within the aBNST when light treatment was withheld (10× magnification) showing low immunoactivity. (J) Expression of ChR2 within the VMN of SF1-Cre⁺ mice (4× magnification). (K) Representative images of VMN^SF1-infected neurons (green), (L) c-Fos (red), and the (M) merged image after photoactivation of VMN^SF1 projections in the aBNST. (Magnification: 10×.) (N) Bilateral view of the VMN showing isolated expression of c-Fos within only the left hemisphere. 3V, third ventricle; c, central; dm, dorsomedial; vl, ventral lateral.

Fig. S2.

Distribution of cholera toxin B throughout the brain after aBNST injection. (A) Schematic of cholera toxin B (CTB) injection into the aBNST of WT mice and tracing of the Alexa-488 fluorophore throughout the brain. See corresponding Fig. 6 for images of mediobasal hypothalamic areas. CTB within (B) the anterior olfactory nucleus (OFN), (C) the lateral septal nucleus (LSN), and (D) the medial preoptic nucleus (MPO) (at both 4× and 10× magnification). (E) CTB at the injection site within the BNST (4× magnification), (F) the medial amygdala (mAMG), (G) the paraventricular thalamic nucleus (PVT), and (H) the central gray (CG) (at both 4× and 10× magnification).

Fig. 7.

aBNST→VMN^SF1 afferents originate within the PBN. (A) Schematic of CTB distribution after VMN injection. LPBN, lateral parabrachial nucleus; LSN, lateral septal nucleus; mAMG, medial amygdala; MPO, medial preoptic nucleus; PP, peripeduncular nucleus. (B) Microphotograph of CTB at the injection site within the VMN. (C) Representative image showing the presence of CTB in the lateral PBN 4 d after VMN injection. (Magnification: Left, 4×; Right, 10×.) Additional microphotographs of CTB spread are included in Fig. S3. (D) Diagram showing procedure and timeline of the modified rabies approach (both virus and mode of action). (E) Back propagated modified rabies virus within the VMN from aBNST projecting terminals at 4× (Left) and 10× (Right) magnification. (F) Representative image of modified rabies virus in VMN^SF1 afferent neurons found within the LPBN at 4× (Left) and 10× (Right) magnification.

Fig. S3.

Distribution of cholera toxin B in upstream brain regions after a VMN injection. (A) Schematic of cholera toxin B (CTB) injection into the VMN of WT mice and tracing of the Alexa-488 fluorophore throughout the brain. See corresponding Fig. 7 for images for lateral PBN. CTB within (B) the lateral septal nucleus (LSN), (C) the medial preoptic nucleus (MPO), (D) the BNST, (E) the medial amygdala (mAMG), and (F) the peripeduncular nucleus (PP) (at both 4× and 10× magnification).

Fig. S4.

Distribution of rabies virus throughout the brain after infection of aBNST-VMN^SF1 efferent neurons. (A) Schematic of rabies virus procedure and outcome. See corresponding Fig. 7 for images of VMN^SF1 efferent neurons located within the LPBN. Rabies virus within (B) the lateral septal nucleus (LSN) (4× and 10×), (C) the premammillary nucleus (PMN) (4× and 10×), (D) the peripeduncular nucleus (PP), and subiculum (S) (4× and 10×), and (E) the PVN, anterior hypothalamic area (AHA), and the medial amygdala (mAMG) (4× shows all three regions simultaneously, and 10× of each individually).

Fig. S5.

Model for a physiological role for VMN^SF1 neurons in glucose homeostasis. Our data suggest that activation of VMN^SF1 neurons is necessary for counterregulatory responses to insulin-induced hypoglycemia, including increases in corticosterone and glucagon secretion and inhibition of glucose-induced insulin secretion. In addition, activation of VMN^SF1 neurons is sufficient to elicit hyperglycemia in otherwise normal mice. Moreover, these neurons receive ascending input from neurons in the lateral parabrachial nucleus (LPBN) and in turn control blood glucose levels via projections selectively to the anterior bed nucleus of the stria terminalis (aBNST).