Distinct Neuronal Projections From the Hypothalamic Ventromedial Nucleus Mediate Glycemic and Behavioral Effects

Abstract

The hypothalamic ventromedial nucleus (VMN) is implicated both in autonomic control of blood glucose and in behaviors including fear and aggression, but whether these divergent effects involve the same or distinct neuronal subsets and their projections is unknown. To address this question, we used an optogenetic approach to selectively activate the subset of VMN neurons that express neuronal nitric oxide synthase 1 (VMN^NOS1 neurons) implicated in glucose counterregulation. We found that photoactivation of these neurons elicits 1) robust hyperglycemia achieved by activation of counterregulatory responses usually reserved for the physiological response to hypoglycemia and 2) defensive immobility behavior. Moreover, we show that the glucagon, but not corticosterone, response to insulin-induced hypoglycemia is blunted by photoinhibition of the same neurons. To investigate the neurocircuitry by which VMN^NOS1 neurons mediate these effects, and to determine whether these diverse effects are dissociable from one another, we activated downstream VMN^NOS1 projections in either the anterior bed nucleus of the stria terminalis (aBNST) or the periaqueductal gray (PAG). Whereas glycemic responses are fully recapitulated by activation of VMN^NOS1 projections to the aBNST, freezing immobility occurred only upon activation of VMN^NOS1 terminals in the PAG. These findings support previous evidence of a VMN→aBNST neurocircuit involved in glucose counterregulation and demonstrate that activation of VMN^NOS1 neuronal projections supplying the PAG robustly elicits defensive behaviors.

Figure 1

Strategy for photoactivation of VMN^NOS1 neurons and verification of VMN targeting. Schematic demonstrating unilateral microinjection of the Cre-dependent ChR2-YFP or YFP control virus targeting the VMN of Nos1-Cre⁺ mice (A) and fiberoptic placement dorsal to the injection site (B). C: Representative images indicating unilateral infection and expression of ChR2-EYFP and light-induced c-Fos restricted to the VMN of Nos1-Cre⁺ mice. 3V, third ventricle; ARC, arcuate nucleus.

Figure 2

Photoactivation of VMN^NOS1 neurons elicits both glucose CRRs and defensive freezing immobility. Blood glucose levels during unilateral laser off (mock) and laser-induced stimulation (Stim) of VMN^NOS1 neurons in ChR2-YFP–injected (n = 10) (A) or YFP control–injected (n = 3) (B) animals. Blue shading represents the duration of laser stimulation. Blood glucose values from ChR2-YFP animals at the 60-min time point (C) during which tail blood was collected for measurement of insulin (D), glucagon (E), and corticosterone (F). G: Percentage of photoactivation trials evoking freezing. Percentage of time spent immobile (H), total distance traveled (I), and average velocity (J) during mock and photostimulation. Values are mean ± SEM. P values by two-way ANOVA (A and B) or two-tailed, paired Student t test (C–J). **P < 0.01; ***P < 0.001; ‡P < 0.0001.

Figure 3

Photoinhibition of VMN^NOS1 neurons selectively impairs glucagon responses during insulin-induced hypoglycemia. A: Schematic demonstrating bilateral microinjection of the Cre-dependent inhibitory SwiChR-YFP virus, and fiberoptic placement dorsal to the injection site, targeting the VMN of Nos1-Cre⁺ mice. B: Representative image of EYFP fluorescence showing bilateral infection and expression within the VMN of Nos1-Cre⁺ mice. C: Blood glucose levels in Nos1-Cre⁺ mice during bilateral laser off (Mock) or laser-induced inhibition (Inhib) of VMN^NOS1 neurons. Blue shading represents duration of laser-induced inhibition. D: Changes in glucagon levels during insulin-induced hypoglycemia during mock and photoinhibition of VMN^NOS1 neurons (n = 12 for all). Values are mean ± SEM. P values by two-way ANOVA. *P < 0.05. 3V, third ventricle; ARC, arcuate nucleus.

Figure 4

VMN^NOS1 neurons project to and activate neurons in both the aBNST and the PAG, and insulin-induced hypoglycemia increases c-Fos expression within the aBNST. Fluorescently labeled projections of ChR2-expressing VMN^NOS1 neurons in the aBNST (A, left panel) and PAG (B, left panel). Photoactivation of upstream VMN^NOS1 neurons for 60 min elicits robust c-Fos expression in these regions (A and B, middle panel and merged right panel). C: Quantification of c-Fos⁺ cells in the aBNST and PAG of C57Bl/6J male mice after i.p. saline (n = 4) or insulin (n = 6; 1.5 units/kg). Representative magnification ×10 of c-Fos induction in the aBNST (D and E) and PAG (F and G) after i.p. saline (left) or insulin (right). Magnification 10×. Values are mean ± SEM. Two-tailed, unpaired Student t test for each brain region. **P < 0.01. aco, anterior commissure; aq, cerebral aqueduct.

Figure 5

Photoactivation of VMN^NOS1→aBNST projections selectively promotes hyperglycemia by activating CRRs, without eliciting freezing immobility. A: Schematic for laser off (mock) or laser-induced stimulation (Stim) of VMN^NOS1 terminals in the aBNST (VMN^NOS1→aBNST). B: Blood glucose levels during ipsilateral mock and photoactivation of VMN^NOS1→aBNST terminals. Blue shading represents the duration of laser-induced stimulation. Blood glucose values at the 60-min time point (C) during which tail blood was collected for measurement of insulin (D), glucagon (E), and corticosterone (F). G: Percentage of photoactivation trials evoking freezing. Percentage of time spent immobile (H), total distance traveled (I), and average velocity (J) during mock and photostimulation (n = 8 for all). K: Representative image indicating terminals of ChR2-expressing VMN^NOS1 neurons within the ipsilateral aBNST and light-induced c-Fos within the aBNST. Values are mean ± SEM. P values by two-way ANOVA (B) and two-tailed, paired Student t test (C–J). **P < 0.01; ‡P < 0.0001. aco, anterior commissure; LV, lateral ventricle.

Figure 6

Photoactivation of VMN^NOS1→PAG projections elicits defensive freezing immobility. A: Schematic for laser off (mock) or light-induced stimulation (Stim) of VMN^NOS1 terminals in the PAG (VMN^NOS1→PAG). B: Blood glucose levels during ipsilateral mock and photoactivation of VMN^NOS1→PAG terminals. Blue shading represents the duration of laser-induced stimulation. Blood glucose values at the 60-min time point (C) during which tail blood was collected for measurement of insulin (D), glucagon (E), and corticosterone (F). G: Percentage of photoactivation trials evoking freezing. Percentage of time spent immobile (H), total distance traveled (I), and average velocity (J) during mock and photostimulation (n = 8 for all). K: Representative image indicating terminals of ChR2-expressing VMN^NOS1 neurons in the PAG and light-induced c-Fos within the PAG. Values are mean ± SEM. P values by two-way ANOVA (B) and two-tailed, paired Student t test (C–J). *P < 0.05; **P < 0.01; ***P < 0.001; ‡P < 0.0001. aq, cerebral aqueduct.