Sex-dependent changes in metabolism and behavior, as well as reduced anxiety after eliminating ventromedial hypothalamus excitatory output

Abstract

Objectives: The ventromedial hypothalamic nucleus (VMH) regulates energy homeostasis as well as social and emotional behaviors. Nearly all VMH neurons, including those in the sexually dimorphic ventrolateral VMH (VMHvl) subregion, release the excitatory neurotransmitter glutamate and use the vesicular glutamate transporter 2 (Vglut2). Here, we asked how glutamatergic signaling contributes to the collective metabolic and behavioral responses attributed to the VMH and VMHvl.

Methods: Using Sf1-Cre and a Vglut2 floxed allele, Vglut2 was knocked-out in SF-1 VMH neurons (Vglut2 (Sf1-Cre) ). Metabolic and neurobehavioral assays were carried out initially on Vglut2 (fl/fl) and Vglut2 (Sf1-Cre) mice in a mixed, and then in the C57BL/6 genetic background, which is prone to hyperglycemia and diet induced obesity (DIO).

Results: Several phenotypes observed in Vglut2 (Sf1-Cre) mice were largely unexpected based on prior studies that have perturbed VMH development or VMH glutamate signaling. In our hands, Vglut2 (Sf1-Cre) mice failed to exhibit the anticipated increase in body weight after high fat diet (HFD) or the impaired glucose homeostasis after fasting. Instead, there was a significant sex-dependent attenuation of DIO in Vglut2 (Sf1-Cre) females. Vglut2 (Sf1-Cre) males also display a sex-specific loss of conditioned-fear responses and aggression accompanied by more novelty-associated locomotion. Finally, unlike the higher anxiety noted in Sf1 (Nestin-Cre) mice that lack a fully formed VMH, both male and female Vglut2 (Sf1-Cre) mice were less anxious.

Conclusions: Loss of VMH glutamatergic signaling sharply decreased DIO in females, attenuated aggression and learned fear in males, and was anxiolytic in males and females. Collectively, our findings demonstrate that while glutamatergic output from the VMH appears largely dispensable for counter regulatory responses to hypoglycemia, it drives sex-dependent differences in metabolism and social behaviors and is essential for adaptive responses to anxiety-provoking stimuli in both sexes.

Keywords: Anxiety; Excitatory output; Male aggression; Sex-dependent obesity; VGlut2; VMH.

Graphical abstract

Figure 1

Deletion of Vglut2^fl/flin the VMH does not alter gross anatomical organization or lead to compensatory upregulation of Vglut1 or Vglut3. A) SF-1⁺ neurons in the VMH were identified using Sf1^TauGFP. Expression of the SF-1 reporter (green) and Vglut2 exon 2 mRNA (red) in coronal sections at the level of the VMH was detected simultaneously using DISH (third ventricle = 3V). B) Expression patterns of Vglut2, Vglut1 and Vglut3, as well as Gad67 transcripts as detected by ISH. For Vglut2, higher magnification images of the boxed regions are shown in lower panels. C) Coronal sections showing the major VSOC efferent projection from the VMH in Vglut2^fl/fl (top panel) and mutant Vglut2^Sf1-Cre (lower panel) mice. SF-1 neuronal projections are visualized as described in Materials and Methods and after crossing control and mutant mice with Sf1^TauGFP reporter mice .

Figure 2

Fasting glucose levels on HFD are unaffected in Vglut2^Sf1-Creon a mixed genetic background. Weekly body weight measurements of male (M) and female (F) Vglut2^fl/fl and Vglu2^Sf1-Cre mice from 3 to 18 weeks of age. (A) Body weights of mice on standard chow (left panel) with corresponding blood glucose levels in each cohort measured pre- (Pr, solid bars) and post- (Po, hatched bars) a 24-hour fast (right panel). (B) Body weights of mice on standard chow and placed on HFD (arrow) at 10 weeks (left panel) with corresponding blood glucose levels measured as above and after HFD for 6 wks (right panel). Data are presented as means ± SEM. Statistical significance was determined by 2-way repeated measures ANOVA and Bonferroni post-test. Number of animals is indicated in each panel.

Figure 3

Attenuated Response to HFD in Vglut2^Sf1-Crefemale mice on a pure C57BL/6 genetic background. A) Weekly body weights of adult male (M) and female (F) Vglut2^fl/fl and Vglut2^Sf1-Cre mice on standard chow (NC). Indicated metabolic parameters measured in adult female Vglut2^fl/fl and Vglut2^Sf1-Cre mice on standard chow using CLAMS chambers. B) Hourly heat production during a 24-hour period. C) Average hourly heat production, ambulatory activity, food intake, oxygen consumption (VO2), and respiratory exchange ratio (RER) during the same 24-hour period as shown in panel B. D) Weekly body weights of male (M) and female (F) Vglut2^fl/fl and Vglu2^Sf1-Cre mice fed HFD beginning at 10 weeks of age (arrow). E) Glucose tolerance test in adult (16 wks old) female Vglut2^fl/fl and Vglut2^Sf1-Cre mice on HFD for 6 wks. Data are presented as means ± SEM. For panels A, D, and E, the given p values represent the main effect of genotype as determined by 2-way repeated measures ANOVA, and for each time point results from Bonferroni post-test are indicated with * = p < 0.05; ** = p < 0.01; *** = p < 0.005; **** = p < 0.001. Number of animals is indicated in each panel.

Figure 4

Male and female Vglu2^Sf1-Cremutant mice have reduced levels of anxiety-like behaviors. A) Comparison of nest locations within the home cages of adult Vglut2^fl/fl and Vglut2^Sf1-Cre mice. Percentages reflect the ratio of home cages with centrally located nests built by mutant (top panel) and control (bottom panel) mice. B) Activity in center of open field (upper panel, %) and total ambulatory activity (lower panel, beam breaks) measured in adult male and female Vglut2^fl/fl and Vglut2^Sf1-Cre mice in OF test. C) Percentage of entries, time spent and distance in the open arm (OA) of the elevated plus-maze as well as total distance in open and closed arms (lower panel) for male and female Vglut2^fl/fl and Vglut2^Sf1-Cre mice. Measurements were binned into two 5-min intervals as graphed. D) Serum corticosterone levels measured in adult male Vglut2^fl/fl and Vglut2^Sf1-Cre mice at baseline (Pre) and after (Post) exposure to the indicated stressors. Data are presented as means ± SEM. Statistical significance was determined by unpaired t-tests (panel B) or by 2-way repeated measures ANOVA with Bonferroni post-test (panel C). Number of animals is indicated in each panel.

Figure 5

Male Vglut2^Sf1-Cremice exhibit lowered learned fear and social aggression. A) Performance of male and female Vglut2^fl/fl and Vglut2^Sf1-Cre mice in a cued fear conditioning assay that measures learning and memory associated with an aversive stimuli. Fear was quantified as the percent of total time spent freezing immediately after paired presentation of audible tone and foot shock (Train), and 24 h later in response to the conditioned tone (Test). B) Time engaged in indicated behaviors during exposure to an olfactory component of fox feces, TMT. C) Aggression in male Vglut2^fl/fl and Vglut2^Sf1-Cre mice quantified as the number of attacks toward a resident intruder. Data are shown for all animals tested (All Animals) and only for male mice that displayed aggressive behavior (Aggressors). Data are presented as means ± SEM. Significance was determined by 2-way repeated measures ANOVA with Bonferroni post-test (panel A) or by unpaired t-test (panels B, C). Number of animals is indicated in each panel.