Selective deletion of Bdnf in the ventromedial and dorsomedial hypothalamus of adult mice results in hyperphagic behavior and obesity

Abstract

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB are expressed in several hypothalamic and hindbrain nuclei involved in regulating energy homeostasis, developmentally and in the adult animal. Their depletion during the fetal or early postnatal periods when developmental processes are still ongoing elicits hyperphagic behavior and obesity in mice. Whether BDNF is a chief element in appetite control in the mature brain remains controversial. The required sources of this neurotrophin are also unknown. We show that glucose administration rapidly induced BDNF mRNA expression, mediated by Bdnf promoter 1, and TrkB transcription in the ventromedial hypothalamus (VMH) of adult mice, consistent with a role of this pathway in satiety. Using viral-mediated selective knock-down of BDNF in the VMH and dorsomedial hypothalamus (DMH) of adult mice, we were able to elucidate the physiological relevance of BDNF in energy balance regulation. Site-specific mutants exhibited hyperphagic behavior and obesity but normal energy expenditure. Furthermore, intracerebroventricular administration of BDNF triggered an immediate neuronal response in multiple hypothalamic nuclei in wild-type mice, suggesting that its anorexigenic actions involve short-term mechanisms. Locomotor, aggressive, and depressive-like behaviors, all of which are associated with neural circuits involving the VMH, were not altered in VMH/DMH-specific BDNF mutants. These findings demonstrate that BDNF is an integral component of central mechanisms mediating satiety in the adult mouse and, moreover, that its synthesis in the VMH and/or DMH is required for the suppression of appetite.

Figure 1.

Glucose rapidly induces BDNF mRNA expression in the VMH. A–C, BDNF mRNA in situ hybridization analysis of representative coronal sections from fed (A), fasted (B), and fasted then glucose-treated (systemic treatment; 30 min after treatment) (C) wild-type mice showing BDNF mRNA expression in the VMH (arrows). D, Time course of BDNF mRNA induction after systemic treatment with saline (open bars) or glucose (closed bars) (n = 4–8 per group; *p = 0.0003; **p = 0.01; ***p = 0.05). E, Densitometry of BDNF mRNA signal in the VMH of wild-type mice 30 min after intracerebroventricular (ICV) treatment with saline (S), glucose (G), or insulin (I) (n = 4; *p = 0.005).

Figure 2.

Systemic administration of glucose induces transcription of BDNF directed by promoter 1 in the VMH. A, B, Representative coronal section stained with cresyl violet and containing VMH before (A) and after (B) laser-capture microdissection. C, Content of BDNF mRNA isoforms and full-length TrkB mRNA in glucose-treated fasted mice relative to saline-treated fasted mice using laser-captured VMH tissue determined by quantitative RT-PCR analysis (n = 4–7 per group; *p = 0.03; **p = 0.02; ***p = 0.004). Roman numerals indicate untranslated exon contained in BDNF mRNA isoform. Data are expressed as fold difference of glucose-treated mice relative to saline-treated animals. The mean value for saline-treated mice was set at 1. p values were calculated based on 2^ΔΔCt values for each sample.

Figure 3.

Administration of BDNF into the third ventricle (3V) rapidly induces neuronal activity in several hypothalamic nuclei. A–D, Representative coronal sections showing c-fos signal in the hypothalami of wild-type mice treated with saline (A, C) or BDNF (B, D) 45 min after treatment. Arrows denote c-fos-containing cells in the PVN in D. E, Quantification of total number of c-fos⁺ neurons in the PVN, VMH, DMH, LH, and Arc of wild-type mice treated with saline (open bars) or BDNF (closed bars) (n = 4; *p < 0.01). Counts represent total number of c-fos⁺ cells in three independent coronal levels of each of the studied regions determined from unilateral counts.

Figure 4.

AAV2/8-Cre-treated mice exhibit significant depletion of BDNF mRNA from the basomedial hypothalamus compared with AAV2/8-GFP-treated controls. A–D, In situ hybridization analysis of representative coronal sections obtained from floxed BDNF mice treated with AAV2/8-GFP (A, C) or AAV2/8-Cre (B, D) showing extensive depletion of BDNF mRNA in the VMH (arrows) and DMH (arrowheads) of AAV2/8-Cre-treated mice. C, D, Higher-magnification views of VMH and DMH areas. E, Quantification of BDNF mRNA in situ hybridizations by densitometry demonstrates significant reductions in BDNF mRNA content in the VMH and DMH of AAV2/8-Cre-treated mice (closed bars) relative to AAV2/8-GFP-treated mice (open bars). BDNF mRNA content in the PVN, cortex (Ctx), and hippocampus (Hipp) was comparable in both experimental groups (n = 7; *p = 0.0007; **p = 0.0001).

Figure 5.

Depletion of BDNF in the VMH and DMH induces hyperphagia and significant body weight gain. A, Daily food intake of AAV2/8-GFP- (open triangles) and AAV2/8-Cre (closed triangles)-injected mice. B, Body weight of AAV2/8-GFP- (open triangles) and AAV2/8-Cre (closed triangles)-injected mice after surgery (n = 7; *p < 0.05).

Figure 6.

Obese VMH/DMH-specific BDNF mutants have elevated circulating levels of leptin, insulin and glucose. A, Serum leptin levels of AAV2/8-GFP (G) and AAV2/8-Cre (C) mice (n = 7 per group; *p = 0.002). B, Serum insulin levels of AAV2/8-GFP (G) and AAV2/8-Cre (C) mice (n = 7; *p = 0.008). C, Fasted blood glucose levels of AAV2/8-GFP (G) and AAV2/8-Cre (C) mice (n = 7; *p = 0.001). Lean and obese labels refer to body weights of AAV2/8-cre-treated mice at the time of analysis.

Figure 7.

Mice with BDNF depletion from the VMH and DMH do not exhibit alterations in energy expenditure. Pair feeding AAV2/8-Cre-treated mice (closed triangles) with AAV2/8-GFP-treated mice (open triangles) progressively reduced their body weights until they became comparable with those of the controls (n = 5; *p < 0.05).

Figure 8.

AAV2/8-Cre-injected mice display normal levels of locomotor activity. Total, light cycle, and dark cycle levels of activity of AAV2/8-Cre mutants (closed bars) and AAV2/8-GFP controls (open bars) over a period of 6 d calculated based on beam interruptions in their home cages (n = 7).