Synaptic release of GABA by AgRP neurons is required for normal regulation of energy balance

Abstract

The physiologic importance of GABAergic neurotransmission in hypothalamic neurocircuits is unknown. To examine the importance of GABA release from agouti-related protein (AgRP) neurons (which also release AgRP and neuropeptide Y), we generated mice with an AgRP neuron-specific deletion of vesicular GABA transporter. These mice are lean, resistant to obesity and have an attenuated hyperphagic response to ghrelin. Thus, GABA release from AgRP neurons is important in regulating energy balance.

Figure 1

Generation of mice lacking GABA release from AgRP neurons. All animal care and experimental procedures were approved by the Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committee. (a) Schematic diagram of the wild-type (WT) Vgat allele, the Vgat^flox/flox allele and the allele inactivated by Cre-mediated recombination. (b) Schematic diagram of the Agrp-Ires-cre allele. (c) The expression of Cre activity in the arcuate nucleus of Agrp-Ires-cre mice crossed with Cre-dependent [Au: Please clarify, is Z/EG the name of the mouse strain or is it the name of the transgene? I was unable to find Z/EG listed as a gene in Mouse Genomic Informatics or in Entrez Gene. Z/EG is a abbreviation of LacZ/EGFP, which is a line of transgenic mice. In these mice, the expression of LacZ/EGFP will be turned on with the presence of Cre recombinase. ] GFP reporter Z/EG mice (immunostaining for GFP). (d) Summary of recordings for IPSCs and excitatory postsynaptic currents (EPSCs) from cultured AgRP neurons with autaptic synapses. To permit identification of AgRP neurons, we bred Z/EG GFP reporter mice to Vgat^flox/flox and Agrp-Ires-cre; Vgat^flox/flox mice. 3V, the third ventricle. Scale bar represents 10 μM.

Figure 2

Energy homeostasis in Agrp-Ires-cre; Vgat^flox/flox mice. (a) Body weight of Vgat^flox/flox mice and Agrp-Ires-cre; Vgat^flox/flox mice (males, n = 14−19 animals). (b) O₂ consumption of Vgat^flox/flox mice (7.02 ± 0.09 ml O₂ per h per g of lean mass) and Agrp-Ires-cre; Vgat^flox/flox mice (7.33 ± 0.10) (n = 8 each). O₂ consumption was measured using the Columbus Instruments Comprehensive Lab Animal Monitoring System and was normalized to lean mass. Lean mass was measured by dual-energy x-ray absorptiometry. (c) Body weight of Vgat^flox/flox mice and Agrp-Ires-cre; Vgat^flox/flox mice during HFD feeding (n = 8 each). (d) O₂ consumption of both Vgat^flox/flox mice and Agrp-Ires-cre; Vgat^flox/flox mice during the transition from chow to HFD feeding (n = 6−9). O₂ consumption was normalized to lean mass. The O₂ consumption rates of Vgat^flox/flox mice on 2 consecutive days on chow diet were 6.12 ± 0.07 and 6.16 ± 0.10 ml O₂ per h per g, respectively, and those on the following 2 consecutive days for HFD were 6.63 ± 0.08 and 6.63 ± 0.08 ml O₂ per h per g, respectively. The O₂ consumption rates of Agrp-Ires-cre; Vgat^flox/flox mice on 2 consecutive days on chow were 6.31 ± 0.07 and 6.46 ± 0.09 ml O₂ per h per g, respectively, and on 2 consecutive days on HFD were 7.02 ± 0.07 and 7.21 ± 0.08 ml O₂ per h per g, respectively. The net average increment of O₂ consumption in response to HFD was 0.45 ml O₂ per h per g in Vgat^flox/flox mice versus 0.70 ml O₂ per h per g in Agrp-Ires-cre; Vgat^flox/flox mice. Data are expressed as mean ± s.e.m. * P < 0.05.

Figure 3

Ghrelin-induced food intake in Vgat^flox/flox mice and Agrp-Ires-cre; Vgat^flox/flox mice. (a) Food intake in 0.5-, 2- and 4-h time periods in male Vgat^flox/flox mice and Agrp-Ires-cre; Vgat^flox/flox mice after intraperitoneal administration of saline (S) or ghrelin (G, 500 μg per kg of body weight, n = 6−10). For the 2-h period, food intake increased from 0.22 ± 0.05 g to 0.46 ± 0.06 g by ghrelin in Vgat^flox/flox mice versus 0.22 ± 0.05 g to 0.25 ± 0.05 g by ghrelin in Agrp-Ires-cre; Vgat^flox/flox mice. (b) Food intake in females using the same conditions as in a. For the 2-h period, food intake increased from 0.14 ± 0.04 g to 0.61 ± 0.06 g by ghrelin in Vgat^flox/flox mice versus 0.11 ± 0.05 g to 0.31 ± 0.05 g by ghrelin in Agrp-Ires-cre; Vgat^flox/flox mice. (c) IPSCs in POMC neurons of Vgat^flox/flox mice and Agrp-Ires-cre; Vgat^flox/flox mice. To permit identification of POMC neurons, Pomc-gfp transgenic mice were bred to Vgat^flox/flox and Agrp-Ires-cre; Vgat^flox/flox mice. Postsynaptic currents were recoded from GFP neurons in brain slices and IPSCs were isolated by eliminating glutamate-mediated EPSCs using d-APV and CNQX. (d) Summary of IPSC recordings in c (n = 8 each). Firing rate was increased from 1.44 ± 0.33 Hz to 2.60 ± 0.26 Hz by ghrelin in Vgat^flox/flox mice versus 1.04 ± 0.33 Hz to 1.23 ± 0.36 Hz in Agrp-Ires-cre; Vgat^flox/flox mice. d-APV and CNQX are glutamate receptor antagonists. Data are expressed as mean ± s.e.m. * P < 0.05.