Growth hormone secretagogue receptor in GABA neurons modulates homeostatic and hedonic feeding

CORNEJO MP, FERNANDEZ G, CABRAL A, HEREDIA F, BARRILE F, TOLOSA MJ, GARCÍA ROMERO G, P ERELLO M

Conferencia; Gordon Research Conference; 2023

Frontiers of Science

The growth hormone secretagogue receptor (GHSR) is a G-protein coupled receptor that acts as the receptor for ghrelin, a stomach-derived hormone with the most potent orexigenic effect. GHSR is mainly expressed in the brain and different neuronal populations express GHSR, including neurons that produce gammaaminobutiric acid (GABA neurons) of the hypothalamic arcuate nucleus (ARH) and other brain nuclei. Here, we aim to test if the selective expression of GHSR in GABA neurons mediates different aspects of feeding that are modulated by ghrelin.Animals: experiments were performed in adult (12-20 weeks-old) male mice. Mouse strains included: wild-type (WT) C57BL6/J mice; Gad2-Cre (Taniguchi et al 2011) mice; R26-eGFP (Mao et al 2001) mice; Ghsr-deficient (Zigman et al 2005) mice. Gad2-Cre mice express Cre recombinase under the promoter of Gad2 gene, used as marker of GABA neurons. Cre activity in Gad2-Cre mice depends on the induction with tamoxifen, as it is fusioned with estrogen receptor. R26-eGFP and Ghsr-deficient mice have transcription blocking cassettes flanked by loxP sequences that impair eGFP and Ghsr expression, respectively. Cleveage of transcription blocking cassettes by Cre recombinase allows the expression of eGFP (Gad2-eGFP mice) and Ghsr (Gad2-Ghsr mice).Stereotaxic surgeries: mice were anesthetized and guide cannulas were implanted in the lateral ventricle using t h e c o o r d i n a t e s : A P : - 0 . 3 4 m m , M L : - 1 . 0 0 m m a n d D V : - 2 . 3 0 m m . c-Fos immunostaining: for fluorescent immunostainings, coronal sections of mouse brains were washed with phosphate-saline buffer (PBS) and sequentially incubated with blocking solution (adult horse serum in PBSTriton X), rabbit anti-c-Fos antibody (1:2,000; Calbiochem). Then, sections were incubated with a Alexa594-conjugated anti-rabbit antibody (1:1,000; Invitrogen). For chromogenic immunostainings, coronal sections were sequentially incubated with H O (0.5% in PBS), blocking solution and anti-c-Fos antibody (1:20,000). 2 2 Then, sections were incubated with a biotynilated anti-rabbit antibody (1:3,000; Vector Labs), avidinperoxidase complex (Vector Labs) and the reaction was revealed with 2,2'-diaminobencidine+nickel chloride to generate nuclear black/purple precipitates.Binge-like eating protocol: satiated mice had access to a pre-weighed high fat diet (HFD, 20% w/w fat) pellet for 2 h (9:30 am to 11:30 am) during 4 consecutive days.A subset of GABA neurons of the ARH binds Fr-ghrelin and responds to peripherally-administeredghrelin.GHSR expression exclusively in GABA neurons mediates ghrelin-induced ARH activation.GHSR expression exclusively in GABA neurons seems to mediate HFD intake in a binge-like eatingprotocol.GHSR expression exclusively in GABA neurons does neither mediate ghrelin-induced food intakenor fasting-induced hypothalamic activation.