The novel Ghsr IRES Cre knock in mouse
The novel Ghsr-IRES-Cre knock-in mouse line allowed us to deconstruct the involvement of MBH GHSR-expressing neurons in mediating food intake responses using chemogenetic modulation of their neuronal activity. In our studies, hM4Di DREADD virus injections to inhibit GHSR neurons targeted several nuclei within the MBH area including the Arc, VMH, DMH, and the PMV, all of which are involved in the regulation of food intake , , , , , , . Within these regions, we predict that the inhibition of GHSR-expressing Arc AgRP/Neuropeptide Y (NPY) neurons is likely key to the reduced food intake response following fasting. This prediction is based on the known function of Arc AgRP/NPY neurons in homeostatic and fasting-induced rebound feeding and the previously-described high responsiveness of Arc ARP/NPY neurons to ghrelin, as discussed below. Arc AgRP/NPY neurons are activated during fasted conditions , ,  and are essential for feeding, as ablation of these neurons in adult animals leads to cessation of eating , . Numerous other studies have indicated key roles for Arc AgRP/NPY neurons in food intake, including those in which optogenetic and chemogenetic methodologies were used to stimulate them , . In contrast, chemogenetic inhibition of Arc AgRP neurons reduces food intake  and diphtheria toxin-induced ablation of AgRP neurons from neonates significantly blunts fasting-induced rebound feeding of standard chow . GHSR expression and c-fos induction – a marker of neuronal activation – by ghrelin or ghrelin mimetics are very high in these orexigenic Arc AgRP/NPY neurons , , , , . As would be expected based on this high level of GHSR expression within and ghrelin responsiveness of Arc AgRP/NPY neurons, several studies also have implicated Arc AgRP/NPY neurons as direct mediators of ghrelin's orexigenic actions , , , , , , , , , , , . For example, chemical ablation of the Arc and selective ablation of Arc AgRP neurons both abolish ghrelin-induced feeding , . Also, selective expression of GHSRs in Arc AgRP neurons reestablishes the acute orexigenic response to peripherally-administered ghrelin in animals that are otherwise completing lacking GHSR , while selective trp channel of GHSR expression from Arc AgRP neurons inhibits the orexigenic response to ghrelin . Importantly, many parallels in the food intake responses exist between our current studies targeting MBH GHSR neurons and the above-described previous studies targeting Arc AgRP/NPY neurons. For example, our studies suggest that chemogenetic activation of the MBH GHSR neurons is sufficient to increase food intake, as observed with optogenetic and chemogenetic stimulation of Arc AgRP neurons , . Also, chemogenetic inhibition of the MBH GHSR neurons reduced food intake, as shown for Arc AgRP neurons . Moreover, chemogenetic inhibition of MBH GHSR neurons blunted fasting-induced rebound feeding, as described for mice with AgRP neuronal ablation . Hypothalamic and extrahypothalamic axonal projection sites of the Cre expressing MBH GHSR neurons revealed here, which include the PVN, PAG, BNST, mPOA, and LH, mirror the axonal projection sites observed for Arc AgRP neurons , , . Collectively, the above similarities point to the likely involvement of Arc AgRP/NPY feeding circuits in mediating the food intake responses that we observe with modification of the MBH GHSR neuronal activity. Further studies are needed to dissect out the roles of the other targeted GHSR-expressing MBH neuronal populations. As introduced above, an area of disagreement in the literature is the significance of the endogenous ghrelin system as it relates to food intake , , , , , , . There is no doubt that ghrelin can dose-dependently increase food intake acutely when administered peripherally or centrally; however, some studies argue that this occurs only when doses resulting in supraphysiological plasma ghrelin levels are used . However, other studies suggest that food intake does occur when administered ghrelin leads to similar plasma ghrelin levels as achieved physiologically upon caloric restriction or psychosocial stress . Furthermore, several models with genetic deletion of ghrelin, GOAT, or GHSR exhibit no or only minimal reductions in body weight and/or food intake , , , , , . These stand in contrast to other studies in which GHSR deletion lowered both body weight and food intake , . Even in those studies in which there was a decrease in body weight, the effect on food intake was minimal . It could be argued that the lack of a food intake phenotype in some loss-of-function models could be a result of developmental or physiological adaptations to the lack of GHSR and/or ghrelin. That said, even in a mouse model in which ghrelin cells were ablated from adult mice − presumably after feeding circuits had developed normally − acute effects on food intake and body weight were not observed .