Ghrelin Action in the PVH of Male Mice: Accessibility, Neuronal Targets, and CRH Neurons Activation.

The hormone ghrelin displays several well-characterized functions, including some with pharmaceutical interest. The receptor for ghrelin, the growth hormone secretagogue receptor (GHSR), is expressed in the hypothalamic paraventricular nucleus (PVH), a critical hub for the integration of metabolic, neuroendocrine, autonomic, and behavioral functions. Here, we performed a neuroanatomical and functional characterization of the neuronal types mediating ghrelin actions in the PVH of male mice. We found that fluorescent ghrelin mainly labels PVH neurons immunoreactive for nitric oxide synthase 1 (NOS1), which catalyze the production of nitric oxide [NO]). Centrally injected ghrelin increases c-Fos in NOS1 PVH neurons and NOS1 phosphorylation in the PVH. We also found that a high dose of systemically injected ghrelin increases the ghrelin level in the cerebrospinal fluid and in the periventricular PVH, and induces c-Fos in NOS1 PVH neurons. Such a high dose of systemically injected ghrelin activates a subset of NOS1 PVH neurons, which do not express oxytocin, via an arcuate nucleus-independent mechanism. Finally, we found that pharmacological inhibition of NO production fully abrogates ghrelin-induced increase of calcium concentration in corticotropin-releasing hormone neurons of the PVH whereas it partially impairs ghrelin-induced increase of plasma glucocorticoid levels. Thus, plasma ghrelin can directly target a subset of NO-producing neurons of the PVH that is involved in ghrelin-induced activation of the hypothalamic-pituitary-adrenal neuroendocrine axis.

The controversial role of the vagus nerve in mediating ghrelin's actions: gut feelings and beyond.

Ghrelin is a stomach-derived peptide hormone that acts via the growth hormone secretagogue receptor (GHSR) and displays a plethora of neuroendocrine, metabolic, autonomic and behavioral actions. It has been proposed that some actions of ghrelin are exerted via the vagus nerve, which provides a bidirectional communication between the central nervous system and peripheral systems. The vagus nerve comprises sensory fibers, which originate from neurons of the nodose and jugular ganglia, and motor fibers, which originate from neurons of the medulla. Many anatomical studies have mapped GHSR expression in vagal sensory or motor neurons. Also, numerous functional studies investigated the role of the vagus nerve mediating specific actions of ghrelin. Here, we critically review the topic and discuss the available evidence supporting, or not, a role for the vagus nerve mediating some specific actions of ghrelin. We conclude that studies using rats have provided the most congruent evidence indicating that the vagus nerve mediates some actions of ghrelin on the digestive and cardiovascular systems, whereas studies in mice resulted in conflicting observations. Even considering exclusively studies performed in rats, the putative role of the vagus nerve in mediating the orexigenic and growth hormone (GH) secretagogue properties of ghrelin remains debated. In humans, studies are still insufficient to draw definitive conclusions regarding the role of the vagus nerve mediating most of the actions of ghrelin. Thus, the extent to which the vagus nerve mediates ghrelin actions, particularly in humans, is still uncertain and likely one of the most intriguing unsolved aspects of the field.

Growth hormone secretagogue receptor signaling in the supramammillary nucleus targets nitric oxide-producing neurons and controls recognition memory in mice.

Ghrelin is a stomach-derived hormone that acts via the growth hormone secretagogue receptor (GHSR). Recent evidence suggests that some of ghrelin's actions may be mediated via the supramammillary nucleus (SuM). Not only does ghrelin bind to cells within the mouse SuM, but ghrelin also activates SuM cells and intra-SuM ghrelin administration induces feeding in rats. In the current study, we aimed to further characterize ghrelin action in the SuM. We first investigated a mouse model expressing enhanced green fluorescent protein (eGFP) under the promoter of GHSR (GHSR-eGFP mice). We found that the SuM of GHSR-eGFP mice contains a significant amount of eGFP cells, some of which express neuronal nitric oxide synthase. Centrally-, but not systemically-, injected ghrelin reached the SuM, where it induced c-Fos expression. Furthermore, a 5-day 40% calorie restriction protocol, but not a 2-day fast, increased c-Fos expression in non-eGFP+ cells of the SuM of GHSR-eGFP mice, whereas c-Fos induction by calorie restriction was not observed in GHSR-deficient mice. Exposure of satiated mice to a binge-like eating protocol also increased c-Fos expression in non-eGFP+ cells of the SuM of GHSR-eGFP mice in a GHSR-dependent manner. Finally, intra-SuM-injected ghrelin did not acutely affect food intake, locomotor activity, behavioral arousal or spatial memory but increased recognition memory. Thus, we provide a compelling neuroanatomical characterization of GHSR SuM neurons and its behavioral implications in mice.

The ups and downs of growth hormone secretagogue receptor signaling.

The growth hormone secretagogue receptor (GHSR) has emerged as one of the most fascinating molecules from the perspective of neuroendocrine control. GHSR is mainly expressed in the pituitary and the brain, and plays key roles regulating not only growth hormone secretion but also food intake, adiposity, body weight, glucose homeostasis and other complex functions. Quite atypically, GHSR signaling displays a basal constitutive activity that can be up- or downregulated by two digestive system-derived hormones: the octanoylated-peptide ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2), which was recently recognized as an endogenous GHSR ligand. The existence of two ligands with contrary actions indicates that GHSR activity can be tightly regulated and that the receptor displays the capability to integrate such opposing inputs in order to provide a balanced intracellular signal. This article provides a summary of the current understanding of the biology of ghrelin, LEAP2 and GHSR and discusses the reconceptualization of the cellular and physiological implications of the ligand-regulated GHSR signaling, based on the latest findings.

THE INTRIGUING LIGAND-DEPENDENT AND LIGAND-INDEPENDENT ACTIONS OF THE GROWTH HORMONE SECRETAGOGUE RECEPTOR ON REWARD-RELATED BEHAVIORS.

The growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor (GPCR) highly expressed in the brain, and also in some peripheral tissues. GHSR activity is evoked by the stomach-derived peptide hormone ghrelin and abrogated by the intestine-derived liver-expressed antimicrobial peptide 2 (LEAP2). In vitro, GHSR displays ligand-independent actions, including a high constitutive activity and an allosteric modulation of other GPCRs. Beyond its neuroendocrine and metabolic effects, cumulative evidence shows that GHSR regulates the activity of the mesocorticolimbic pathway and modulates complex reward-related behaviors towards different stimuli. Here, we review current evidence indicating that ligand-dependent and ligand-independent actions of GHSR enhance reward-related behaviors towards appetitive stimuli and drugs of abuse. We discuss putative neuronal networks and molecular mechanisms that GHSR would engage to modulate such reward-related behaviors. Finally, we briefly discuss imaging studies showing that ghrelin would also regulate reward processing in humans. Overall, we conclude that GHSR is a key regulator of the mesocorticolimbic pathway that influences its activity and, consequently, modulates reward-related behaviors via ligand-dependent and ligand-independent actions.

Inter-individual Variability for High Fat Diet Consumption in Inbred C57BL/6 Mice.

Since inbred C57BL/6 mice are known to show inter-individual phenotypic variability for some traits, we tested the hypothesis that inbred C57BL/6 mice display a different tendency to consume a high fat (HF) diet. For this purpose, we used a compilation of HF intake data from an experimental protocol in which satiated mice were exposed to a HF pellet every morning for 2-h over 4 consecutive days. We found that mice displayed a large degree of variability in HF intake. Since day 1 HF intake significantly correlated with HF intake in successive days, we applied a hierarchical clustering algorithm on HF intake measurements in days 2, 3, and 4 in order to classify mice into "low" or "high" HF intake groups. "Low" HF intake group showed a day 1 HF intake similar to that seen in mice exposed to regular chow, while "high" HF intake group showed a higher day 1 HF intake as compared to "low" HF intake group. Both groups of mice increased HF consumption over the successive days, but "high" HF intake group always displayed a higher HF consumption than the "low" HF intake group. As compared to "low" HF intake group, "high" HF intake group showed a higher number of dopamine neurons positive for c-Fos in the VTA after the last event of HF intake. Thus, inbred C57BL/6 mice show inter-individual variability for HF intake and such feature may be linked to a different response to the rewarding properties of the HF diet.

Brain accessibility delineates the central effects of circulating ghrelin.

Ghrelin is a hormone produced in the gastrointestinal tract that acts via the growth hormone secretagogue receptor. In the central nervous system, ghrelin signalling is able to recruit different neuronal targets that regulate the behavioural, neuroendocrine, metabolic and autonomic effects of the hormone. Notably, several studies using radioactive or fluorescent variants of ghrelin have found that the accessibility of circulating ghrelin into the mouse brain is both strikingly low and restricted to some specific brain areas. A variety of studies addressing central effects of systemically injected ghrelin in mice have also provided indirect evidence that the accessibility of plasma ghrelin into the brain is limited. Here, we review these previous observations and discuss the putative pathways that would allow plasma ghrelin to gain access into the brain together with their physiological implications. Additionally, we discuss some potential features regarding the accessibility of plasma ghrelin into the human brain based on the observations reported by studies that investigate the consequences of ghrelin administration to humans.

Circulating Ghrelin Acts on GABA Neurons of the Area Postrema and Mediates Gastric Emptying in Male Mice.

Ghrelin is known to act on the area postrema (AP), a sensory circumventricular organ located in the medulla oblongata that regulates a variety of important physiological functions. However, the neuronal targets of ghrelin in the AP and their potential role are currently unknown. In this study, we used wild-type and genetically modified mice to gain insights into the neurons of the AP expressing the ghrelin receptor [growth hormone secretagogue receptor (GHSR)] and their role. We show that circulating ghrelin mainly accesses the AP but not to the adjacent nucleus of the solitary tract. Also, we show that both peripheral administration of ghrelin and fasting induce an increase of c-Fos, a marker of neuronal activation, in GHSR-expressing neurons of the AP, and that GHSR expression is necessary for the fasting-induced activation of AP neurons. Additionally, we show that ghrelin-sensitive neurons of the AP are mainly γ-aminobutyric acid (GABA)ergic, and that an intact AP is required for ghrelin-induced gastric emptying. Overall, we show that the capacity of circulating ghrelin to acutely induce gastric emptying in mice requires the integrity of the AP, which contains a population of GABA neurons that are a target of plasma ghrelin.

Escalation in high fat intake in a binge eating model differentially engages dopamine neurons of the ventral tegmental area and requires ghrelin signaling.

Binge eating is a behavior observed in a variety of human eating disorders. Ad libitum fed rodents daily and time-limited exposed to a high-fat diet (HFD) display robust binge eating events that gradually escalate over the initial accesses. Intake escalation is proposed to be part of the transition from a controlled to a compulsive or loss of control behavior. Here, we used a combination of behavioral and neuroanatomical studies in mice daily and time-limited exposed to HFD to determine the neuronal brain targets that are activated--as indicated by the marker of cellular activation c-Fos--under these circumstances. Also, we used pharmacologically or genetically manipulated mice to study the role of orexin or ghrelin signaling, respectively, in the modulation of this behavior. We found that four daily and time-limited accesses to HFD induce: (i) a robust hyperphagia with an escalating profile, (ii) an activation of different sub-populations of the ventral tegmental area dopamine neurons and accumbens neurons that is, in general, more pronounced than the activation observed after a single HFD consumption event, and (iii) an activation of the hypothalamic orexin neurons, although orexin signaling blockage fails to affect escalation of HFD intake. In addition, we found that ghrelin receptor-deficient mice fail to both escalate the HFD consumption over the successive days of exposure and fully induce activation of the mesolimbic pathway in response to HFD consumption. Current data suggest that the escalation in high fat intake during repeated accesses differentially engages dopamine neurons of the ventral tegmental area and requires ghrelin signaling.