GHSR in a Subset of GABA Neurons Controls Food Deprivation-Induced Hyperphagia in Male Mice.

The growth hormone secretagogue receptor (GHSR), primarily known as the receptor for the hunger hormone ghrelin, potently controls food intake, yet the specific Ghsr-expressing cells mediating the orexigenic effects of this receptor remain incompletely characterized. Since Ghsr is expressed in gamma-aminobutyric acid (GABA)-producing neurons, we sought to investigate whether the selective expression of Ghsr in a subset of GABA neurons is sufficient to mediate GHSR's effects on feeding. First, we crossed mice that express a tamoxifen-dependent Cre recombinase in the subset of GABA neurons that express glutamic acid decarboxylase 2 (Gad2) enzyme (Gad2-CreER mice) with reporter mice, and found that ghrelin mainly targets a subset of Gad2-expressing neurons located in the hypothalamic arcuate nucleus (ARH) and that is predominantly segregated from Agouti-related protein (AgRP)-expressing neurons. Analysis of various single-cell RNA-sequencing datasets further corroborated that the primary subset of cells coexpressing Gad2 and Ghsr in the mouse brain are non-AgRP ARH neurons. Next, we crossed Gad2-CreER mice with reactivable GHSR-deficient mice to generate mice expressing Ghsr only in Gad2-expressing neurons (Gad2-GHSR mice). We found that ghrelin treatment induced the expression of the marker of transcriptional activation c-Fos in the ARH of Gad2-GHSR mice, yet failed to induce food intake. In contrast, food deprivation-induced refeeding was higher in Gad2-GHSR mice than in GHSR-deficient mice and similar to wild-type mice, suggesting that ghrelin-independent roles of GHSR in a subset of GABA neurons is sufficient for eliciting full compensatory hyperphagia in mice.

Cell-to-cell transmitted alpha-synuclein recapitulates experimental Parkinson's disease.

Parkinson's disease is characterized by a progressive accumulation of alpha-Synuclein (αSyn) neuronal inclusions called Lewy bodies in the nervous system. Lewy bodies can arise from the cell-to-cell propagation of αSyn, which can occur via sequential steps of secretion and uptake. Here, by fusing a removable short signal peptide to the N-terminus of αSyn, we developed a novel mouse model with enhanced αSyn secretion and cell-to-cell transmission. Expression of the secreted αSyn in the mouse brain was under the control of a novel hybrid promoter in combination with adeno-associated virus serotype 9 (AAV9). This combination of promoter and viral vector induced a robust expression in neurons but not in the glia of injected mice. Biochemical characterization of the secreted αSyn revealed that, in cultured cells, this protein is released to the extracellular milieu via conventional secretion. The released αSyn is then internalized and processed by acceptor cells via the endosome-lysosome pathway indicating that the secreted αSyn is cell-to-cell transmitted. The secreted αSyn is aggregation-prone and amyloidogenic, and when expressed in the brain of wild-type non-transgenic mice, it induces a Parkinson's disease-like phenotype that includes a robust αSyn pathology in the substantia nigra, neuronal loss, neuroinflammation, and motor deficits, all the key features of experimental animal models of Parkinson's disease. In summary, a novel animal model of Parkinson's disease based on enhanced cell-to-cell transmission of αSyn was developed. The neuron-produced cell-to-cell transmitted αSyn triggers all phenotypic features of experimental Parkinson's disease in mice.

Systemic Ghrelin Treatment Induces Rapid, Transient, and Asymmetric Changes in the Metabolic Activity of the Mouse Brain.

INTRODUCTION: Ghrelin regulates a variety of functions by acting in the brain. The targets of ghrelin in the mouse brain have been mainly mapped using immunolabeling against c-Fos, a transcription factor used as a marker of cellular activation, but such analysis has several limitations. Here, we used positron emission tomography in mice to investigate the brain areas responsive to ghrelin. METHODS: We analyzed in male mice the brain areas responsive to systemically injected ghrelin using positron emission tomography imaging of 18F-fluoro-2-deoxyglucose (18F-FDG) uptake, an indicator of metabolic rate. Additionally, we studied if systemic administration of fluorescent ghrelin or native ghrelin displays symmetric accessibility or induction of c-Fos, respectively, in the brain of male mice. RESULTS: Ghrelin increased 18F-FDG uptake in few specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain at 0-10-min posttreatment. At the 10-20 and 20-30 min posttreatment, ghrelin induced mixed changes in 18F-FDG uptake in specific areas of the isocortex, striatum, pallidum, thalamus, and midbrain, as well as in areas of the olfactory areas, hippocampal and retrohippocampal regions, hypothalamus, pons, medulla, and even the cerebellum. Ghrelin-induced changes in 18F-FDG uptake were transient and asymmetric. Systemically administrated fluorescent-ghrelin-labeled midline brain areas known to contain fenestrated capillaries and the hypothalamic arcuate nucleus, where a symmetric labeling was observed. Ghrelin treatment also induced a symmetric increased c-Fos labeling in the arcuate nucleus. DISCUSSION/CONCLUSION: Systemically injected ghrelin transiently and asymmetrically affects the metabolic activity of the brain of male mice in a wide range of areas, in a food intake-independent manner. The neurobiological bases of such asymmetry seem to be independent of the accessibility of ghrelin into the brain.

Growth hormone secretagogue receptor in dopamine neurons controls appetitive and consummatory behaviors towards high-fat diet in ad-libitum fed mice.

Growth hormone secretagogue receptor (GHSR), the receptor for ghrelin, is expressed in key brain nuclei that regulate food intake. The dopamine (DA) pathways have long been recognized to play key roles mediating GHSR effects on feeding behaviors. Here, we aimed to determine the role of GHSR in DA neurons controlling appetitive and consummatory behaviors towards high fat (HF) diet. For this purpose, we crossed reactivable GHSR-deficient mice with DA transporter (DAT)-Cre mice, which express Cre recombinase under the DAT promoter that is active exclusively in DA neurons, to generate mice with GHSR expression limited to DA neurons (DAT-GHSR mice). We found that DAT-GHSR mice show an increase of c-Fos levels in brain areas containing DA neurons after ghrelin treatment, in a similar fashion as seen in wild-type mice; however, they did not increase food intake or locomotor activity in response to systemically- or centrally-administered ghrelin. In addition, we found that satiated DAT-GHSR mice displayed both anticipatory activity to scheduled HF diet exposure and HF intake in a binge-like eating protocol similar to those in wild-type mice, whereas GHSR-deficient mice displayed impaired responses. We conclude that GHSR expression in DA neurons is sufficient to both mediate increased anticipatory activity to a scheduled HF diet exposure and fully orchestrate binge-like HF intake, but it is insufficient to restore the acute orexigenic or locomotor effects of ghrelin treatment. Thus, GHSR in DA neurons affects appetitive and consummatory behaviors towards HF diet that take place in the absence of caloric needs.

Evidence Supporting a Role for the Blood-Cerebrospinal Fluid Barrier Transporting Circulating Ghrelin into the Brain.

The stomach-derived hormone ghrelin mainly acts in the brain. Studies in mice have shown that the accessibility of ghrelin into the brain is limited and that it mainly takes place in some circumventricular organs, such as the median eminence. Notably, some known brain targets of ghrelin are distantly located from the circumventricular organs. Thus, we hypothesized that ghrelin could also access the brain via the blood-cerebrospinal fluid (CSF) barrier, which consists of the choroid plexus and the hypothalamic tanycytes. Using systemic injection of ghrelin or fluorescent-ghrelin in mice, we found that cells of the blood-CSF barrier internalize these molecules. In time-response studies, we found that peripherally injected fluorescent-ghrelin quickly reaches hypothalamic regions located in apposition to the median eminence and more slowly reaches the periventricular hypothalamic parenchyma, adjacent to the dorsal part of the third ventricle. Additionally, we found that CSF ghrelin levels increase after the systemic administration of ghrelin, and that central infusions of either an anti-ghrelin antibody, which immuno-neutralizes CSF ghrelin, or a scrambled version of ghrelin, which is also internalized by cells of the blood-CSF barrier, partially impair the orexigenic effect of peripherally injected ghrelin. Thus, current evidence suggests that the blood-CSF barrier can transport circulating ghrelin into the brain, and that the access of ghrelin into the CSF is required for its full orexigenic effect.

Ghrelin Recruits Specific Subsets of Dopamine and GABA Neurons of Different Ventral Tegmental Area Sub-nuclei.

Ghrelin is a stomach-derived hormone that regulates rewarding behaviors and reinforcement by acting on the ventral tegmental area (VTA). The VTA is a complex midbrain structure mainly comprised of dopamine (DA) and gamma-aminobutiric acid (GABA) neurons that are distributed in several VTA sub-nuclei. Here, we investigated the neuroanatomical distribution and chemical phenotype of ghrelin-responsive neurons within the VTA. In wild-type mice, we found that: (1) ghrelin binding cells are present in most VTA sub-nuclei but not in its main target, the nucleus accumbens (Acb); (2) systemically injected ghrelin increases food intake but does neither affect locomotor activity nor the levels of the marker of neuronal activation c-Fos in the VTA sub-nuclei; (3) centrally injected ghrelin increases food intake, locomotor activity and c-Fos levels in non-DA neurons of all VTA sub-nuclei; (4) intra-VTA-injected ghrelin increases food intake, locomotor activity and c-Fos levels in non-DA neurons of all VTA sub-nuclei; (5) both centrally and intra-VTA-injected ghrelin increase c-Fos levels in DA neurons of the parabrachial pigmented VTA sub-nucleus. In genetically modified mice in which a subset of GABA neurons expresses the red fluorescent protein tdTomato, we found that centrally injected ghrelin increases c-Fos levels in GABA neurons of the interfascicular VTA sub-nucleus. These results suggest that ghrelin can recruit specific subsets of VTA neurons in order to modulate food intake and locomotor activity.

Ghrelin receptor signaling targets segregated clusters of neurons within the nucleus of the solitary tract.

Ghrelin is a stomach-derived hormone that regulates a variety of biological functions such as food intake, gastrointestinal function and blood glucose metabolism, among others. Ghrelin acts via the growth hormone secretagogue receptor (GHSR), a G-protein-coupled receptor located in key brain areas that mediate specific actions of the hormone. GHSR is highly expressed in the nucleus of the solitary tract (NTS), which is located in the medulla oblongata and controls essential functions, including orofacial, autonomic, neuroendocrine and behavioral responses. Here, we used a mouse model, in which the expression of enhanced green fluorescent protein (eGFP) is controlled by the promoter of GHSR (GHSR-eGFP mice), to gain neuroanatomical and functional insights of the GHSR-expressing neurons of the NTS. We found that GHSR-expressing neurons of the NTS are segregated in clusters that were symmetrically distributed to the midline: (1) a pair of rostral clusters, and (2) a caudal and medially located cluster. We also identified that a subset of GHSR neurons of the caudal NTS are GABAergic. Finally, we found that rostral NTS GHSR neurons increase the levels of the marker of neuronal activation c-Fos in mice exposed to fasting/refeeding or high-fat diet bingeing protocols, while caudal NTS GHSR neurons increase the levels of c-Fos in mice exposed to gastric distension or LiCl-induced malaise protocols. Thus, current data provide evidence that ghrelin receptor signaling seems to target segregated clusters of neurons within the NTS that, in turn, may be activated by different stimuli.

A simple strategy for culturing morphologically-conserved rat hypothalamic tanycytes.

Hypothalamic tanycytes are specialized bipolar ependymal cells that line the floor of the third ventricle. Given their strategic location, tanycytes are believed to play several key functions including being a selective barrier and controlling the amount of hypothalamic-derived factors reaching the anterior pituitary. The in vitro culture of these cells has proved to be difficult. Here, we report an improved method for the generation of primary cultures of rat hypothalamic tanycytes. Ependymal cultures were derived from tissue dissected out of the median eminence region of 10-day-old rats and cultured in a chemically defined medium containing DMEM:F12, serum albumin, insulin, transferrin and the antibiotic gentamycin. After 7 days in vitro, ∼30% of the cultured cells exhibited morphological features of tanycytes as observed by phase contrast or scanning electron microscopy. Tanycyte-like cells were strongly immuno-reactive for vimentin and dopamine-cAMP-regulated phospho-protein (DARPP-32) and weakly immune-reactive for glial fibrillary acidic protein. Tanycyte-like cells displayed a stable negative resting plasma membrane potential and failed to show spiking properties in response to current injections. When exposed to fluorescent beads in the culture medium, tanycyte-like cells exhibited a robust endocytosis. Thus, the present method effectively yields cultures containing tanycyte-like cells that resemble in vivo tanycytes in terms of morphologic features and molecular markers as well as electrical and endocytic activity. To our knowledge, this is the first protocol that allows the culturing of tanycyte-like cells that can be individually identified and that conserve the morphology of tanycytes in their natural physiological environment.