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.

GHSR controls food deprivation-induced activation of CRF neurons of the hypothalamic paraventricular nucleus in a LEAP2-dependent manner.

OBJECTIVE: Prolonged fasting is a major challenge for living organisms. An appropriate metabolic response to food deprivation requires the activation of the corticotropin-releasing factor-producing neurons of the hypothalamic paraventricular nucleus (PVHCRF neurons), which are a part of the hypothalamic-pituitary-adrenal axis (HPA), as well as the growth hormone secretagogue receptor (GHSR) signaling, whose activity is up- or down-regulated, respectively, by the hormones ghrelin and the liver-expressed antimicrobial peptide 2 (LEAP2). Since ghrelin treatment potently up-regulates the HPA axis, we studied the role of GHSR in mediating food deprivation-induced activation of the PVHCRF neurons in mice. METHODS: We estimated the activation of the PVHCRF neurons, using immuno-staining against CRF and the marker of neuronal activation c-Fos in brain sections, and assessed plasma levels of corticosterone and glucose in different pharmacologically or genetically manipulated mouse models exposed, or not, to a 2-day food deprivation protocol. In particular, we investigated ad libitum fed or food-deprived male mice that: (1) lacked GHSR gene expression, (2) had genetic deletion of the ghrelin gene, (3) displayed neurotoxic ablation of the hypothalamic arcuate nucleus, (4) were centrally treated with an anti-ghrelin antibody to block central ghrelin action, (5) were centrally treated with a GHSR ligand that blocks ghrelin-evoked and constitutive GHSR activities, or (6) received a continuous systemic infusion of LEAP2(1-12). RESULTS: We found that food deprivation results in the activation of the PVHCRF neurons and in a rise of the ghrelin/LEAP2 molar ratio. Food deprivation-induced activation of PVHCRF neurons required the presence and the signaling of GHSR at hypothalamic level, but not of ghrelin. Finally, we found that preventing the food deprivation-induced fall of LEAP2 reverses the activation of the PVHCRF neurons in food-deprived mice, although it has no effect on body weight or blood glucose. CONCLUSION: Food deprivation-induced activation of the PVHCRF neurons involves ghrelin-independent actions of GHSR at hypothalamic level and requires a decrease of plasma LEAP2 levels. We propose that the up-regulation of the actions of GHSR associated to the fall of plasma LEAP2 level are physiologically relevant neuroendocrine signals during a prolonged fasting.

Circulating ghrelin crosses the blood-cerebrospinal fluid barrier via growth hormone secretagogue receptor dependent and independent mechanisms.

Ghrelin is a peptide hormone mainly secreted from gastrointestinal tract that acts via the growth hormone secretagogue receptor (GHSR), which is highly expressed in the brain. Strikingly, the accessibility of ghrelin to the brain seems to be limited and restricted to few brain areas. Previous studies in mice have shown that ghrelin can access the brain via the blood-cerebrospinal fluid (CSF) barrier, an interface constituted by the choroid plexus and the hypothalamic tanycytes. Here, we performed a variety of in vivo and in vitro studies to test the hypothesis that the transport of ghrelin across the blood-CSF barrier occurs in a GHSR-dependent manner. In vivo, we found that the uptake of systemically administered fluorescent ghrelin in the choroid plexus epithelial (CPE) cells and in hypothalamic tanycytes depends on the presence of GHSR. Also, we detected lower levels of CSF ghrelin after a systemic ghrelin injection in GHSR-deficient mice, as compared to WT mice. In vitro, the internalization of fluorescent ghrelin was reduced in explants of choroid plexus from GHSR-deficient mice, and unaffected in primary cultures of hypothalamic tanycytes derived from GHSR-deficient mice. Finally, we found that the GHSR mRNA is detected in a pool of CPE cells, but is nearly undetectable in hypothalamic tanycytes with current approaches. Thus, our results suggest that circulating ghrelin crosses the blood-CSF barrier mainly by a mechanism that involves the GHSR, and also possibly via a GHSR-independent mechanism.

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.

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.

Therapeutic potential of IGF-I on hippocampal neurogenesis and function during aging.

In rats, learning and memory performance decline during normal aging, which is paralleled by a severe reduction of the levels of neurogenesis in the hippocampal dentate gyrus (DG). A promising therapeutic strategy to restore neurogenesis in the hippocampus of old rats and their spatial memory involves the use of insulin-like growth factor-I (IGF-I). The peptide exerts pleiotropic effects in the brain, regulating multiple cellular processes. Thus, 4-week intracerebroventricular (ICV) perfusion of IGF-I significantly restored spatial memory and hippocampal neurogenesis in old male rats. Similar results were achieved by ICV IGF-I gene therapy in aging female rats. Thus, the treatment seemed to increase the number of immature neurons in the DG of 28 mo old rats, which was paralleled by an increase in the accuracy of the animals to remember specific patterns, which is known as pattern separation memory. The DG is thought to be the main hippocampal structure involved in pattern separation memory and there is evidence that the level of neurogenesis in the DG is directly related to pattern separation performance in rodents. Summing up, IGF-I emerges as a promising restorative molecule for increasing hippocampal neurogenesis and memory accuracy in aged individuals and possibly, in neurodegenerative pathologies.

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.

Long-lasting training in the Barnes maze prompts hippocampal spinogenesis and habituation in rats.

There is a constant need to assess spatial memory in small rodents to elucidate the basics of cognition in neuroscience experiments. Thus, the significance of the Barnes maze in the biology of hippocampal and cortical neural function cannot be overemphasized. Despite the wide use of the Barnes maze, the effect of maze task training on the structure of hippocampal neurons is yet to be elucidated. Adult Sprague-Dawley rats were subjected to intense training on the Barnes maze (3 months). Subsequently, the hippocampus (cornus ammonis and dentate gyrus) of separate sets of rats was processed for Golgi Colonnier techniques (silver impregnation) and adenoviral-green fluorescent protein labeling (immunohistochemistry). Our results showed that training the animals on the Barne maze increased spinogenesis significantly in the cornus ammonis and dentate gyrus neurons. In addition, we identified a critical time point at which the rats habituated to the trial without escaping box (the probe trial) and could not be tested further in the maze. Taken together, we deduced that a prolonged test on the dry land maze facilitated habituation and caused an increase in hippocampal dendritic spine count. As such, the dry land maze is a suitable paradigm for assessing spatial memory in rats. However, precautions should be taken in selecting suitable experimental controls on the basis of the duration of a study.

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.

Insulin-like growth factor-I gene therapy increases hippocampal neurogenesis, astrocyte branching and improves spatial memory in female aging rats.

In rats, learning and memory performance decline during aging, which makes this rodent species a suitable model to evaluate therapeutic strategies of potential value for correcting age-related cognitive deficits. Some of these strategies involve neurotrophic factors like insulin-like growth factor-I (IGF-I), a powerful neuroprotective molecule in the brain. Here, we implemented 18-day long intracerebroventricular (ICV) IGF-I gene therapy in 28 months old Sprague-Dawley female rats, and assessed spatial memory performance in the Barnes maze. We also studied hippocampal morphology using an unbiased stereological approach. Adenovectors expressing the gene for rat IGF-I or the reporter DsRed were used. Cerebrospinal fluid (CSF) samples were taken and IGF-I levels determined by radioimmunoassay. At the end of the study, IGF-I levels in the CSF were significantly higher in the experimental group than in the DsRed controls. After treatment, the IGF-I group showed a significant improvement in spatial memory accuracy as compared with DsRed counterparts. In the dentate gyrus (DG) of the hippocampus, the IGF-I group showed a higher number of immature neurons than the DsRed controls. The treatment increased hippocampal astrocyte branching and reduced their number in the hippocampal stratum radiatum. We conclude that the ependymal route is an effective approach to increase CSF levels of IGF-I and that this strategy improves the accuracy of spatial memory in aging rats. The favorable effect of the treatment on DG neurogenesis and astrocyte branching in the stratum radiatum may contribute to improving memory performance in aging rats.