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The hypothalamus has long been known to control food intake and energy metabolism through a complex network of primary and secondary neurons and glial cells. Anorexia nervosa being a complex disorder characterized by abnormal feeding behavior and food aversion, it is thus quite surprising that not much is known concerning potential hypothalamic modifications in this disorder. In this chapter, we review the recent advances in the fields of genetics, epigenetics, structural and functional imaging, and brain connectivity, as well as neuroendocrine findings and emerging animal models, which have begun to unravel the importance of hypothalamic adaptive processes to our understanding of the pathology of eating disorders.

Introduction: While the vast majority of research investigating the role of ghrelin or its receptor, GHS-R1a, in growth, feeding, and metabolism has been conducted in male rodents, very little is known about sex differences in this system. Furthermore, the role of GHS-R1a signaling in the control of pulsatile GH secretion and its link with growth or metabolic parameters has never been characterized. Methods: We assessed the sex-specific contribution of GHS-R1a signaling in the activity of the GH/IGF-1 axis, metabolic parameters, and feeding behavior in adolescent (5–6 weeks old) or adult (10–19 weeks old) GHS-R KO (Ghsr−/−) and WT (Ghsr+/+) male and female mice. Results: Adult Ghsr−/− male and female mice displayed deficits in weight and linear growth that were correlated with reduced GH pituitary contents in males only. GHS-R1a deletion was associated with reduced meal frequency and increased meal intervals, as well as reduced hypothalamic GHRH and NPY mRNA in males, not females. In adult, GH release from Ghsr−/− mice pituitary explants ex vivo was reduced independently of the sex. However, in vivo pulsatile GH secretion decreased in adult but not adolescent Ghsr−/− females, while in males, GHS-R1a deletion was associated with reduction in pulsatile GH secretion during adolescence exclusively. In males, linear growth did not correlate with pulsatile GH secretion, but rather with ApEn, a measure that reflects irregularity of the rhythmic secretion. Fat mass, plasma leptin concentrations, or ambulatory activity did not predict differences in GH secretion. Discussion/Conclusion: These results point to a sex-dependent dimorphic effect of GHS-R1a signaling to modulate pulsatile GH secretion and meal pattern in mice with different compensatory mechanisms occurring in the hypothalamus of adult males and females after GHS-R1a deletion. Altogether, we show that GHS-R1a signaling plays a more critical role in the regulation of pulsatile GH secretion during adolescence in males and adulthood in females.

The neuroendocrine system (NES) plays a crucial role in synchronizing the physiology and behavior of the whole organism in response to environmental constraints. The NES consists of a hypothalamic-pituitary-target organ axis that acts in coordination to regulate growth, reproduction, stress and basal metabolism. The growth (or somatotropic), hypothalamic-pituitary-gonadal (HPG), hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-thyroid (HPT) axes are therefore finely tuned by the hypothalamus through the successive release of hypothalamic and pituitary hormones to control the downstream physiological functions. These functions rely on a complex set of mechanisms requiring tight synchronization between peripheral organs and the hypothalamic-pituitary complex, whose functionality can be altered during aging. Here, we review the results of research on the effects of aging on the NES of nonhuman primate (NHP) species in wild and captive conditions. A focus on the age-related dysregulation of the master circadian pacemaker, which, in turn, alters the synchronization of the NES with the organism environment, is proposed. Finally, practical and ethical considerations of using NHP models to test the effects of nutrition-based or hormonal treatments to combat the deterioration of the NES are discussed.

International audience; Age-associated cognitive impairment is a major health and social issue because of increasing aged population. Cognitive decline is not homogeneous in humans and the determinants leading to differences between subjects are not fully understood. In middle-aged healthy humans, fasting blood glucose levels in the upper normal range are associated with memory impairment and cerebral atrophy. Due to a close evolutional similarity to Man, non-human primates may be useful to investigate the relationships between glucose homeostasis, cognitive deficits and structural brain alterations. In the grey mouse lemur, $Microcebus\ murinus$, spatial memory deficits have been associated with age and cerebral atrophy but the origin of these alterations have not been clearly identified. Herein, we showed that, on 28 female grey mouse lemurs (age range 2.4-6.1 years-old), age correlated with impaired fasting blood glucose (r$_s$=0.37) but not with impaired glucose tolerance or insulin resistance. In middle-aged animals (4.1-6.1 years-old), fasting blood glucose was inversely and closely linked with spatial memory performance (r$_s$=0.56) and hippocampus (r$_s$=-0.62) or septum (r$_s$=-0.55) volumes. These findings corroborate observations in humans and further support the grey mouse lemur as a natural model to unravel mechanisms which link impaired glucose homeostasis, brain atrophy and cognitive processes.

Recent data confirmed the efficiency of caloric restriction for promoting both healthspan and lifespan in primates, but also revealed potential adverse effects at the central level. This paper proposes perspectives and future directions to counterbalance potential adverse effects. Efforts should be made in combining nutrition-based clinical protocols with therapeutic and/or behavioral interventions to aim for synergetic effects, and therefore delay the onset of age-related diseases without adverse effects., Fabien Pifferi et al. discuss the latest research in using caloric restriction for promoting healthspan and lifespan in primates. Their Comment touches on their previous study, addressing how to combine nutrition-based clinical protocols with interventions to delay the onset of age-related diseases.

International audience; Vesicular glutamate transporters (VGLUT1-3) mediate the uptake of glutamate into synaptic vesicles. VGLUTs are pivotal actors of excitatory transmission and of almost all brain functions. Their implication in various pathologies has been clearly documented. Despite their functional importance, the pharmacology of VGLUTs is limited to a few dyes such as Trypan Blue, Rose Bengal or Brilliant Yellow type. Here, we report the design and evaluation of new potent analogs based on Trypan Blue scaffold. Our best compound, named LSP5-2157, has an EC50 of 50 nM on glutamate vesicular uptake. Using a 3D homology model of VGLUT1 and docking experiments, we determined its putative binding subdomains within vesicular glutamate transporters and validated the structural requirement for VGLUT inhibition. To better estimate the specificity and potency of LSP5-2157, we also investigated its ability to block glutamatergic transmission in autaptic hippocampal cells. Neither glutamate receptors nor GABAergic transmission or transmission machinery were affected by LSP5-2157. Low doses of compound reversibly reduce glutamatergic neurotransmission in hippocampal autpases. LSP5-2157 had a low and depressing effect on synaptic efficacy in hippocampal slice. Furthermore, LSP5-2157 had no effect on NMDA-R- mediated fEPSP but reduce synaptic plasticity induced by 3 trains of 100 Hz. Finally, LSP5-2157 had the capacity to inhibit VGLUT3-dependent auditory synaptic transmission in the guinea pig cochlea. In this model, it abolished the compound action potential of auditory nerve at high concentration showing the limited permeation of LSP5-2157 in an in-vivo model. In summary, the new ligand LSP5-2157, has a high affinity and specificity for VGLUTs and shows some permeability in isolated neuron, tissue preparations or in vivo in the auditory system. These findings broaden the field of VGLUTs inhibitors and open the way to their use to assess glutamatergic functions in vitro and in vivo.

Ghrelin is a potent orexigenic peptide hormone that acts through the growth hormone secretagogue receptor (GHSR), a G protein-coupled receptor highly expressed in the hypothalamus. In vitro studies have shown that GHSR displays a high constitutive activity, whose physiological relevance is uncertain. As GHSR gene expression in the hypothalamus is known to increase in fasting conditions, we tested the hypothesis that constitutive GHSR activity at the hypothalamic level drives the fasting-induced hyperphagia. We found that refed wild-type (WT) mice displayed a robust hyperphagia that continued for 5 days after refeeding and changed their food intake daily pattern. Fasted WT mice showed an increase in plasma ghrelin levels, as well as in GHSR expression levels and ghrelin binding sites in the hypothalamic arcuate nucleus. When fasting-refeeding responses were evaluated in ghrelin- or GHSR-deficient mice, only the latter displayed an ∼15% smaller hyperphagia, compared with WT mice. Finally, fasting-induced hyperphagia of WT mice was significantly smaller in mice centrally treated with the GHSR inverse agonist K-(D-1-Nal)-FwLL-NH2, compared with mice treated with vehicle, whereas it was unaffected in mice centrally treated with the GHSR antagonists D-Lys3-growth hormone-releasing peptide 6 or JMV2959. Taken together, genetic models and pharmacological results support the notion that constitutive GHSR activity modulates the magnitude of the compensatory hyperphagia triggered by fasting. Thus, the hypothalamic GHSR signaling system could affect the set point of daily food intake, independently of plasma ghrelin levels, in situations of negative energy balance., Instituto Multidisciplinario de Biología Celular

This study investigated whether sst2 gene deletion interacts with age and chronic stress exposure to produce exacerbated emotional and cognitive ageing. Middle-aged (10-12 month) sst2 knockout (sst2KO) and wild-type (WT) mice underwent an unpredictable chronic mild stress (UCMS) procedure for 6 weeks or no stress for control groups. This was followed by a battery of tests to assess emotional and cognitive functions and neuroendocrine status (CORT level). A re-evaluation was performed 6 months later (i.e. with 18-month-old mice). UCMS reproduced neuroendocrine and behavioral features of stress-related disorders such as elevated circulating CORT levels, physical deteriorations, increased anxiety- and depressive-like behaviors and working memory impairments. sst2KO mice displayed behavioral alterations which were similar to stressed WT and exhibited exacerbated changes following UCMS exposure. The evaluations performed in the older mice showed significant long-term effects of UCMS exposure. Old sst2KO mice previously exposed to UCMS exhibited spatial learning and memory accuracy impairments and high levels of anxiety-like behaviors which drastically added to the effects of normal ageing. Spatial abilities and emotionality scores (mean z-scores) measured both at the UCMS outcome and 6 months later were correlated with the initially measured CORT levels in middle-age. The present findings indicate that the deletion of the sst2 receptor gene produces chronic hypercorticosteronemia and exacerbates sensitivity to stressors which over time, have consequences on ageing brain function processes.

Ghrelin is an octanoylated peptide that acts via its specific receptor, the growth hormone secretagogue receptor type 1a (GHSR-1a), and regulates a vast variety of physiological functions. It is well established that ghrelin is predominantly synthesized by a distinct population of endocrine cells located within the gastric oxyntic mucosa. In addition, some studies have reported that ghrelin could also be synthesized in some brain regions, such as the hypothalamus. However, evidences of neuronal production of ghrelin have been inconsistent and, as a consequence, it is still as a matter of debate if ghrelin can be centrally produced. Here, we provide a comprehensive review and discussion of the data supporting, or not, the notion that the mammalian central nervous system can synthetize ghrelin. We conclude that no irrefutable and reproducible evidence exists supporting the notion that ghrelin is synthetized, at physiologically relevant levels, in the central nervous system of adult mammals., Instituto Multidisciplinario de Biología Celular

International audience; Nutrition during the perinatal period programs body growth. Growth hormone (GH) secretion from the pituitary regulates body growth and is controlled by Growth Hormone Releasing Hormone (GHRH) neurons located in the arcuate nucleus of the hypothalamus. We observed that dietary restriction during the early postnatal period (i.e. lactation) in mice influences postnatal growth by permanently altering the development of the somatotropic axis in the pituitary gland. This alteration may be due to a lack of GHRH signaling during this critical developmental period. Indeed, underfed pups showed decreased insulin-like growth factor I (IGF-I) plasma levels, which are associated with lower innervation of the median eminence by GHRH axons at 10 days of age relative to normally fed pups. IGF-I preferentially stimulated axon elongation of GHRH neurons in in vitro arcuate explant cultures from 7 day-old normally fed pups. This IGF-I stimulating effect was selective since other arcuate neurons visualized concomitantly by neurofilament labeling, or AgRP immunochemistry, did not significantly respond to IGF-I stimulation. Moreover, GHRH neurons in explants from age-matched underfed pups lost the capacity to respond to IGF-I stimulation. Molecular analyses indicated that nutritional restriction was associated with impaired activation of AKT. These results highlight a role for IGF-I in axon elongation that appears to be cell selective and participates in the complex cellular mechanisms that link underfeeding during the early postna-tal period with programming of the growth trajectory.

Chantier qualité GA; International audience; Objective: Since a reduction of the insulin/IGF-1 signaling cascade extends life span in many species and IGF-1 signaling might partly mediate the effects of caloric restriction (CR), an experimental intervention for increasing longevity, the purpose of the present study was to use quantitative trait loci (QTL) analysis, an unbiased genetic approach, to identify particular regions of the genome influencing plasma IGF-1 levels in an F2 intercross between F344 and LOU/C rats; the latter being an inbred strain of Wistar origin, considered as a model of healthy aging since it resists to age (and diet)-induced obesity. Design: F1 hybrids were obtained by crossbreeding LOU/C with F344 rats, and then F1 were bred inter se to obtain the F2 population, of which 93 males and 94 females were studied. Total plasma IGF-1 levels were determined by radioimmunoassay. A genome scan of the F2 population was made with 100 microsatellite markers) selected for their polymorphism between LOU/C and F344 strains (and by covering evenly the whole genome. Results: By simple interval mapping sex-dependent QTLs were found on chromosome 17 in males and on chromosome 18 in females. By multiple interval mapping, additional QTLs were found on chromosomes 1, 4, 5, 6, 12, 15 and 19 in males and on chromosomes 3, 5, 6, 12 and 17 in females. Only the markers D1Rat196 and D12Mgh5 were found in both males and females. The majority of QTLs corresponded to metabolic syndrome (cardiac function: n = 45 (30%), obesity/diabetes: n = 22 (15%), inflammation: n = 19 (13%) and only a limited number to body weight: n = 13 (9%), proliferation (n = 10 (7%) or ossification: n = 7 (5%). Ninety-six candidate genes were located on the different QTLs. A significant proportion of these genes are connected to IGF-1 production and receptor pathways (n = 18) or metabolic syndrome (n = 11). Conclusions: Subsequent studies are necessary to determine whether the genetic networks underscored are also involved in age-associated obesity, diabetes and inflammation as well as cardiovascular impairments.

International audience; Altered brain somatostatin functions recently appeared as key elements for the pathogenesis of stress-related neuropsychiatric disorders. The hippocampus exerts an inhibitory feedback on stress but the mechanisms involved remain unclear. We investigated herein the role of hippocampal somatostatin receptor subtypes in both stress response and behavioral emotionality using C57BL/6, wild type and sst2 or sst4 knockout mice. Inhibitory effects of hippocampal infusions of somatostatin agonists on stress-induced hypothalamo-pituitary-adrenal axis (HPA) activity were tested by monitoring peripheral blood and local hippocampus corticosterone levels, the latter by using microdialysis. Anxiolytic and antidepressant-like effects were determined in the elevated-plus maze, open field, forced swimming, and stress-sensitive beam walking tests. Hippocampal injections of somatostatin analogs and sst2 or sst4, but not sst1 or sst3 receptor agonists produced rapid and sustained inhibition of HPA axis. sst2 agonists selectively produced anxiolytic-like behaviors whereas both sst2 and sst4 agonists had antidepressant-like effects. Consistent with these findings, high corticosterone levels and anxiety were found in sst2KO mice and depressive-like behaviors observed in both sst2KO and sst4KO strains. Both hippocampal sst2 and sst4 receptors selectively inhibit stress-induced HPA axis activation but mediate anxiolytic and antidepressive effects through distinct mechanisms. Such results are to be accounted for in development of pathway-specific somatostatin receptor agents in the treatment of hypercortisolism (Cushing's disease) and stress-related neuropsychiatric disorders.

This article reviews the main findings that emerged in the intervening years since the previous volume on hormonal control of growth in the section on the endocrine system of the Handbook of Physiology concerning the intra- and extrahypothalamic neuronal networks connecting growth hormone releasing hormone (GHRH) and somatostatin hypophysiotropic neurons and the integration between regulators of food intake/metabolism and GH release. Among these findings, the discovery of ghrelin still raises many unanswered questions. One important event was the application of deconvolution analysis to the pulsatile patterns of GH secretion in different mammalian species, including Man, according to gender, hormonal environment and ageing. Concerning this last phenomenon, a great body of evidence now supports the role of an attenuation of the GHRH/GH/Insulin-like growth factor-1 (IGF-1) axis in the control of mammalian aging.

Fasting results in the mobilization of adipose stores and the elevation of levels of free fatty acids (FFA). In humans, this process is driven by a release in GH. Little is known regarding the role of GH in modulating this process during early stages of fasting in the mouse. Confirmation of the role of GH in modulating FFA release in the fasting mouse is of particular importance given the frequent use of mouse models to study metabolic mechanisms. Here, we correlate the initial release of FFA throughout fasting in mice with pulsatile GH secretion. Observations illustrate the rapid release of FFA in response to food withdrawal. This does not correlate with a rise in GH secretion. Rather, we observed a striking loss in pulsatile secretion of GH throughout the first 6 h of fasting, suggesting that GH does not modulate the initial release of FFA in the mouse in response to fasting. This was confirmed in GH receptor knockout mice, in which we observed a robust fasting-induced rise in FFA. We further illustrate the dynamic relationship between the orexigenic and anorexigenic hormones ghrelin and leptin during fasting in the mouse. Our findings show an initial suppression of leptin and the eventual rise in circulating levels of acyl-ghrelin with fasting. However, altered acyl-ghrelin and leptin secretion occurs well after the rise in FFA and the suppression of GH secretion. Consequently, we conclude that although acyl-ghrelin and leptin may modulate the physiological response to drive food intake, these changes do not contribute to the initial loss of pulsatile GH secretion. Rather, it appears that the suppression of GH secretion in fasting may occur in response to an elevation in fasting levels of FFA or physiological stress. Observations highlight a divergent role for GH in modulating FFA release between man and mouse.

Among the factors playing a crucial role in the regulation of energy metabolism, gastro-intestinal peptides are essential signals to maintain energy homeostasis as they relay to the central nervous system the informations about the nutritional status of the body. Among these factors, preproghrelin is a unique prohormone as it encodes ghrelin, a powerful GH secretagogue and the only orexigenic signal from the gastrointestinal tract and obestatin, a proposed functional ghrelin antagonist. These preproghrelin-derived peptides may contribute to balance energy intake, metabolism and body composition by regulating the activity of the GH/IGF-1 axis and appetite. Whereas the contribution of ghrelin has been well characterized, the role of the more recently identified obestatin, in this regulatory process is still controversial. In this chapter, we describe the contribution of these different preproghrelin-derived peptides and their receptors in the regulation of GH secretion and feeding. Data obtained from pharmacological approaches, mutant models and evaluation of the hormones in animal and human models are discussed.

The memory deficits associated with Alzheimer's disease result to a great extent from hippocampal network dysfunction. The coordination of this network relies on theta (symbol) oscillations generated in the medial septum. Here, we investigated in rats the impact of hippocampal amyloid beta (Abeta) injections on the physiological and cognitive functions that depend on the septohippocampal system. Hippocampal Abeta injections progressively impaired behavioral performances, the associated hippocampal theta power, and theta frequency response in a visuospatial recognition test. These alterations were associated with a specific reduction in the firing of the identified rhythmic bursting GABAergic neurons responsible for the propagation of the theta rhythm to the hippocampus, but without loss of medial septal neurons. Such results indicate that hippocampal Abeta treatment leads to a specific functional depression of inhibitory projection neurons of the medial septum, resulting in the functional impairment of the temporal network.

Somatostatin (SS14) has been implicated in various cognitive disorders, and converging evidence from animal studies suggests that SS14 neurons differentially regulate hippocampal- and striatal-dependent memory formation. Four SS14 receptor subtypes (SSTR1-4) are expressed in the hippocampus, but their respective roles in memory processes remain to be determined.In the present study, effects of selective SSTR1-4 agonists on memory formation were assessed in a water-maze task which can engage either hippocampus-dependent "place" and/or striatum-dependent "cue" memory formation.Mice received an intrahippocampal injection of one of each of the selective agonists and were then trained to locate an escape platform based on either distal cues (place memory) or a visible proximal cue (cue memory). Retention was tested 24 h later on probe trials aimed at identifying which memory strategy was preferentially retained.Both SS14 and the SSTR4 agonist (L-803,087) dramatically impaired place memory formation in a dose-dependent manner, whereas SSTR1 (L-797,591), SSTR2 (L-779,976), or SSTR3 (L-796,778) agonists did not yield any behavioral effects. However, unlike SS14, the SSTR4 agonist also dose-dependently enhanced cue-based memory formation. This effect was confirmed in another striatal-dependent memory task, the bar-pressing task, where L-803,087 improved memory of the instrumental response, whereas SS14 was once again ineffective.These data suggest that hippocampal SSTR4 are selectively involved in the selection of memory strategies by switching from the use of hippocampus-based multiple associations to the use of simple dorsal striatum-based behavioral responses. Possible neural mechanisms and functional implications are discussed.

Somatostatin is abundantly expressed in mammalian brain. The peptide binds with high affinity to six somatostatin receptors, sst1, sst2A and B, sst3 to 5, all belonging to the G-protein-coupled receptor family. Recent advances in the neuroanatomy of somatostatin neurons and cellular distribution of sst receptors shed light on their functional roles in the neuronal network. Beside their initially described neuroendocrine role, somatostatin systems subserve neuromodulatory roles in the brain, influencing motor activity, sleep, sensory processes and cognitive functions, and are altered in brain diseases like affective disorders, epilepsia and Alzheimer's disease.

In adults, the adipocyte-derived hormone, leptin, regulates food intake and body weight principally via the hypothalamic arcuate nucleus (ARC). During early postnatal development, leptin functions to promote the outgrowth of neuronal projections from the ARC, whereas a selective insensitivity to the effects of leptin on food intake appears to exist. To investigate the mechanisms underlying the inability of leptin to regulate food intake during early development, leptin signaling was analyzed both in vitro using primary cultures of rat embryonic ARC neurones and in vivo by challenging early postnatal rats with leptin. In neuronal cultures, despite the presence of key components of the leptin signaling pathway, no detectable activation of either signal transducer and activator of transcription 3 or the MAPK pathways by leptin was detected. However, leptin down-regulated mRNA levels of proopiomelanocortin and neuropeptide Y and decreased somatostatin secretion. Leptin challenge in vivo at postnatal d (P) 7, P14, P21, and P28 revealed that, in contrast to adult and P28 rats, mRNA levels of neuropeptide Y, proopiomelanocortin, agouti-related peptide and cocaine- and amphetamine-regulated transcript were largely unaffected at P7, P14, and P21. Furthermore, leptin stimulation increased the suppressor of cytokine signaling-3 mRNA levels at P14, P21, and P28 in several hypothalamic nuclei but not at P7, indicating that selective leptin insensitivity in the hypothalamus is coupled to developmental shifts in leptin receptor signaling. Thus, the present study defines the onset of leptin sensitivity in the regulation of energy homeostasis in the developing hypothalamus.

The secretion of growth hormone (GH) is inhibited by hypothalamic somatostatin (SRIF) in somatotropes through five subtypes of the somatostatin receptor (SSTR1–SSTR5). We aimed to characterize the subtype(s) of SSTRs involved in the Ca2+current reduction in GH3 somatotrope cells using specific SSTR subtype agonists. We used nystatin-perforated patch clamp to record voltage-gated Ca2+currents, using a holding potential of −80 mV in the presence of K+and Na+channel blockers. We first established the presence of T-, L-, N-, and P/Q-type Ca2+currents in GH3 cells using a variety of channel blockers (Ni+, nifedipine, ω-conotoxin GVIA, and ω-agatoxin IVA). SRIF (200 nM) reduced L- and N-type but not T- or P/Q-type currents in GH3 cells. A range of concentrations of each specific SSTR agonist was tested on Ca2+currents to find the maximal effective concentration. Activation of SSTR2 with 10−7and 10−8M L-797,976 decreased the voltage-gated Ca2+current and abolished any further decrease by SRIF. SSTR1, SSTR3, SSTR4, and SSTR5 agonists at 10−7M did not modify the voltage-gated Ca2+current and did not affect the Ca2+current response to SRIF. These results indicate that SSTR2 is involved mainly in regulating voltage-gated Ca2+currents by SRIF, which contributes to the decrease in intracellular Ca2+concentration and GH secretion by SRIF.

Growth hormone (GH) secretion decreases spontaneously during lifespan, and the resulting GH deficiency participates in aging-related morbidity. This deficiency appears to involve a defect in the activity of hypothalamic GH-releasing hormone (GHRH) neurons. Here, we investigated this hypothesis, as well as the underlying mechanisms, in identified GHRH neurons from adult ( approximately 13 weeks old) and aged ( approximately 100 weeks old) transgenic GHRH-green fluorescent protein mice, using morphological, biochemical and electrophysiological methods. Surprisingly, the spontaneous action potential frequency was similar in adult and aged GHRH neurons studied in brain slices. This was explained by a lack of change in the intrinsic excitability, and simultaneous increases in both stimulatory glutamatergic- and inhibitory GABAergic-synaptic currents of aged GHRH neurons. Aging did not decrease GHRH and enhanced green fluorescent protein contents, GHRH neuronal number or GHRH-fibre distribution, but we found a striking enlargement of GHRH-positive axons, suggesting neuropeptide accumulation. Unlike in adults, autophagic vacuoles were evident in aged GHRH-axonal profiles using electron microscopy. Thus, GHRH neurons are involved in aging of the GH axis. Aging had a subtle effect at the nerve terminal level in GHRH neurons, contrasting with the view that neuronal aging is accompanied by more widespread damage.

In the present work, we took advantage of a recently described model of GHRH-enhanced green fluorescent protein (eGFP) transgenic mice to evaluate the extent of co-localization of GHRH neurons with galanin (GAL), neurotensin (NT) and tyrosine hydroxylase (TH) in 3- and 8-month-old male and female mice. The total number of GHRH-eGFP neurons along the rostro-caudal axis of the arcuate nucleus did not differ according to gender or age. GAL-immunoreactivity was present in 40–44% of 3-month-old GHRH-eGFP neurons in male and female arcuate nucleus, respectively, but only 25–22% in 8-month-old mice. TH immunoreactivity occurred in 36–35% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice and these proportions increased to 40 and 45% in 8-month-old mice. NT immunoreactivity was present in 14 and 24% of GHRH-eGFP neurons in male and female arcuate nucleus from 3-month-old mice up to 28 and 26% in 8-month-old mice. Thus, co-localization of peptides and enzyme in GHRH-eGFP neurons displays a sexual dimorphism at 3-month of age for NT, and at 8-month for TH, while the total number of GHRH-eGFP neurons does not exhibit gender difference at either age. In summary, it appears that changes in co-localized (and presumably co-released) peptides, rather than GHRH per se , may contribute to the changes in sexually dimorphic GH secretion with aging in the mouse.

Ghrelin, a gut hormone originating from the post-translational cleavage of preproghrelin, is the endogenous ligand of growth hormone secretagogue receptor 1a (GHS-R1a). Within the growth hormone (GH) axis, the biological activity of ghrelin requires octanoylation by ghrelin-O-acyltransferase (GOAT), conferring selective binding to the GHS-R1a receptor via acylated ghrelin. Complete loss of preproghrelin-derived signalling (through deletion of the Ghrl gene) contributes to a decline in peak GH release; however, the selective contribution of endogenous acyl-ghrelin to pulsatile GH release remains to be established. We assessed the pulsatile release of GH in ad lib. fed male germline goat(-/-) mice, extending measures to include mRNA for key hypothalamic regulators of GH release, and peripheral factors that are modulated relative to GH release. The amount of GH released was reduced in young goat(-/-) mice compared to age-matched wild-type mice, whereas pulse frequency and irregularity increased. Altered GH release did not coincide with alterations in hypothalamic Ghrh, Srif, Npy or Ghsr mRNA expression, or pituitary GH content, suggesting that loss of Goat does not compromise canonical mechanisms that contribute to pituitary GH production and release. Although loss of Goat resulted in an irregular pattern of GH release (characterised by an increase in the number of GH pulses observed during extended secretory events), this did not contribute to a change in the expression of sexually dimorphic GH-dependent liver genes. Of interest, circulating levels of insulin-like growth factor (IGF)-1 were elevated in goat(-/-) mice. This rise in circulating levels of IGF-1 was correlated with an increase in GH pulse frequency, suggesting that sustained or increased IGF-1 release in goat(-/-) mice may occur in response to altered GH release patterning. Our observations demonstrate that germline loss of Goat alters GH release and patterning. Although the biological relevance of altered GH secretory patterning remains unclear, we propose that this may contribute to sustained IGF-1 release and growth in goat(-/-) mice.

Cocaine- and amphetamine-regulated transcript (CART) mRNA and peptides are abundant in the adenohypophysis, but their role in pituitary function has not yet been elucidated. CART peptides were recently shown to colocalise with luteinising hormone (LH) or prolactin in rat anterior pituitary, and contradictory results concerning the peptide effects on pituitary hormonal secretions were obtained in vitro from pituitary cell cultures. Thus, we reinvestigated the expression of CART mRNA within the pituitary. Immunohistochemistry for pituitary hormones was performed on sections from adult male Wistar rats followed by in situ hybridisation using CART mRNA antisense 35S-labelled probes. The most represented CART-expressing cells were lactotrophs (42 +/- 1% of CART cells) and gonadotrophs (32 +/- 3%), followed by thyrotrophs (10 +/- 2%), corticotrophs (7 +/- 2%) and somatotrophs (6 +/- 1%). In the pars tuberalis, CART mRNA was easily detectable in gonadotrophs and lactotrophs and, to a lesser extent, in corticotrophs and thyrotrophs. CART peptide was quickly and potently released from perifused pituitary by depolarisation (K+ 30 mM for 15 min; 465 +/- 37% over basal release, n = 5). Gonadotrophin-releasing hormone and thyrotrophin-releasing hormone (0.1 microM) were also active to a lesser extent (138 +/- 11% and 71 +/- 17, n = 7, respectively). CART (0.1 microM) did not modify basal LH or prolactin release but selectively inhibited K+-induced LH release without affecting K+-induced prolactin secretion. Pituitary CART mRNA and content were sex dependent and varied during the oestrous cycle, being lower in dioestrous 2. Pituitary CART content also varied widely amongst rat strains being five to six-fold higher in Wistar and Fischer rats compared to Brown Norway and Lou C rats. Ageing differentially affected pituitary CART mRNA and content, resulting in a marked decrease in Lou C and an increase in Wistar and Sprague-Dawley rats. Taken together, these results suggest that pituitary CART expression is dependent of the sex steroid environment and may be physiologically involved in LH secretion.

GABAergic septohippocampal neurons play a major role in the generation of hippocampal theta rhythm, but modulatory factors intervening in this function are poorly documented. The neuropeptide somatostatin (SST) may be one of these factors, because nearly all hippocampal GABAergic neurons projecting to the medial septum/diagonal band of Broca (MS-DB) express SST. In this study, we took advantage of the high and selective expression of the SST receptor sst2Ain MS-DB to examine its possible role on theta-related activity. Immunohistochemical experiments demonstrated that sst2Areceptors were selectively targeted to the somatodendritic domain of neurons expressing the GABAergic marker GAD67 but were not expressed by cholinergic neurons. In addition, a subpopulation of GABAergic septohippocampal projecting neurons expressing parvalbumin (PV) also displayed sst2Areceptors. Usingin vivojuxtacellular recording and labeling with neurobiotin, we showed that a number of bursting and nonbursting neurons exhibiting high discharge rates and brief spikes were immunoreactive for PV or GAD67 and expressed the sst2Areceptor. Microiontophoresis applications of SST and the sst2Aagonist octreotide (OCT) showed that sst2Areceptor activation decreased the discharge rate of both nonbursting and bursting MS-DB neurons and lessened the rhythmic activity of the latter. Finally, intraseptal injections of OCT and SST in freely moving rats reduced the power of hippocampal EEG in the theta band. Together, thesein vivoexperiments suggest that SST action on MS-DB GABAergic neurons, through sst2Areceptors, represents an important modulatory mechanism in the control of theta activity.

The physiological actions of somatostatin-14 (SRIF: somatotrophin release inhibitory factor) receptor subtypes (sst(1)-sst(5)), which are endogenously expressed in growth cells (GC cells), have not yet been elucidated, although there is evidence that sst(2) receptors are negatively coupled to cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and adenosine 3,5'-cyclic monophosphate (cAMP) accumulation. In addition, both sst(1) and sst(2) receptors are negatively coupled to growth hormone (GH) secretion in GC cells. Here we report on studies concerning the expression, the pharmacology and the functional role of native SRIF receptors in GC cells with the use of five nonpeptidyl agonists, highly selective for each of the SRIF receptors. Radioligand binding studies show that sst(2) and sst(5) receptors are present at different relative densities, while the presence of sst(3) and sst(4) receptors appears to be negligible. The absence of sst(1) receptor binding was unexpected in view of sst(1) receptor functional effects on GH secretion. This suggests very efficient receptor-effector coupling of a low-density population of sst(1) receptors. Functionally, only sst(2) receptors are coupled to the inhibition of [Ca(2+)](i) and cAMP accumulation and the selective activation of sst(5) receptors facilitates the stimulation of adenylyl cyclase activity through G(i/o) proteins. This effect was not observed when sst(2) and sst(5) receptors were simultaneously activated, suggesting that there is a functional interaction between sst(2) and sst(5) receptors. In addition, sst(1), sst(2) and sst(5) receptor activation inhibits GH release, further indicating that SRIF can modulate GH secretion in GC cells through mechanisms both dependent and independent on [Ca(2+)](i) and cAMP-dependent pathways. The present data suggest SRIF-mediated functional effects in GC cells to be very diverse and provides compelling arguments to propose that multiple native SRIF receptors expressed in the same cells are not simply redundant, but contribute to marked signalling diversity.

Numerous physiological studies as well as in situ hybridization and PCR experiments concur in reporting a role for the sst2A receptor in transducing somatostatin (SRIF) actions in the rat hypothalamus. However, the distribution of this receptor protein is not known within this structure. Regional and cellular localization of the sst2A receptor was therefore examined in the rat hypothalamus using highly sensitive immunohistochemical techniques. In close correspondence with the distribution of SRIF-immunoreactive fibers, numerous hypothalamic areas displayed sst2A receptor immunoreactivity. Receptor labeling was, however, diffusely distributed over the tissue, and few immunopositive cells were apparent. Unraveling the distribution of receptor-expressing cells was achieved through acute in vivo agonist stimulation and subsequent receptor internalization. At the cellular level, double-immunolabeling experiments with synaptophysin and microtubule-associated protein 2 demonstrated that sst2A receptors were predominantly internalized in perikarya and dendrites. Double-labeling experiments with SRIF revealed that 93% of arcuate, but only 18% of periventricular, SRIF-positive neurons expressed internalized receptors. Taken together, these results demonstrate for the first time that the sst2A receptor protein is widely, but selectively, distributed in the hypothalamus, and that postsynaptic sst2A auto- and heteroreceptors are well poised to play an important role in the somatostatinergic regulation of hypothalamic endocrine and metabolic processes.

A somatostatin deficit occurs in the cerebral cortex of Alzheimer's disease patients without a major loss in somatostatin-containing neurons. This deficit could be related to a reduction in the rate of proteolytic processing of peptide precursors. Since the two proprotein convertases (PC)1 and PC2 are responsible for the processing of neuropeptide precursors directed to the regulated secretory pathway, we examined whether they are involved first in the proteolytic processing of prosomatostatin in mouse and human brain and secondly in somatostatin defect associated with Alzheimer's disease. By size exclusion chromatography, the cleavage of prosomatostatin to somatostatin-14 is almost totally abolished in the cortex of PC2 null mice, while the proportions of prosomatostatin and somatostatin-28 are increased. By immunohistochemistry, PC1 and PC2 were localized in many neuronal elements in human frontal and temporal cortex. The convertases levels were quantified by Western blot, as well as the protein 7B2 which is required for the production of active PC2. No significant change in PC1 levels was observed in Alzheimer's disease. In contrast, a marked decrease in the ratio of the PC2 precursor to the total enzymatic pool was observed in the frontal cortex of Alzheimer patients. This decrease coincides with an increase in the binding protein 7B2. However, the content and enzymatic activity of the PC2 mature form were similar in Alzheimer patients and controls. Therefore, the cortical somatostatin defect is not due to convertase alteration occuring during Alzheimer's disease. Further studies will be needed to assess the mechanisms involved in somatostatin deficiency in Alzheimer's disease.

We have investigated the role of somatostatin receptor subtypes sst 2 and sst 4 in limbic seizures and glutamate-mediated neurotransmission in mouse hippocampus. As compared to wild-type littermates, homozygous mice lacking sst 2 receptors showed a 52% reduction in EEG ictal activity induced by intrahippocampal injection of 30 ng kainic acid (P < 0.05). The number of behavioural tonic-clonic seizures was reduced by 50% (P < 0.01) and the time to onset of seizures was doubled on average (P < 0.05). Seizure-associated neurodegeneration was found in the injected hippocampus (CA1, CA3 and hilar interneurons) and sporadically in the ipsilateral latero-dorsal thalamus. This occurred to a similar extent in wild-type and sst 2 knock-out mice. Intrahippocampal injection of three selective sst 2 receptor agonists in wild-type mice (Octreotide, BIM 23120 and L-779976, 1.5-6.0 nmol) did not affect kainate seizures while the same compounds significantly reduced seizures in rats. L-803087 (5 nmol), a selective sst4 receptor agonist, doubled seizure activity in wild-type mice on average. Interestingly, this effect was blocked by 3 nmol octreotide. It was determined, in both radioligand binding and cAMP accumulation, that octreotide had no direct agonist or antagonist action at mouse sst4 receptors expressed in CCl39 cells, up to micromolar concentrations. In hippocampal slices from wild-type mice, octreotide (2 μM) did not modify AMPA-mediated synaptic responses while facilitation occurred with L-803087 (2 μM). Similarly to what was observed in seizures, the effect of L-803087 was reduced by octreotide. In hippocampal slices from sst 2 knock-out mice, both octreotide and L-803087 were ineffective on synaptic responses. Our findings show that, unlike in rats, sst 2 receptors in mice do not mediate anticonvulsant effects. Moreover, stimulation of sst 4 receptors in the hippocampus of wild-type mice induced excitatory effects which appeared to depend on the presence of sst 2 subtypes, suggesting these receptors are functionally coupled.

Ghrelin, an endogenous ligand for the GHS receptor, stimulates GH secretion and gastrointestinal motility and has orexigenic effects. In this study, the relationships between ghrelin, GH secretion, feeding behavior, and sleep-wake patterns were investigated in adult male rats. The half-life of exogenous ghrelin (10 microg i.v.) in plasma was about 30 min. Repeated administration of ghrelin at 3- to 4-h intervals (one during lights-on and two during lights-off periods) increased GH release and feeding activity, and decreased rapid eye movement sleep duration. Endogenous plasma ghrelin levels exhibited pulsatile variations that were smaller and less regular compared with those of GH. No significant correlation between GH and ghrelin circulating levels was found, although mean interpeak intervals and pulse frequencies were close for the two hormones. In contrast, ghrelin pulse variations were correlated with food intake episodes in the lights off period, and plasma ghrelin concentrations decreased by 26% in the 20 min following the end of the food intake periods. A positive correlation between ghrelin levels and active wake was found during the first 3 h of the dark period only. In conclusion, ghrelin, in addition to affecting GH secretion, gastrointestinal motility, and feeding activity, also modifies sleep-wake patterns. However, a direct action of ghrelin per se or the indirect effects of feeding (and all of its attendant metabolic sequelae) on sleep cannot be differentiated. Moreover, ghrelin secretion is pulsatile and directly related to feeding behavior only.

The peptide hormone/neurotransmitter somatostatin (somatotropin release inhibiting factor; SRIF) and its receptors (sst(1)-sst(5)) appear to regulate many physiological functions in the CNS. Semi-quantitative analysis of the densities of mRNA expression for sst(1-5) receptors and SRIF receptor binding sites were established in sst(2) receptor knock-out (KO) mice. Patterns of sst(1-5) receptor mRNA expression were largely conserved for sst(1,3,4) and sst(5) selective oligonucleotide probes; whereas sst(2) signals were completely absent in KO mouse brain. Autoradiographic analysis demonstrated [(125)I]LTT SRIF(28), [(125)I]CGP 23996 (two radioligands known to label all five recombinant SRIF receptors) and [(125)I]Tyr(3)-octreotide (sst(2) and sst(5) receptor selective) binding in wild type (WT) mouse brain sections; yet no specific binding of [(125)I]Tyr(3)-octreotide in KO mice. In contrast, [(125)I]LTT SRIF(28) and [(125)I]CGP 23996 binding was still present in a number of brain areas in KO mice, although to a lesser degree than in those regions where [(125)I]Tyr(3)-octreotide binding was found, in WT animals. The present data suggest first, that both sst(2) receptor protein and mRNA were completely absent in the brain of these KO animals. Second, there was little evidence of compensatory regulation, at the mRNA level, of the other SRIF receptors as a consequence of the sst(2) KO. Third, the absence of any [(125)I]Tyr(3)-octreotide binding, in KO mice, suggests that this particular ligand is selective for the sst(2) receptor subtype (under the conditions utilised); or that sst(5) receptors are only marginally expressed in brain. Fourth, there were regions where the binding of [(125)I]LTT SRIF(28) and [(125)I]CGP 23996 were moderately affected by the sst(2) KO, suggesting that additional SRIF receptors may well contribute to the binding of the aforementioned radioligands. Finally, since the relative distribution of these two ligands were not entirely superimposable, it suggests that their respective selectivity profiles towards the different SRIF receptor subtypes in situ are not identical.

Ghrelin is a gut hormone processed from the proghrelin peptide acting as the endogenous ligand of the GH secretagogue receptor 1a. The regulatory role of endogenous ghrelin on pulsatile GH secretion and linear growth had to be established. The aim of the present study was to delineate the endogenous actions of preproghrelin on peripheral and central components of the GH axis. Accordingly, the ultradian pattern of GH secretion was measured in young and old preproghrelin-deficient males. Blood samples were collected by tail bleeding every 10 minutes over a period of 6 hours. Analysis of the GH pulsatile pattern by deconvolution showed that GH was secreted in an ultradian manner in all genotypes, with major secretory peaks occurring at about 3-hour intervals. In older mice, the peak number was reduced and secretion was less irregular compared with younger animals. Remarkably, in young Ghrl−/− mice, the amplitude of GH secretory bursts was significantly reduced. In older mice, however, genotype differences were less significant. Changes in GH pulsatility in young Ghrl−/− mice were associated with a tendency for reduced GH pituitary contents and plasma IGF-I concentrations, but with only a minor impact on linear growth. In Ghrl+/− mice, despite reduced Acyl ghrelin to des-acyl ghrelin ratio, GH secretion was not impaired. Ghrelin deficiency was not associated with a reduction in hypothalamic GHRH content or altered response to GHRH stimulation. Therefore, reduction in GHRH production and/or sensitivity do not primarily account for the altered GH pulsatile secretion of young Ghrl−/− mice. Instead, GHRH expression was elevated in young but not old Ghrl−/− mice, suggesting that differential compensatory responses resulting from the absence of endogenous ghrelin is occurring according to age. These results show that endogenous ghrelin is a regulator of GH pulse amplitude in growing mice but does not significantly modulate linear growth.

Somatostatin (SOM) cortical levels decline in Alzheimer's disease (AD) in correlation with cognitive impairment severity, the latter being closely related to the presence of neurofibrillary tangles. Impaired olfaction is another hallmark of AD tightly related to tau pathology in the olfactory pathways. Recent studies showed that SOM modulates olfactory processing, suggesting that alterations in SOM levels participate to olfactory deficits in AD. Herein, we first observed that human olfactory peduncle and cortex are enriched in SOM cells and fibers, in aged postmortem brains. Then, the possible link between SOM alterations and olfactory deficits was evaluated by exploring the impact of age and tau hyperphosphorylation on olfactory SOM networks and behavioral performances in THY-Tau22 mice, a tauopathy transgenic model. Distinct molecular repertoires of SOM peptide and receptors were associated to sensory or cortical olfactory processing structures. Aging mainly affected SOM neurotransmission in piriform and entorhinal cortex in wild-type mice, although olfactory performances decreased. However, no further olfactory impairment was evidenced in THY-Tau22 mice until 12 months although tau pathology early affected olfactory cortical structures. Thus, tau hyperphosphorylation per se has a limited impact on olfactory performances in THY-Tau22 mice.

The grey mouse lemur (Microcebus murinus) is a non-human primate used to study the ageing process. Resveratrol is a polyphenol that may increase lifespan by delaying age-associated pathologies. However, no information about resveratrol absorption and metabolism is available for this primate. Resveratrol and its metabolites were qualitatively and quantitatively analyzed in male mouse-lemur plasma (after 200 mg.kg−1 of oral resveratrol) by ultra-high performance liquid chromatography (UHPLC), coupled to a quadrupole-time-of-flight (Q-TOF) mass spectrometer used in full-scan mode. Data analyses showed, in MSE mode, an ion common to resveratrol and all its metabolites: m/z 227.072, and an ion common to dihydro-resveratrol metabolites: m/z 229.08. A semi-targeted study enabled us to identify six hydrophilic resveratrol metabolites (one diglucurono-conjugated, two monoglucurono-conjugated, one monosulfo-conjugated and two both sulfo- and glucurono-conjugated derivatives) and three hydrophilic metabolites of dihydro-resveratrol (one monoglucurono-conjugated, one monosulfo-conjugated, and one both sulfo- and glucurono-conjugated derivatives). The presence of such metabolites has been already detected in the mouse, rat, pig, and humans. Free resveratrol was measurable for several hours in mouse-lemur plasma, and its two main metabolites were trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate. Free dihydro-resveratrol was not measurable whatever the time of plasma collection, while its hydrophilic metabolites were present at 24 h after intake. These data will help us interpret the effect of resveratrol in mouse lemurs and provide further information on the inter-species characteristics of resveratrol metabolism.

To determine whether cellular compartmentalization of somatostatin receptors can be regulated in vivo, we examined the immunocytochemical distribution of the sst2A receptor (sst2AR) after stereotaxical injections of somatostatin analogs into the rat parietal cortex. Whereas CH-275, a sst1R agonist, failed to induce changes in the diffuse sst2AR immunostaining pattern characteristic of control animals, somatodendritic profiles displaying intracytoplasmic immunoreactive granules became apparent short-term after injection of either somatostatin or the sst2R agonist octreotide. Confocal microscopy revealed that 90% of sst2AR-immunoreactive endosome-like organelles displayed transferrin receptor immunoreactivity. At the electron microscopic level, the percentage of sst2AR immunoparticles dramatically decreased at the plasmalemma of perikarya and dendrites after octreotide injection. Conversely, it significantly increased in endosomes-like organelles. These results demonstrate that sst2ARs undergo, in vivo, rapid and massive internalization into the endocytic recycling compartment in response to acute agonist stimulation and provide important clues toward elucidating somatostatin receptor signaling in the mammalian brain.

Ghrelin (Ghr), a 28 amino acid gastric peptide with an n-octanoylation on Ser 3, has recently been identified as an endogenous ligand of the growth hormone secretagogue (GHS) receptor. A cDNA was also isolated from a mouse stomach library encoding a protein named prepromotilin-related peptide (ppMTLRP) which shares sequence similarities with prepromotilin. Mouse and rat ppMTLRP sequences (rGhr) are identical and show 89% identity with human ghrelin (hGhr). By analogy with promotilin, cleavage of proMTLRP into an 18 amino acid endogenous processed peptide can be assumed on the basis of a conserved dibasic motif in position 9–10 of its sequence. In the present work, we compared the GH-releasing activity of rGhr28/MTLRP and of hGhr28/MTRLP with that of a shorter form of the peptide, hGhr18. A short peptide devoid of Ser-3 n-octanoylation hGhr18[–] was also tested. Addition of rGhr28, hGhr28 and hGhr18 stimulated GH release to the same extent from superfused pituitaries. The effect was dose dependent in a 10–8 to 10–6M concentration range. In contrast, hGhr 18[–] was inactive. In freely moving animals, both rGhr28 and hGhr28 (10 µg, i.v.) stimulated GH release, whereas the same dose of hGhr18 or of hGhr18[–] was ineffective. After rGhr28, GH plasma levels increased as early as 5 min after injection and returned to basal values within 40–60 min. Expressed as percent stimulation, administration of rGhr28 was equally effective when injected during troughs or peaks of GH. Plasma concentrations of prolactin, adrenocorticotropin and leptin were not modified. Spontaneous GH secretory episodes were no longer observed within 3 h of rGhr28 treatment, but repeated administration of the secretagogue at 3- to 4-hour intervals resulted in a similar GH response. Activation of somatostatin (SRIH) release by ether stress did not blunt the GH response to rGhr28. This suggests that the secretagogue acts in part by inhibiting endogenous SRIH, as further substantiated by the ability of rGhr28 (10–6M), to decrease the amplitude of 25 mM K+-induced SRIH release from perifused hypothalami. In conclusion, (1) n-octanoylation of Ghrs and the shorter form hGhr18 is essential for the direct pituitary GH-releasing effect of this new family of endogenous GHSs; (2) only the longer forms are active in vivo and (3) inhibition of SRIH release appears involved in the mechanism of Ghr action.

In vitro studies using various cell systems have provided conflicting results regarding homologous regulation of somatostatin (SRIH) receptors, and information on whether SRIH regulates the expression of its own receptors in vivo is lacking. In the present study we examined, by in situ hybridization, the effects of pretreatment with the sst2-preferring SRIH analog, octreotide, in vivo, on mRNA levels of two SRIH receptor subtypes, sst1 and sst2, in rat brain and pituitary. 125I-[DTrp8]-SRIH binding was also measured in these regions. Three hours after the iv injection of 50 µg octreotide to conscious adult male rats, there was a 46% increase (p < 0.01) in the labeling density of sst2 mRNA-expressing cells in the hypothalamic arcuate nucleus compared to normal saline-pretreated controls, but not in any of the other brain regions examined. Computer-assisted image analysis revealed that 3 h exposure to octreotide significantly (p < 0.01) augmented both the number and labeling density of sst2 mRNA-expressing cells in the arcuate nucleus, compared to those in saline-treated controls. By contrast, within the anterior pituitary gland, in vivo exposure to octreotide did not affect the expression of sst2 mRNA. No changes in sst1 mRNA-expressing cells were observed after octreotide treatment in any of the regions measured, indicating that the observed effects were homologous, i.e. specific of the receptor subtype stimulated. Octreotide pretreatment was also without effect on the density of 125I-[DTrp8]-SRIH binding in either the arcuate nucleus or pituitary. These results demonstrate, for the first time, that SRIH preexposure in vivo upregulates the expression of a subtype of its own receptors, sst2, within the central nervous system. They further suggest that pretreatment with SRIH in vivo does not cause sst2 receptor desensitization in arcuate nucleus and pituitary. Such homologous regulatory mechanisms may play an important role in the neuroendocrine control of growth hormone (GH) secretion by the arcuate nucleus.

Aging of the rat pituitary is often accompanied by the occurrence of adenomas. We asked whether complementary DNA hybridization array was adapted to identify gene expression patterns linked to aging and associated spontaneous adenomas. Thus, [32P]dATP-labeled cDNAs were prepared from pituitaries of three month-old rats (Y) and tumor-bearing 20-28-month-old rats (OT). The cDNAs were hybridized to identical membrane arrays allowing to study simultaneously 588 known genes (Clontech 7738-1). Among the 79 genes detected, the GH gene was predominantly expressed in both groups. Twenty-eight genes in the OT group and 15 in the Y group were found to be expressed at a higher level. The largest differences were of about 17 fold and were observed for the galanin and glutathione S transferase genes in the Y and OT groups, respectively. Relative RT-PCR was applied to validate the OT versus Y expression pattern obtained via cDNA array hybridization. The results were consistent for 14 out the 15 genes tested. In the light of these results, differential membrane array hybridization appears suitable to identify gene expression profiles associated with pituitary aging.

Somatostatin was originally characterised as a hypothalamic neurohormone responsible for the inhibition of pituitary Growth Hormone secretion. In mammals two genes encode for somatostatin-related peptides, somatostatin 14 and 28, and cortistatins, respectively. All peptides bind with similar affinities to the five cloned somatostatin receptors (sst), which belong to the GPCR family. Despite numerous studies, no clear behavioural function has yet been attributed to somatostatin-related peptides. This is due to the lack of good pharmacological tools (selective antagonists) and animal models. This review will focus on the recent development of such tools.

Gene trapping was used in embryonic stem (ES) cells in an attempt to inactivate genes involved in development. The Emk (ELKL motif kinase) gene has been disrupted and a mutant mouse line derived. Previous work had shown that EMK kinases, called MARK in the rat, exert a major control on microtubule stability by phosphorylating microtubule-associated proteins and that genes homologous to Emk in yeast or Caenorhabditis elegans are essential for cell and embryonic polarity. Although we found the Emk gene to be active in the preimplantation mouse embryo and then to show a widespread expression, Emk-null mice had no embryonic defect and were viable. They show an overall proportionate dwarfism and a peculiar hypofertility: homozygotes are not fertile when intercrossed, but are fertile in other types of crosses. Insulin-like growth factor I (IGF I) and IGF-binding protein 3 (IGFBP3) were reduced in the plasma of homozygotes of both sexes. A direct implication of the EMK kinase in IGF I plasmatic production is unlikely because the Emk gene does not seem to be expressed in hepatocytes. Nevertheless, GH assayed at arbitrary times in plasma did not show differences between genotypes and GH concentrations in pituitary extracts were not found to be altered in homozygotes. Our results, though, do not exclude the possibility that in the mutants the overall quantity of GH secreted daily is reduced. Our observation of a smaller size of the pituitaries of the mutants is in favor of this hypothesis. The prolactin concentration in the pituitaries was much lowered in homozygous females, but it was normal in males. The possible involvement of EMK protein kinase in hormone secretion in the pituitary and/or the hypothalamus, via the microtubule network, is discussed.

Somatostatin (somatotropin release-inhibiting factor, SRIF) interacts with G-protein coupled receptors (sst) designated sst1 through sst5. In PC12 and GC cells, SRIF binding sites were identified and mRNA receptor expression was evaluated. SRIF binding sites were expressed at a much lower density in PC12 (Kd = 21.2 +/- 3.9 nM; Bmax = 31 +/- 8 fmol/mg protein) than in GC cells (Kd = 6.4 +/- 1.6 nM; Bmax = 643 +/- 29 fmol/mg protein). sst3 receptor mRNA (81% of the total) was mainly expressed in PC12 cells, while sst1/2 receptor mRNAs were mostly expressed in GC cells (64 and 29%, respectively). In PC12 cells, adenylyl cyclase (AC) activity was unaffected by SRIF-14 (binding all SRIF receptors), octreotide (specific for sst2/3/5 receptors), BIM 23056 (binding sst3/5 receptors) or CH275 (specific for sst1 receptors), 1 microM each. In GC cells, SRIF-14 or octreotide, but not the two other peptides, significantly inhibited AC activity.

In the present study, we tested whether 17beta-estradiol (E2)-induced PRL sensitivity to somatostatin-14 (SRIF) involves selective up-regulation of discrete somatostatin receptor subtypes (ssts) in primary cultures of female rat pituitary cells. The efficacy of the endogenous peptide SRIF to inhibit GH and PRL secretion and cAMP accumulation was compared with those of octreotide (OCT), BIM-23052, BIM-23056, and BIM-23268, which have been reported to be relatively selective for rat sst2, sst3, and sst5. Experiments were performed in steroid-depleted media supplemented or not with 1 nM E2 for 96 h. SRIF, OCT, and BIM-23052 inhibited cAMP accumulation and GH release independently of E2. In contrast, all three agonists affected PRL release in E2-treated cultures only. Inhibition of cAMP accumulation by SRIF, OCT, and BIM-23052 was enhanced by exposure of cells to E2. The rank of potency of the agonists, OCT = SRIFBIM-23052, was similar for GH and PRL inhibition. BIM-23268 was a weak agonist on GH, but not on PRL, secretion. BIM-23056 had no effect on the release of either hormone, but slightly inhibited cAMP formation in E2-treated cells. To verify whether SRIF receptor gene expression correlated with these observations, messenger RNA (mRNA) transcripts corresponding to the five ssts were measured by quantitative RT-PCR in the presence or absence of E2. Control cells expressed predominantly sst2 and sst3 transcripts; sst1 mRNA was present in moderate amounts, whereas sst4 and sst5 were only weakly expressed. E2 had a differential effect on distinct ssts; it increased mRNA concentrations corresponding to sst2 and sst3, and decreased that of sst1. These results indicate that sst2 and sst3 receptors are the major somatostatin receptors expressed in the female rat pituitary, and that both of them are positively regulated by estradiol. However, the capacity of lactotropes to respond to SRIF after exposure to E2 seems to depend more upon E2-induced up-regulation of the sst2 than of the sst3 receptor subtype.

The aim of this work was to compare the localization of galanin and galanin receptors in the guinea pig median eminence at the light and electron microscopic level. Concerning galanin the highest labeling was shown in the external part of the median eminence. At the ultrastructural level, galanin immunoreactivity was observed only in nerve terminals containing granular vesicles of approximately 120 nm in diameter. Light microscopic autoradiographs of semithin sections exhibited a moderate labeling in the external part of the median eminence. Galanin receptors were labeled in vitro on semithin sections (2 microm) using the highly specific radioligand [125I]galanin. Ultrastructural data showed that most of galanin binding sites overlaid membrane appositions between nerve terminals and also between nerve terminal and tanycyte. By considering the percentages in the distribution of the binding it appeared that galanin receptors were located on some nerve ending membranes. Our observations were not really in favor of a presence of receptors in tanycytes. The presence of galanin nerve endings in the external part suggests that like in the rat the peptide may have a direct hypophysiotrophic role. In contrast, the occurrence of numerous binding sites gives additional arguments in favor of a local action (paracrine and/or autocrine) of galanin occurring via galanin receptors located essentially on the pericapillary nerve terminals in the guinea pig median eminence.

Somatostatin binding sites have been localized and quantified in the sheep brain using 125I-Tyr0-DTrp8-somatostatin, by quantitative high resolution light microscopic autoradiography. Sections were analyzed by densitometry on radioautographic film, and subsequently on slides coated with photoemulsion. Specific somatostatin binding sites were concentrated in the medial habenula, superior colliculus, dorsal motor nucleus of the vagus nerve, inferior olive, spinal trigeminal nucleus, and cerebellum. In competition experiments, octreotide, a sst2/sst3/sst5 selective agonist only partially displaced 125I-Tyr0-DTrp8-somatostatin in the three cerebellar layers while it was fully active as compared to somatostatin 14 and 28 in the deeper layers of the parietal cortex. Moderate to low somatostatin receptor densities were present in the mesencephalic periaqueductal gray, dorsal raphe, thalamic paraventricular nucleus, interpeduncular nucleus, pineal gland, dorsal tegmental, dorsolateral tegmental and parabrachial nuclei, nucleus of the solitary tract. The distribution of somatostatin binding sites generally correlates with the data obtained on slides dipped in photoemulsion which provided better resolution and more precise localization. In most of the labeled areas, 125I-Tyr0-DTrp8-somatostatin receptor binding was distributed between both neuropil and perikarya. Perikarya bearing 125I-Tyr0-DTrp8-somatostatin receptors were observed in areas which did not display detectable binding sites on film such as the preoptic-anterior hypothalamic complex and arcuate nucleus and in the locus coeruleus. In conclusion, the distribution of 125I-Tyr0-DTrp8-somatostatin binding sites in sheep brain is very reminiscent of other mammals being closer to the human than to rodents.

Involvement of endogenous corticotropin releasing hormone (CRH) in the regulation of spontaneous growth hormone (GH) secretion was investigated. A CRH antagonist, α helical CRH 9–41, was intracerebroventricularly infused for 36 h at a rate of 1 μg/0·5 μl/h to freely moving, cannulated adult male rats. Serial blood samples were drawn every 20 min for the last 8 hours of α helical CRH 9–41 infusion. The treatment induced a marked increase in GH peak amplitude without affecting either trough levels or numbers of peaks. In parallel, levels of growth hormone releasing hormone (GHRH) mRNA in the arcuate nucleus, but not of somatotropin release inhibiting hormone (SRIH) mRNA in the periventricular and arcuate nuclei, were increased. These data suggest that, in addition to its action in the stress-induced inhibition of GH secretion through regulation of periventricular SRIH neurons, CRH can also act as a modulator of endogenous GH secretion through regulation of arcuate GHRH neurons. Whether the modulatory effects of CRH on GHRH neurons are direct or indirect remains to be established. Journal of Endocrinology (1997) 152, 431–436

Amyloid precursor protein (APP) is a transmembrane glycoprotein which is believed to promote neural cell adhesion, neural survival, and neuritogenesis. The present study was undertaken to determine whether APP could be detected within different types of astroglial cells present in the central nervous system (CNS) of neonatal or adult rats. The localization of this protein within glial cells was studied by using a monoclonal antibody (Mab22C11) that recognizes all APP isoforms and in addition cross-reacts with APP-like proteins. In the brain of neonatal rats, Mab22C11 immunostaining was associated with numerous elongated radial glia-like structures. In the intact brain and spinal cord of adult rats, Mab22C11 immunostaining was associated with (i) numerous neuron-like structures and (ii) glial structures immunostained for glial fibrillary acidic protein (GFAP) and/or vimentin, including tanycytes mostly located in the mediobasal hypothalamus, fibrous astrocytes located in the white matter and ependymocytes bordering the ventricles. On the other hand, all the GFAP-immunostained astrocytes located in the grey matter were Mab22C11 negative. In the lesioned brain and spinal cord of adult rats, Mab22C11 immunostaining was associated with intensely GFAP-immunostained reactive astrocytes located close to a surgical lesion, but not with those induced by Wallerian degeneration that appear at a distance from a lesion. Electron microscopic observations further indicated that in all these labeled astroglial cells, Mab22C11 immunostaining was mainly localized to the limiting plasma membrane and the membrane of intracytoplasmic cisternae and vesicles. These data indicate that Mab22C11 antibody induces strong immunostaining of specific astroglial cells of the neonatal and adult rat CNS that support axonal outgrowth, therefore suggesting that an APP-like protein associated with these cells participates in their axonal outgrowth promoting properties.

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology. ispartof: Molecular Psychiatry vol:18 issue:11 pages:1225-1234 ispartof: location:England status: published

Abnormally high GH/IGF-I levels, most often caused by adenomas arising from pituitary somatotrophs, generate deleterious effects. We recently described a targeted secretion inhibitor (SXN101742) comprising a GHRH domain and the endopeptidase domain of botulinum toxin serotype D (GHRH-light chain endopeptidase type D domain [LC/D] associated to a heavy chain translocation domain [HN]) able to down-regulate the GH/IGF-I axis. In the present study, we compared the effect of a single iv bolus of a related molecule developed for clinical studies (SXN101959, 1 mg/kg) with a sc infusion of the somatostatin analog octreotide (SMS201–995, 10 μg/kg·h) to lower GH/IGF-I activity in growing male rats. Ten days after administration of SXN101959 or initiation of the octreotide infusion, body and pituitary weights, body length, GH peaks, and IGF-I production were reduced by both treatments but to a greater extent with SXN101959. In contrast to unaltered GH gene expression and increased GH storage in pituitaries from octreotide-treated rats, the inhibition of GH secretion was associated with a collapse of both GH mRNA and protein level in pituitaries from SXN101959-treated rats, in line with a specific decrease in hypothalamic GHRH production, not observed with octreotide. SXN101959 did not induce major apoptotic events in anterior pituitary and exhibited a reversible mode of action with full recovery of somatotroph cell functionality 30 days after treatment. Octreotide infusion permanently decreased ghrelin levels, whereas SXN101959 only transiently attenuated ghrelinemia. Both treatments limited bone mass acquisition and altered specifically tissues development. In conclusion, SXN101959 exerts a powerful and reversible inhibitory action on the somatotropic axis. Specific features of SXN101959, including long duration of action coupled to a strong inhibition of pituitary GH synthesis, represent advantages when treating overproduction of GH.

To test the hypothesis of the involvement of centrally expressed rat growth hormone receptors (rGH-R) in the ultradian rhythmicity of pituitary GH secretion, adult male rats were submitted to a 60 hr intracerebroventricular infusion of an antisense (AS) oligodeoxynucleotide (ODN) complementary to the sequence of rGH-R mRNA. Eight hour (10 A.M.–6 P.M.) GH secretory profiles, obtained from freely moving male rats infused with 2.0 nmol/hr of rGH-R AS, revealed a marked increase in GH peak amplitude (150 ± 12 vs 101 ± 10 ng/ml), trough levels (16.2 ± 3.0 vs 5.4 ± 1.4 ng/ml), and number of peaks (2.9 ± 0.3 vs 1.8 ± 0.2). No change was observed in rats treated with an ODN complementary to the prolactin receptor mRNA sequence (2.0 nmol/hr). Infusion of increasing ODN concentrations resulted in a dose-dependent stimulation of GH release. In parallel, somatogenic binding sites in the choroid plexus were decreased by 40%, and levels of rGH-R mRNA were increased in the periventricular nucleus (PeV) but unchanged in the arcuate nucleus (ARC). Levels of somatostatin mRNA, in the PeV but not in the ARC, were lowered by the treatment. Levels of GH-releasing hormone mRNA in the ARC were not affected. These data suggest that GH negative feedback results from a direct effect on central GH receptors and a subsequent activation of hypophysiotropic somatostatin neurons located in the anterior periventricular hypothalamus.

Galanin is a 29-amino acid peptide widely distributed in the mammalian central nervous system. Galanin receptors in the guinea pig brain were visualized using [125I]galanin by in vitro receptor quantitative autoradiography. Scatchard analysis of [125I]galanin binding to slide-mounted sections revealed saturable binding to a single class of high affinity receptors with a KD of approximately 1 nM. Specific [125I]galanin binding sites were detected in a large number of brain areas (concentration range: from non detectable to 99.32 fmol/mg of tissular proteins). The anatomical mapping revealed high densities essentially in the telencephalon (e.g. lateral septal nuclei, amygdala, hippocampal dentate gyrus) and the diencephalon (e.g. the anterodorsal and medial habenular thalamic nuclei, the paraventricular, dorsomedian and median mammillary hypothalamic nuclei, the posterior lobe of the pituitary). Addition of Mg2+ and GTP increased binding in some areas such as the zona incerta, the median eminence and the arcuate nucleus, and decreased it in other areas such as the amygdala, the hippocampus and the mammillary nuclei. This regional heterogeneity in the effect of Mg2+ and GTP can be interpreted as: (1) different rates of galanin receptor occupancy by endogenous peptide; (2) a differential coupling of GTP binding proteins to galanin receptors in the brain structures; and (3) a different nature of receptors. At any rate, this study provides evidence for a specific GTP-sensitive galanin receptor in guinea pig brain with an extensive distribution suggesting various physiological implications. Comparison with studies performed in several mammals shows that the overall distribution of galanin receptors is well preserved among species. These data suggest that galanin may posses similar functional properties in the different species tested so far. Nevertheless, very distinct differences were found in some areas like the cortex, the hippocampus and the pituitary.