Characterization of Sonic Hedgehog transcripts in the adult mouse brain: co-expression with neuronal and oligodendroglial markers.

In the adult mammalian brain, astrocytes are proposed to be the major Sonic Hedgehog (Shh)-responsive cells. However, the sources of the Shh molecule mediating activation of the pathway are still poorly characterized. The present work investigates the distribution and phenotype of cells expressing Shh mRNA in the adult mouse brain. Using single-molecule fluorescent in situ hybridization (smfISH), we report much broader expression of Shh transcripts in almost all brain regions than originally reported. We identify Shh mRNA in HuC/D+ neuronal populations, including GABAergic (glutamic acid decarboxylase 67, Gad67), cholinergic (choline acetyltransferase, ChAT), dopaminergic (tyrosine hydroxylase, TH), nitrergic (neuronal nitric oxide synthase, nNOS), and in a small population of oligodendroglial cells expressing Sox10 and Olig2 mRNA transcription factors. Further analysis of Shh mRNA in cerebral cortical and hypothalamic neurons suggests that Shh is also expressed by glutamatergic neurons. Interestingly, we did not observe substantial Desert Hedgehog and Indian Hedgehog mRNA signals, nor Shh signals in S100ß+ astrocytes and Iba1+ microglial cells. Collectively, the present work provides the most robust central map of Shh-expressing cells to date and underscores the importance of nitrergic neurons in regulating Shh availability to brain cells. Thus, our study provides a framework for future experiments aimed at better understanding of the functions of Shh signaling in the brain in normal and pathological states, and the characterization of novel regulatory mechanisms of the signaling pathway.

Dietary fatty acid composition drives neuroinflammation and impaired behavior in obesity.

Nutrient composition in obesogenic diets may influence the severity of disorders associated with obesity such as insulin-resistance and chronic inflammation. Here we hypothesized that obesogenic diets rich in fat and varying in fatty acid composition, particularly in omega 6 (ω6) to omega 3 (ω3) ratio, have various effects on energy metabolism, neuroinflammation and behavior. Mice were fed either a control diet or a high fat diet (HFD) containing either low (LO), medium (ME) or high (HI) ω6/ω3 ratio. Mice from the HFD-LO group consumed less calories and exhibited less body weight gain compared to other HFD groups. Both HFD-ME and HFD-HI impaired glucose metabolism while HFD-LO partly prevented insulin intolerance and was associated with normal leptin levels despite higher subcutaneous and perigonadal adiposity. Only HFD-HI increased anxiety and impaired spatial memory, together with increased inflammation in the hypothalamus and hippocampus. Our results show that impaired glucose metabolism and neuroinflammation are uncoupled, and support that diets with a high ω6/ω3 ratio are associated with neuroinflammation and the behavioral deterioration coupled with the consumption of diets rich in fat.

Long-COVID cognitive impairments and reproductive hormone deficits in men may stem from GnRH neuronal death.

BACKGROUND: We have recently demonstrated a causal link between loss of gonadotropin-releasing hormone (GnRH), the master molecule regulating reproduction, and cognitive deficits during pathological aging, including Down syndrome and Alzheimer's disease. Olfactory and cognitive alterations, which persist in some COVID-19 patients, and long-term hypotestosteronaemia in SARS-CoV-2-infected men are also reminiscent of the consequences of deficient GnRH, suggesting that GnRH system neuroinvasion could underlie certain post-COVID symptoms and thus lead to accelerated or exacerbated cognitive decline. METHODS: We explored the hormonal profile of COVID-19 patients and targets of SARS-CoV-2 infection in post-mortem patient brains and human fetal tissue. FINDINGS: We found that persistent hypotestosteronaemia in some men could indeed be of hypothalamic origin, favouring post-COVID cognitive or neurological symptoms, and that changes in testosterone levels and body weight over time were inversely correlated. Infection of olfactory sensory neurons and multifunctional hypothalamic glia called tanycytes highlighted at least two viable neuroinvasion routes. Furthermore, GnRH neurons themselves were dying in all patient brains studied, dramatically reducing GnRH expression. Human fetal olfactory and vomeronasal epithelia, from which GnRH neurons arise, and fetal GnRH neurons also appeared susceptible to infection. INTERPRETATION: Putative GnRH neuron and tanycyte dysfunction following SARS-CoV-2 neuroinvasion could be responsible for serious reproductive, metabolic, and mental health consequences in long-COVID and lead to an increased risk of neurodevelopmental and neurodegenerative pathologies over time in all age groups. FUNDING: European Research Council (ERC) grant agreements No 810331, No 725149, No 804236, the European Union Horizon 2020 research and innovation program No 847941, the Fondation pour la Recherche Médicale (FRM) and the Agence Nationale de la Recherche en Santé (ANRS) No ECTZ200878 Long Covid 2021 ANRS0167 SIGNAL, Agence Nationale de la recherche (ANR) grant agreements No ANR-19-CE16-0021-02, No ANR-11-LABEX-0009, No. ANR-10-LABEX-0046, No. ANR-16-IDEX-0004, Inserm Cross-Cutting Scientific Program HuDeCA, the CHU Lille Bonus H, the UK Medical Research Council (MRC) and National Institute of Health and care Research (NIHR).

Cell proliferation and glial cell marker expression in the wall of the third ventricle in the tuberal region of the male mouse hypothalamus during postnatal development.

The third ventricle (3 V) wall of the tuberal hypothalamus is composed of two types of cells; specialized ependymoglial cells called tanycytes located ventrally and ependymocytes dorsally, which control the exchanges between the cerebrospinal fluid and the hypothalamic parenchyma. By regulating the dialogue between the brain and the periphery, tanycytes are now recognized as central players in the control of major hypothalamic functions such as energy metabolism and reproduction. While our knowledge of the biology of adult tanycytes is progressing rapidly, our understanding of their development remains very incomplete. To gain insight into the postnatal maturation of the 3 V ependymal lining, we conducted a comprehensive immunofluorescent study of the mouse tuberal region at four postnatal ages (postnatal day (P) 0, P4, P10, and P20). We analyzed the expression profile of a panel of tanycyte and ependymocyte markers (vimentin, S100, connexin-43 [Cx43], and glial fibrillary acidic protein [GFAP]) and characterized cell proliferation in the 3 V wall using the thymidine analog bromodeoxyuridine. Our results show that most changes in marker expression occur between P4 and P10, with a switch from a 3 V mostly lined by radial cells to the emergence of a tanycytic domain ventrally and an ependymocytic domain dorsally, a drop in cell proliferation and increased expression of S100, Cx43, and GFAP that acquire a mature profile at P20. Our study thus identifies the transition between the first and the second postnatal week as a critical time window for the postnatal maturation of the 3 V wall ependymal lining.

Selective Depletion of Adult GFAP-Expressing Tanycytes Leads to Hypogonadotropic Hypogonadism in Males.

In adult mammals, neural stem cells are localized in three neurogenic regions, the subventricular zone of the lateral ventricle (SVZ), the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the hypothalamus. In the SVZ and the SGZ, neural stem/progenitor cells (NSPCs) express the glial fibrillary acidic protein (GFAP) and selective depletion of these NSPCs drastically decreases cell proliferation in vitro and in vivo. In the hypothalamus, GFAP is expressed by α-tanycytes, which are specialized radial glia-like cells in the wall of the third ventricle also recognized as NSPCs. To explore the role of these hypothalamic GFAP-positive tanycytes, we used transgenic mice expressing herpes simplex virus thymidine kinase (HSV-Tk) under the control of the mouse Gfap promoter and a 4-week intracerebroventricular infusion of the antiviral agent ganciclovir (GCV) which kills dividing cells expressing Tk. While GCV significantly reduced the number and growth of hypothalamus-derived neurospheres from adult transgenic mice in vitro, it causes hypogonadotropic hypogonadism in vivo. The selective death of dividing tanycytes expressing GFAP indeed results in a marked decrease in testosterone levels and testicular weight, as well as vacuolization of the seminiferous tubules and loss of spermatogenesis. Additionally, GCV-treated GFAP-Tk mice show impaired sexual behavior, but no alteration in food intake or body weight. Our results also show that the selective depletion of GFAP-expressing tanycytes leads to a sharp decrease in the number of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons and a blunted LH secretion. Overall, our data show that GFAP-expressing tanycytes play a central role in the regulation of male reproductive function.

The polygamous GnRH neuron: Astrocytic and tanycytic communication with a neuroendocrine neuronal population.

To ensure the survival of the species, hypothalamic neuroendocrine circuits controlling fertility, which converge onto neurons producing gonadotropin-releasing hormone (GnRH), must respond to fluctuating physiological conditions by undergoing rapid and reversible structural and functional changes. However, GnRH neurons do not act alone, but through reciprocal interactions with multiple hypothalamic cell populations, including several glial and endothelial cell types. For instance, it has long been known that in the hypothalamic median eminence, where GnRH axons terminate and release their neurohormone into the pituitary portal blood circulation, morphological plasticity displayed by distal processes of tanycytes modifies their relationship with adjacent neurons as well as the spatial properties of the neurohemal junction. These alterations not only regulate the capacity of GnRH neurons to release their neurohormone, but also the activation of discrete non-neuronal pathways that mediate feedback by peripheral hormones onto the hypothalamus. Additionally, a recent breakthrough has demonstrated that GnRH neurons themselves orchestrate the establishment of their neuroendocrine circuitry during postnatal development by recruiting an entourage of newborn astrocytes that escort them into adulthood and, via signalling through gliotransmitters such as prostaglandin E2, modulate their activity and GnRH release. Intriguingly, several environmental and behavioural toxins perturb these neuron-glia interactions and consequently, reproductive maturation and fertility. Deciphering the communication between GnRH neurons and other neural cell types constituting hypothalamic neuroendocrine circuits is thus critical both to understanding physiological processes such as puberty, oestrous cyclicity and aging, and to developing novel therapeutic strategies for dysfunctions of these processes, including the effects of endocrine disruptors.

GnRH neurons recruit astrocytes in infancy to facilitate network integration and sexual maturation.

Neurons that produce gonadotropin-releasing hormone (GnRH), which control fertility, complete their nose-to-brain migration by birth. However, their function depends on integration within a complex neuroglial network during postnatal development. Here, we show that rodent GnRH neurons use a prostaglandin D2 receptor DP1 signaling mechanism during infancy to recruit newborn astrocytes that 'escort' them into adulthood, and that the impairment of postnatal hypothalamic gliogenesis markedly alters sexual maturation by preventing this recruitment, a process mimicked by the endocrine disruptor bisphenol A. Inhibition of DP1 signaling in the infantile preoptic region, where GnRH cell bodies reside, disrupts the correct wiring and firing of GnRH neurons, alters minipuberty or the first activation of the hypothalamic-pituitary-gonadal axis during infancy, and delays the timely acquisition of reproductive capacity. These findings uncover a previously unknown neuron-to-neural-progenitor communication pathway and demonstrate that postnatal astrogenesis is a basic component of a complex set of mechanisms used by the neuroendocrine brain to control sexual maturation.

Neurogenesis in the adult hypothalamus: A distinct form of structural plasticity involved in metabolic and circadian regulation, with potential relevance for human pathophysiology.

The adult brain harbors specific niches where stem cells undergo substantial plasticity and, in some regions, generate new neurons throughout life. This phenomenon is well known in the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampus and has recently also been described in the hypothalamus of several rodent and primate species. After a brief overview of preclinical studies illustrating the pathophysiologic significance of hypothalamic neurogenesis in the control of energy metabolism, reproduction, thermoregulation, sleep, and aging, we review current literature on the neurogenic niche of the human hypothalamus. A comparison of the organization of the niche between humans and rodents highlights some common features, but also substantial differences, e.g., in the distribution and extent of the hypothalamic neural stem cells. Exploring the full dynamics of hypothalamic neurogenesis in humans raises a formidable challenge however, given among others, inherent technical limitations. We close with discussing possible functional role(s) of the human hypothalamic niche, and how gaining more insights into this form of plasticity could be relevant for a better understanding of pathologies associated with disturbed hypothalamic function.

Sonic Hedgehog receptor Patched deficiency in astrocytes enhances glucose metabolism in mice.

OBJECTIVE: Astrocytes are glial cells proposed as the main Sonic hedgehog (Shh)-responsive cells in the adult brain. Their roles in mediating Shh functions are still poorly understood. In the hypothalamus, astrocytes support neuronal circuits implicated in the regulation of energy metabolism. In this study, we investigated the impact of genetic activation of Shh signaling on hypothalamic astrocytes and characterized its effects on energy metabolism. METHODS: We analyzed the distribution of gene transcripts of the Shh pathway (Ptc, Gli1, Gli2, and Gli3) in astrocytes using single molecule fluorescence in situ hybridization combined with immunohistofluorescence of Shh peptides by Western blotting in the adult mouse hypothalamus. Based on the metabolic phenotype, we characterized Glast-CreERT2-YFP-Ptc-/- (YFP-Ptc-/-) mice and their controls over time and under a high-fat diet (HFD) to investigate the potential effects of conditional astrocytic deletion of the Shh receptor Patched (Ptc) on metabolic efficiency, insulin sensitivity, and systemic glucose metabolism. Molecular and biochemical assays were used to analyze the alteration of key pathways modulating energy metabolism, insulin sensitivity, glucose uptake, and inflammation. Primary astrocyte cultures were used to evaluate a potential role of Shh signaling in astrocytic glucose uptake. RESULTS: Shh peptides were the highest in the hypothalamic extracts of adult mice and a large population of hypothalamic astrocytes expressed Ptc and Gli1-3 mRNAs. Characterization of Shh signaling after conditional Ptc deletion in the YFP-Ptc-/- mice revealed heterogeneity in hypothalamic astrocyte populations. Interestingly, activation of Shh signaling in Glast+ astrocytes enhanced insulin responsiveness as evidenced by glucose and insulin tolerance tests. This effect was maintained over time and associated with lower blood insulin levels and also observed under a HFD. The YFP-Ptc-/- mice exhibited a lean phenotype with the absence of body weight gain and a marked reduction of white and brown adipose tissues accompanied by increased whole-body fatty acid oxidation. In contrast, food intake, locomotor activity, and body temperature were not altered. At the cellular level, Ptc deletion did not affect glucose uptake in primary astrocyte cultures. In the hypothalamus, activation of the astrocytic Shh pathway was associated with the upregulation of transcripts coding for the insulin receptor and liver kinase B1 (LKB1) after 4 weeks and the glucose transporter GLUT-4 after 32 weeks. CONCLUSIONS: Here, we define hypothalamic Shh action on astrocytes as a novel master regulator of energy metabolism. In the hypothalamus, astrocytic Shh signaling could be critically involved in preventing both aging- and obesity-related metabolic disorders.

C9C5 positive mature oligodendrocytes are a source of Sonic Hedgehog in the mouse brain.

In the mature rodent brain, Sonic Hedgehog (Shh) signaling regulates stem and progenitor cell maintenance, neuronal and glial circuitry and brain repair. However, the sources and distribution of Shh mediating these effects are still poorly characterized. Here, we report in the adult mouse brain, a broad expression pattern of Shh recognized by the specific monoclonal C9C5 antibody in a subset (11-12%) of CC1+ mature oligodendrocytes that do not express carbonic anhydrase II. These cells express also Olig2 and Sox10, two oligodendrocyte lineage-specific markers, but not PDGFRα, a marker of oligodendrocyte progenitors. In agreement with oligodendroglial cells being a source of Shh in the adult mouse brain, we identify Shh transcripts by single molecule fluorescent in situ hybridization in a subset of cells expressing Olig2 and Sox10 mRNAs. These findings also reveal that Shh expression is more extensive than originally reported. The Shh-C9C5-associated signal labels the oligodendroglial cell body and decorates by intense puncta the processes. C9C5+ cells are distributed in a grid-like manner. They constitute small units that could deliver locally Shh to its receptor Patched expressed in GFAP+ and S100ß+ astrocytes, and in HuC/D+ neurons as shown in PtcLacZ/+ reporter mice. Postnatally, C9C5 immunoreactivity overlaps the myelination peak that occurs between P10 and P20 and is down regulated during ageing. Thus, our data suggest that C9C5+CC1+ oligodendroglial cells are a source of Shh in the mouse postnatal brain.

Non-secreting pituitary tumours characterised by enhanced expression of YAP/TAZ.

Tumours of the anterior pituitary can manifest from all endocrine cell types but the mechanisms for determining their specification are not known. The Hippo kinase cascade is a crucial signalling pathway regulating growth and cell fate in numerous organs. There is mounting evidence implicating this in tumour formation, where it is emerging as an anti-cancer target. We previously demonstrated activity of the Hippo kinase cascade in the mouse pituitary and nuclear association of its effectors YAP/TAZ with SOX2-expressing pituitary stem cells. Here, we sought to investigate whether these components are expressed in the human pituitary and if they are deregulated in human pituitary tumours. Analysis of pathway components by immunofluorescence reveals pathway activity during normal human pituitary development and in the adult gland. Poorly differentiated pituitary tumours (null-cell adenomas, adamantinomatous craniopharyngiomas (ACPs) and papillary craniopharyngiomas (PCPs)), displayed enhanced expression of pathway effectors YAP/TAZ. In contrast, differentiated adenomas displayed lower or absent levels. Knockdown of the kinase-encoding Lats1 in GH3 rat mammosomatotropinoma cells suppressed Prl and Gh promoter activity following an increase in YAP/TAZ levels. In conclusion, we have demonstrated activity of the Hippo kinase cascade in the human pituitary and association of high YAP/TAZ with repression of the differentiated state both in vitro and in vivo. Characterisation of this pathway in pituitary tumours is of potential prognostic value, opening up putative avenues for treatments.

A recurrent point mutation in PRKCA is a hallmark of chordoid gliomas.

Chordoid glioma (ChG) is a characteristic, slow growing, and well-circumscribed diencephalic tumor, whose mutational landscape is unknown. Here we report the analysis of 16 ChG by whole-exome and RNA-sequencing. We found that 15 ChG harbor the same PRKCA D463H mutation. PRKCA encodes the Protein kinase C (PKC) isozyme alpha (PKCα) and is mutated in a wide range of human cancers. However the hot spot PRKCA D463H mutation was not described in other tumors. PRKCA D463H is strongly associated with the activation of protein translation initiation (EIF2) pathway. PKCαD463H mRNA levels are more abundant than wild-type PKCα transcripts, while PKCαD463H is less stable than the PCKαWT protein. Compared to PCKαWT, the PKCαD463H protein is depleted from the cell membrane. The PKCαD463H mutant enhances proliferation of astrocytes and tanycytes, the cells of origin of ChG. In conclusion, our study identifies the hallmark mutation for chordoid gliomas and provides mechanistic insights on ChG oncogenesis.

The Versatile Tanycyte: A Hypothalamic Integrator of Reproduction and Energy Metabolism.

The fertility and survival of an individual rely on the ability of the periphery to promptly, effectively, and reproducibly communicate with brain neural networks that control reproduction, food intake, and energy homeostasis. Tanycytes, a specialized glial cell type lining the wall of the third ventricle in the median eminence of the hypothalamus, appear to act as the linchpin of these processes by dynamically controlling the secretion of neuropeptides into the portal vasculature by hypothalamic neurons and regulating blood-brain and blood-cerebrospinal fluid exchanges, both processes that depend on the ability of these cells to adapt their morphology to the physiological state of the individual. In addition to their barrier properties, tanycytes possess the ability to sense blood glucose levels, and play a fundamental and active role in shuttling circulating metabolic signals to hypothalamic neurons that control food intake. Moreover, accumulating data suggest that, in keeping with their putative descent from radial glial cells, tanycytes are endowed with neural stem cell properties and may respond to dietary or reproductive cues by modulating hypothalamic neurogenesis. Tanycytes could thus constitute the missing link in the loop connecting behavior, hormonal changes, signal transduction, central neuronal activation and, finally, behavior again. In this article, we will examine these recent advances in the understanding of tanycytic plasticity and function in the hypothalamus and the underlying molecular mechanisms. We will also discuss the putative involvement and therapeutic potential of hypothalamic tanycytes in metabolic and fertility disorders.

A comparative study of the neural stem cell niche in the adult hypothalamus of human, mouse, rat and gray mouse lemur (Microcebus murinus).

The adult brain contains niches of neural stem cells that continuously add new neurons to selected circuits throughout life. Two niches have been extensively studied in various mammalian species including humans, the subventricular zone of the lateral ventricles and the subgranular zone of the hippocampal dentate gyrus. Recently, studies conducted mainly in rodents have identified a third neurogenic niche in the adult hypothalamus. In order to evaluate whether a neural stem cell niche also exists in the adult hypothalamus in humans, we performed multiple immunofluorescence labeling to assess the expression of a panel of neural stem/progenitor cell (NPC) markers (Sox2, nestin, vimentin, GLAST, GFAP) in the human hypothalamus and compared them with the mouse, rat and a non-human primate species, the gray mouse lemur (Microcebus murinus). Our results show that the adult human hypothalamus contains four distinct populations of cells that express the five NPC markers: (a) a ribbon of small stellate cells that lines the third ventricular wall behind a hypocellular gap, similar to that found along the lateral ventricles, (b) ependymal cells, (c) tanycytes, which line the floor of the third ventricle in the tuberal region, and (d) a population of small stellate cells in the suprachiasmatic nucleus. In the mouse, rat and mouse lemur hypothalamus, co-expression of NPC markers is primarily restricted to tanycytes, and these species lack a ventricular ribbon. Our work thus identifies four cell populations with the antigenic profile of NPCs in the adult human hypothalamus, of which three appear specific to humans.

When Size Matters: How Astrocytic Processes Shape Metabolism.

The hypothalamic control of metabolism appears to be a puzzle that cannot be explained by neuronal function alone. Zhang and colleagues (2017) add a few new pieces by demonstrating that astrocytes critically modulate neural circuits controlling energy homeostasis through nutritional-status-dependent morphological plasticity and IKKß/NF-κB signaling, which modulate extracellular neurotransmitter bioavailability.

A driver role for GABA metabolism in controlling stem and proliferative cell state through GHB production in glioma.

Cell populations with differing proliferative, stem-like and tumorigenic states co-exist in most tumors and especially malignant gliomas. Whether metabolic variations can drive this heterogeneity by controlling dynamic changes in cell states is unknown. Metabolite profiling of human adult glioblastoma stem-like cells upon loss of their tumorigenicity revealed a switch in the catabolism of the GABA neurotransmitter toward enhanced production and secretion of its by-product GHB (4-hydroxybutyrate). This switch was driven by succinic semialdehyde dehydrogenase (SSADH) downregulation. Enhancing GHB levels via SSADH downregulation or GHB supplementation triggered cell conversion into a less aggressive phenotypic state. GHB affected adult glioblastoma cells with varying molecular profiles, along with cells from pediatric pontine gliomas. In all cell types, GHB acted by inhibiting α-ketoglutarate-dependent Ten-eleven Translocations (TET) activity, resulting in decreased levels of the 5-hydroxymethylcytosine epigenetic mark. In patients, low SSADH expression was correlated with high GHB/α-ketoglutarate ratios, and distinguished weakly proliferative/differentiated glioblastoma territories from proliferative/non-differentiated territories. Our findings support an active participation of metabolic variations in the genesis of tumor heterogeneity.

[The stem cell niche in glioblastoma: from fundamental aspects to targeted therapies]. / La niche des cellules souches tumorales dans le glioblastome : des aspects fondamentaux au ciblage thérapeutique.

The concept of cancer stem cell (CSC) was established from models of leukemogenesis explaining tumor repopulation by the clonogenic properties of this specific population of tumoral cells. Among solid tumors, glioblastoma are currently the most documented models. Cancer stem cells reside in specific locations within tumors called niches. Anatomically, two complementary niches have been described in glioblastoma. The first one is a perivascular niche composed of vessels (endothelial cells, pericytes) and their microenvironment (integrins, interleukins) constitutive the nest of "normal" neural stem cells and cancer stem cells. The second one is a hypoxic niche found in regions with low oxygen tension such as the core of the tumor. In these niches, mutual interactions between CSC and their microenvironment involving the activation of multiple signaling pathways promote stemness maintenance and tumor propagation. The median overall survival of glioblastoma does not exceed 15 months despite an aggressive multimodal treatment, thus the therapeutic targeting of these niches, by systemic agents or radiotherapy, in order to inhibit the signaling pathways involved in the maintenance of the CSC niches, represents a major challenge. The combination of these two strategies appears promising and many clinical trials are underway.

Role of glia in the regulation of gonadotropin-releasing hormone neuronal activity and secretion.

Gonadotropin-releasing hormone (GnRH) neurons are the final common pathway for the central control of reproduction. The coordinated and timely activation of these hypothalamic neurons, which determines sexual development and adult reproductive function, lies under the tight control of a complex array of excitatory and inhibitory transsynaptic inputs. In addition, research conducted over the past 20 years has unveiled the major contribution of glial cells to the control of GnRH neurons. Glia use a variety of molecular and cellular strategies to modulate GnRH neuronal function both at the level of their cell bodies and at their nerve terminals. These mechanisms include the secretion of bioactive molecules that exert paracrine effects on GnRH neurons, juxtacrine interactions between glial cells and GnRH neurons via adhesive molecules and the morphological plasticity of the glial coverage of GnRH neurons. It now appears that glial cells are integral components, along with upstream neuronal networks, of the central control of GnRH neuronal function. This review attempts to summarize our current knowledge of the mechanisms used by glial cells to control GnRH neuronal activity and secretion.

Isolation and culture of human astrocytes.

Although rodent models have been essential to unveil the emerging functions of astrocytes, the existence of interspecies differences calls for caution in extrapolating data from rodent to human astrocytes. We have developed highly enriched primary astrocyte cultures from human fetuses and adult cerebro-cortical biopsies from neurosurgery patients. Immunocytochemical characterization shows that cultures are composed of more than 95% of cells expressing in vitro astrocytic markers. Examination of the morphological and proliferative properties of cultures derived from the cerebral cortex and the hypothalamus both in untreated conditions and after treatment with EGF-related ligands illustrates the high plasticity of human astrocytes and their functional heterogeneity according to the cerebral region of origin. Our preparation offers the opportunity to characterize human astrocyte functions in vitro and also provides a valuable tool for studying the functional heterogeneity of human astrocytes isolated from distinct brain regions.