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.

Developmental genes controlling neural circuit formation are expressed in the early postnatal hypothalamus and cellular lining of the third ventricle.

The arcuate nucleus of the hypothalamus is central in the regulation of body weight homeostasis through its ability to sense peripheral metabolic signals and relay them, through neural circuits, to other brain areas, ultimately affecting physiological and behavioural changes. The early postnatal development of these neural circuits is critical for normal body weight homeostasis, such that perturbations during this critical period can lead to obesity. The role for peripheral regulators of body weight homeostasis, including leptin, insulin and ghrelin, in this postnatal development is well described, yet some of the fundamental processes underpinning axonal and dendritic growth remain unclear. Here, we hypothesised that molecules known to regulate axonal and dendritic growth processes in other areas of the developing brain would be expressed in the postnatal arcuate nucleus and/or target nuclei where they would function to mediate the development of this circuitry. Using state-of-the-art RNAscope® technology, we have revealed the expression patterns of genes encoding Dcc/Netrin-1, Robo1/Slit1 and Fzd5/Wnt5a receptor/ligand pairs in the early postnatal mouse hypothalamus. We found that individual genes had unique expression patterns across developmental time in the arcuate nucleus, paraventricular nucleus of the hypothalamus, ventromedial nucleus of the hypothalamus, dorsomedial nucleus of the hypothalamus, median eminence and, somewhat unexpectedly, the third ventricle epithelium. These observations indicate a number of new molecular players in the development of neural circuits regulating body weight homeostasis, as well as novel molecular markers of tanycyte heterogeneity.

Third Ventricular Injection of CCL2 in Rat Embryo Stimulates CCL2/CCR2 Neuroimmune System in Neuroepithelial Radial Glia Progenitor Cells: Relation to Sexually Dimorphic, Stimulatory Effects on Peptide Neurons in Lateral Hypothalamus.

Clinical and animal studies show maternal alcohol consumption during pregnancy causes in offspring persistent alterations in neuroimmune and neurochemical systems known to increase alcohol drinking and related behaviors. Studies in lateral hypothalamus (LH) demonstrate in adolescent offspring that maternal oral administration of ethanol stimulates the neuropeptide, melanin-concentrating hormone (MCH), together with the inflammatory chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 which are increased in most MCH neurons. These effects, consistently stronger in females than males, are detected in embryos, not only in LH but hypothalamic neuroepithelium (NEP) along the third ventricle where neurons are born and CCL2 is stimulated within radial glia progenitor cells and their laterally projecting processes that facilitate MCH neuronal migration toward LH. With ethanol's effects similarly produced by maternal peripheral CCL2 administration and blocked by CCR2 antagonist, we tested here using in utero intracerebroventricular (ICV) injections whether CCL2 acts locally within the embryonic NEP. After ICV injection of CCL2 (0.1 µg/µl) on embryonic day 14 (E14) when neurogenesis peaks, we observed in embryos just before birth (E19) a significant increase in endogenous CCL2 within radial glia cells and their processes in NEP. These auto-regulatory effects, evident only in female embryos, were accompanied by increased density of CCL2 and MCH neurons in LH, more strongly in females than males. These results support involvement of embryonic CCL2/CCR2 neuroimmune system in radial glia progenitor cells in mediating sexually dimorphic effects of maternal challenges such as ethanol on LH MCH neurons that colocalize CCL2 and CCR2.

Low-dose infusions of leptin into the nucleus of the solitary tract increase sensitivity to third ventricle leptin.

Previous studies suggest that weight loss occurs when leptin receptors in both the forebrain and hindbrain are activated. Experiments described here tested whether this integration is mediated through a neural connection or by leptin diffusion through the subarachanoid space. If the hypothalamus and hindbrain communicated through a neural pathway, then a very low dose of leptin infused directly into the nucleus of the solitary tract (NTS) would enhance the response to third ventricle (3V) leptin but would have no effect if infused into the fourth ventricle (4V). A 12-day infusion of 10 ng/24 h into the 4V or the NTS reduced body fat. Leptin at 5 ng/24 h into the 4V or NTS had no effect on food intake or body composition, but infusion of 5 ng of leptin/24 h into the NTS combined with a 3V injection of 0.1 µg of leptin inhibited food intake between 6 and 12 h after injection. Cumulative intake was inhibited for up to 36 h. 3V leptin had no effect on food intake of rats receiving the 4V leptin infusion. Similar results were found using infusions of 5 ng leptin/24 h and a 3V injection of 0.025 µg leptin. These data suggest that activation of leptin receptors in the NTS lowers the threshold for response to leptin in the forebrain through a neural network.

One-week exposure to a free-choice high-fat high-sugar diet does not disrupt blood-brain barrier permeability in fed or overnight fasted rats.

Objectives: The hypothalamus lies adjacent to the third ventricle and is in close proximity with the median eminence (ME), a circumventricular organ with an incomplete blood-brain barrier (BBB) which controls direct entry of nutrients into the brain. The blood-CSF barrier of the hypothalamus shows dynamic changes upon neuroendocrine events and adjusts permeability with the tight junction (TJ) complex. It has been shown that chronic exposure to a high-fat diet (HFD) affects BBB permeability. HFD also induces leptin resistance and alters neuropeptide expression in the arcuate nucleus (Arc) of the hypothalamus starting early during overnutrition. We hypothesized altered integrity of the BBB to occur after exposing rats to a free-choice high-fat high-sugar (fcHFHS) diet for 1 week. Methods: We measured diffusion of Evans blue dye over the ME and assessed expression of the TJ proteins ZO-1, claudin-5, and occludin in the tanycytic wall of the third ventricle. Furthermore, we assessed protein expression of glucose transporter 1 (GLUT-1), which is highly expressed in the Arc-ME complex and facilitates glucose transport over the BBB. Results: fcHFHS-fed rats increased caloric intake compared to control, however, there was no effect of the fcHFHS diet on permeability of the BBB, nor changes in protein expression of tight TJ proteins or GLUT-1. Fasting acutely affects the BBB and we hypothesized that exposure to the fcHFHS diet affects the BBB differently compared to chow after fasting. We did not, however, find any differences in Evans blue diffusion nor protein expression between chow- and fcHFHS-fed rats when fasted overnight. Conclusions: We conclude that short-term consumption of a fcHFHS diet does not change permeability or diffusion in the hypothalamus barrier in ad libitum fed or fasted rats.

Sustained alterations of hypothalamic tanycytes during posttraumatic hypopituitarism in male mice.

Traumatic brain injury is a leading cause of hypopituitarism, which compromises patients' recovery, quality of life, and life span. To date, there are no means other than standardized animal studies to provide insights into the mechanisms of posttraumatic hypopituitarism. We have found that GH levels were impaired after inducing a controlled cortical impact (CCI) in mice. Furthermore, GHRH stimulation enhanced GH to lower level in injured than in control or sham mice. Because many characteristics were unchanged in the pituitary glands of CCI mice, we looked for changes at the hypothalamic level. Hypertrophied astrocytes were seen both within the arcuate nucleus and the median eminence, two pivotal structures of the GH axis, spatially remote to the injury site. In the arcuate nucleus, GHRH neurons were unaltered. In the median eminence, injured mice exhibited unexpected alterations. First, the distributions of claudin-1 and zonula occludens-1 between tanycytes were disorganized, suggesting tight junction disruptions. Second, endogenous IgG was increased in the vicinity of the third ventricle, suggesting abnormal barrier properties after CCI. Third, intracerebroventricular injection of a fluorescent-dextran derivative highly stained the hypothalamic parenchyma only after CCI, demonstrating an increased permeability of the third ventricle edges. This alteration of the third ventricle might jeopardize the communication between the hypothalamus and the pituitary gland. In conclusion, the phenotype of CCI mice had similarities to the posttraumatic hypopituitarism seen in humans with intact pituitary gland and pituitary stalk. It is the first report of a pathological status in which tanycyte dysfunctions appear as a major acquired syndrome.

Blockade of the cerebral aqueduct in rats provides evidence of antagonistic leptin responses in the forebrain and hindbrain.

Previously, we reported that low-dose leptin infusions into the fourth ventricle produced a small but significant increase in body fat. These data contrast with reports that injections of higher doses of leptin into the fourth ventricle inhibit food intake and weight gain. In this study, we tested whether exogenous leptin in the fourth ventricle opposed or contributed to weight loss caused by third ventricle leptin infusion by blocking diffusion of CSF from the third to the fourth ventricle. Male Sprague-Dawley rats received third ventricle infusions of PBS or 0.3 µg leptin/24 h from miniosmotic pumps. After 4 days, rats received a 3-µl cerebral aqueduct injection of saline or of thermogelling nanoparticles (hydrogel) that solidified at body temperature. Third ventricle leptin infusion inhibited food intake and caused weight loss. Blocking the aqueduct exaggerated the effect of leptin on food intake and weight loss but had no effect on the weight of PBS-infused rats. Leptin reduced both body fat and lean body mass but did not change energy expenditure. Blocking the aqueduct decreased expenditure of rats infused with PBS or leptin. Infusion of leptin into the third ventricle increased phosphorylated STAT3 in the VMHDM of the hypothalamus and the medial NTS in the hindbrain. Blocking the aqueduct did not change hypothalamic p-STAT3 but decreased p-STAT3 in the medial NTS. These results support previous observations that low-level activation of hindbrain leptin receptors has the potential to blunt the catabolic effects of leptin in the third ventricle.

Rax regulates hypothalamic tanycyte differentiation and barrier function in mice.

The wall of the ventral third ventricle is composed of two distinct cell populations: tanycytes and ependymal cells. Tanycytes regulate many aspects of hypothalamic physiology, but little is known about the transcriptional network that regulates their development and function. We observed that the retina and anterior neural fold homeobox transcription factor (Rax) is selectively expressed in hypothalamic tanycytes, and showed a complementary pattern of expression to markers of hypothalamic ependymal cells, such as Rarres2 (retinoic acid receptor responder [tazarotene induced] 2). To determine whether Rax controls tanycyte differentiation and function, we generated Rax haploinsufficient mice and examined their cellular and molecular phenotype in adulthood. These mice appeared grossly normal, but careful examination revealed a thinning of the third ventricular wall and reduction of both tanycyte and ependymal markers. These experiments show that Rax is required for hypothalamic tanycyte and ependymal cell differentiation. Rax haploinsufficiency also resulted in the ectopic presence of ependymal cells in the α2 tanycytic zone, where few ependymal cells are normally found, suggesting that Rax is selectively required for α2 tanycyte differentiation. These changes in the ventricular wall were associated with reduced diffusion of Evans Blue tracer from the ventricle to the hypothalamic parenchyma, with no apparent repercussion on the gross anatomical or behavioral phenotype of these mice. In conclusion, we have provided evidence that Rax is required for the normal differentiation and patterning of hypothalamic tanycytes and ependymal cells, as well as for maintenance of the cerebrospinal fluid-hypothalamus barrier.

Neuronal circuits in the hypothalamus controlling gonadotrophin-releasing hormone release: the neuroanatomical projections of kisspeptin neurons.

The hypothalamus regulates the key hormonal signalling events essential for reproduction and fertility in mammals. The gonadotrophin-releasing hormone (GnRH) neurons are the principal neurons of the complex neuronal network that co-ordinates multiple internal homeostatic and external factors necessary for fertility. Kisspeptin neurons are one of the major regulators of GnRH neuronal activity, but the ways in which kisspeptin neurons, located in the arcuate nucleus (ARN) and rostral periventricular area of the third ventricle (RP3V), control GnRH neurons are poorly understood. This study focused on the use of anterograde and retrograde tracing techniques to establish the neuronal projection patterns of kisspeptin cells in the female mouse brain. Both anterograde and retrograde tracing studies highlight the complexity of the kisspeptin neuronal system and indicate that both ARN and RP3V kisspeptin neurons may participate in a variety of limbic functions. In relationship to the GnRH neuronal network, these investigations demonstrate that rostral ARN kisspeptin neurons may also project to GnRH neuronal cell bodies. However, we found no anatomical evidence to suggest that kisspeptin neurons innervate GnRH nerve terminals in the external layer of the median eminence. These studies provide a neuroanatomical framework for the further elucidation of the functions of the ARN and RP3V kisspeptin neuron populations.

Photoperiodic expression of two RALDH enzymes and the regulation of cell proliferation by retinoic acid in the rat hypothalamus.

Retinoic acid (RA) has been found to regulate hypothalamic function, but precisely where it acts is unknown. This study shows expression of retinaldehyde dehydrogenase (RALDH) enzymes in tanycytes that line the third ventricle in an area overlapping with the site of hypothalamic neural stem cells. The influence of RA was examined on the proliferation of progenitors lining the third ventricle using organotypic slice cultures. As has been shown in other regions of neurogenesis, RA was found to inhibit proliferation. Investigations of the dynamics of RALDH1 expression in the rat hypothalamus have shown that this enzyme is in tanycytes under photoperiodic control with highest levels during long versus short days. In parallel to this shift in RA synthesis, cell proliferation in the third ventricle was found to be lowest during long days when RA was highest, implying that RALDH1 synthesized RA may regulate neural stem cell proliferation. A second RA synthesizing enzyme, RALDH2 was also present in tanycytes lining the third ventricle. In contrast to RALDH1, RALDH2 showed little change with photoperiodicity, but surprisingly the protein was present in the apparent absence of mRNA transcript and it is hypothesized that the endocytic tanycytes may take this enzyme up from the cerebrospinal fluid (CSF).

Kisspeptin neurons co-express met-enkephalin and galanin in the rostral periventricular region of the female mouse hypothalamus.

It is now well established that the kisspeptin neurons of the hypothalamus play a key role in regulating the activity of gonadotropin-releasing hormone (GnRH) neurons. The population of kisspeptin neurons residing in the rostral periventricular region of the third ventricle (RP3V), encompassing the anteroventral periventricular (AVPV) and periventricular preoptic nuclei (PVpo), are implicated in the generation of the preovulatory GnRH surge mechanism and puberty onset in female rodents. The present study examined whether these kisspeptin neurons may express other neuropeptides in the adult female mouse. Initially, the distribution of galanin, neurotensin, met-enkephalin (mENK), and cholecystokinin (CCK)-immunoreactive cells was determined within the RP3V of colchicine-treated mice. Subsequent experiments, using a new kisspeptin-10 antibody raised in sheep, examined the relationship of these neuropeptides to kisspeptin neurons. No evidence was found for expression of neurotensin or CCK by RP3V kisspeptin neurons, but subpopulations of kisspeptin neurons were observed to express galanin and mENK. Dual-labeled RP3V kisspeptin/galanin cells represented 7% of all kisspeptin and 21% of all galanin neurons whereas dual-labeled kisspeptin/mENK cells represented 28-38% of kisspeptin neurons and 58-68% of the mENK population, depending on location within the AVPV or PVpo. Kisspeptin neurons in the arcuate nucleus were also found to express galanin but not mENK. These observations indicate that, like the kisspeptin population of the arcuate nucleus, kisspeptin neurons in the RP3V also co-express a range of neuropeptides. This pattern of co-expression should greatly increase the dynamic range with which kisspeptin neurons can modulate the activity of their afferent neurons.

Acute food deprivation reduces expression of diazepam-binding inhibitor, the precursor of the anorexigenic octadecaneuropeptide ODN, in mouse glial cells.

In the central nervous system of mammals, the gene encoding diazepam-binding inhibitor (DBI) is exclusively expressed in glial cells. Previous studies have shown that central administration of a DBI processing product, the octadecaneuropeptide ODN, causes a marked inhibition of food consumption in rodents. Paradoxically, however, the effect of food restriction on DBI gene expression has never been investigated. Here, we show that in mice, acute fasting dramatically reduces DBI mRNA levels in the hypothalamus and the ependyma bordering the third and lateral ventricles. I.p. injection of insulin, but not of leptin, selectively stimulated DBI expression in the lateral ventricle area. These data support the notion that glial cells, through the production of endozepines, may relay peripheral signals to neurons involved in the central regulation of energy homeostasis.

Ghrelin decreases food intake in juvenile rainbow trout (Oncorhynchus mykiss) through the central anorexigenic corticotropin-releasing factor system.

Ghrelin stimulates pituitary growth hormone (GH) release, and has a key role in the regulation of food intake and adiposity in vertebrates. To investigate the central effect of native rainbow trout ghrelin (rtghrelin) on food intake in rainbow trout, as well as its possible mode of action, four groups of fish received a single injection into the third brain ventricle (i.c.v. injection): (1) control group (physiological saline) (2) ghrelin-treated group (2.0 ng rtghrelin g bwt(-1)), (3) group given the corticotropin-releasing hormone receptor antagonist alpha-helical CRF 9-41 (ahCRF) (4.0 ng g bwt(-1)) and (4) group receiving the same dose of both ghrelin and ahCRF. Food intake was assessed 1h after treatment. In addition, the presence of the GHS-R (the ghrelin receptor) in the rainbow trout CNS was examined with Western blot. To investigate peripheral effects of ghrelin, rainbow trout received an intraperitoneal cholesterol-based implant with or without rtghrelin, and daily food intake was measured during 14 days. Weight and length were measured at the start and termination of the experiment and specific growth rates were calculated. Mesenteric fat stores, muscle and liver lipid content were analysed after the treatment period. Central ghrelin injections decreased food intake compared with controls, and treatment with ahCRF abolished the ghrelin-effect. Western blot analysis of the GHS-R revealed a single band at around 60 kDa in pituitary, hypothalamus, brain and stomach. Long-term peripheral ghrelin treatment decreased daily food intake compared with controls. This was reflected in a ghrelin-induced decrease in weight growth rate (p<0.06). There was no effect of ghrelin on plasma GH levels or tissue fat stores. The conclusion from this study is that the GHS-R is indicated in the CNS in rainbow trout and that ghrelin may act there as an anorexigenic hormone, through a CRF-mediated pathway. Elevated peripheral ghrelin levels also seem to lead to decreased feed intake in the longer term.

Anorexia in rats caused by a valine-deficient diet is not ameliorated by systemic ghrelin treatment.

Rodents exhibit aversive behavior toward a diet that lacks at least one of the essential amino acids. We sought to determine whether the particular form of anorexia caused by such diets could be ameliorated by the administration of orexigenic peptides while simultaneously analyzing the neural mechanisms underlying anorexia. Rats were fed a valine-deficient diet, which induced severe anorexia (reducing food consumption by 80%). The severe anorexia was associated with a significant decrease in the cerebrospinal fluid valine concentration and hyper-ghrelinemia. Between 6 and 12 days after initiation of the valine-deficient diet, we injected rats twice daily with valine and/or an orexigenic peptide (ghrelin, neuropeptide Y, or agouti-related protein) either i.p. or i.c.v.. We then measured dietary intake. An i.c.v. valine injection allowed earlier food intake compared with an i.p valine injection and increased the density of c-Fos-positive ependymal cells lining the third ventricle. Whereas an i.c.v. injection of ghrelin or neuropeptide Y increased consumption of the valine-deficient diet, i.p injection of ghrelin or i.c.v. injection of agouti-related protein did not. Following i.c.v. administration of either valine or ghrelin, we did not observe complete recovery of consumption of the valine-deficient diet. This may be due to the ineffectiveness of peripheral ghrelin and central agouti-related protein and/or to conditioned aversion to the valine-deficient diet. Since ghrelin is known to be involved in food anticipatory activities, whether the hyper-ghrelinemia observed in valine-deficient rats play role in foraging behavior other than food intake is the future study to be investigated.

Developmental changes in hypothalamic leptin receptor: relationship with the postnatal leptin surge and energy balance neuropeptides in the postnatal rat.

In the adult brain, leptin regulates energy homeostasis primarily via hypothalamic circuitry that affects food intake and energy expenditure. Evidence from rodent models has demonstrated that during early postnatal life, leptin is relatively ineffective in modulating these pathways, despite the high circulating levels and the presence of leptin receptors within the central nervous system. Furthermore, in recent years, a neurotrophic role for leptin in the establishment of energy balance circuits has emerged. The precise way in which leptin exerts these effects, and the site of leptin action, is unclear. To provide a detailed description of the development of energy balance systems in the postnatal rat in relation to leptin concentrations during this time, endogenous leptin levels were measured, along with gene expression of leptin receptors and energy balance neuropeptides in the medial basal hypothalamus, using in situ hybridization. Expression of leptin receptors and both orexigenic and anorexigenic neuropeptides increased in the arcuate nucleus during the early postnatal period. At postnatal day 4 (P4), we detected dense leptin receptor expression in ependymal cells of the third ventricle (3V), which showed a dramatic reduction over the first postnatal weeks, coinciding with marked morphological changes in this region. An acute leptin challenge robustly induced suppressor of cytokine signaling-3 expression in the 3V of P4 but not P14 animals, revealing a clear change in the location of leptin action over this period. These findings suggest that the neurotrophic actions of leptin may involve signaling at the 3V during a restricted period of postnatal development.

Behavioral effects of systemic transforming growth factor-alpha in Syrian hamsters.

The growth factor, transforming growth factor-alpha (TGF-alpha) is strongly expressed in the hypothalamic circadian pacemaker, the suprachiasmatic nucleus (SCN). TGF-alpha is one of several SCN peptides recently suggested to function as a circadian output signal for the regulation of locomotor activity rhythms in nocturnal rodents. When infused in the brain, TGF-alpha suppresses activity. TGF-alpha suppresses other behaviors as well including feeding, resulting in weight loss. Elevated TGF-alpha is correlated with some cancers, and it is possible the TGF-alpha and its receptor, the epidermal growth factor receptor (EGFR), mediate fatigue and weight loss associated with cancer. If true for cancers outside of the brain, then systemic TGF-alpha should also affect behavior. We tested this hypothesis in hamsters with intraperitoneal injections or week-long subcutaneous infusions of TGF-alpha. Both treatments suppressed activity and infusions caused reduced food consumption and weight loss. To identify areas of the brain that might mediate these effects of systemic TGF-alpha, we used immunohistochemistry to localize cells with an activated MAP kinase signaling pathway (phosphorylated ERK1). Cells were activated in two hypothalamic areas, the paraventricular nucleus and a narrow region surrounding the third ventricle. These sites could not only be targets of TGF-alpha produced in the SCN but could also mediate effects of elevated TGF-alpha from tumors both within and outside the central nervous system.

Hypothalamic activity during altered salt and water balance in the snake Bothrops jararaca.

The effects of water and salt overload on the activities of the supraoptic and paraventricular nuclei and the adjacent periventricular zone of the hypothalamus of the snake Bothrops jararaca were investigated by measurements of Fos-like immunoreactivity (Fos-ir). Both water and salt overload resulted in changes in body mass, plasma osmolality, and plasma concentrations of sodium, potassium, and chloride. Hyper-osmolality increased Fos immunoreactivity in the rostral supraoptic nucleus (SON), the paraventricular nucleus (PVN), and adjacent periventricular areas. Both hyper- and hypo-osmolality increased Fos immunoreactivity in the intermediate SON, but not in other areas of the hypothalamus. Immunostaining was abundant in cerebrospinal fluid (CSF)-contacting tanycyte-like cells in the ependymal layer of the third ventricle. These data highlight some features of regional distribution of Fos immunoreactivity that are consistent with vasotocin functioning as a hormone, and support the role of hypothalamic structures in the response to disruption of salt and water balance in this snake.

New insights on Saccus vasculosus evolution: a developmental and immunohistochemical study in elasmobranchs.

The saccus vasculosus (SV) is a circumventricular organ of the hypothalamus of many jawed fishes whose functions have not yet been clarified. It is a vascularized neuroepithelium that consists of coronet cells, cerebrospinal fluid-contacting (CSF-c) neurons and supporting cells. To assess the organization, development and evolution of the SV, the expression of glial fibrillary acidic protein (GFAP) and the neuronal markers gamma-aminobutyric acid (GABA), glutamic acid decarboxylase (GAD; the GABA synthesizing enzyme), neuropeptide Y (NPY), neurophysin II (NPH), tyrosine hydroxylase (TH; the rate-limiting catecholamine-synthesizing enzyme) and serotonin (5-HT), were investigated by immunohistochemistry in developing and adult sharks. Coronet cells showed GFAP immunoreactivity from embryos at stage 31 to adults, indicating a glial nature. GABAergic CSF-c neurons were evidenced just when the primordium of the SV becomes detectable (at stage 29). Double immunolabeling revealed colocalization of NPY and GAD in these cells. Some CSF-c cells showed TH immunoreactivity in postembryonic stages. Saccofugal GABAergic fibers formed a defined SV tract from the stage 30 and scattered neurosecretory (NPH-immunoreactive) and monoaminergic (5-HT- and TH-immunoreactive) saccopetal fibers were first detected at stages 31 and 32, respectively. The early differentiation of GABAergic neurons and the presence of a conspicuous GABAergic saccofugal system are shared by elasmobranch and teleosts (trout), suggesting that GABA plays a key function in the SV circuitry. Monoaminergic structures have not been reported in the SV of bony fishes, and were probably acquired secondarily in sharks. The existence of saccopetal monoaminergic and neurosecretory fibers reveals reciprocal connections between the SV and hypothalamic structures which have not been previously detected in teleosts.

Anteroventral third ventricular N-methyl-D-aspartate receptors, but not metabotropic glutamate receptors are involved in hemorrhagic AVP secretion.

This study aimed to investigate the roles of glutamate (Glu) receptors in the anteroventral third ventricular region (AV3V), a pivotal area for water, cardiovascular and neuroendocrine regulations, in causing vasopressin (AVP) secretion and other phenomena in response to bleeding. The effects of intracerebral infusions of MK-801 [a N-methyl-D-aspartate (NMDA) receptor antagonist] or a metabotropic Glu receptor antagonist (MCPG) on plasma levels of AVP, electrolytes, osmolality and glucose, heart rate and arterial pressure following AV3V administration with NMDA or bleeding stimuli were analyzed in conscious rats. NMDA provoked prominent rises of plasma AVP, osmolality, glucose and arterial pressure, without changing plasma electrolytes or heart rate significantly. All the effects of NMDA were blocked by pre-administration of MK-801 into the same loci. Removal through a femoral arterial line of 10 ml blood per kg body weight did not affect arterial pressure or other variables significantly, although plasma AVP and angiotensin II (ANG II) tended to increase. When bleeding was repeated after 10 min (B2), arterial pressure dropped promptly, and plasma AVP, ANG II, osmolality and glucose augmented remarkably. MK-801 applied 35 min preceding B2, to loci such as the median preoptic nucleus, periventricular nucleus and medial preoptic area inhibited the response of plasma AVP significantly, without exerting any effects on other variables. When MK-801 was administered intracerebroventricularly, or when MCPG was infused into the AV3V, significant alterations did not occur in B2-evoked responses of plasma AVP nor in those of the other variables. In rats given sham bleeding after AV3V infusions of MK-801 or MCPG or intracerebroventricular applications of MK-801, all monitored variables roughly remained at stable levels throughout the experiments. We conclude that NMDA receptors in AV3V, but not metabotropic Glu receptors, may facilitate AVP secretion in hypotensive hypovolemia.

Sodium vitamin C cotransporter SVCT2 is expressed in hypothalamic glial cells.

Kinetic analysis of vitamin C uptake demonstrated that different specialized cells take up ascorbic acid through sodium-vitamin C cotransporters. Recently, two different isoforms of sodium-vitamin C cotransporters (SVCT1/SLC23A1 and SVCT2/SLC23A2) have been cloned. SVCT2 was detected mainly in choroidal plexus cells and neurons; however, there is no evidence of SVCT2 expression in glial and endothelial cells of the brain. Certain brain locations, including the hippocampus and hypothalamus, consistently show higher ascorbic acid values compared with other structures within the central nervous system. However, molecular and kinetic analysis addressing the expression of SVCT transporters in cells isolated from these specific areas of the brain had not been done. The hypothalamic glial cells, or tanycytes, are specialized ependymal cells that bridge the cerebrospinal fluid with different neurons of the region. Our hypothesis postulates that SVCT2 is expressed selectively in tanycytes, where it is involved in the uptake of the reduced form of vitamin C (ascorbic acid), thereby concentrating this vitamin in the hypothalamic area. In situ hybridization and optic and ultrastructural immunocytochemistry showed that the transporter SVCT2 is highly expressed in the apical membranes of mouse hypothalamic tanycytes. A newly developed primary culture of mouse hypothalamic tanycytes was used to confirm the expression and function of the SVCT2 isoform in these cells. The results demonstrate that tanycytes express a high-affinity transporter for vitamin C. Thus, the vitamin C uptake mechanisms present in the hypothalamic glial cells may perform a neuroprotective role concentrating vitamin C in this specific area of the brain.