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.

Role of estradiol in the dynamic control of tanycyte plasticity mediated by vascular endothelial cells in the median eminence.

In the ever-changing physiological context of the neuroendocrine brain, the mechanisms by which cellular events involving neurons, astroglia, and vascular cells are coordinated to bring forth the appropriate neuronal signaling is not yet known but is amenable to examination. In the median eminence of the hypothalamus, endothelial cells are key players in the plasticity of tanycytes (specialized astroglia) and neuroendocrine synapse efficacy. Here we report that estradiol acts on both purified endothelial cells and isolated tanycytes to trigger endothelial-to-glial communication that leads to a sudden and massive retraction of tanycyte processes. The blockade of endothelial nitric oxide synthase by in vitro adenoviral-mediated gene transfer of a dominant-negative form of endothelial nitric oxide synthase abrogates the estradiol-induced tanycyte plasticity mediated by endothelial cells. In parallel, increases in prostaglandin-E(2) (PGE(2)) due to changes in cyclooxygenase (COX)-1 and COX-2 expression induced by the exposure of tanycytes to estradiol promote acute tanycyte plasticity. We also demonstrate by electron microscopy that the administration of PGE(2) to median eminence explants induces rapid neuroglial plasticity at the neurovascular junction of neurons that release GnRH (the neuropeptide controlling reproduction). Conversely, preventing local PGE(2) synthesis in the median eminence of adult female rats with the COX inhibitor indomethacin impairs the ovarian cycle, a process that requires a pulsatile, coordinated delivery of GnRH into the hypothalamo-hypophyseal portal system. Taken together, our findings show that estradiol controls the dialog between endothelial cells and astroglia to regulate neuroglial plasticity in the neuroendocrine brain.

Coupling of neuronal nitric oxide synthase to NMDA receptors via postsynaptic density-95 depends on estrogen and contributes to the central control of adult female reproduction.

Considerable research has been devoted to the understanding of how nitric oxide (NO) influences brain function. Few studies, however, have addressed how its production is physiologically regulated. Here, we report that protein-protein interactions between neuronal NO synthase (nNOS) and glutamate NMDA receptors via the scaffolding protein postsynaptic density-95 (PSD-95) in the hypothalamic preoptic region of adult female rats is sensitive to cyclic estrogen fluctuation. Coimmunoprecipitation experiments were used to assess the physical association between nNOS and NMDA receptor NR2B subunit in the preoptic region of the hypothalamus. We found that nNOS strongly interacts with NR2B at the onset of the preovulatory surge at proestrus (when estrogen levels are highest) compared with basal-stage diestrous rats. Consistently, estrogen treatment of gonadectomized female rats also increases nNOS/NR2B complex formation. Moreover, endogenous fluctuations in estrogen levels during the estrous cycle coincide with changes in the physical association of nNOS to PSD-95 and the magnitude of NO release in the preoptic region. Finally, temporary and local in vivo suppression of PSD-95 synthesis by using antisense oligodeoxynucleotides leads to inhibition of nNOS activity in the preoptic region and disrupted estrous cyclicity, a process requiring coordinated activation of neurons containing gonadotropin-releasing hormone (the neuropeptide controlling reproductive function). In conclusion, our findings identify a novel steroid-mediated molecular mechanism that enables the adult mammalian brain to control NO release under physiological conditions.

Progesterone receptor and dopamine synthesizing enzymes in hypothalamic neurons of the guinea pig: an immunohistochemical triple-label analysis.

Interactions among gonadal steroid hormones and the dopamine synthesizing enzymes, tyrosine hydroxylase (TH) or aromatic L-amino acid decarboxylase (AADC), participate in hypothalamic functions. Several findings suggest that the expression patterns of the progesterone receptor (PR), TH and AADC overlap in the guinea pig brain. However, it remained to be determined whether or not these two enzymes coexist in the same neurons which contain the PR. To test this hypothesis and quantify these colocalization relationships in the hypothalamus, we used a triple-labeling immunofluorescence procedure. Only PR/AADC-immunoreactive cells were seen in the preoptic area but no PR/TH cells and, therefore, no triple immunoreactive cells were found. An occasional colocalization between PR and the two enzymes was observed throughout the rostrocaudal extent of the arcuate nucleus with the greatest concentration of triple-labeled cells in the medial subdivision. In this region, quantitative estimation of cellular immunoreactivity showed that the triple immunoreactive cells represented about 29% of PR/TH cells, 9% of PR/AADC cells and 22% of TH/AADC cells in spite of a very low percentage in relation to total populations of neurons expressing only PR, TH or AADC. Thus, the PR are only present in monoenzymatic AADC expressing neurons in the preoptic area while they can be observed in neurons expressing both enzymes in the arcuate nucleus.

Comparative distribution of estrogen receptor alpha and beta immunoreactivities in the forebrain and the midbrain of the female guinea pig.

Estrogen plays an important role in regulating gonadotropin secretion and reproductive behavior. The estrogen receptor alpha (ERalpha) was believed to be the only receptor which mediated the actions of the hormone until the identification of a novel ER called ERbeta. In the present study, the map of ERalpha immunoreactive (IR) neurons was compared with the distribution pattern of ERbeta-IR neurons in the forebrain and midbrain of ovariectomized guinea pigs using immunocytochemistry. The immunoreactivities appeared to be mainly nuclear in their subcellular distribution. Both ERalpha- and ERbeta-like immunoreactivities were highly expressed in the bed nucleus of the stria terminalis and the ventrolateral hypothalamic nucleus but were found to be differentially expressed in discrete subregions of the amygdaloid complex. A large number of intensely labeled ERalpha cells were observed throughout the rostrocaudal extent of the preoptic region, whereas only a few ERbeta-IR neurons were found in the periventricular preoptic nucleus bordering the third ventricle or scattered in the medial preoptic area. In contrast, only ERalpha-immunoreactivity was seen in the septum, and in the magnocellular supraoptic, paraventricular, arcuate, and premammillary nuclei. In the midbrain, neurons containing ERalpha were observed throughout the rostrocaudal extent of the gray matter, whereas ERbeta was only detected within the dorsal raphe nucleus. These observations provide evidence of a distinct neuroanatomical pattern for the two subtypes of the ER which may have different roles in regulating behavior and the neuroendocrine mechanisms of reproduction. Species similarities and differences in the distributions of ERalpha and ERbeta immunoreactivities are discussed.

AMPA glutamate receptor subunits in the guinea pig hypothalamus: distribution and colocalization with progesterone receptor.

Excitatory amino acids (EAAs), particularly glutamate, have been implicated in the control of luteinizing hormone (LH) secretion through facilitation of gonadotropin-releasing hormone release. The effects of EAAs are mediated by means of ionotropic glutamate receptors, which are divided into N-methyl-D-aspartate (NMDA) and non-NMDA (kainate and AMPA) subtypes. Moreover, ovarian steroids are responsible for inducing the preovulatory surge of LH and are involved in the actions of EAAs on LH release. Progesterone is directly involved in the potentiating effect of ovarian steroids on the stimulating effect of AMPA neurotransmission on gonadotropin secretion. To broaden our understanding of the role of hypothalamic AMPA receptors in the steroid-induced LH surge, we determined the cellular localization of AMPA receptors in the hypothalamus of guinea pigs by using antibodies that recognize the GluR1, GluR2, GluR2/3, or GluR4 subunits, and then we examined the neuroanatomic relationships between these receptors and the progesterone receptor (PR). Different patterns of immunostaining within the preoptic area and hypothalamus were evident with the antibodies to the four subunits with marked contrasts between moderate staining for GluR1, intensely stained structures for GluR2 and GluR2/3, and little specific staining for GluR4. Immunoreactive (IR) neurons were visualized in many regions, including the two regions known to contain a dense population of estradiol-induced PR-IR cells: the preoptic periventricular and ventrolateral hypothalamic nuclei. Approximately 60% of GluR1-IR and 39% of GluR2-IR cells in the preoptic region possessed PR, whereas 46% of GluR1-IR and 54% of GluR2-IR cells in the ventrolateral nucleus expressed PR. These neuroanatomic results suggest that the coordinated actions of progesterone and glutamatergic inputs on mammalian reproductive functions are integrated at the cellular level.