The Localization of R-Spondin1 and R-Spondin 3 Peptides in Rat Hypothalamus: An Immunohistochemical Study / Localización de los Péptidos R-Spondin1 y R-Spondin3 en el Hipotálamo de Rata: Un Estudio Inmunohistoquímico

SUMMARY: The R-spondin protein family is a group of proteins that enhance Wnt/b-catenin signaling and have pleiotropic functions in stem cell growth and development. In the literature reviews, there is no histomorphological study showing the localization and distribution of R-spondins in different hypothalamic nuclei. For this reason, the purpose of this study was to determine the localization, distribution characteristics, and densities in the hypothalamic nuclei of neurons expressing Rspo1 and Rspo3 proteins. The free-floating brain sections of the male rats who were not exposed to any treatment were stained with the indirect immunoperoxidase method using the relevant antibodies. As a result of the immunohistochemical studies, it was determined that neurons expressing the Rspo1 protein were found in large numbers in the supraoptic nucleus (SON), the suprachiasmatic nucleus (SCh), anterior paraventricular nucleus, periventricular hypothalamic nucleus (PeV), anterior hypothalamic area, magnocellular preoptic nucleus (MCPO) and the lateral hypothalamic area (LH) from the hypothalamic nuclei, while they were localized in fewer numbers in the arcuate nucleus (ARC). Rspo3 protein expression was found in neurons localized in the hypothalamic nuclei SON, paraventricular nucleus (PVN), PeV, ARC, ventromedial nucleus (VMH), LH, anterior parvicellular nucleus, and zona inserta (ZI). In addition, neurons synthesizing both peptides were found in the cortex and hippocampus regions (H). Rspo1 and 3 proteins are expressed in hypothalamic energy homeostatic areas, thus these proteins may be involved in the regulation of food intake.

La familia de proteínas R-espondina es un grupo de proteínas que mejoran la señalización de Wnt/b-catenina y tienen funciones pleiotrópicas en el crecimiento y desarrollo de las células madre. En las revisiones de la literatura no existen estudios histomorfológicos que muestren la localización y distribución de las R-espondinas en diferentes núcleos hipotalámicos. Por esta razón, el propósito de este estudio fue determinar la localización, características de distribución y densidades en los núcleos hipotalámicos de neuronas que expresan las proteínas Rspo1 y Rspo3. Secciones de cerebro flotantes de ratas macho que no fueron expuestas a ningún tratamiento se tiñeron con el método de inmunoperoxidasa indirecta utilizando los anticuerpos pertinentes. Como resultado de los estudios inmunohistoquímicos, se determinó que las neuronas que expresan la proteína Rspo1 se encontraron en gran número en el núcleo supraóptico (SON), el núcleo supraquiasmático (SCh), el núcleo paraventricular anterior, el núcleo hipotalámico periventricular (PeV), el núcleo hipotalámico anterior área, núcleo preóptico magnocelular (MCPO) y el área hipotalámica lateral (LH) de los núcleos hipotalámicos, mientras que se localizaron en menor número en el núcleo arqueado (ARC). La expresión de la proteína Rspo3 se encontró en neuronas localizadas en los núcleos hipotalámicos SON, núcleo paraventricular (PVN), PeV, ARC, núcleo ventromedial (VMH), LH, núcleo parvicelular anterior y zona inserta (ZI). Además, se encontraron neuronas que sintetizan ambos péptidos en las regiones de la corteza y el hipocampo (H). Las proteínas Rspo1 y 3 se expresan en áreas homeostáticas de energía hipotalámicas, por lo que estas proteínas pueden estar involucradas en la regulación de la ingesta de alimentos.

Expression of the ionotropic glutamate receptors on neuronostatin neurons in the periventricular nucleus of the hypothalamus.

BACKGROUND: Neuronostatin, a newly identified peptide, is accepted as an anorexigenic peptide since it suppresses food intake when given intracerebroventricularly. Although the effect mechanisms of neuronostatin have been shown in different studies, there are no reports in the literature describing the mechanisms controlling neuronostatin neurons. In this study, we aimed to determine the presence of the ionotropic glutamate receptor subunits (iGluRs) in neuronostatin neurons in the periventricular nucleus of the hypothalamus. MATERIALS AND METHODS: The presence of glutamate receptors in neuronostatin neurons was investigated by dual immunohistochemistry. Immunohistochemistry was performed on 40 µm thick coronal brain sections with antibodies against AMPA (GluA1-4), kainate (GluK1/2/3, and GluK5), and NMDA (GluN1 and GluN2A) receptor subunits. RESULTS: The results showed that the neuronostatin neurons expressed most of the NMDA and non-NMDA receptor subunits. The neuronostatin neurons in the anterior hypothalamic periventricular nucleus were particularly immunopositive for GluA1, GluA4, GluK1/2/3, GluK5 and GluN1 antibodies. No expression was observed for GluA2, GluA3 and GluN2A antibodies. CONCLUSIONS: For the first time in the literature, our study demonstrated that the neuronostatin neurons express glutamate receptor subunits which may form homomeric or heteromeric functional receptor complexes. Taken together, these results suggest that multiple subunits of iGluRs are responsible for glutamate transmission on neuronostatin neurons in the anterior hypothalamic periventricular nucleus.

Glutamate receptor antagonist suppresses the activation of nesfatin-1 neurons following refeeding or glucose administration.

BACKGROUND: Nesfatin-1 is a newly identified satiety peptide that has regulatory effects on food intake and glucose metabolism, and is located in the hypothalamic nuclei, including the supraoptic nucleus (SON). In this study, we have investigated the hypothesis that nesfatin-1 neurons are activated by refeeding and intraperitoneal glucose injection and that the glutamatergic system has regulatory influences on nesfatin-1 neurons in the SON. MATERIALS AND METHODS: The first set of experiments analysed activation of nesfatin-1 neurons after refeeding as a physiological stimulus and the effectiveness of the glutamatergic system on this physiological stimulation. The subjects were randomly divided into three groups: fasting group, refeeding group and antagonist (CNQX + refeeding) group. The second set of experiments analysed activation of nesfatin-1 neurons by glucose injection as a metabolic stimulus and the effectiveness of the glutamatergic system on this metabolic stimulation. The subjects were randomly divided into three groups: saline group, glucose group and antagonist (CNQX + glucose) group. RESULTS: Refeeding significantly increased the number of activated nesfatin-1 neurons by approximately 66%, and intraperitoneal glucose injection activated these neurons by about 55%, compared to the fasting and saline controls. The injections of glutamate antagonist (CNQX) greatly decreased the number of activated nesfatin-1 neurons. CONCLUSIONS: This study suggested that nesfatin-1 neurons were activated by peripheral and/or metabolic signals and that this effect was mediated through the glutamatergic system.

Nesfatin-1 and neuronostatin neurons are co-expressed with glucocorticoid receptors in the hypothalamus.

Nesfatin-1 and neuronostatin in the central nervous system participate in regulating stress responses. Glucocorticoid hormones affect the brain through glucocorticoid receptors (GR). We investigated in the rat the possibility of co-localizing nesfatin-1 and neuronostatin neurons in hypothalamic areas with GR. using immunohistochemistry. We counted nesfatin-1 and neuronostatin stained neurons. We counted GR positive nesfatin-1 neurons in the arcuate nucleus (ARC) and paraventricular nucleus (PVN) and GR positive neuronostatin neurons in the periventricular nucleus (PeN). The percentage of nesfatin-1 neurons that expressed GR was 38.4% in female rats and 21.9% in male rats in the ARC, and 33.3% in female rats and 29.2% in male rats in the PVN. The percentage of neuronostatin neurons that expressed GR was 39.1% in female rats and 39.9% in male rats in the PeN. We found that a substantial portion of nesfatin-1 and neuronostatin neurons were stained for GR. We speculate that the pattern of GR might permit secretion of neuropeptides to be stimulated by peripheral glucocorticoid signals. Stress can suppress food intake, in part, through the GR in neurons that express nesfatin-1, which is a satiety molecule, and in neurons that express neuronostatin, which is an anorexigenic peptide.

Identification of neurones in the female rat hypothalamus that express oestrogen receptor-alpha and vesicular glutamate transporter-2.

Oestrogen exerts its effects in the brain by binding to and activating two members of the nuclear receptor family, oestrogen receptor (ER)-alpha and ER-beta. Evidence suggests that oestrogen-receptive neurones participate in the generation of reproductive behaviours and that they convey the oestrogen message to gonadotropin-releasing hormone (GnRH) neurones. The aim of the present study was to identify the neurochemical phenotype of a subset of oestrogen receptor-expressing neurones. To this aim, we focused on the glutamate neuronal system, which is one of the most important stimulators of GnRH synthesis and release. We used the presence of vesicular glutamate transporter-2 (VGLUT2) mRNA as a specific marker to identify glutamate neurones and employed dual in situ hybridization to localize ERalpha mRNA-(35S-labelling) and VGLUT2 mRNA-(digoxigenin-labelling) expressing neurones within the hypothalamus. The results show that the overall distribution of VGLUT2 mRNA and ERalpha mRNA are consistent with previous data in the literature. Dual-labelled neurones were localized in the ventrolateral part of the ventromedial nucleus where 81.3 +/- 3.4% of the ERalpha mRNA containing neurones expressed VGLUT2 mRNA, in the anteroventral periventricular nucleus (30% colocalization) and in the medial preoptic nucleus (19% colocalization). Only 4.4% of the ERalpha expressing neurones in the arcuate nucleus contained VGLUT2 mRNA. These findings reveal that certain subpopulations of oestrogen-receptive neurones are glutamatergic in select hypothalamic areas that are known to regulate reproductive behaviour and GnRH neurones in the female rat. Thus, the oestrogen signal could be propagated through glutamate neurones to distant sites and influence the activity of the postsynaptic neurones.

Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus.

The excitatory amino acid neurotransmitter glutamate participates in the control of most (and possibly all) neuroendocrine systems in the hypothalamus. This control is exerted by binding to two classes of membrane receptors, the ionotropic and metabotropic receptor families, which differ in their structure and mechanisms of signal transduction. To gain a better understanding about the precise sites of action of glutamate and the subunit compositions of the receptors involved in the glutamatergic neurotransmission in the hypothalamus and septum, in situ hybridization was used with 35S-labeled cRNA probes for the different ionotropic receptor subunits, including glutamate receptor subunits 1-4 (GluR1-GluR4), kainate-2, GluR5-GluR7, N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), and NMDAR2A-NMDAR2D. The results showed that subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-preferring, kainate-preferring, and NMDA-preferring receptor subunits are distributed widely but heterogeneously and that the GluR1, GluR2, kainate-2, NMDAR1, NMDAR2A, and NMDAR2B subunits are the most abundant in the hypothalamus. Thus, GluR1 subunit mRNA was prominent in the lateral septum, preoptic area, mediobasal hypothalamus, and tuberomammillary nucleus, whereas kainate-2 subunit mRNA was abundant in the medial septum-diagonal band, median and anteroventral preoptic nuclei, and supraoptic nuclei as well as the magnocellular portion of the posterior paraventricular nucleus. Regions that contained the highest levels of NMDAR1 subunit mRNA included the septum, the median preoptic nucleus, the anteroventral periventricular nucleus, and the supraoptic and suprachiasmatic nuclei as well as the arcuate nucleus. Together, the extensive distribution of the different GluR subunit mRNAs strengthen the view that glutamate is a major excitatory neurotransmitter in the hypothalamus. The overlap in the distribution of the various subunit mRNAs suggests that many neurons can express GluR channels that belong to different families, which would allow a differential regulation of the target neurons by glutamate.

Expression of ionotropic glutamate receptor subunit mRNAs in the hypothalamic paraventricular nucleus of the rat.

The hypopthalamic paraventricular nucleus (PVN) coordinates multiple aspects of homeostatic regulation, including pituitary-adrenocortical function, cardiovascular tone, metabolic balance, fluid/electrolyte status, parturition and lactation. In all cases, a substantial component of this function is controlled by glutamate neurotransmission. In this study, the authors performed a high-resolution in situ hybridization analysis of ionotropic glutamate receptor subunit expression in the PVN and its immediate surround. N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), NMDAR2A, and NMDAR2B mRNAs were expressed highly throughout the PVN and its perinuclear region as well as in the subparaventricular zone. NMDAR2C/2D expression was limited to subsets of neurons in magnocellular and hypophysiotrophic regions. In contrast with NMDA subunit localization, AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate)-preferring and kainate (KA)-preferring receptor subunit mRNAs were expressed heterogeneously in the PVN and surround. Glutamate receptor 1 (GluR1) mRNA labeling was most intense in preautonomic subregions, whereas GluR2, GluR4, GluR5, and KA2 were expressed in hypophysiotrophic cell groups. It is noteworthy that GluR5 mRNA expression was particularly robust in the dorsolateral region of the medial parvocellular PVN, suggesting localization in corticotropin-releasing hormone neurons. All four AMPA subunits and GluR6 and GluR7 mRNAs were expressed highly in the perinuclear PVN region and the subparaventricular zone. These data suggest the capacity for multifaceted regulation of PVN function by glutamate, with magnocellular neurons preferentially expressing NMDA subunits, preautonomic neurons preferentially expressing AMPA subunits, and hypophysiotrophic neurons preferentially expressing KA subunits. Localization of all species in the perinuclear PVN suggests that glutamate input to the immediate region of the PVN may modulate its function, perhaps by communication with local gamma-aminobutyric acid neurons.

Kainate receptor subunit-positive gonadotropin-releasing hormone neurons express c-Fos during the steroid-induced luteinizing hormone surge in the female rat.

During the preovulatory and estradiol-progesterone-induced GnRH-LH surge, a subpopulation of GnRH neurons transiently expresses the transcription factor c-fos, which is a useful marker of cell activation. To further characterize this subpopulation of GnRH neurons, multiple immunohistochemical procedures were applied to visualize GnRH, c-Fos, KA2, GluR5, GluR6, and GluR7 receptor subunits during different phases of the estrogen-progesterone-induced LH surge. The results show that the LH surge begins at 1400 h and peaks at 1600 h before returning to baseline late in the evening. At 1400 h, about 50% of the GnRH neurons contained c-Fos, and this percentage remained high at 65% at 1600 and 2000 h. During the surge, 50% of the c-Fos-positive GnRH neurons contained KA2 receptor subunit protein at 1400 h, 65% of the c-Fos-positive GnRH neurons expressed the KA2 subunit at 1600 h, and 50% of the c-Fos-positive GnRH neurons expressed the KA2 subunit at 2000 h. As KA2 subunits require other kainate-preferring subunits to form functional receptor channels, we examined GnRH neurons for the presence of GluR5, GluR6, and GluR7 messenger RNA (mRNA) and protein. The results show that the KA2-containing GnRH neurons also contain GluR5 receptor subunit mRNA and protein, and that these GnRH neurons are c-Fos positive during the steroid-induced LH surge. To determine whether administration of kainate is sufficient to induce c-Fos in GnRH neurons, steroid-primed animals received iv injections of subseizure-inducing amounts of kainic acid and were processed for immunohistochemistry and in situ hybridization. The results show that kainic acid causes a significant increase in circulating LH; however, it does not induce c-Fos in GnRH neurons, nor does it cause an increase in GnRH mRNA. Together, the results suggest that a large subset of GnRH neurons expresses KA2 as well as GluR5 receptor subunits, which would allow the formation of functional glutamate receptor channels, and that this subset of GnRH neurons is activated during the steroid-induced LH surge.

Identification of kainate-preferring glutamate receptor subunit GluR7 mRNA and protein in the rat median eminence.

In situ hybridization and immunohistochemistry were used to determine the presence of kainate-preferring glutamate receptor subunits GluR6 and GluR7 mRNA and protein in the median eminence of the rat. The results show that most tanycytes lining the ventral third ventricle and many astrocytes within the median eminence contain the GluR7 receptor subunit mRNA but not the GluR5 and GluR6 receptor subunit mRNA. Immunohistochemical stainings show that the GluR6/7 receptor protein was localized to tanycytic cell bodies, their basal processes and to many other astrocytes in different layers of the median eminence. The results suggest that glutamate can act directly on the glial cells in the median eminence by binding to the GluR7 subunit which may be important for the control of the secretion of releasing and inhibiting hormones from axon terminals in the external layer. In order to determine if these receptor subunits are functional, kainic acid was injected and c-fos expression monitored. Results show that kainic acid induced c-fos synthesis in most of these glial cells.

Expression of glutamate receptor subunit mRNAs in gonadotropin-releasing hormone neurons during the sexual maturation of the female rat.

Excitatory amino acids, particularly glutamate, are thought to be important for the maturation of the brain-pituitary-gonadal axis and the induction of puberty in the rat. We have previously shown that, in the female rat, GnRH neurons preferentially express the KA2 and NMDAR2A receptor subunit mRNAs, but not AMPA or NMDAR1 mRNA. The aim of the present study was to determine whether the onset or rate of KA2 and NMDAR2A receptor expression in GnRH neurons is correlated with the onset of puberty. Dual in situ hybridization using digoxigenin-labeled GnRH cRNA probes and 35S-labeled glutamate receptor subunit probes, followed by autoradiography and image analysis were used to measure the KA2 or NMDAR2A mRNA content in GnRH neurons in 20- to 50-day-old female rats which were sacrificed at 08.00 or 17.00 h. The results show that: (a) the KA2 mRNA content of GnRH neurons and the number of GnRH neurons expressing KA2 mRNA increase progressively in the morning hours between postnatal days 20 and 40; (b) the diurnal pattern of KA2 mRNA levels in GnRH neurons changes between days 40 and 50 from high KA2 levels in the morning hours before day 40 to high KA2 mRNA levels in the afternoon in 45- and 50-day-old animals; (c) while the high levels of KA2 mRNA in GnRH neurons in the morning hours of 20- to 40-day-old animals are paralleled by an overall increase in KA2 expression in the preoptic area, the rise in KA2 mRNA in GnRH neurons in the afternoon of 45- and 50-day-old animals appears to be specific for the GnRH neurons, and (d) no significant differences were detected for the NMDAR2A mRNA content in GnRH neurons among the different age groups and the morning and afternoon values. Since the gradual increase in the KA2 mRNA content in GnRH neurons of animals reaching puberty as well as the reversal of diurnal rhythmicity in KA2 receptor mRNA content of GnRH neurons coincide with the times of vaginal opening and first ovulation, it is suggested that glutamate, acting through KA2 receptors directly on GnRH neurons is, at least in part, an important factor in the excitatory regulation of the postnatal sexual development of the female rat. In contrast, expression of the NMDA-preferring receptor, NMDAR2A, in GnRH neurons appeared to be unchanged during this development.

Brain gonadotropin releasing hormone receptors: localization and regulation.

In this review, the current information about the location of GnRH receptor protein and GnRH receptor mRNA in the rat central nervous system is summarized as well as the changes that occur in the GnRH receptor mRNA levels during different endocrine conditions of the animals. The results of these studies show that GnRH receptor protein and mRNA levels change in parallel in the hippocampus, suggesting that pretranscriptional factors control the synthesis of the receptor. In the arcuate and ventromedial nuclei of the hypothalamus, GnRH receptor mRNA levels are highest during the early morning of proestrus and during the morning of an estrogen-progesterone-induced LH surge. The timing of the changes in GnRH receptor mRNA levels indicates that increasing levels of estradiol are responsible for the increase in GnRH receptor synthesis. Binding of GnRH agonist to the brain GnRH receptor causes a dose-dependent increase in inositol phosphates as well as changes in intracellular Ca++ levels of the target neurons. Together, it is suggested that GnRH functions in the brain as a neurotransmitter and/or modulator linking the peripheral endocrine effects of GnRH to actions of the peptide inside the central nervous system where it can facilitate, for example, reproductive behaviors.

Identification of glutamate receptor subtype mRNAs in gonadotropin-releasing hormone neurons in rat brain.

The aims of the present study were to determine: 1. If glutamate neurites can provide input to gonadotropin-releasing hormone (GnRH) neurons; 2. Which glutamate receptor subtype mRNAs are expressed in GnRH neurons; and 3. If GnRH neurons synthesize kainate 2 receptor (KA(2)) protein. Immunohistochemical double stainings for GnRH and glutamate or for GnRH and KA(2)-receptor protein were applied to rat brain sections containing the medial septum-diagonal band and preoptic area or the median eminence; in addition, dualin situ hybridization studies were carried out with digoxygenin-labeled cRNA probes encoding GnRH in combination with(35)S-labeled cRNA probes encoding the glutamate receptor subtypes GluR(1-4), KA(2), NMDA R(1), or NMDA R(2A-D). The results show that GnRH neurons are surrounded by glutaminergic neurites, which form puncta-like close appositions with the GnRH perikarya, and that an extensive overlap exists in the distribution of GnRH-positive axon terminals and glutaminergic neurites in the median eminence. Similarly, KA(2)-receptor immunoreactivity is present in the perikarya of many GnRH neurons and in their axon terminals in the median eminence. Dualin situ hybridization experiments show that about 32% of all digoxygenin-labeled GnRH neurons also contain KA(2)-receptor mRNA, 17% contain NMDA R(2A) mRNA, 8% contain NMDR R(1), whereas <5% of the GnRH neurons express measurable amounts of GluR(1-4) or NMDAR(2B-D) mRNA. The results suggest that glutaminergic neurons innervate the GnRH neuronal system directly through activation of KA(2) receptors on GnRH neurons, whereas the effects of AMPA and NMDA on GnRH release are likely to be exerted indirectly through interneurons.