Differential brain distribution of gonadotropin-releasing hormone receptors in the goldfish

The present study describes the differential distributions in the brain of the two goldfish gonadotropin-releasing hormone (GnRH) receptors, using both immunohistochemistry and in situ hybridization approaches. The goldfish GnRH GfA and GfB receptors are variant forms of the same receptor subtype, although with distinct differences in ligand binding characteristics, and differential distributions in the pituitary and body tissues [Proc. Natl. Acad. Sci. USA 96 (1999) 2526]. The goldfish GnRH GfA receptor was found to be widespread throughout the brain, with neurons showing immunoreactivity in the olfactory bulbs, telencephalon, preoptic region, ventro-basal hypothalamus, thalamus, midbrain, motor neurons of the fifth, seventh, and tenth cranial nerves, reticular formation, cerebellum, and motor zone of the vagal lobes. The tracts in the posterior commissure, optic tectum, and motor zone of the vagal lobes also demonstrated immunoreactivity. While the brain was not systematically surveyed for in situ hybridization, hybridization was found in similar locations in the telencephalon, preoptic region, ventro-basal hypothalamus, cerebellum, and optic tectum. Hybridization was additionally found in the medial hypothalamus. The goldfish GnRH GfB receptor was found to have a more restricted distribution in the brain, with neurons showing immunoreactivity in the telencephalon, preoptic region, and ventro-basal hypothalamus. In situ hybridization demonstrated a somewhat wider distribution of expression of the receptor, with hybridization occurring in the preoptic region, ventro-basal and medial hypothalamus, as well as in the thalamus, epithalamus, and optic tectum. The widespread distribution of GnRH GfA receptor, and in particular its localization in the midbrain tegmentum in the region of the GnRH-II neurons, suggests that this receptor may be involved in the behavioral actions of GnRH peptides in the goldfish.     

Gonadotropin-releasing hormone-immunoreactive neurons and associated nicotinamide adenine dinucleotide phosphate-diaphorase-positive neurons in the brain of a teleost, Rhodeus amarus

Using combined nicotinamide adenine dinucleotide phosphate-diaphorase (NADPHd) histochemistry and salmon gonadotropin-releasing hormone (sGnRH) immunocytochemistry, it is reported for the first time that possible potential contacts occur between the nitric oxide (NO)- and the GnRH-containing neurons in the brain of a freshwater teleost, Rhodeus amarus. GnRH-immunoreactive (ir) neurons were observed in the olfactory nerve (OLN), olfactory bulb (OB), medial olfactory tract (MOT), ventral telencephalon (VT), nucleus preopticus periventricularis (NPP), nucleus lateralis tuberis (NLT), and midbrain tegmentum (MT). Although NADPHd neurons were widely distributed in the brain, only those having an association with GnRH-ir neurons are described. Based on the nature of the association between the GnRH and the NADPHd neurons, the former were classified into three types. The Type I GnRH neurons were characterized by the presence of NADPHd-positive granules in the perikarya and processes and occurred in the OLN, OB, MOT, and VT. The Type II GnRH neurons, having soma-soma or soma-process contacts with the NADPHd neurons, were restricted to the MT; the long processes of NADPHd cells crossed over either the perikarya or the thick processes of GnRH cells. However, the Type III GnRH neurons, found in the NPP and NLT, did not show direct contact, but a few NADPHd fibers were present in the vicinity. The terminal-soma contacts in the olfactory system and the VT and the soma-soma contacts in the MT represent the sites of possible potential contacts indicating a direct NO involvement in GnRH function, although NO action by diffusion remains possible. NO may influence the NPP and NLT GnRH cells by diffusion only, since a direct contact was not observed.     

Gonadotropin-inhibitory hormone neurons interact directly with gonadotropin-releasing hormone-I and -II neurons in European starling brain

Gonadotropin-inhibitory hormone (GnIH) is a hypothalamic dodecapeptide (SIKPSAYLPLRF-NH(2)) that directly inhibits gonadotropin synthesis and release from quail pituitary. The action of GnIH is mediated by a novel G-protein coupled receptor. This gonadotropin-inhibitory system may be widespread in vertebrates, at least birds and mammals. In these higher vertebrates, histological evidence suggests contact of GnIH immunoreactive axon terminals with GnRH neurons, thus indicating direct regulation of GnRH neuronal activity by GnIH. In this study we investigated the interaction of GnIH and GnRH-I and -II neurons in European starling (Sturnus vulgaris) brain. Cloned starling GnIH precursor cDNA encoded three peptides that possess characteristic LPXRF-amide (X = L or Q) motifs at the C termini. Starling GnIH was further identified as SIKPFANLPLRF-NH(2) by mass spectrometry combined with immunoaffinity purification. GnIH neurons, identified by in situ hybridization and immunocytochemistry (ICC), were clustered in the hypothalamic paraventricular nucleus. GnIH immunoreactive fiber terminals were present in the external layer of the median eminence in addition to the preoptic area and midbrain, where GnRH-I and GnRH-II neuronal cell bodies exist, respectively. GnIH axon terminals on GnRH-I and -II neurons were shown by GnIH and GnRH double-label ICC. Furthermore, the expression of starling GnIH receptor mRNA was identified in both GnRH-I and GnRH-II neurons by in situ hybridization combined with GnRH ICC. The cellular localization of GnIH receptor has not previously been identified in any vertebrate brain. Thus, GnIH may regulate reproduction of vertebrates by directly modulating GnRH-I and GnRH-II neuronal activity, in addition to influencing the pituitary gland.     

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.     

Mineralocorticoid receptors: evolutionary and pathophysiological considerations

Mineralocorticoid receptors (MR), glucocorticoid receptors (GR), progesterone receptors (PR), and androgen receptors (AR) comprise a closely related subfamily within the human 49-member nuclear receptor family. These receptors and their cognate ligands play major roles in homeostasis, reproduction, growth, and development, despite which their evolution and diversification remains incompletely understood. Several conflicting models have been advanced for the evolution of this subfamily. We have thus undertaken Bayesian and maximum likelihood phylogenetic analyses of this subfamily. The Bayesian consensus and maximum likelihood trees support a basal position for MR, with the PR and AR forming a sister clade. We next performed analyses using topological constraints to directly contrast the likelihood of seven phylogenetic models. In these analyses, three models have similar support: one proposes two sister clades (MR and GR, PR and AR); the other two propose a different subfamily member (MR or GR) to be the first to have diverged. Ancestral state reconstructions at sites critical for physiological function show that the S810L mutation in the MR, which results in the MR being similar to estrogen receptors and the more distantly related retinoic acid receptor-α is likely to reflect the ancestral receptor sequence before the divergence of this subfamily and provides further support for MR having been the first of the subfamily to diverge. Finally, we drew on pathophysiological comparisons to help to distinguish the different models. On the basis of our phylogenetic analyses and pathophysiological considerations, we propose that the MR was the first to diverge from the ancestral gene lineage from which this subfamily derived.     

Close juxtapositions between luteinizing hormone-releasing hormone-immunoreactive neurons and corticotropin-releasing factor-immunoreactive axons in the human diencephalon

Gonadal functions are modulated by corticotropin-releasing factor (CRF) in the rat via direct suppression of LH-releasing hormone (LHRH) release. Although there is evidence of direct morphological contacts between the LHRH and CRF-immunoreactive (-IR) structures in the rat hypothalamus, little is known about the morphological base of CRF-influenced LHRH release in man. Thus, we studied the distribution of the CRF-IR and LHRH-IR systems in the human diencephalon and revealed putative CRF-LHRH juxtapositions using double label immunohistochemistry. LHRH-IR cells were present mainly in the infundibular region and the medial preoptic area. CRF-IR neuronal structures were observed in the periventricular area, paraventricular nucleus, infundibular region, and median eminence. CRF-LHRH juxtapositions were found mainly in the infundibulum and median eminence. Few juxtapositions were detected in the medial preoptic area. In these regions, black diaminobenzidine/silver-labeled CRF-IR fibers abutted fusiform brown diaminobenzidine-labeled LHRH neurons, usually forming multiple contacts. Examination of semithin sections of these close associations with the aid of oil immersion revealed no cleft between CRF-IR nerve terminals contacting LHRH-IR structures. These findings suggest that the juxtapositions between the LHRH-IR and CRF-IR neurons may be functional synapses forming the morphological substrate of the CRF-controlled LHRH secretion. Moreover, the wide distribution of CRF-IR elements suggests that CRF controls other diencephalic functions as well.     

Tryptase: Genetic and functional considerations

Tryptase is one of the main proteases located in the secretory granules of the mast cells, and is released through degranulation. It is therefore assumed to play an important role in inflammatory and allergic processes. Four genes are known to encode for these enzymes, with different alleles that give rise to different types of tryptases. The term “tryptase” generally refers to β-tryptase, which in vivo is a heterotetramer, possessing a structure of vital importance for enabling drug and substrate access to the active site of the molecule. Tryptase has been reported to possess antagonistic functions, since it plays an important role both in inflammatory phenomena and as a protector against infection. In allergic processes it is associated to bronchial hyperresponsiveness in asthmatic patients, where PAR-2 is of great importance as an airway receptor. Lastly, the genes that encode for tryptase are highly polymorphic and complex. As a result, it is important to establish a relationship between genotype and phenotype in disorders such as asthma, and to identify mutations that are presumably of pharmacological relevance.     

Development of functional human embryonic stem cell-derived neurons in mouse brain

Human embryonic stem cells are pluripotent entities, theoretically capable of generating a whole-body spectrum of distinct cell types. However, differentiation of these cells has been observed only in culture or during teratoma formation. Our results show that human embryonic stem cells implanted in the brain ventricles of embryonic mice can differentiate into functional neural lineages and generate mature, active human neurons that successfully integrate into the adult mouse forebrain. Moreover, this study reveals the conservation and recognition of common signals for neural differentiation throughout mammalian evolution. The chimeric model will permit the study of human neural development in a live environment, paving the way for the generation of new models of human neurodegenerative and psychiatric diseases. The model also has the potential to speed up the screening process for therapeutic drugs.     

Colocalization of kisspeptin and gonadotropin-releasing hormone in the ovine brain

Kisspeptin is a peptide that has been implicated in the regulation of GnRH cells in the brain. Immunohistochemical studies were undertaken to examine the distribution of kisspeptin-immunoreactive (IR) cells in the ovine diencephalon and determine the effect of ovariectomy in the ewe. We report that kisspeptin colocalizes to a high proportion of GnRH-IR cells in the preoptic area, which is a novel finding. A high level of colocalization of kisspeptin and GnRH was also seen in varicose neuronal fibers within the external, neurosecretory zone of the median eminence. Apart from the kisspeptin/GnRH cells, a population of single-labeling kisspeptin-IR cells was also observed in the preoptic area. Within the hypothalamus, kisspeptin-IR cells were found predominantly in the arcuate nucleus, and there was an increase in the number of immunohistochemically identified cell within this nucleus after ovariectomy. Kisspeptin-IR cells were also found in the periventricular nucleus of the hypothalamus, but the number observed was similar in gonad-intact and ovariectomized ewes. The colocalization of GnRH and kisspeptin within cells of the preoptic area and GnRH neurosecretory terminals of the median eminence suggests that the two peptides might be cosecreted into the hypophyseal portal blood to act on the pituitary gland. Effects of ovariectomy on the non-GnRH, Kisspeptin-IR cells of the hypothalamus suggest that kisspeptin production is negatively regulated by ovarian steroids.     

Two new forms of gonadotropin-releasing hormone in a protochordate and the evolutionary implications.

The neuropeptide gonadotropin-releasing hormone (GnRH) is the major regulator of reproduction in vertebrates. Our goal was to determine whether GnRH could be isolated and identified by primary structure in a protochordate and to examine its location by immunocytochemistry. The primary structure of two novel decapeptides from the tunicate Chelyosoma productum (class Ascidiacea) was determined. Both show significant identity with vertebrate GnRH. Tunicate GnRH-I (pGlu-His-Trp-Ser-Asp-Tyr-Phe-Lys-Pro-Gly-NH2) has 60% of its residues conserved, compared with mammalian GnRH, whereas tunicate GnRH-II (pGlu-His-Trp-Ser-Leu-Cys-His-Ala-Pro-Gly-NH2) is unusual in that it was isolated as a disulfide-linked dimer. Numerous immunoreactive GnRH neurons lie within blood sinuses close to the gonoducts and gonads in both juveniles and adults, implying that the neuropeptide is released into the bloodstream. It is suggested that in ancestral chordates, before the evolution of the pituitary, the hormone was released into the bloodstream and acted directly on the gonads.     

Minireview: kisspeptin neurons as central processors in the regulation of gonadotropin-releasing hormone secretion

The Kiss1 gene encodes a family of peptides called kisspeptins, which bind to the G protein-coupled receptor GPR54. Kisspeptin(s) and its receptor are expressed in the forebrain, and the discovery that mice and humans lacking a functional GPR54 fail to undergo puberty and exhibit hypogonadotropic hypogonadism implies that kisspeptin signaling plays an essential role in reproduction. Studies in several mammalian species have shown that kisspeptins stimulate the secretion of gonadotropins from the pituitary by stimulating the release of GnRH from the forebrain after the activation of GPR54, which is expressed by GnRH neurons. Kisspeptin is expressed abundantly in the arcuate nucleus (Arc) and the anteroventral periventricular nucleus (AVPV) of the forebrain. Both estradiol and testosterone regulate the expression of the Kiss1 gene in the Arc and AVPV; however, the response of the Kiss1 gene to these steroids is exactly opposite between these two nuclei. Estradiol and testosterone down-regulate Kiss1 mRNA in the Arc and up-regulate its expression in the AVPV. Thus, kisspeptin neurons in the Arc may participate in the negative feedback regulation of gonadotropin secretion, whereas kisspeptin neurons in the AVPV may contribute to generating the preovulatory gonadotropin surge in the female. Hypothalamic levels of Kiss1 and GPR54 mRNA increase dramatically at puberty, suggesting that kisspeptin signaling could mediate the neuroendocrine events that trigger the onset of puberty. Together, these observations demonstrate that kisspeptin-GPR54 signaling in the brain serves as an important conduit for controlling GnRH secretion in the developing and adult animal.     

Comparative distribution of vasopressin and oxytocin neurons in the rat brain using a double-label procedure

The distribution of vasopressin (VP) and oxytocin (OT) neurons in the rat supraoptic (SON), paraventricular (PVN), and accessory magnocellular (AMN) nuclei was studied by localizing both peptides on the same section with a double immunocytochemical staining procedure employing specific monoclonal antibodies (MAB). This procedure allows us to visualize the distribution of one cell type relative to the other. In the rostral SON, VP cells lie dorsal and medial to the OT cells. Near the mid-point of the nucleus along its rostral-caudal length, there is a transition zone in which the two cell types are mixed. Proceeding caudalward, the relative locations of OT and VP cells are exchanged so that most of VP cells are located in the ventral and medial sector of the nucleus, whereas the OT cells are situated dorsal and lateral. However, there is no absolute segregation of the two types of cells anywhere in the nucleus. In the anterior part of the PVN a rostral group (rPVN) of cells composed of a medial portion and a lateral wing can be recognized. Nearly all of the cells in the rPVN are oxytocin-containing. The rPVN is separated from the next group, the middle PVN (mPVN), by a cell poor zone of about 100-150 micron. The mPVN contains both OT and VP neurons. As one proceeds caudally, the OT cells extend in the rostrocaudal direction from an anterior and ventromedial location, forming a shell around a core of VP neurons. In the most caudal PVN (cPVN), a triangular cell group characterized by fusiform cells with long-beaded processes can be distinguished from the more rounded cells of the remaining PVN. Many fusiform cells in the cPVN appear to send their axons to the posterior perifornical nucleus and the nucleus of the medial forebrain bundle. Other fusiform cells of the cPVN are oriented in a rostral-caudal plane and are situated more medially in this subdivision. The dendrites of these cells project into the mPVN while their posterior processes, most of which also appear to be dendrites, project caudally along a medial route.     

Glutamatergic and GABAergic innervation of human gonadotropin-releasing hormone-I neurons

Amino acid (aa) neurotransmitters in synaptic afferents to hypothalamic GnRH-I neurons are critically involved in the neuroendocrine control of reproduction. Although in rodents the major aa neurotransmitter in these afferents is γ-aminobutyric acid (GABA), glutamatergic axons also innervate GnRH neurons directly. Our aim with the present study was to address the relative contribution of GABAergic and glutamatergic axons to the afferent control of human GnRH neurons. Formalin-fixed hypothalamic samples were obtained from adult male individuals (n = 8) at autopsies, and their coronal sections processed for dual-label immunohistochemical studies. GABAergic axons were labeled with vesicular inhibitory aa transporter antibodies, whereas glutamatergic axons were detected with antisera against the major vesicular glutamate transporter (VGLUT) isoforms, VGLUT1 and VGLUT2. The relative incidences of GABAergic and glutamatergic axonal appositions to GnRH-immunoreactive neurons were compared quantitatively in two regions, the infundibular and paraventricular nuclei. Results showed that GABAergic axons established the most frequently encountered type of axo-somatic apposition. Glutamatergic contacts occurred in significantly lower numbers, with similar contributions by their VGLUT1 and VGLUT2 subclasses. The innervation pattern was different on GnRH dendrites where the combined incidence of glutamatergic (VGLUT1 + VGLUT2) contacts slightly exceeded that of the GABAergic appositions. We conclude that GABA represents the major aa neurotransmitter in axo-somatic afferents to human GnRH neurons, whereas glutamatergic inputs occur somewhat more frequently than GABAergic inputs on GnRH dendrites. Unlike in rats, the GnRH system of the human receives innervation from the VGLUT1, in addition to the VGLUT2, subclass of glutamatergic neurons.     

Estrogen and estrogen receptor-{beta} (ER{beta})-selective ligands induce galanin expression within gonadotropin hormone-releasing hormone-immunoreactive neurons in the female rat brain

Among the many factors that integrate the activity of the GnRH neuronal system, estrogens play the most important role. In females, estrogen, in addition to the negative feedback, also exhibits a positive feedback influence upon the activity and output of GnRH neurons to generate the preovulatory LH surge and ovulation. Until recently, the belief has been that the GnRH neurons do not contain estrogen receptors (ERs) and that the action of estrogen upon GnRH neurons is indirect involving several, estrogen-sensitive neurotransmitter and neuromodulator systems that trans-synaptically regulate the activity of the GnRH neurons. Based on our recent findings that GnRH neurons of the female rat coexpress galanin, that galanin is a potent GnRH-releasing peptide, and that ERbeta is present in GnRH neurons, we have evaluated the effect of 17beta-estradiol and two ERbeta-selective agonists (WAY-200070, WAY-166818) on the expression of galanin within GnRH neurons. By combining immunocytochemistry for GnRH and in situ hybridization histochemistry for galanin, we demonstrate that 17beta-estradiol (20 mug/kg, sc) stimulates galanin expression within GnRH-immunoreactive neurons in a time-dependent manner. A significant increase was observed 2 h after its administration to ovariectomized rats. However, a more robust expression required 3-d treatment regimen. Treatment with the beta-selective ligands resulted in similar observations, although no statistical analysis is available for the 2 hr survival. These observations strongly suggest that estrogen and the ERbeta-selective ligands stimulate galanin expression within GnRH neurons via ERbeta, although an indirect mechanism via interneurons still cannot be ruled out.     

Differential decrease in the rate of dopamine synthesis in several dopaminergic neurons of aged rat brain

The purpose of this study was to investigate the in vivo rate of dopamine (DA) synthesis in dopaminergic neurons of the brain related to motor disturbances observed in aged rats. Aged rats of both sexes showed a decrease in spontaneous motor activity during a dark period as compared with those of mature rats. The in vivo rate of DA synthesis, as reflected in DOPA accumulation, in the striatum and nucleus accumbens, was slower in aged than in mature rats, whereas in the olfactory tubercle there was no significant difference between them. This suggests a differential vulnerability of dopaminergic neurons in the extrapyramidal motor areas of aged animals. After DA receptor blockade by haloperidol, increases in DA synthesis in the striatum, nucleus accumbens and olfactory tubercle were similar in both aged and mature rats, suggesting that there may be no age-related change in inhibitory regulation of in vivo DA synthesis mediated by presynaptic DA autoreceptors and/or a neuronal feedback mechanism via postsynaptic DA receptors in the striatum and mesolimbic DA regions.