Gonadotropin-releasing Hormone Cells Research Articles

γ-MSH increases intracellular cAMP accumulation and GnRH release in vitro and LH release in vivo

The roles of the melanocortin 3 receptor (MC3-R) and its agonist, γ 2-melanocyte-stimulating hormone (γ 2-MSH) in the regulation of the hypothalamo-pituitary-gonadal (HPG) axis are poorly understood. Here we show γ 2-MSH stimulated intracellular cAMP accumulation and gonadotrophin-releasing hormone (GnRH) secretion in the immortalised GnRH cell line GT 1-7. The MC3/4-R antagonist Agrp blocked these actions. Reverse transcriptase polymerase chain reaction demonstrated GT 1-7 cells express MC3-R mRNA. γ 2-MSH also stimulated GnRH release from hypothalamic explants. In vivo, γ 2-MSH administration into the medial preoptic area significantly increased plasma luteinising hormone. MC3-R and γ 2-MSH may modulate the HPG axis.

Gonadotropin-releasing hormone (gnrh) cells arise from cranial neural crest and adenohypophyseal regions of the neural plate in the zebrafish, danio rerio

The olfactory placodes generate the primary sensory neurons of the olfactory sensory system. Additionally, the olfactory placodes have been proposed to generate a class of neuroendocrine cells containing gonadotropin-releasing hormone (GnRH). GnRH is a multifunctional decapeptide essential for the development of secondary sex characteristics in vertebrates as well as a neuromodulator within the central nervous system. Here, we show that endocrine and neuromodulatory GnRH cells arise from two separate, nonolfactory regions in the developing neural plate. Specifically, the neuromodulatory GnRH cells of the terminal nerve arise from the cranial neural crest, and the endocrine GnRH cells of the hypothalamus arise from the adenohypophyseal region of the developing anterior neural plate. Our findings are consistent with cell types generated by the adenohypophysis, a source of endocrine tissue in vertebrate animals, and by neural crest, a source of cells contributing to the cranial nerves. The adenohypophysis arises from a region of the anterior neural plate flanked by the olfactory placode fields at early stages of development, and premigratory cranial neural crest lies adjacent to the caudal edge of the olfactory placode domain [Development 127 (2000), 3645]. Thus, the GnRH cells arise from tissue closely associated with the developing olfactory placode, and their different developmental origins reflect their different functional roles in the adult animal.

Role of Na+ and Ca2+ channels in the preoptic LH surge generating mechanism in proestrous rats.

We studied whether Na+ and Ca2+ channels are involved in the neural mechanism responsible for the surge of gonadotropin-releasing hormone (GnRH) in proestrous rats. In experiment 1, female rats in proestrus were i.p. injected at 1345 h with pentobarbital sodium (35 mg/kg) to block spontaneous surge of LH and electrical stimulation was applied between 1400 and 1600 h to the preoptic area (POA) together with POA injection of 0.5 microl saline containing the Na+ channel blocker tetrodotoxin (TTX) at a concentration of 1 microM, 2 microM, or 5 microM. Since 5 microM TTX completely blocked the increase in serum LH concentrations evoked by the POA stimulation, we used this concentration in experiment 2 to observe the TTX effect on the spontaneous LH surge. In experiment 2, bilateral injections of 1.5 microl of 5 microM TTX at 1430 h in the POA in proestrous rats postponed the peak time and reduced the peak level of the LH surge. In experiment 3, bilateral injections of 1.5 microl of 5 microM L-type Ca2+ channel blocker nifedipine at 1430 h in the POA completely blocked the LH surge. Since the cell bodies of GnRH neurons are primarily concentrated in the POA in rats, these results suggest that both voltage-sensitive Na+ channels and Ca2+ channels contribute to the generation of action potentials at GnRH cell bodies for the surge release of GnRH.

Phosphodiesterase expression targeted to gonadotropin-releasing hormone neurons inhibits luteinizing hormone pulses in transgenic rats.

Experiments in the GT1 gonadotropin-releasing hormone (GnRH) cell line have shown that the cAMP signaling pathway plays a central role in regulating the excitability of the cells. Lowering cAMP levels by expressing the constitutively active cAMP-specific phosphodiesterase PDE4D1 in GT1 cells inhibited spontaneous Ca2+ oscillations and intrinsic pulsatile GnRH secretion. To address the role of cAMP levels in endogenous GnRH neurons, we genetically targeted expression of PDE4D1 (P) to GnRH neurons in transgenic rats (R) by using the GnRH gene promoterenhancer regions (G). Three lines of transgenic rats, GPR-2, -4, and -5, were established. In situ hybridization and RT-PCR studies demonstrated that transgene expression was specifically targeted to GnRH neurons. Decreased fertility was observed in female but not in male rats from all three lines. The mean luteinizing hormone (LH) levels in ovariectomized rats were significantly reduced in the GPR-4 and -5 lines but not in the GPR-2 line. In castrated male and female GPR-4 rats, the LH pulse frequency was dramatically reduced. Six of twelve GPR-4 females studied did not ovulate and had polycystic ovaries. The remaining six females ovulated, but the magnitude of the preovulatory LH surge was inhibited by 63%. These findings support the hypothesis that cAMP signaling may play a central role in regulating excitability of GnRH neurons in vivo. The GPR-4 line of transgenic rats provides a genetic model for the understanding of the role of pulsatile gonadotropin release in follicular development.

Vasoactive intestinal polypeptide contacts on gonadotropin-releasing hormone neurones increase following puberty in female rats.

Successful reproduction requires precise temporal coordination among various endocrine and behavioural events. The circadian system regulates daily temporal organization in behaviour and physiology, including neuroendocrine rhythms. The main circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus. The SCN sends direct efferents to the reproductive axis via monosynaptic projections to gonadotropin-releasing hormone (GnRH) neurones. This communication generates circadian endocrine rhythms as well as the preovulatory luteinizing hormone (LH) surge necessary for successful ovulation. One SCN peptide thought to be important for the regulation of oestrous cycles is vasoactive intestinal polypeptide (VIP). VIP neurones from the SCN contact GnRH cells, and these cells are preferentially activated during an LH surge in rats. Unlike adult rats, prepubertal females do not exhibit oestrous cycles, nor do they exhibit an LH surge in response to oestradiol positive-feedback. The present study was undertaken to determine the extent to which the development of a 'mature' reproductive axis in female rats is associated with modifications in VIP contacts on GnRH neurones. The brains of diestrus adult (approximately 60 days of age) and prepubertal (21 days of age) female rats were examined using double-label fluorescence immunohistochemistry for VIP and GnRH, with light and confocal microscopy. Although the total number of GnRH-immunoreactive neurones did not differ between adult and prepubertal females, adults had a significant increase in the percentage of GnRH cells receiving VIP contacts compared to juveniles. These data suggest that the development of reproductive hormone rhythms and oestrous cyclicity may be, in part, due to modifications of VIP input to the GnRH system.

Neuroendocrine plasticity in GnRH release during rat estrous cycle: correlation with molecular markers of synaptic remodeling

Morphological changes in the gonadotropin releasing hormone (GnRH) neurons in the preoptic area (POA) and their terminals in the median eminence-arcuate (ME-ARC) region are reported to occur during ovarian cycle that may be involved in the GnRH release into the portal blood during preovulatory surge. However, the neuronal substrates participating in altered GnRH neuronal plasticity are poorly understood. The present study was designed to determine whether morphological changes occurring in the GnRH neuron cell bodies in the POA and their terminals in the ME-ARC region of hypothalamus with pulsatile GnRH release in cycling female rats are associated with expression of intrinsic determinants of neuronal plasticity. The plasticity markers studied are polysialylated neural cell adhesion molecule (PSA-NCAM), high molecular weight isoforms of NCAM, growth associated protein (GAP-43), glial fibrillary acidic protein (GFAP) and synaptophysin. Regularly cycling female rats were sacrificed at diestrous, i.e., when GnRH release is low, and at proestrous, i.e., when preovulatory GnRH surge occurs, using perfusion fixation method for immunohistochemical staining of GnRH cells. GnRH cell bodies and their terminals from the POA and ME-ARC region respectively, were localized using immunohistochemical technique in proestrous and diestrous phase of estrous cycle and our results showed a marked increase in the GnRH nerve terminals length and immunoreactivity in the ME-ARC region from proestrous phase rats as compared to diestrous rats. Immunoblot analyses of the POA and ME-ARC region of the hypothalamus revealed a significant increase in the content of PSA-NCAM, NCAM-180, NCAM-140, GAP-43 and synaptophysin from proestrous phase rats as compared to diestrous phase rats. The ME-ARC region showed more pronounced increase in the protein expression of these markers of neuronal plasticity as compared to the POA, whereas, hippocampal region did not show any significant change in the content of these markers showing specificity of the changes to the GnRH system. GFAP content was significantly decreased in the POA with a marginal increase in the GFAP level from the ME-ARC region. These results demonstrate the involvement of synaptic proteins in the dynamic plasticity of the ME-ARC region of hypothalamus, allowing GnRH nerve terminals to release the neurohormone into the pituitary portal blood on the day of proestrous.

Molecular mechanisms for migration of placodally derived GnRH neurons.

Gonadotropin-releasing hormone (GnRH) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain along nasal axons. GnRH neurons appear to diverge from olfactory sensory cells during early stages of nasal placode differentiation. However, GnRH neurons rely on olfactory/vomeronasal axons as their pathway to the central nervous system (CNS). A novel factor, termed nasal embryonic luteinizing hormone-releasing hormone factor (NELF), was discovered in a differential screen of migrating versus nonmigrating GnRH neurons. NELF is expressed in olfactory sensory cells and GnRH cells in nasal areas. Antisense experiments demonstrated that knock-down of NELF decreased olfactory axon outgrowth and GnRH neuronal migration. These results indicate that NELF plays a role as a guidance molecule for olfactory axon projections and migration of GnRH cells. We hypothesize that NELF acts via a homophilic interaction and that NELF expression is critical for reproduction by insuring that GnRH cells reach the CNS. Furthermore, down-regulation of NELF on GnRH cells as they enter the telencephalon may allow GnRH cells to distinguish a different pathway(s) in the CNS (from those leading to olfactory regions) and thereby facilitate establishment of the appropriate adult-like GnRH distribution.

Development of gonadotropin-releasing hormone-1 neurons

Gonadotropin releasing hormone-1 (GnRH-1) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain during prenatal development. Once within the brain, GnRH-1 cells become integral components of the CNS–pituitary–gonadal axis, essential for reproductive maturation and maintenance of reproductive function in adults. This review focuses on the lineage and development of the GnRH-1 neuroendocrine system. Although the migration of these cells from nose to brain has been well documented in a variety of species, many questions remain concerning the melecules and cues directing GnRH-1 cell differentiation, migration, axon targeting, and establishment and control of GnRH-1 secretion. These process most likely involve multiple and redundant cues because if these mechanisms fail, reproduction dysfunction will ensue and guarantee that this defect does not remain in the gene pool.

Mechanisms of differentiation and migration of olfactory progenitors: an overview.

A current focus of research in neurobiology is to define the mechanisms by which neurons reach their final destination and acquire their differentiated phenotype. An ideal model for these studies is the olfactory system, where nasal placodally derived gonadotropin-releasing hormone (GnRH) neurons, olfactory receptor neurons and interneurons of the olfactory bulb (OB)show unique migratory behaviors and phenotypic regulation. Peripheral olfactory receptor cells and OB interneurons, both granule and periglomerular cells, are continuously generated even in adult animals, permitting the comparison of migratory and differentiation phenomena in developing and adult animals. Recent advances in defining the molecular mechanisms directing appropriate targeting and differentiation of both GnRH neurons and OB interneurons were the subject of a symposium at the 23rd annual AChemS meeting. The following four reviews summarize presentations that addressed the intrinsic and extrinsic regulation of pathfinding, cell division and phenotype in migrating progenitor cells. Susan Wray posits that GnRH cells derive from an area of the nasal placode associated with the respiratory system—not, as previously assumed, with the region generating olfactory neuroepithial cells. She also presents data demonstrating a role for the recently identified factor, nasal embryonic luteinizing hormone-releasing hormone factor, in the derivation and migration of GnRH-expressing cells from the olfactory epithelium into the preoptic area and hypothalamus during early gestation. Stewart Anderson addresses issues related to the early derivation of cortical and OB interneurons, including granule and periglomerular cells. OB interneurons derive from the anterior subventricular zone (SVZa)and migrate through the rostral migratory stream (RMS)to populate the granule and glomerular cell layers. Genes required for neuronal fate determination of bulbar and cortical interneuron precursors are identified. Data are also presented to support the hypothesis that early telencephalic dorsal/ventral expression of the signaling molecules, bone morphogenic proteins (BMPs), retinoids and sonic hedgehog pattern the expression of transcription factors that determine neurotransmitter phenotype in the cortex and OB. Aldo Fasolo describes glia- and neuron-associated molecules in the RMS required for directed migration of cells into the OB. Data presented from different species suggest that during early postnatal development glial tubes are not required for appropriate migration of progenitors through the RMS. The authors conclude that glia in the RMS may be implicated in the compartmentalization of a microenvironment necessary for persistent neurogenesis and migration in adults. Molecules regulating the dynamics of stem cell proliferation and diffentiation in the SVZa and in the RMS are the focus of the contribution by Marla Luskin. Specifically discussed is the role of BMP signaling in regulating the cell cycle inhibitor p19 INK4d . The latter is expressed at highest levels in regions where proliferation is low—specifically, in the ventricular ependymal zone, in more anterior aspects of the RMS and in the OB. In sum, the data described in these reviews address current concepts on a broad range of issues related to olfactory system development.

Increased galanin synapses onto activated gonadotropin-releasing hormone neuronal cell bodies in normal female mice and in functional preoptic area grafts in hypogonadal mice.

Galanin synaptic input onto gonadotropin-releasing hormone (GnRH) neuronal cell bodies was analysed in female mice using the presynaptic vesicle-specific protein, synaptophysin (Syn) as a marker. In the first experiment, forebrain sections from normal ovariectomized ovarian steroid-primed mice exhibiting a surge of luteinizing hormone were processed for immunohistochemical labelling for GnRH, synaptophysin, galanin and Fos. Two representative sections from each brain, one passing through the anterior septum (anterior section) and the other through the organum vasculosum lamina terminalis-preoptic area (posterior section), were analysed under the confocal microscope. None of the GnRH cells analysed in the anterior sections were Fos immunoreactive (IR) or received input from galanin-IR fibres. In contrast, the majority of GnRH cells in the posterior sections analysed were Fos-positive. The number of galanin synapses onto the Fos-positive GnRH cells was significantly higher than that in the Fos-negative cells in this area of the brain, even though the number of Syn-IR appositions was comparable to each other. Transplantation of preoptic area (POA) into the third cerebral ventricle of hypogonadal (HPG) mice corrects deficits in the reproductive system. In the second experiment, synaptic input to GnRH cells was compared between HPG/POA mice with (functional graft) or without (nonfunctional graft) gonadal development. The mean numbers of Syn-IR appositions and galanin synapses per GnRH cell and the proportion of GnRH cells with galanin input were significantly higher in the functional than in the nonfunctional grafts. The results suggest that galanin can act directly on the GnRH cell bodies and may have an important regulatory role on the GnRH system.

Redefining gonadotropin-releasing hormone (GnRH) cell groups in the male Syrian hamster: testosterone regulates GnRH mRNA in the tenia tecta.

Gonadotropin-releasing hormone (GnRH) regulates the production of testosterone via the hypothalamic-pituitary-gonadal axis and testosterone, in turn, regulates the GnRH system via negative feedback. We compared testosterone regulation of GnRH mRNA expression in four anatomically defined GnRH cell groups in juvenile and adult male Syrian hamsters, including a rostral population of GnRH cells in the tenia tecta. In situ hybridization histochemistry (ISHH) was used to measure GnRH mRNA in brains from castrated juveniles and adults treated with 0 mg or 2.5 mg testosterone pellets for one week. ISHH was performed on coronal sections using a 35S-cRNA probe generated from Syrian hamster GnRH cDNA. Testosterone treatment resulted in a significant reduction in mean area of GnRH neurones covered by silver grains within the tenia tecta, but only a trend toward decreased GnRH mRNA in the diagonal band of Broca/organum vasculosum of the lamina terminalis (DBB/OVLT), medial septum (MS), and caudal preoptic area (cPOA). The effects of testosterone were independent of age. Frequency distribution analyses unveiled a significant reduction in the number of heavily labelled cells following testosterone treatment within the tenia tecta and MS. Simple regression analyses revealed a significant positive correlation between plasma luteinizing hormone concentrations and GnRH mRNA only in the tenia tecta. These data indicate that, overall, GnRH mRNA is modestly reduced by testosterone, and the most robust attenuation of GnRH mRNA occurs within the tenia tecta. This is the first report to link mechanisms of steroid negative feedback with tenia tecta GnRH neurones, providing a new focus for investigating brain region-specific steroidal regulation of GnRH synthesis.

Age‐Related Changes in Hypothalamic Gonadotropin‐Releasing Hormone and N‐Methyl‐d‐Aspartate Receptor Gene Expression, and their Regulation by Oestrogen, in the Female Rat

During reproductive ageing, the oestrous cycles of female rats become irregular and eventually cease. The mechanisms for reproductive senescence in rodents are believed to involve changes in hypothalamic neurones, including gonadotropin-releasing hormone (GnRH) cells and their afferent inputs. In addition, effects of oestrogen on hypothalamic function may vary in animals of different ages. These issues were addressed using young (aged 4-5 months), middle-aged (12-14 months) and old (24-26 months) female Sprague-Dawley rats. Animals were ovariectomized and given oestrogen or vehicle replacement. They were killed and the preoptic area-anterior hypothalamus (POA-AH) and the medial basal hypothalamus-median eminence (MBH-ME) were dissected out, RNA extracted, and RNase protection assay used to quantify gene expression of several hypothalamic molecules. In the first experiment, GnRH RNA levels were measured in the POA-AH. No effects of ageing or oestrogen were observed on GnRH gene expression. This finding suggests that ageing and oestrogen may affect GnRH release from neuroterminals independently of de novo biosynthesis, and that this may involve other neurones that affect GnRH neurosecretory function. In the second experiment, we investigated changes in N-methyl-D-aspartate (NMDA) receptor subunit mRNA levels. These receptors play an important regulatory role in mediating effects of glutamate on GnRH function, and are themselves regulated by oestrogen and ageing. NMDA receptor subunit (NR) 1, 2a and 2b mRNA levels were quantified in the POA-AH and MBH-ME, the sites of GnRH perikarya and neuroterminals, respectively. In general, oestrogen had inhibitory effects on NR1 and NR2a, and differential effects on NR2b subunit mRNA levels. NMDA receptor subunit mRNA levels also changed during ageing: age-related decreases in NR1 mRNA occurred in the MBH-ME, and an age-related increase in NR2b mRNA occurred in the POA-AH. Taken together, these results demonstrate subunit- and region-specific changes in hypothalamic NMDA receptor subunit gene expression with oestrogen and ageing. These alterations could have implications for the physiological effects of glutamate on its NMDA receptor, and impact the regulation of reproductive and other neuroendocrine and autonomic functions by hypothalamic glutamatergic inputs.

Distribution of gonadotropin-releasing hormone neurones in the chick forebrain is independent of lineage relationships among cells of the early nasal placode.

The regulation of reproduction depends upon the successful migration of gonadotropin-releasing hormone (GnRH) neurones from the nasal placode to the ventral forebrain during embryogenesis. Within the central nervous system (CNS), these neurones migrate to stereotyped, highly reproducible locations in septal, preoptic and hypothalamic nuclei. We postulated that lineage relationships (descent from a common precursor) might predict the final location of these neurones. To test this hypothesis, a complex retroviral library was used to label dividing cells in the placode and subsequently to identify them by the presence of the alkaline phosphatase marker. GnRH was detected immunocytochemically and lineage relationships determined by single cell polymerase chain reaction and sequencing of the degenerate oligonucleotide component of the retrovirus. GnRH-positive and GnRH-negative neurones were confined to the side ipsilateral to the injection; many cells derived from the placode that entered the CNS did not contain GnRH. This precise method of identifying and mapping the progeny of single neurones revealed that GnRH cells in any given area were derived from multiple precursors. This developmental pattern may contribute to assuring that all CNS locations critical to the orchestration of reproductive events will be populated by GnRH neurones.

Evidence in favour of a direct input from the ventromedial nucleus to gonadotropin-releasing hormone neurones in the ewe: an anterograde tracing study.

The mechanism by which oestrogen activates the gonadotropin releasing hormone (GnRH) neurones to induce the preovulatory luteinizing hormone (LH) surge is not understood. Previous work in the ewe has suggested that the primary site of action for oestradiol in stimulating the GnRH neurones was in the region of the ventromedial nucleus (VMN) within the mediobasal hypothalamus (MBH). In the present study, we used anterograde tracing techniques in the ewe to investigate whether direct neuronal projections may exist from neurones located in the region of the VMN to the GnRH neurones. Following the injection of biotinylated dextran amine into the VMN of four ewes, anterogradely labelled fibres were found located principally within the ipsilateral diagonal band of Broca (DBB), septum, preoptic and anterior hypothalamic areas, and periventricular, paraventricular, dorsomedial and arcuate nuclei of the MBH. Dual-labelling for GnRH revealed that fibres containing anterograde tracer were adjacent to the soma and/or dendrites of approximately 50% of all ipsilateral GnRH neurones located throughout the DBB and hypothalamus. Few anterogradely labelled fibres were detected within the median eminence. Although such studies cannot define the presence of direct synaptic connections between VMN neurones and the GnRH cells, these observations support further the hypothesis that oestrogen-sensitive VMN neurones represent a direct transsynaptic input to the GnRH cell bodies which are involved in the generation of the LH surge in the ewe.

Evidence that orexin-containing neurones provide direct input to gonadotropin-releasing hormone neurones in the ovine hypothalamus.

Orexins A and B (ORX) have been added recently to the growing list of neuropeptides implicated in feeding and drinking behaviour as well as neuroendocrine function. In the present study, we have used single and dual labelling immunohistochemistry and a rabbit polyclonal anti-orexin-A antibody, which recognizes both ORX A and B, to examine ORX pathways in the sheep hypothalamus. ORX immunoreactive cells were distributed in the dorsomedial hypothalamic nucleus, lateral hypothalamic area, zona incerta and perifornical area; a few cells were also observed in the anterior hypothalamic area. In contrast to distribution in the rat brain, most of the ORX immunoreactive cells are localized to the dorsomedial hypothalamic nucleus and perifornical area; scattered cells are found in lateral hypothalamic area. ORX immunoreactive fibres were widely distributed throughout the hypothalamus and preoptic area with dense innervation of the medial preoptic area and bed nucleus of stria terminalis. Dual labelling demonstrated widespread expression of the long form of the leptin receptor within all ORX cells that were examined. Thirty percent of the gonadotropin releasing hormone (GnRH) cells that were examined had ORX immunoreactive terminals in close contact with no regional or sex differences. FluoroGold injections into the preoptic area retrogradely labelled a subpopulation of ORX cells in the lateral hypothalamic/perifornical area, showing ORX cells of this region project to the preoptic and could potentially provide input to GnRH cells. These findings suggest an integral role for ORX in the regulation of GnRH cells in the sheep and thus provide evidence of a novel mechanism whereby leptin can influence reproductive neuroendocrine function.

Molecular and cellular properties of GnRH neurons revealed through transgenics in the mouse

Recent advances in the use of gonadotropin-releasing hormone (GnRH) promoter-driven transgenics in the mouse are beginning to open up the once elusive GnRH neuronal phenotype to detailed molecular and cellular investigation. This review highlights progress in the development of GnRH promoter transgenic constructs and the understanding of murine gene sequences required for the correct temporal and spatial targeting of transgenes to the GnRH phenotype in vivo. Strategies enabling the identification of single, living GnRH neurons in the acute brain slice preparation are allowing gene profiling and electrophysiological experiments to be undertaken. Results so far indicate that, like other neurons, GnRH cells express a variety of sodium, potassium and calcium channels as well as GABAergic and glutamatergic receptors which are responsible for determining the membrane properties and firing characteristics of the GnRH neuron. Many of these receptors and channels appear to be expressed heterogeneously within the GnRH phenotype. Furthermore, several display distinct postnatal developmental expression profiles which are likely to be of consequence to the development of synchronized, pulsatile GnRH secretion in the adult animal.

Galanin synaptic input to gonadotropin-releasing hormone perikarya in juvenile and adult female mice: implications for sexual maturity

Changes in connectivity of the gonadotropin-releasing hormone (GnRH) neuronal system are believed to occur during the transition from juvenile to adulthood in females. Experiments were designed to investigate whether there is any difference in the number of galanin inputs to GnRH cells located in the organum vasculosum of lamina terminalis-rostral preoptic area (OVLT-rPOA) between juvenile (2 weeks old) and adult (10 weeks old) female mice. Triple label immunofluorescence staining of brain sections for galanin, GnRH and the presynaptic vesicle marker synaptophysin coupled with confocal microscopy was employed to identify galanin synapses to GnRH perikarya. The number of galanin synapses to GnRH cells and the proportion of GnRH cells with galanin input were significantly higher in adults than in juvenile mice. In adult mice, the proportions of GnRH cells with 0, 1, 2, 3, 4, 5 or 6 galanin synapses/cell were comparable to each other whereas in the juveniles the vast majority of them received no galanin synaptic input. A greater number of galanin synapses in adult as compared with juvenile female mice suggests a functional role for galanin in the maturation of the GnRH system.

Appositions between cocaine and amphetamine-related transcript- and gonadotropin releasing hormone-immunoreactive neurons in the hypothalamus of the Siberian hamster

Recent in vitro studies have provided evidence that cocaine and amphetamine-related transcript (CART) pathways in the hypothalamus mediate the effects of leptin upon gonadotropin releasing hormone (GnRH) secretion. The aim of the current study was to use dual label immunofluorescence to investigate the anatomical basis of such a pathway. CART-ir processes were found extensively in regions where GnRH cell bodies where located. Analysis using confocal microscopy showed that the majority of GnRH neurons (62%) had close appositions from CART-ir processes. The proportion of GnRH-ir perikarya with CART-ir appositions was significantly higher ( P<0.05) in neurons located in the diagonal band of Broca (70%) compared to those more caudally located in the preoptic area (53%). This anatomical evidence for close appositions between CART-ir processes and GnRH cell bodies supports the hypothesis that one mechanism by which leptin causes its effect on the GnRH pulse generator is indirectly via CART neurons, thus allowing information about nutritional status and body fat stores to be conveyed to the reproductive system.

Subsets of gonadotropin-releasing hormone (GnRH) neurons are activated during a steroid-induced luteinizing hormone surge and mating in mice: a combined retrograde tracing double immunohistochemical study

The decapeptide gonadotropin-releasing hormone (GnRH) plays a pivotal role in reproduction and is synthesized by GnRH-producing cell bodies in the basal forebrain. Experiments were designed to investigate whether GnRH cells projecting outside the blood brain barrier or those projecting within the brain are activated during the steroid-induced LH surge or mating in female mice. Retrograde uptake of intraperitoneally administered fluorogold (FG) by GnRH cells and double immunostaining for GnRH and Fos was employed for this purpose. The number of GnRH cells with FG uptake was comparable among the surged, mated and control mice. However, the number of Fos-positive GnRH cells was significantly higher in the steroid-induced LH surge group than in the mated mice. The number of Fos+FG-positive GnRH cells was higher and the number of FG-only GnRH cells was lower in mice with a steroid-induced LH surge as compared with the mated mice. This suggests the existence of a subgroup of GnRH cells projecting outside the blood–brain barrier activated during the steroid-induced LH surge but not during mating. The activation of similar proportions of GnRH cells without FG uptake in both the mated and the surge group indicate that nonneuroendocrine GnRH cells are not silent but can be activated by both mating and steroid hormones. Thus, functional subgroups may exist within the GnRH system with considerable overlap in the input to these cells.

Oestrogen receptor beta-immunoreactivity in gonadotropin releasing hormone-expressing neurones: regulation by oestrogen.

Double-label immunohistochemistry was employed to establish whether immunoreactivity for the beta subtype of the oestrogen receptor (ER beta-IR) is present in gonadotropin releasing hormone (GnRH)-containing cells. In the immortalized GnRH cell line, GT1-7, almost all nuclei were immunoreactive for ER beta. In the preoptic area of ovariectomized rats, more than one-half of the GnRH neurones (52.0-63.5%) contained ER beta-IR within the nucleus; a smaller proportion of these neurones (5-10%) displayed a particularly intense nuclear signal for ER beta. The presence of ER beta-IR in the nuclei of GT1-7 cells and GnRH neurones is consistent with recent reports of ER beta mRNA in these cells. Oestrogen treatment reduced the percentage of GnRH neurones with detectable ER beta-IR. The range of signal intensity for ER beta and the incidence of the ER beta signal in GnRH neurones were comparable following double-label immunohistochemistry involving either bright field or fluorescent techniques. These findings raise the possibility that ER beta receptors mediate direct effects of oestrogen on GnRH neurones.