Gonadotropin-releasing Hormone Nerve Terminals Research Articles

Neural Control of Sexual Behavior in Fish.

Many vertebrate species show breeding periods and exhibit series of characteristic species-specific sexual behaviors only during the breeding period. Here, secretion of gonadal sex hormones from the mature gonads has been considered to facilitate sexual behaviors. Thus, the sexual behavior has long been considered to be regulated by neural and hormonal mechanisms. In this review, we discuss recent progress in the study of neural control mechanisms of sexual behavior with a focus on studies using fish, which have often been the favorite animals used by many researchers who study instinctive animal behaviors. We first discuss control mechanisms of sexual behaviors by sex steroids in relation to the anatomical studies of sex steroid-concentrating neurons in various vertebrate brains, which are abundantly distributed in evolutionarily conserved areas such as preoptic area (POA) and anterior hypothalamus. We then focus on another brain area called the ventral telencephalic area, which has also been suggested to contain sex steroid-concentrating neurons and has been implicated in the control of sexual behaviors, especially in teleosts. We also discuss control of sex-specific behaviors and sexual preference influenced by estrogenic signals or by olfactory/pheromonal signals. Finally, we briefly summarize research on the modulatory control of motivation for sexual behaviors by a group of peptidergic neurons called terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which are known to be especially developed in fishes among various vertebrate species.

Co-existing Neuropeptide FF and Gonadotropin-Releasing Hormone 3 Coordinately Modulate Male Sexual Behavior.

Animals properly perform sexual behaviors by using multiple sensory cues. However, neural mechanisms integrating multiple sensory cues and regulating motivation for sexual behaviors remain unclear. Here, we focused on peptidergic neurons, terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons, which receive inputs from various sensory systems and co-express neuropeptide FF (NPFF) in addition to GnRH. Our behavioral analyses using knockout medaka of GnRH (gnrh3) and/or NPFF (npff) demonstrated that some sexual behavioral repertoires were delayed, not disrupted, in gnrh3 and npff single knockout males, while the double knockout appeared to alleviate the significant defects that were observed in single knockouts. We also found anatomical evidence to show that both neuropeptides modulate the sexual behavior-controlling brain areas. Furthermore, we demonstrated that NPFF activates neurons in the preoptic area via indirect pathway, which is considered to induce the increase in motivation for male sexual behaviors. Considering these results, we propose a novel mechanism by which co-existing peptides of the TN-GnRH neurons, NPFF, and GnRH3 coordinately modulate certain neuronal circuit for the control of behavioral motivation. Our results may go a long way toward understanding the functional significance of peptidergic neuromodulation in response to sensory information from the external environments.

Developing neurites from mouse basal forebrain gonadotropin-releasing hormone neurons use Sonic hedgehog to modulate their growth

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are required for fertility in all mammalian species studied to date. GnRH neuron cell bodies reside in the basal forebrain, and most extend long neurites in the caudal direction to terminate at the median eminence (ME), the site of hormone secretion.Using in vitro neurite growth assays, histological methods, and genetic deletion strategies in mice we have analysed the role of the morphogen and neurite growth and guidance molecule, Sonic hedgehog (Shh), in the growth of GnRH neurites to their target. Immunohistochemistry revealed that Shh was present in the basal forebrain, the preoptic area (POA) and mediobasal hypothalamus (MBH) at gestational day 14.5 (GD 14.5), a time when GnRH neurites grow towards the ME. Furthermore, in situ hybridization revealed that mRNA encoding the Shh receptor, Smoothened (Smo), was present in GnRH neurons from GD 15.5, when the first GnRH neurites are extending towards the MBH. In vitro neurite growth assays using hypothalamic explants from GD 15.5 fetuses in 3-D collagen gels showed that Shh was able to significantly stimulate GnRH neurite outgrowth. Finally, genetic deletion of Smo specifically from GnRH neurons in vivo, using Cre-loxP technology, resulted in a significant decrease in GnRH neurites innervating the ME.These experiments demonstrate that GnRH neurites use Shh for their neurite development, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional hypothalamic neuronal population for which Shh/Smo signaling is developmentally important.

The Role of Kiss1 Neurons As Integrators of Endocrine, Metabolic, and Environmental Factors in the Hypothalamic-Pituitary-Gonadal Axis.

Kisspeptin–GPR54 signaling in the hypothalamus is required for reproduction and fertility in mammals. Kiss1 neurons are key regulators of gonadotropin-releasing hormone (GnRH) release and modulation of the hypothalamic–pituitary–gonadal (HPG) axis. Arcuate Kiss1 neurons project to GnRH nerve terminals in the median eminence, orchestrating the pulsatile secretion of luteinizing hormone (LH) through the intricate interaction between GnRH pulse frequency and the pituitary gonadotrophs. Arcuate Kiss1 neurons, also known as KNDy neurons in rodents and ruminants because of their co-expression of neurokinin B and dynorphin represent an ideal hub to receive afferent inputs from other brain regions in response to physiological and environmental changes, which can regulate the HPG axis. This review will focus on studies performed primarily in rodent and ruminant species to explore potential afferent inputs to Kiss1 neurons with emphasis on the arcuate region but also considering the rostral periventricular region of the third ventricle (RP3V). Specifically, we will discuss how these inputs can be modulated by hormonal, metabolic, and environmental factors to control gonadotropin secretion and fertility. We also summarize the methods and techniques that can be used to study functional inputs into Kiss1 neurons.

Juvenile-Specific Burst Firing of Terminal Nerve GnRH3 Neurons Suggests Novel Functions in Addition to Neuromodulation.

Peptidergic neurons are suggested to play a key role in neuromodulation of animal behaviors in response to sensory cues in the environment. Terminal nerve gonadotropin-releasing hormone 3 (TN-GnRH3) neurons are thought to be one of the peptidergic neurons important for such neuromodulation in adult vertebrates. On the other hand, it has been reported that TN-GnRH3 neurons are labeled by a specific GnRH3 antibody from early developmental stages to adulthood and are thus suggested to produce mature GnRH3 peptide even in the early developmental stages. However, it remains unknown when TN-GnRH3 neurons show spontaneous burst firing, which is suggested to be involved in neuropeptide release. Using a whole-brain in vitro preparation of gnrh3:enhanced green fluorescent protein (EGFP) medaka fish, we first recorded spontaneous firings of TN-GnRH3 neurons after hatching to adulthood. Contrary to what one would expect from their neuromodulatory functions-that TN-GnRH3 neurons are more active in adulthood-TN-GnRH3 neurons in juveniles showed spontaneous burst firing more frequently than in adulthood (juvenile-specific burst firing). Ca2+ imaging of TN-GnRH3 neurons in juveniles may further suggest that juvenile-specific burst firing triggers neuropeptide release. Furthermore, juvenile-specific burst firing was suggested to be induced by blocking persistent GABAergic inhibition to the glutamatergic neurons, which leads to an increase in glutamatergic synaptic inputs to TN-GnRH3 neurons. The present study reports that peptidergic neurons show juvenile-specific burst firing involved in triggering peptide release and suggests that juvenile TN-GnRH3 neurons have novel functions, in addition to neuromodulation.

Neuropeptide RFRP inhibits the pacemaker activity of terminal nerve GnRH neurons

The terminal nerve gonadotropin-releasing hormone (TN-GnRH) neurons show spontaneous pacemaker activity whose firing frequency is suggested to regulate the release of GnRH peptides and control motivation for reproductive behaviors. Previous studies of the electrophysiological properties of TN-GnRH neurons reported excitatory modulation of pacemaker activity by auto/paracrine and synaptic modulations, but inhibition of pacemaker activity has not been reported to date. Our recent study suggests that neuropeptide FF, a type of Arg-Phe-amide (RFamide) peptide expressed in TN-GnRH neurons themselves, inhibits the pacemaker activity of TN-GnRH neurons in an auto- and paracrine manner. In the present study, we examined whether RFamide-related peptides (RFRPs), which are produced in the hypothalamus, modulate the pacemaker activity of TN-GnRH neurons as candidate inhibitory synaptic modulators. Bath application of RFRP2, among the three teleost RFRPs, decreased the frequency of firing of TN-GnRH neurons. This inhibition was diminished by RF9, a potent antagonist of GPR147/74, which are candidate RFRP receptors. RFRP2 changed the conductances for Na(+) and K(+). The reversal potential for RFRP2-induced current was altered by inhibitors of the transient receptor potential canonical (TRPC) channel (La(3+) and 2-aminoethoxydiphenyl borate) and by a less selective blocker of voltage-independent K(+) channels (Ba(2+)). By comparing the current-voltage relationship in artificial cerebrospinal fluid with that under each drug, the RFRP2-induced current was suggested to consist of TRPC channel-like current and voltage-independent K(+) current. Therefore, synaptic release of RFRP2 from hypothalamic neurons is suggested to inhibit the pacemaker activity of TN-GnRH neurons by closing TRPC channels and opening voltage-independent K(+) channels. This novel pathway may negatively regulate reproductive behaviors.

Netrin-1 Stimulates Developing GnRH Neurons to Extend Neurites to the Median Eminence in a Calcium- Dependent Manner

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are required for fertility in all mammalian species studied to date. In rodents, GnRH neuron cell bodies reside in the rostral hypothalamus, and most extend a single long neuronal process in the caudal direction to terminate at the median eminence (ME), the site of hormone secretion. The molecular cues that GnRH neurites use to grow and navigate to the ME during development, however, remain poorly described. Reverse transcription-PCR (RT-PCR) identified mRNAs encoding Netrin-1, and its receptor, DCC, in the fetal preoptic area (POA) and mediobasal hypothalamus (MBH), respectively, from gestational day 12.5 (GD12.5), a time when the first GnRH neurites extend toward the MBH. Moreover, a subpopulation of GnRH neurons from GD14.5 through GD18.5 express the Netrin-1 receptor, DCC, suggesting a role for Netrin-1/DCC signaling in GnRH neurite growth and/or guidance. In support of this notion, when GD15.5 POA explants, containing GnRH neurons actively extending neurites, were grown in three-dimensional collagen gels and challenged with exogenous Netrin-1 (100 ng/ml or 400 ng/ml) GnRH neurite growth was stimulated. In addition, Netrin-1 provided from a fixed source was able to stimulate outgrowth, although it did not appear to chemoattract GnRH neurites. Finally, the effects of Netrin-1 on the outgrowth of GnRH neurites could be inhibited by blocking either L-type voltage-gated calcium channels (VGCCs) with nifedipine (10 µM), or ryanodine receptors with ryanodine (10 µM). This is consistent with the role of Ca2+ from extra- and intracellular sources in Netrin-1/DCC-dependent growth cone motility in other neurons. These results indicate that Netrin-1 directly stimulates the growth of a subpopulation of GnRH neurites that express DCC, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional neuronal population whose neurites utilize Netrin-1/DCC signaling for their development.

Gliotransmission by Prostaglandin E2: A Prerequisite for GnRH Neuronal Function?

Over the past four decades it has become clear that prostaglandin E2 (PGE2), a phospholipid-derived signaling molecule, plays a fundamental role in modulating the gonadotropin-releasing hormone (GnRH) neuroendocrine system and in shaping the hypothalamus. In this review, after a brief historical overview, we highlight studies revealing that PGE2 released by glial cells such as astrocytes and tanycytes is intimately involved in the active control of GnRH neuronal activity and neurosecretion. Recent evidence suggests that hypothalamic astrocytes surrounding GnRH neuronal cell bodies may respond to neuronal activity with an activation of the erbB receptor tyrosine kinase signaling, triggering the release of PGE2 as a chemical transmitter from the glia themselves, and, in turn, leading to the feedback regulation of GnRH neuronal activity. At the GnRH neurohemal junction, in the median eminence of the hypothalamus, PGE2 is released by tanycytes in response to cell–cell signaling initiated by glial cells and vascular endothelial cells. Upon its release, PGE2 causes the retraction of the tanycyte end-feet enwrapping the GnRH nerve terminals, enabling them to approach the adjacent pericapillary space and thus likely facilitating neurohormone diffusion from these nerve terminals into the pituitary portal blood. In view of these new insights, we suggest that synaptically associated astrocytes and perijunctional tanycytes are integral modulatory elements of GnRH neuronal function at the cell soma/dendrite and nerve terminal levels, respectively.

Kisspeptin Signaling Is Required for Peripheral But Not Central Stimulation of Gonadotropin-Releasing Hormone Neurons by NMDA

NMDA and kisspeptins can stimulate gonadotropin-releasing hormone (GnRH) release after peripheral or central administration in mice. To determine whether these agonists act independently or through a common pathway, we have examined their ability to stimulate GnRH/luteinizing hormone (LH) release after peripheral or central administration in Kiss1- or Gpr54 (Kiss1r)-null mutant mice. Peripheral injection of NMDA failed to stimulate GnRH/LH release in prepubertal or gonadally intact mutant male mice. Dual-labeling experiments indicated a direct activation of Kiss1-expressing neurons in the arcuate nucleus. In contrast, central injection of NMDA into the lateral ventricle increased plasma LH levels in both Kiss1 and Gpr54 mutant male mice similar to the responses in wild-type mice. Central injection of NMDA stimulated c-Fos expression throughout the hypothalamus but not in GnRH neurons, suggesting an action at the nerve terminals only. In contrast, kisspeptin-10 stimulated LH release after both central and peripheral injection but induced c-Fos expression in GnRH neurons only after central administration. Finally, central injection of NMDA induces c-Fos expression in catecholamine- and nitric oxide-producing neurons in the hypothalamus of mutant mice, indicating a possible kisspeptin-independent GnRH/LH release by NMDA through activation of these neurons. Thus, NMDA may act at both GnRH cell bodies (kisspeptin-independent) and nerve terminals (kisspeptin-dependent) in a dual way to participate in the GnRH/LH secretion in the male mouse.

Three‐dimensional properties of GnRH neuroterminals in the median eminence of young and old rats

The decapeptide gonadotropin-releasing hormone (GnRH), which regulates reproduction in all vertebrates, is stored in, and secreted from, large dense-core secretory vesicles in nerve terminals in the median eminence. GnRH is released from these terminals with biological rhythms that are critical for the maintenance of normal reproduction. During reproductive aging in female rats, there is a loss of GnRH pulses and a diminution of the GnRH surge. However, information about the specific role of GnRH nerve terminals is lacking, particularly in the context of aging. We sought to gain novel ultrastructural information about GnRH neuroterminals by performing three-dimensional (3D) reconstructions of GnRH neuroterminals and their surrounding microenvironment in the median eminence of young (4-5 months) and old (22-24 months) ovariectomized Sprague-Dawley female rats. Median eminence tissues were freeze-plunge embedded and serial ultrathin sections were collected on slot grids for immunogold labeling of GnRH immunoreactivity. Sequential images were used to create 3D models of GnRH terminals. These reconstructions provided novel perspectives into the morphological properties of GnRH terminals and their neural and glial environment. We also noted that the cytoarchitectural features of the median eminence became disorganized with aging. Quantitative measures showed a significant decrease in the apposition between GnRH terminal membranes and glial cells. Our data suggest reproductive aging in rats is characterized by structural organizational changes to the GnRH terminal microenvironment in the median eminence.

Kisspeptin Signaling in Rodents: Insights from Knock-Out Mice.William H. Colledge, Ph.D.

Initiation of gonadotropin-releasing hormone (GnRH) secretion from the hypothalamus at puberty stimulates luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from the anterior pituitary to galvanize maturation of the gonads. A key molecular event that initiates this activation of GnRH secretion has recently been identified with the discovery that kisspeptin neuropeptides are potent agonists of GnRH release. Kisspeptins are a family of overlapping amidated peptides encoded by the Kiss1 gene that signal through the G-protein coupled receptor GPR54 (KISS1R). Five transgenic mouse lines have been generated with mutations in the Gpr54 gene and two lines with mutations in the Kiss1 gene. The phenotypes of these mutant mice are broadly similar, suggesting no obvious functional compensation by other ligands or receptors. The mutant mice fail to undergo puberty and have poor gonadal development and low sex steroid and gonadotropic hormone levels (hypogonadotropic hypogonadism). Spermatogenesis and ovulation are severely impaired and mutant females do not show normal estrous cyclicity. The gonads and the anterior pituitary retain functional responses to hormonal stimulation, however, consistent with the primary defect being a failure to secrete GnRH from the hypothalamus to activate the hypothalamic-pituitary-gonadal axis. These mutant mice are being used to interrogate the function of GPR54 and kisspeptins in key aspects of kisspeptin signaling including the role of these proteins in regulating the neuroendocrine responses of GnRH neurons. Kisspeptin expression is regulated by gonadal steroids (testosterone or estrogen) and this provides a possible mechanism by which these steroid hormones control GnRH secretion. We have shown that kisspeptin-GPR54 signaling is essential for GnRH neuronal activation and the pre-ovulatory LH surge. Using an ovariectomized, gonadal steroid-replacement regimen, which reliably generates an LH surge, 30% of kisspeptin neurons express c-FOS in surging mice compared with 0% in non-surging controls. A strong correlation was found between the percentage of c-FOS positive kisspeptin neurons and the percentage of c-FOS-positive GnRH neurons. To evaluate whether kisspeptin and/or GPR54 are essential for GnRH neuron activation and the LH surge, Gpr54 and Kiss1 null mice were examined. Wild-type mice all exhibited LH surges and c-FOS in ~50% of their GnRH neurons, but none of the mutant mice from either line showed an LH surge or any GnRH neurons with c-FOS. These observations provide the first evidence that kisspeptin-GPR54 signaling is essential for GnRH neuron activation that initiates ovulation. We have also shown that kisspeptin can stimulate GnRH secretion by a direct action at GnRH nerve terminals in addition to its likely action at the cell bodies. These mutant mice provide a useful resource to further understand the hypothalamic regulation of mammalian reproduction, its integration with the pituitary-gonadal axis and to study the potential function of Gpr54 and Kiss1 in peripheral tissues. This research was funded by Takeda Cambridge and BBSRC Grant BB/C003861/1.

Kisspeptin Can Stimulate Gonadotropin-Releasing Hormone (GnRH) Release by a Direct Action at GnRH Nerve Terminals

The G protein-coupled receptor GPR54, and its peptide ligand kisspeptin (Kp), are crucial for the induction and maintenance of mammalian reproductive function. GPR54 is expressed by GnRH neurons and is directly activated by Kp to stimulate GnRH release. We hypothesized that Kp may be able to act at the GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region. To test this hypothesis, we used organotypic culture of MBH explants challenged with Kp, followed by RIA to detect GnRH released into the cultured medium. Kp stimulation for 1 h induced GnRH release from wild-type male MBH in a dose-dependent manner, whereas this did not occur in MBH explants isolated from Gpr54 null mice. Continuous Kp stimulation caused a sustained GnRH release for 4 h, followed by a decrease of GnRH release, suggesting a desensitization of GPR54 activity. Tetrodotoxin did not alter the Kp-induced GnRH release, indicating that Kp can act directly at the GnRH nerve terminals. To localize Gpr54 expression within the MBH, we used transgenic mice, in which Gpr54 expression is tagged with an IRES-LacZ reporter gene and can be visualized by beta-galactosidase staining. Gpr54 expression was detected outside of the median eminence, in the pars tuberalis. In conclusion, our results provide evidence for a potent stimulating effect of Kp at GnRH nerve terminals in the MBH of the mouse. This study suggests a new point at which Kp can act on GnRH neurons.

Development of GnRH cells: Setting the stage for puberty

Cells containing gonadotropin-releasing hormone (GnRH) are essential not only for reproduction but also for neuromodulatory functions in the adult animal. A variety of studies have hinted at multiple origins for GnRH-containing cells in the developing embryo. We have shown, using zebrafish as a model system, that GnRH cells originate from precursors lying outside the olfactory placode: the region of the anterior pituitary gives rise to hypothalamic GnRH cells and the cranial neural crest gives rise to the GnRH cells of the terminal nerve and midbrain. Cells of both the forming anterior pituitary and cranial neural crest are closely apposed to the precursors of the olfactory epithelium during early development. Disruption of kallmann gene function results in loss of the hypothalamic but not the terminal nerve GnRH cells during early development. The GnRH proteins are expressed early in development and this expression is mirrored by the onset of GnRH receptor (GnRH-R) expression during early development. Thus the signaling of the GnRH neuronal circuitry is set up early in development laying the foundation for the GnRH network that is activated at puberty leading to reproductive function in the mature animal.

Presence of delta opioid receptors on a subset of hypothalamic gonadotropin releasing hormone (GnRH) neurons

Opioid peptides exert an inhibitory effect on hypothalamic gonadotropin releasing hormone (GnRH) secretion mainly by interacting with μ-opioid receptors. Although a direct role for opioids via δ-opioid receptors (DORs) has been suggested, the presence of these receptors on GnRH neurons has never been demonstrated. In the present study, we determined the distribution of DORs in the basal hypothalamus of rat with special focus on their relation to GnRH neurons. Double-labelling immunofluorescence and confocal microscopy revealed that DORs are exclusively present in a subpopulation of GnRH nerve terminals, with the highest density in the external layer of the median eminence. We then studied the functional characteristics of DORs in an immortalized GnRH-secreting neuronal cell line (GT1-1) known to endogenously express this receptor. Here, pertussis toxin pretreatment abolished the δ-agonist (DPDPE) inhibitory effect on cAMP accumulation. We also analyzed the type of G proteins involved in the signal transduced by the DOR and showed that GT1-1 cells express the inhibitory Go and Gi2 alpha-subunits. However, only Go was down-regulated under chronic DPDPE exposure. Finally, since DOR is expressed postnatally in brain, we compared GnRH neuronal cells immortalized at different developmental stages (the more mature GT1-1 and GT1-7 cells, versus the more immature GN11 cells), evidencing that only mature neurons express DOR. In conclusion, our study indicates that a direct control of opioids via δ-receptors occurs on GnRH neurons and validates the use of GT1 cells to further investigate the nature of the DOR present on GnRH neurons.

A role forfoxd3andsox10in the differentiation of gonadotropin-releasing hormone (GnRH) cells in the zebrafishDanio rerio

Gonadotropin-releasing hormone (GnRH) is found in a wide range of vertebrate tissues, including the nervous system. In general, GnRH has two functions: endocrine, acting as a releasing hormone; and neuromodulatory, affecting neural activity in the peripheral and central nervous system. The best understood population of GnRH cells is that of the hypothalamus, which is essential for reproduction. Less well understood are the populations of GnRH cells found in the terminal nerve and midbrain, which appear to be neuromodulatory in function. The GnRH-containing cells of the midbrain are proposed to arise from the mesencephalic region of the neural tube. Previously, we showed that neuromodulatory GnRH cells of the terminal nerve arise from cranial neural crest. To test the hypothesis that neuromodulatory GnRH cells of the midbrain also arise from neural crest, we used gene knockdown experiments in zebrafish to disrupt neural crest development. We demonstrate that decrement of the function of foxd3 and/or sox10, two genes important for the development and specification of neural crest, resulted in a reduction and/or loss of GnRH cells of the midbrain, as well as a reduction in the number of terminal nerve GnRH cells. Therefore, our data support a neural crest origin for midbrain GnRH cells. Additionally, we demonstrate that knockdown of kallmann gene function resulted in the loss of endocrine GnRH cells of the hypothalamus, but not of neuromodulatory GnRH cells of the midbrain and terminal nerve, thus providing additional evidence for separate pathways controlling the development of neuromodulatory and endocrine GnRH cells.

Sexually dimorphic hormonal regulation of the gap junction protein, CX43, in rats and altered female reproductive function in CX43+/− mice

Astrocytic gap junctional communication is important in steroid hormone regulation of reproductive processes at the level of the hypothalamus, including estrous cyclicity and sexual behavior. We examined the effects of estradiol and progesterone on the abundance of the gap junctional protein, connexin 43 (CX43), which is highly expressed in astrocytes. Gonadectomized rats received hormone treatments that induce maximal sexual behavior and gonadotropin surges in females (estrogen for 48 h followed by progesterone, estrogen alone or progesterone alone). Control animals received vehicle (oil) injections. In the female rat preoptic area (POA), containing the gonadotropin-releasing hormone (GnRH) cell bodies, treatment with estrogen, progesterone or estrogen + progesterone significantly increased CX43 protein levels in immunoblots. In contrast, estrogen + progesterone significantly decreased CX43 levels in the male rat POA. This sexually dimorphic hormonal regulation of CX43 was not evident in the hypothalamus, which contains primarily GnRH nerve terminals. Treatment with estrogen + progesterone significantly decreased CX43 levels in both the male and female hypothalamus. To examine the role of CX43 in female reproductive function, we studied heterozygous female CX43 (CX43+/−) mice. Most mutant mice did not show normal estrous cycles. In addition, when compared to wild type females, CX43+/− mice had reduced lordosis behavior. These data suggest that hypothalamic CX43 expression is regulated by steroid hormones in a brain-region-specific and sexually dimorphic manner. Therefore, gap junctional communication in the POA and hypothalamus may be a factor regulating the estrous cycle and sexual behavior in female rodents.

Seasonal morphological changes in the neuro-glial interaction between gonadotropin-releasing hormone nerve terminals and glial endfeet in Japanese quail.

In a previous study we showed that photoperiodically generated T3 in the hypothalamus is critical for the photoperiodic response of gonads in Japanese quail. The expression of thyroid hormone receptors in the median eminence (ME) suggested that photoperiodically generated T3 acts on the ME. Because thyroid hormone is known to play a critical role in the development and plasticity of the central nervous system, in the present study we have examined ultrastructure of the ME in Japanese quail kept in short-day and long-day environments. Immunoelectron microscopy revealed that GnRH nerve terminals are in close proximity to the basal lamina under long-day conditions, and conventional transmission electron microscopy demonstrated the encasement of the terminals by the endfeet of glia under short-day conditions. These morphological changes may regulate photoperiodic GnRH secretion.

The role of GabaA receptors in the neural systems of the ventromedial hypothalamus-nucleus infundibular region in the control of GnRH release in ewes during follicular phase.

To examine the role of the GABAA receptor mediating systems in the control of gonadotropin releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/NI) of the hypothalamus of ewes during the follicular phase of the estrous cycle, the extracellular concentrations of GnRH, beta-endorphin (B-END), noradrenaline (NE), dopamine (DA), and their metabolites MHPG, DOPAC and concentration of luteinizing hormone (LH) in blood plasma were quantified during local stimulation or blockade of GABAA receptors with muscimol and bicuculline, respectively. Stimulation of GABAA receptors attenuated GnRH and LH release, increased beta-endorphin outflow and dopaminergic activity but had no evident effect on noradrenergic activity. Blockade of GABAA receptors decreased beta-endorphin release but had no evident effect on the extracellular concentration of GnRH, LH levels in the blood and catecholaminergic activity. It is suggested that suppression of GnRH/LH release under muscimol treatment may result from activation of GABAA receptors on GnRH nerve terminals and through GABAA receptor mechanism activated beta-endorphinergic and dopaminergic neurons in the VEN/NI. Lack of changes in NE and MHPG concentration during stimulation or blockade of GABAA receptors suggests, that during the follicular phase of the estrous cycle the noradrenergic system in the VEN/NI is not involved in the control of GnRH/LH release by GABA.

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.

Effects of Olfactory Tract Section on the Immunohistochemical Distribution of Brain GnRH Fibers in the Female Goldfish, Carassius auratus

Abstract The organization of the gonadotropin-releasing hormone (GnRH) system originating from the terminal nerve (TN) neuron in the olfactory bulb was examined immunohistochemically by tracing the changes in the distribution of salmon type (sGnRH) and chicken-II type (cGnRH-II) GnRH fibers after olfactory tract section (OTX) in the female goldfish. Following OTX, which blocks the axonal transport of GnRH from the TN to other brain regions, the density of sGnRH-ir fibers in various brain regions decreased from 7 days to 28 days, with the exception of rather restricted areas surrounding cell bodies in the ventral telencephalon, preoptic area, and hypothalamus. The density of cGnRH-II-ir fibers decreased only in the telencephalon from 7 days to 14 days. In spite of the decrease of GnRH-ir fibers in several brain areas, neither type of GnRH fibers showed marked changes in the pituitary gland during the experiment. These results indicate that the TN GnRH system project fibers to wide brain areas and that most...