Activity Of GnRH Neurons Research Articles

Functional relationship between obesity and male reproduction: from humans to animal models

The increase in the incidence of obesity has a substantial societal health impact. Contrasting reports have been published on whether overweight and obesity affect male fertility. To clarify this, we have reviewed published data on the relation between overweight/obesity, semen parameters, endocrine status and human male fertility. Subsequently, we have used results obtained in animal models of obesity to explain the human data. Pubmed, Scopus, Web of Science and Google Scholar databases were searched between September 2009 and October 2010 for a comprehensive publication record. Available studies on adult human males were examined. The included animal studies examined obesity and fertility, and focused on leptin, leptin receptor signaling, kisspeptins and/or NPY. Most overweight/obese men do not experience significant fertility problems, despite the presence of reduced testosterone alongside normal gonadotrophin levels. Only a subgroup of subjects suffers from hypogonadotropic hypogonadism. Animal models offer several explanations and show that reduced leptin signaling leads to reduced GnRH neuronal activity. This may be due to decreased hypothalamic Kiss1 expression, a potent regulator of GnRH/LH/FSH release. As the Kiss1 neurons express leptin receptors, the Kiss1 system may participate in transmitting metabolic information to the GnRH neurons, thus providing a bridge between metabolic regulation and fertility. Infertility in overweight/obese males may be explained by leptin insensitivity. This implies a possible role for the KISS1 system in human obesity-related male infertility. If substantiated, it will pave the way for methods to restore fertility in these subjects.

Projections of Arcuate Nucleus and Rostral Periventricular Kisspeptin Neurons in the Adult Female Mouse Brain

The important role of kisspeptin neurons in the regulation of GnRH neuron activity is now well accepted. However, the ways in which kisspeptin neurons located in the arcuate nucleus (ARN) and rostral periventricular area of the third ventricle (RP3V) control GnRH neurons are poorly understood. The present study used anterograde and retrograde tracing techniques to establish the neuronal projection patterns of kisspeptin cell populations in the female mouse brain. Anterograde tracing studies revealed that kisspeptin neurons in the ARN innervated a wide number of hypothalamic and associated limbic region nuclei, whereas RP3V kisspeptin neurons projected to a smaller number of mostly medially located hypothalamic nuclei. Retrograde tracing confirmed a major projection of RP3V kisspeptin neurons to the ARN and showed that kisspeptin neurons located in the rostral half of the ARN projected to the rostral preoptic area. Peripheral administration of Fluorogold was found to label the majority of GnRH neurons but no kisspeptin neurons. Together, these studies highlight the complexity of the brain kisspeptin neuronal system and indicate that both ARN and RP3V kisspeptin neurons participate in a variety of limbic functions. In relation to the GnRH neuronal network, these investigations demonstrate that, alongside the RP3V kisspeptin cells, rostral ARN kisspeptin neurons may also project to GnRH neuron cell bodies. However, no kisspeptin neurons innervate GnRH nerve terminals in the external layer of the median eminence. These studies provide a neuroanatomical framework for the further elucidation of the functions of the ARN and RP3V kisspeptin neuron populations.

Glucosensing by GnRH Neurons: Inhibition by Androgens and Involvement of AMP-Activated Protein Kinase

GnRH neurons integrate steroidal and metabolic cues to regulate fertility centrally. Central glucoprivation reduces LH secretion, which is governed by GnRH release, suggesting GnRH neuron activity is modulated by glucose availability. Here we tested whether GnRH neurons can sense changes in extracellular glucose, and whether glucosensing is altered by the steroids dihydrotestosterone (DHT) and/or estradiol (E). Extracellular recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice ± DHT and/or E implants. Firing rate was reduced by a switch from 4.5 to 0.2 mm glucose in cells from OVX, OVX+E, and OVX+DHT+E mice, but not OVX+DHT mice. This suggests that androgens reduce the sensitivity of GnRH neurons to changes in extracellular glucose, but E mitigates this effect. Next we investigated potential mechanisms. In the presence of the ATP-sensitive potassium channel antagonist tolbutamide, glucosensing persisted. In contrast, glucosensing was attenuated in the presence of compound C, an antagonist of AMP-activated protein kinase (AMPK), suggesting a role for AMPK in glucosensing. The AMPK activator N1-(b-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR) mimicked the effect of low glucose and was less effective in cells from DHT-treated mice. The effect of DHT to diminish responses to low glucose and AICAR was abolished by blockade of fast synaptic transmission. Both AICAR and low glucose activated a current with a reversal potential near -50 mV, suggesting a nonspecific cation current. These studies indicate that glucosensing is one mechanism by which GnRH neurons sense fuel availability and point to a novel role for AMPK in the central regulation of fertility.

Increase of Anteroventral Periventricular Kisspeptin Neurons and Generation of E2-Induced LH-Surge System in Male Rats Exposed Perinatally to Environmental Dose of Bisphenol-A

Perinatal exposure to environmental levels of bisphenol-A (BPA) impairs sexually dimorphic behaviors in rodents. Kisspeptin neurons in anteroventral periventricular nucleus (AVPV), which plays an important role in the activation of GnRH neurons and the initiation of LH-surge, have been suggested to be sexual dimorphism in rats. This study focused on exploring the influence of a perinatal exposure to an environmental dose of BPA on the development and maturation of male AVPV kisspeptin neurons and hypothalamus-pituitary-gonadal axis. Female rats were injected sc with 2 μg BPA/kg·d from gestation d 10 through lactation d 7. Anatomical and functional changes in AVPV kisspeptin neurons and hypothalamus-pituitary-gonadal axis were examined in prepubertal, pubertal, and adult male rats exposed perinatally to BPA (BPA-rats). Here, we show that in postnatal d (PND)30/50/90 BPA-rats, the number of AVPV kisspeptin-immunoreactive cells was persistently increased in comparison with age-matched control male rats. The number of GnRH-immunoreactive cells in PND30 BPA-rats declined approximately 40% compared with control male rats, whereas that in PND50/90 BPA-rats was increased in a G protein-coupled receptor 54-dependent manner. Estradiol could induce a stable LH-surge in PND90 BPA-rats and control female rats, which was sensitive to the G protein-coupled receptor 54 inhibitor. In PND30/50 BPA-rats, plasma level of LH was higher, but the level of testosterone was lower than control male rats. These findings provide evidence that perinatal exposure to an environmental dose of BPA causes a sustained increase in AVPV kisspeptin neurons in male rats, leading to the generation of estradiol-induced LH-surge system.

Rapid action of estradiol in primate GnRH neurons: The role of estrogen receptor alpha and estrogen receptor beta

Estrogens play a pivotal role in the control of female reproductive function. Recent studies using primate GnRH neurons derived from embryonic nasal placode indicate that 17β-estradiol (E 2) causes a rapid stimulatory action. E 2 (1 nM) stimulates firing activity and intracellular calcium ([Ca 2+] i) oscillations of primate GnRH neurons within a few min. E 2 also stimulates GnRH release within 10 min. However, the classical estrogen receptors, ERα and ERβ, do not appear to play a role in E 2-induced [Ca 2+] i oscillations or GnRH release, as the estrogen receptor antagonist, ICI 182,780, failed to block these responses. Rather, this rapid E 2 action is, at least in part, mediated by a G-protein coupled receptor GPR30. In the present study we further investigate the role of ERα and ERβ in the rapid action of E 2 by knocking down cellular ERα and ERβ by transfection of GnRH neurons with specific siRNA for rhesus monkey ERα and ERβ. Results indicate that cellular knockdown of ERα and ERβ failed to block the E 2-induced changes in [Ca 2+] i oscillations. It is concluded that neither ERα nor ERβ is required for the rapid action of E 2 in primate GnRH neurons.

Progesterone Treatment Inhibits and Dihydrotestosterone (DHT) Treatment Potentiates Voltage-Gated Calcium Currents in Gonadotropin-Releasing Hormone (GnRH) Neurons

GnRH neurons are central regulators of fertility, and their activity is modulated by steroid feedback. In normal females, GnRH secretion is regulated by estradiol and progesterone (P). Excess androgens present in hyperandrogenemic fertility disorders may disrupt communication of negative feedback signals from P and/or independently stimulate GnRH release. Voltage-gated calcium channels (VGCCs) are important in regulating excitability and hormone release. Estradiol alters VGCCs in a time-of-day-dependent manner. To further elucidate ovarian steroid modulation of GnRH neuron VGCCs, we studied the effects of dihydrotestosterone (DHT) and P. Adult mice were ovariectomized (OVX) or OVX and treated with implants containing DHT (OVXD), estradiol (OVXE), estradiol and DHT (OVXED), estradiol and P (OVXEP), or estradiol, DHT, and P (OVXEDP). Macroscopic calcium current (I(Ca)) was recorded in the morning or afternoon 8-12 d after surgery using whole-cell voltage-clamp. I(Ca) was increased in afternoon vs. morning in GnRH neurons from OVXE mice but this increase was abolished in cells from OVXEP mice. I(Ca) in cells from OVXD mice was increased regardless of time of day; there was no additional effect in OVXED mice. P reduced N-type and DHT potentiated N- and R-type VGCCs; P blocked the DHT potentiation of N-type-mediated current. These data suggest P and DHT have opposing actions on VGCCs in GnRH neurons, but in the presence of both steroids, P dominates. VGCCs are targets of ovarian steroid feedback modulation of GnRH neuron activity and, more specifically, a potential mechanism whereby androgens could activate GnRH neuronal function.

Neuroendocrine and Genetic Control of Seasonal Reproduction in Sheep and Goats

Goats and sheep generally express seasonal variations in their sexual behaviour, spermatogenic activity (from moderate decrease to very low sperm production), gamete quality (variations in fertilization rates and embryo survival), ovulation frequency (presence or absence of ovulation), and ovulation rate (number of eggs shed per ovulation period). This induces seasonal availability of derived, fresh animal products (meat, milk and cheese) because of a more or less marked seasonal distribution of births. A complex combination of an endogenous circannual rhythm driven and synchronized by light and melatonin, which controls the pulsatile activity of GnRH neurons in the preoptic-mediobasal hypothalamus, is responsible for these changes. Dramatic and long-term neuroendocrine changes, involving different neuromediator systems and neuronal plasticity, have been shown to play a role in these processes. A strong variability between breeds exists in both species regarding the dates of onset and end of the breeding season, with a gradient of seasonality from southern to northern latitudes. Within a breed, seasonal traits are heritable; thus, genetic selection could be one way to decrease seasonality in sheep and goats in the future.

251 Potent and efficient testosterone suppression by chronic administration of novel metastin analogues, TAK-448 and TAK-683, in male rats

Metastin is a novel peptide that has been isolated from the human placenta as the cognate ligand of the G-protein-coupled receptor OT7T175 (or GPR54). However, its physiological functions have not yet been fully investigated. In the present study, we show that subcutaneous administration of metastin increased the plasma levels of gonadotropins (follicle-stimulating hormone and luteinizing hormone) and induced ovulation in prepubertal female rats that had been pretreated with pregnant mare serum gonadotropin to induce follicle maturation. Furthermore, metastin administration drastically increased the plasma levels of gonadotropins in male rats. This action was abolished by pretreatment with a GnRH antagonist, and was accompanied by induction of c-Fos immunoreactivity in GnRH neurons. These results suggest that s.c. administered metastin induces the release of gonadotropin via activation of the hypothalamic GnRH neurons.

Hypothalamic Insulin-Like Growth Factor-I Receptors Are Necessary for Hormone-Dependent Luteinizing Hormone Surges: Implications for Female Reproductive Aging

Brain IGF-I receptors are required for maintenance of estrous cycles in young adult female rats. Circulating and hypothalamic IGF-I levels decrease with aging, suggesting a role for IGF-I in the onset of reproductive senescence. Therefore, the present study investigated potential mechanisms of action of brain IGF-I receptors in the regulation of LH surges in young adult and middle-aged rats. We continuously infused IGF-I, the selective IGF-I receptor antagonist JB-1, or vehicle into the third ventricle of ovariectomized young adult and middle-aged female rats primed with estradiol and progesterone. Pharmacological blockade of IGF-I receptors attenuated and delayed the LH surge in young adult rats, reminiscent of the LH surge pattern that heralds the onset of reproductive senescence in middle-aged female rats. Infusion of IGF-I alone had no effect on the LH surge but reversed JB-1 attenuation of the surge in young females. In middle-aged rats, infusion of low doses of IGF-I partially restored LH surge amplitude, and infusion of JB-1 completely obliterated the surge. Intraventricular infusion of IGF-I or JB-1 did not modify pituitary sensitivity to exogenous GnRH or GnRH peptide content in the anterior or mediobasal hypothalamus in either young or middle-aged rats. These findings support the hypothesis that brain IGF-I receptor signaling is necessary for GnRH neuron activation under estrogen-positive feedback conditions and that decreased brain IGF-I signaling in middle-aged females contributes, in part, to LH surge dysfunction by disrupting estradiol-sensitive processes that affect GnRH neuron activation and/or GnRH release.

Insights into the mechanism by which kisspeptin stimulates a preovulatory LH surge and ovulation in seasonally acyclic ewes: Potential role of estradiol

We have previously demonstrated that a constant intravenous infusion of kisspeptin (Kp) for 48 h in anestrous ewes induces a preovulatory luteinizing hormone (LH) surge followed by ovulation in approximately 75% of animals. The mechanisms underlying this effect are unknown. In this study, we investigated whether Kp-induced preovulatory LH surges in anestrous ewes were the result of the general activation of the whole gonadotropic axis or of the direct activation of central GnRH neurons required for the GnRH/LH surge. In the first experiment, a constant iv infusion of ovine kisspeptin 10 (Kp; 15.2 nmol/h) was given to 11 seasonally acyclic ewes over 43 h. Blood samples were taken every 10 min for 15 h, starting 5 h before the infusion, and then hourly until the end of the infusion. We found that the infusion of Kp induced a well-synchronized LH surge (around 22 h after the start of the Kp infusion) in 82% of the animals. In all ewes with an LH surge, there was an immediate but transient increase in the plasma concentrations of LH, follicle-stimulating hormone (FSH), and growth hormone (GH) at the start of the Kp infusion. Mean (± SEM) concentrations for the 5-h periods preceding and following the start of the Kp infusion were, respectively, 0.33 ± 0.09 vs 2.83 ± 0.49 ng/mL ( P = 0.004) for LH, 0.43 ± 0.05 vs 0.55 ± 0.03 ng/mL ( P = 0.015) for FSH, and 9.34 ± 1.01 vs 11.51 ± 0.92 ng/mL ( P = 0.004) for GH. In the first experiment, surges of LH were observed only in ewes that also had a sustained rise in plasma concentrations of estradiol (E 2) in response to Kp. Therefore, a second experiment was undertaken to determine the minimum duration of Kp infusion necessary to induce such a pronounced and prolonged increase in plasma E 2 concentration. Kisspeptin (15.2 nmol/h) was infused for 6, 12, or 24 h in seasonally acyclic ewes (N = 8), and blood samples were collected hourly for 28 h (beginning 5 h before the start of infusion), then every 2 h for the following 22 h. Kisspeptin infused for 24 h induced LH surges in 75% of animals, and this percentage decreased with the duration of the infusion (12 h = 50%; 6 h = 12.5%). The plasma concentration of E 2 was greater in ewes with an LH surge compared to those without LH surges; mean (± SEM) concentrations for the 5-h period following the Kp infusion were, respectively, 2.23 ± 0.16 vs 1.27 ± 0.13 pg/mL ( P < 0.001). Collectively, our results strongly suggest that the systemic delivery of Kp induced LH surges by activating E 2-positive feedback on gonadotropin secretion in acyclic ewes.

Decreased Expression of A-Kinase Anchoring Protein 150 in GT1 Neurons Decreases Neuron Excitability and Frequency of Intrinsic Gonadotropin-Releasing Hormone Pulses

The frequency of intrinsic pulsatile GnRH secretion from endogenous GnRH neurons and GT1 GnRH cell lines is stimulated by increased intracellular cAMP levels. The downstream molecules comprising the cAMP signaling pathway are organized in microdomains by a family of scaffolding proteins, A-kinase anchoring proteins (AKAPs). These molecules tether protein kinase A, cAMP-specific phosphodiesterases, phosphatases to known substrates. In neurons AKAP150 organizes many of the signaling molecules known to regulate the excitability and intrinsic pulsatile activity of GnRH neurons. AKAP150 was expressed in both the GT1-1 and GT1-7 cells. We determined the role of AKAP150 in coordinating GT1-1 cell excitability and intrinsic GnRH pulsatile secretion by lowering AKAP150 levels with a small interfering RNA (siRNA) adenovirus construct to AKAP150 (Ad-AKAP150-siRNA). Infection with Ad-AKAP150-siRNA specifically decreased AKAP150 mRNA levels by 74% and protein levels by 53% relative to uninfected cells or cells infected with a luciferase control adenovirus siRNA vector. In GT1 cells, spontaneous Ca(2+) oscillations, an index of neuron excitability, are stimulated by increased levels of intracellular cAMP and lowered by decreased levels. The frequency of spontaneous Ca(2+) oscillations in Ad-AKAP150-siRNA-treated GT1-1 cells decreased by 47.2% relative to controls. A dramatic decrease in the number of spontaneous GnRH pulses was also observed after infection with Ad-AKAP150-siRNA. The interpulse interval increased to 143 +/- 20.25 min in Ad-AKAP150-siRNA infected cells from 32.2 +/- 7.3 min in luciferase control adenovirus siRNA vector-infected cells. These data demonstrate an important role of AKAP150 in coordinating signaling events regulating the frequency of intrinsic pulsatile GnRH secretion.

Interactions between neurotensin and GnRH neurons in the positive feedback control of GnRH/LH secretion in the mouse

In female mammals, increased ovarian estradiol (E(2)) secretion triggers GnRH release from neurons in the basal forebrain, which drives LH secretion from the pituitary and subsequently induces ovulation. However, the neural circuits that activate this preovulatory GnRH/LH surge remain unidentified. Neurotensin is expressed in neurons of the anteroventral periventricular nucleus (AVPV), a region thought to be critical for generating the preovulatory GnRH/LH surge. E(2) induces neurotensin (Nts) gene expression in this region, and blockade of neurotensin signaling reduces the LH surge in the rat. We postulated that neurotensin signaling plays a similar role in generating the E(2)-induced GnRH/LH surge in mice. We used in situ hybridization (ISH) to determine whether E(2) induces Nts expression in the mouse and found evidence to support this proposition. Next, we determined that the neurotensin receptor (Ntsr2) is present in many GnRH-expressing neurons. Since the kisspeptin gene (Kiss1) is expressed in the AVPV and is responsive to E(2), we predicted that some neurons in this region express both Kiss1 and Nts; however, by double-label ISH, we observed no coexpression of the two mRNAs. We also postulated that Nts mRNA expression would increase in parallel with the E(2)-induced LH surge and that the central (icv) administration of neurotensin would stimulate LH secretion and activation of GnRH neurons but found no evidence to support either of these hypotheses. Together, these findings suggest that, although neurotensin neurons in the AVPV are targets for regulation by E(2), neurotensin does not appear to play a direct role in generating the GnRH/LH surge in the mouse.

Male-Biased Effects of Gonadotropin-Releasing Hormone Neuron-Specific Deletion of the Phosphoinositide 3-Kinase Regulatory Subunit p85α on the Reproductive Axis

GnRH neurosecretion is subject to regulation by insulin, IGF-I, leptin, and other neuroendocrine modulators whose effects may be conveyed by activation of phosphoinositide 3-kinase (PI3K)-mediated pathways. It is not known, however, whether any of these regulatory actions are exerted directly, via activation of PI3K in GnRH neurons, or whether they are primarily conveyed via effects on afferent circuitries governing GnRH neurosecretion. To investigate the role of PI3K signaling in GnRH neurons, we used conditional gene targeting to ablate expression of the major PI3K regulatory subunit, p85alpha, in GnRH neurons. Combined in situ hybridization and immunohistochemistry confirmed reduction of p85alpha mRNA expression in GnRH neurons of GnRH-p85alpha knockout (KO) animals. Females of both genotypes exhibited estrous cyclicity and had comparable serum LH, estradiol-17beta, and FSH levels. In male GnRH-p85alphaKO mice, serum LH, testosterone, and sperm counts were significantly reduced compared with wild type. To investigate the role of the other major regulatory subunit, p85beta, on the direct control of GnRH neuronal function, we generated mice with a GnRH-neuron-specific p85alpha deletion on a global betaKO background. No additional reproductive effects in male or female mice were found, suggesting that p85beta does not substitute p85 activity toward PI3K function in GnRH neurons. Our results suggest that p85alpha, and thus PI3K activity, participates in the control of GnRH neuronal activity in male mice. The sex-specific phenotype in these mice raises the possibility that PI3K activation during early development may establish sex differences in GnRH neuronal function.

The Excitatory Peptide Kisspeptin Restores the Luteinizing Hormone Surge and Modulates Amino Acid Neurotransmission in the Medial Preoptic Area of Middle-Aged Rats

Reproductive success depends on a robust and appropriately timed preovulatory LH surge. The LH surge, in turn, requires ovarian steroid modulation of GnRH neuron activation by the neuropeptide kisspeptin and glutamate and gamma-aminobutyric acid (GABA) neurotransmission in the medial preoptic area (mPOA). Middle-aged females exhibit reduced excitation of GnRH neurons and attenuated LH surges under estrogen-positive feedback conditions, in part, due to increased GABA and decreased glutamate neurotransmission in the mPOA. This study tested the hypothesis that altered kisspeptin regulation by ovarian steroids plays a role in age-related LH surge dysfunction. We demonstrate that middle-aged rats exhibiting delayed and attenuated LH surges have reduced levels of Kiss1 mRNA in the anterior hypothalamus under estrogen-positive feedback conditions. Kisspeptin application directly into the mPOA rescues total LH release and the LH surge amplitude in middle-aged rats and increases glutamate and decreases GABA release to levels seen in the mPOA of young females. Moreover, the N-methyl-D-aspartate receptor antagonist MK801 blocks kisspeptin reinstatement of the LH surge. These observations suggest that age-related LH surge dysfunction results, in part, from reduced kisspeptin drive under estrogen-positive feedback conditions and that kisspeptin regulates GnRH/LH release, in part, through modulation of mPOA glutamate and GABA release.

Postnatal Development of an Estradiol-Kisspeptin Positive Feedback Mechanism Implicated in Puberty Onset

The regulation of GnRH neurons by kisspeptin is critical for normal puberty onset in mammals. In the rodent the kisspeptin neurons innervating GnRH neurons are thought to reside in the rostral periventricular area of the third ventricle (RP3V). Using kisspeptin immunocytochemistry we show that kisspeptin peptide expression in the RP3V of female mice begins around postnatal d 15 (P15) and rapidly increases to achieve adult-like levels by P30, the time of puberty onset. Ovariectomy of female pups at P15 resulted in a 70-90% reduction (P < 0.01) in kisspeptin peptide expression within the RP3V of P30 or P60 mice. Replacement of 17-beta-estradiol (E2) in P15-ovariectomized mice from P15-30 or P22-30 resulted in a complete restoration of kisspeptin peptide expression in the RP3V (P < 0.01). Kisspeptin-immunoreactive fibers throughout the hypothalamus, including the arcuate nucleus, followed the same pattern of estrogen-dependent expression. To test the absolute necessity of estrogen for kisspeptin expression in the RP3V, aromatase knockout mice were examined. Kisspeptin-immunoreactive cells were detected in the arcuate nucleus, but there was a complete absence of kisspeptin peptide in RP3V neurons of aromatase knockout adult females. These results demonstrate that E2 is essential for the prepubertal development of kisspeptin peptide within RP3V neurons and suggest that an E2-kisspeptin positive feedback mechanism exists before puberty. This implies that RP3V kisspeptin neurons are E2-dependent amplifiers of GnRH neuron activity in the prepubertal period.

RFamide-Related Peptide-3, a Mammalian Gonadotropin-Inhibitory Hormone Ortholog, Regulates Gonadotropin-Releasing Hormone Neuron Firing in the Mouse

The recent discovery that an RFamide termed gonadotropin-inhibitory hormone is likely to be a hypophysiotrophic gonadotropin release-inhibiting hormone in birds has generated interest into the role of LPXRFamide neuropeptides in the control of gonadotropin secretion in mammals. Recent immunocytochemical studies in birds and mammals have suggested that neurons expressing the mammalian LPXRFamides, RFamide-related peptides (RFRPs) 1 and 3, may innervate and regulate GnRH neurons directly. We used cell-attached electrophysiology in adult male and female GnRH-green fluorescent protein-tagged neurons to examine whether RFRP-3 modulated the electrical excitability of GnRH neurons. RFRP-3 was found to exhibit rapid and repeatable inhibitory effects on the firing rate of 41% of GnRH neurons. A small population of GnRH neurons (12%) increased their firing rate in response to RFRP-3, and the remainder was unaffected. No difference was detected in the RFRP-3 responses of GnRH neurons from male, diestrous, or proestrus female mice. The suppressive effect of RFRP-3 was maintained when amino acid transmission was blocked, suggesting a possible direct effect of RFRP-3 upon GnRH neurons. To evaluate the effects of other RFamide neuropeptides on GnRH neurons, we tested the actions of prolactin-releasing peptide-20 and -31. Neither compounds altered the firing rate of GnRH neurons. These studies demonstrate that RFRP-3 has a likely direct suppressive action on the excitability of GnRH neurons, indicating a role for RFRPs in the regulation of gonadotropin secretion in mammals through modulation of GnRH neuron activity.

Central and Peripheral Effects of RFamide-Related Peptide-3 on Luteinizing Hormone and Prolactin Secretion in Rats

Hypothalamic RFamide-related peptide-3 (RFRP-3) neurons inhibit LH secretion via a central action. A direct hypophysiotropic action on the gonadotropes has also been suggested. To assess central RFRP-3 effects on the GnRH/LH surge that induces ovulation, ovariectomized rats were subjected to an estradiol plus progesterone surge-induction protocol. Chronic infusion of RFRP-3 (2.5 or 25 ng/h, intracerebroventricularly) caused a dose-dependent 50-60% inhibition of GnRH neuronal activation (assessed by colocalization with the immediate early gene c-Fos) at the surge peak compared with vehicle-treated controls. RFRP-3 also suppressed neuronal activation in the anteroventral periventricular region, which provides stimulatory input to GnRH neurons, by 50-80% compared with control values. To test whether centrally administered RFRP-3 inhibits pulsatile GnRH/LH secretion, chronically ovariectomized, low-level estradiol-treated rats without surge induction were blood sampled every 10 min for 4 h. Bolus injection of RFRP-3 (0, 2.5, or 25 microg, intracerebroventricularly) after 1.5 h did not affect subsequent LH pulse frequency, pulse amplitude, or the mean concentrations of LH or prolactin. RFRP-3 treatment of isolated anterior pituitary cells at moderate doses of up to 10(-7) m did not significantly inhibit LH release, either with or without GnRH cotreatment. These data reveal a central inhibitory effect of RFRP-3 on the hypothalamo-pituitary gonadal axis specifically during the estradiol-induced GnRH/LH surge. This effect may include actions of RFRP-3 on GnRH neurons and/or their anteroventral periventricular afferent inputs but is unlikely to involve direct inhibition of LH secretion at the level of the gonadotrope.

Suppression of Basal Spontaneous Gonadotropin-Releasing Hormone Neuronal Activity during Lactation: Role of Inhibitory Effects of Neuropeptide Y

Increased neuropeptide Y (NPY) activity drives the chronic hyperphagia of lactation and may contribute to the suppression of GnRH activity. The majority of GnRH neurons are contacted by NPY fibers, and GnRH cells express NPY Y5 receptor (Y5R). Therefore, NPY provides a neurocircuitry for information about food intake/energy balance to be directly transmitted to GnRH neurons. To investigate the effects of lactation on GnRH neuronal activity, hypothalamic slices were prepared from green fluorescent protein-GnRH transgenic rats. Extracellular loose-patch recordings determined basal GnRH neuronal activity from slices of ovariectomized control and lactating rats. Compared with controls, hypothalamic slices from lactating rats had double the number of quiescent GnRH neurons (14.51 +/- 2.86 vs. 7.04 +/- 2.84%) and significantly lower firing rates of active GnRH neurons (0.25 +/- 0.02 vs. 0.37 +/- 0.03 Hz). To study the NPY-postsynaptic Y5R system, whole-cell current-clamp recordings were performed in hypothalamic slices from control rats to examine NPY/Y5R antagonist effects on GnRH neuronal resting membrane potential. Under tetrodotoxin treatment, NPY hyperpolarized GnRH neurons from -56.7 +/- 1.94 to -62.1 +/- 1.83 mV; NPY's effects were blocked by Y5R antagonist. To determine whether increased endogenous NPY tone contributes to GnRH neuronal suppression during lactation, hypothalamic slices were treated with Y5R antagonist. A significantly greater percentage of GnRH cells were activated in slices from lactating rats (52%) compared with controls (28%). These results suggest that: 1) basal GnRH neuronal activity is suppressed during lactation; 2) NPY can hyperpolarize GnRH neurons via postsynaptic Y5R; and 3) increased inhibitory NPY tone during lactation is a component of the mechanisms responsible for suppression of GnRH neuronal activity.

Courtship interactions stimulate rapid changes in GnRH synthesis in male ring doves

Many birds and mammals show changes in the hypothalamo-pituitary-gonadal (HPG) axis in response to social or sexual interactions between breeding partners. While alterations in GnRH neuronal activity play an important role in stimulating these changes, it remains unclear if acute behaviorally-induced alterations in GnRH release are accompanied by parallel changes in GnRH synthesis. To investigate this relationship, we examined changes in the activity of GnRH neurons in the brains of male ring doves following brief periods of courtship interactions with females. Such interactions have been previously shown to increase plasma LH in courting male doves at 24 h, but not at 1 h, after pairing with females. In the first study, males allowed to court females for 2 h had 60% more cells that showed immunocytochemical labeling for GnRH-I in the preoptic area (POA) of the hypothalamus than did control males that remained isolated from females. To determine whether an increase in GnRH gene expression preceded this increase in GnRH immunoreactivity in the POA, changes in the number of cells with detectable GnRH-I mRNA in the POA were measured by in situ hybridization following a 1 h period of courtship interactions with females. In this second study, courting males exhibited 40% more cells with GnRH-I in this region than did isolated control males. GnRH-immunoreactive neurons in two other diencephalic regions failed to show these courtship-induced changes. Plasma LH was not elevated after 1 or 2 h of courtship. These results demonstrate that the release of GnRH-I in the POA that is presumably responsible for courtship-induced pituitary and gonadal activation is accompanied by a rapid increase in GnRH synthesis that occurs before plasma LH levels increase. We suggest that this increase in GnRH synthesis is necessary to support the extended period of HPG axis activation that is seen in this species during the 5–10 day period of courtship and nest building activity.

Critical Roles for Fast Synaptic Transmission in Mediating Estradiol Negative and Positive Feedback in the Neural Control of Ovulation

A switch in the balance of estradiol feedback actions from negative to positive initiates the GnRH surge, triggering the LH surge that causes ovulation. Using an ovariectomized, estradiol-treated (OVX+E) mouse model that exhibits daily switches between negative in the morning and positive feedback in the evening, we investigated the roles of fast synaptic transmission in regulating GnRH neuron firing during negative and positive feedback. Targeted extracellular recordings were used to monitor activity of GnRH neurons from OVX+E and OVX mice in control solution or solution with antagonists to both ionotropic glutamate and gamma-aminobutyric acid receptors (blockade). Blockade had no effect on activity of OVX cells. In contrast, in OVX+E cells in the morning, blockade increased activity compared with control cells, whereas in the evening, blockade decreased activity. In vivo barbiturate sedation of OVX+E mice that blocked LH surge induction prevented the in vitro evening changes in firing rate and response to blockade. These observations suggest at least partial inversion of the negative-to-positive switch in estradiol feedback action and indicate that changes in fast synaptic transmission to GnRH neurons and within the network of cells presynaptic to GnRH neurons are critical for mediating estradiol negative and positive feedback actions on GnRH neurons. Fast synaptic transmission may also affect GnRH neuron activity indirectly through altering release of excitatory and inhibitory neuromodulators onto GnRH neurons at specific times of day. Fast synaptic transmission is thus critical for proper generation and timing of the GnRH surge.