Excitability Of Gonadotropin-releasing Hormone Neurons Research Articles

Linkage and association of variants in the dopamine receptor 2 gene (DRD2) with polycystic ovary syndrome

Polycystic ovarian syndrome (PCOS) is a disorder with a foundation of neuroendocrine dysfunction, characterized by increased gonadotropin-releasing hormone (GnRH) pulsatility, which is antagonized by dopamine. The dopamine receptor 2 (DRD2), encoded by the DRD2 gene, has been shown to mediate dopamine’s inhibition of GnRH neuron excitability through pre- and post-synaptic interactions in murine models. Further, DRD2 is known to mediate prolactin (PRL) inhibition by dopamine, and high blood level of PRL have been found in more than one third of women with PCOS. We recently identified PRL as a gene contributing to PCOS risk and reported DRD2 conferring risk for type 2 diabetes and depression, which can both coexist with PCOS. Given DRD2 mediating dopamine’s action on neuroendocrine profiles and association with metabolic-mental states related to PCOS, polymorphisms in DRD2 may predispose to development of PCOS. Therefore, we aimed to investigate whether DRD2 variants are in linkage to and/or linkage disequilibrium (i.e., linkage and association) with PCOS in Italian families. In 212 Italian families, we tested 22 variants within the DRD2 gene for linkage and linkage disequilibrium with PCOS. We identified five novel variants significantly linked to the risk of PCOS. This is the first study to identify DRD2 as a risk gene in PCOS, however, functional studies are needed to confirm these results.

Hydrogen peroxide suppresses excitability of gonadotropin-releasing hormone neurons in adult mouse.

It has been reported that reactive oxygen species (ROS) derived from oxygen molecule reduction can interfere with the cross-talk between the hypothalamic-pituitary-gonadal (HPG) axis and other endocrine axes, thus affecting fertility. Furthermore, ROS have been linked to GnRH receptor signaling in gonadotropes involved in gonadotropin release. There has been evidence that ROS can interfere with the HPG axis and gonadotropin release at various levels. However, the direct effect of ROS on gonadotropin-releasing hormone (GnRH) neuron remains unclear. Thus, the objective of this study was to determine the effect of hydrogen peroxide (H2O2), an ROS source, on GnRH neuronal excitabilities in transgenic GnRH-green fluorescent protein-tagged mice using the whole-cell patch-clamp electrophysiology. In adults, H2O2 at high concentrations (mM level) hyperpolarized most GnRH neurons tested, whereas low concentrations (pM to μM) caused slight depolarization. In immature GnRH neurons, H2O2 exposure induced excitation. The sensitivity of GnRH neurons to H2O2 was increased with postnatal development. The effect of H2O2 on adult female GnRH neurons was found to be estrous cycle-dependent. Hyperpolarization mediated by H2O2 persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and amino-acids receptor blocking cocktail containing blockers for the ionotropic glutamate receptors, glycine receptors, and GABAA receptors, indicating that H2O2 could act on GnRH neurons directly. Furthermore, glibenclamide, an ATP-sensitive K+ (KATP) channel blocker, completely blocked H2O2-mediated hyperpolarization. Increasing endogenous H2O2 by inhibiting glutathione peroxidase decreased spontaneous activities of most GnRH neurons. We conclude that ROS can act as signaling molecules for regulating GnRH neuron's excitability and that adult GnRH neurons are sensitive to increased ROS concentration. Results of this study demonstrate that ROS have direct modulatory effects on the HPG axis at the hypothalamic level to regulate GnRH neuron's excitabilities.

An Inhibitory Circuit From Brainstem to GnRH Neurons in Male Mice: A New Role for the RFRP Receptor

RFamide-related peptides (RFRPs, mammalian orthologs of gonadotropin-inhibitory hormone) convey circadian, seasonal and social cues to the reproductive system. They regulate gonadotropin secretion by modulating gonadotropin-releasing hormone (GnRH) neurons via the RFRP receptor. Mice lacking this receptor are fertile but exhibit abnormal gonadotropin responses during metabolic challenges such as acute fasting, when the normal drop in gonadotropin levels is delayed. Although it is known that these food intake signals to the reproductive circuit originate in the nucleus tractus solitarius (NTS) in the brainstem, the phenotype of the neurons conveying the signal remains unknown. Given that neuropeptide FF (NPFF), another RFamide peptide, resides in the NTS and can bind to the RFRP receptor, we hypothesized that NPFF may regulate GnRH neurons. To address this question, we used a combination of techniques: cell-attached electrophysiology on GnRH-driven green fluorescent protein-tagged neurons in acute brain slices; calcium imaging on cultured GnRH neurons; and immunostaining on adult brain tissue. We found 1) NPFF inhibits GnRH neuron excitability via the RFRP receptor and its canonical signaling pathway (Gi/o protein and G protein-coupled inwardly-rectifying potassium channels), 2) NPFF-like fibers in the vicinity of GnRH neurons coexpress neuropeptide Y, 3) the majority of NPFF-like cell bodies in the NTS also coexpress neuropeptide Y, and 4) acute fasting increased NPFF-like immunoreactivity in the NTS. Together these data indicate that NPFF neurons within the NTS inhibit GnRH neurons, and thus reproduction, during fasting but prior to the energy deficit.

Dynamic and Sex-Specific Changes in Gonadotropin-Releasing Hormone Neuron Activity and Excitability in a Mouse Model of Temporal Lobe Epilepsy

Reproductive endocrine disorders are prominent comorbidities of temporal lobe epilepsy (TLE) in both men and women. The neural mechanisms underlying these comorbidities remain unclear, but hypothalamic gonadotropin-releasing hormone (GnRH) neurons may be involved. Here, we report the first direct demonstrations of aberrant GnRH neuron function in an animal model of epilepsy. Recordings of GnRH neuron firing and excitability were made in acute mouse brain slices prepared two months after intrahippocampal injection of kainate (KA) or control saline, a well-established TLE model in which most females develop comorbid estrous cycle disruption. GnRH neurons from control females showed elevated firing and excitability on estrus compared with diestrus. By contrast, cells from KA-injected females that developed prolonged, disrupted estrous cycles (KA-long) showed the reverse pattern. Firing rates of cells from KA-injected females that maintained regular cycles (KA-regular) were not different from controls on diestrus, but were reduced on estrus. In KA-injected males, only GnRH neurons in the medial septum displayed elevated firing. In contrast to the diestrus versus estrus and sex-specific changes in firing, GnRH neuron intrinsic excitability was elevated in all KA-injected groups, indicating a role for afferent synaptic and neuromodulatory inputs in shaping overall changes in firing activity. Furthermore, KA-injected females showed cycle-stage-specific changes in circulating sex steroids on diestrus and estrus that also differed between KA-long and KA-regular groups. Together, these findings reveal that the effects of epilepsy on the neural control of reproduction are dynamic across the estrous cycle, distinct in association with comorbid estrous cycle disruption severity, and sex-specific.

Changes in GABAergic Transmission to and Intrinsic Excitability of Gonadotropin-Releasing Hormone (GnRH) Neurons during the Estrous Cycle in Mice.

Gonadotropin-releasing hormone (GnRH) neurons form the final common central output pathway controlling fertility and are regulated by steroid feedback. In females, estradiol feedback action varies between negative and positive; negative feedback typically regulates episodic GnRH release whereas positive feedback initiates a surge of GnRH, and subsequently luteinizing hormone (LH) release ultimately triggering ovulation. During the estrous cycle, changes between estradiol negative and positive feedback occur with cycle stage and time of day, with positive feedback in the late afternoon of proestrus in nocturnal species. To test the hypotheses that synaptic and intrinsic properties of GnRH neurons are regulated by cycle stage and time of day, we performed whole-cell patch-clamp studies of GnRH neurons in brain slices from mice at two times considered negative feedback (diestrous PM and proestrous AM) and during positive feedback (proestrous PM). GABAergic transmission can excite GnRH neurons and was higher in cells from proestrous PM mice than cells from proestrous AM mice and approached traditional significance levels relative to cells from diestrous PM mice. Action potential response to current injection was also greater in cells from proestrous PM mice than the other two groups. Interestingly, the hormonal milieu of proestrous AM provided stronger negative feedback on both GnRH neuron excitability and GABAergic postsynaptic current (PSC) amplitude than diestrous PM. These observations demonstrate elements of both synaptic and intrinsic properties of GnRH neurons are regulated in a cycle-dependent manner and provide insight into the neurobiological mechanisms underlying cyclic changes in neuroendocrine function among states of estradiol negative and positive feedback.

Gonadotropin-Releasing Hormone (GnRH) Neuron Excitability Is Regulated by Estradiol Feedback and Kisspeptin.

Gonadotropin-releasing hormone (GnRH) neurons produce the central output controlling fertility and are regulated by steroid feedback. A switch from estradiol negative to positive feedback initiates the GnRH surge, ultimately triggering ovulation. This occurs on a daily basis in ovariectomized, estradiol-treated (OVX+E) mice; GnRH neurons are suppressed in the morning and activated in the afternoon. To test the hypotheses that estradiol and time of day signals alter GnRH neuron responsiveness to stimuli, GFP-identified GnRH neurons in brain slices from OVX+E or OVX female mice were recorded during the morning or afternoon. No differences were observed in baseline membrane potential. Current-clamp revealed GnRH neurons fired more action potentials in response to current injection during positive feedback relative to all other groups, which were not different from each other despite reports of differing ionic conductances. Kisspeptin increased GnRH neuron response in cells from OVX and OVX+E mice in the morning but not afternoon. Paradoxically, excitability in kisspeptin knock-out mice was similar to the maximum observed in control mice but was unchanged by time of day or estradiol. A mathematical model applying a Markov Chain Monte Carlo method to estimate probability distributions for estradiol- and time of day-dependent parameters was used to predict intrinsic properties underlying excitability changes. A single identifiable distribution of solutions accounted for similar GnRH neuron excitability in all groups other than positive feedback despite different underlying conductance properties; this was attributable to interdependence of voltage-gated potassium channel properties. In contrast, redundant solutions may explain positive feedback, perhaps indicative of the importance of this state for species survival.SIGNIFICANCE STATEMENT Infertility affects 15%-20% of couples; failure to ovulate is a common cause. Understanding how the brain controls ovulation is critical for new developments in both infertility treatment and contraception. Gonadotropin-releasing hormone (GnRH) neurons are the final common pathway for central neural control of ovulation. We studied how estradiol feedback regulates GnRH excitability, a key determinant of neural firing rate using laboratory and computational approaches. GnRH excitability is upregulated during positive feedback, perhaps driving increased neural firing rate at this time. Kisspeptin increased GnRH excitability and was essential for estradiol regulation of excitability. Modeling predicts that multiple combinations of changes to GnRH intrinsic conductances can produce the firing response in positive feedback, suggesting the brain has many ways to induce ovulation.

Frequency-Dependent Recruitment of Fast Amino Acid and Slow Neuropeptide Neurotransmitter Release Controls Gonadotropin-Releasing Hormone Neuron Excitability

The anteroventral periventricular nucleus (AVPV) is thought to play a key role in regulating the excitability of gonadotropin-releasing hormone (GnRH) neurons that control fertility. Using an angled, parahorizontal brain slice preparation we have undertaken a series of electrophysiological experiments to examine how the AVPV controls GnRH neurons in adult male and female mice. More than half (59%) of GnRH neurons located in the rostral preoptic area were found to receive monosynaptic inputs from the AVPV in a sex-dependent manner. AVPV stimulation frequencies <1 Hz generated short-latency action potentials in GnRH neurons with GABA and glutamate mediating >90% of the evoked fast synaptic currents. The AVPV GABA input was dominant and found to excite or inhibit GnRH neurons in a cell-dependent manner. Increasing the AVPV stimulation frequency to 5-10 Hz resulted in the appearance of additional poststimulus inhibitory as well as delayed excitatory responses in GnRH neurons that were independent of ionotropic amino acid receptors. The inhibition observed immediately following the end of the stimulation period was mediated partly by GABA(B) receptors, while the delayed activation was mediated by the neuropeptide kisspeptin. The latter response was essentially absent in Gpr54 knock-out mice and abolished by a Gpr54 antagonist. Together, these studies show that AVPV neurons provide direct amino acid and neuropeptidergic inputs to GnRH neurons. Low-frequency activation generates predominant GABA/glutamate release with higher frequency activation recruiting release of kisspeptin. This frequency-dependent release of amino acid and neuropeptide neurotransmitters greatly expands the range of AVPV control of GnRH neuron excitability.

Hyperpolarization-Activated Currents in Gonadotropin-Releasing Hormone (GnRH) Neurons Contribute to Intrinsic Excitability and Are Regulated by Gonadal Steroid Feedback

Pulsatile release of gonadotropin-releasing hormone (GnRH) is required for fertility and is regulated by steroid feedback. Hyperpolarization-activated currents (I(h)) play a critical role in many rhythmic neurons. We examined the contribution of I(h) to the membrane and firing properties of GnRH neurons and the modulation of this current by steroid milieu. Whole-cell voltage- and current-clamp recordings were made of GFP-identified GnRH neurons in brain slices from male mice that were gonad-intact, castrated, or castrated and treated with estradiol implants. APV, CNQX, and bicuculline were included to block fast synaptic transmission. GnRH neurons (47%) expressed a hyperpolarization-activated current with pharmacological and biophysical characteristics of I(h). The I(h)-specific blocker ZD7288 reduced hyperpolarization-induced sag and rebound potential, decreased GnRH neuron excitability and action potential firing, and hyperpolarized membrane potential in some cells. ZD7288 also altered the pattern of burst firing and reduced the slope of recovery from the after-hyperpolarization potential. Activation of I(h) by hyperpolarization increased spike frequency, whereas inactivation of I(h) by depolarization reduced spike frequency. Castration increased I(h) compared with that in gonad-intact males. This effect was reversed by in vivo estradiol replacement. Together, these data indicate I(h) provides an excitatory drive in GnRH neurons that contributes to action potential burst firing and that estradiol regulates I(h) in these cells. As estradiol is the primary central negative feedback hormone on GnRH neuron firing in males, this provides insight into the mechanisms by which steroid hormones potentially alter the intrinsic properties of GnRH neurons to change their activity.

Sexually Dimorphic Modulation of GABAA Receptor Currents by Melatonin in Rat Gonadotropin-Releasing Hormone Neurons

Gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons in the central control of reproduction. gamma-Amino butyric acid (GABA), one of the major regulators of GnRH neurons, depolarizes GnRH neurons isolated from adult rats via GABA(A) receptors. The presence of GABA(A) receptors in GnRH neurons has also been demonstrated morphologically. Furthermore, the pineal hormone melatonin is involved in the regulation of reproductive function, including the timing of the luteinizing hormone surge. The suprachiasmatic nucleus and the GABAergic system in the medial preoptic area are considered as possible sites of the action of melatonin. Until now, however, a direct action of melatonin on GnRH neurons has not been reported. Therefore we examined the effect of melatonin on GABA(A) receptor currents in GnRH neurons isolated from GnRH-EGFP transgenic rats by means of perforated patch-clamp experiments. The GABA(A) receptor currents were modulated by melatonin in a sex-specific manner. In GnRH neurons from adult males, melatonin augmented these currents in 67% of the neurons examined, but attenuated the currents in only 19% of them. These modulations were blocked by the melatonin receptor antagonist luzindole, suggesting an involvement of melatonin receptors. The modulation by melatonin was not observed in GnRH neurons isolated from infantile rats. These findings indicate that GABA affects the excitability of GnRH neurons in adult rats through GABA(A) receptors, and that melatonin modifies this excitability via melatonin receptors in a sex-specific manner.

Rat GnRH Neurons Exhibit Large Conductance Voltage- and Ca2+-Activated K+ (BK) Currents and Express BK Channel mRNAs

Gonadotropin-releasing hormone (GnRH) neurons form the final common pathway for the central regulation of reproduction. As in other neurons, the discharge pattern of action potentials is important for these neurons to function properly. Therefore it is important to elucidate the expression patterns of various types of ion channels in these neurons because they determine cell excitability. To date, voltage-gated Ca2+ channels and SK channels have been reported to be expressed in rat GnRH neurons. In this study, we focused on K+ channels and analyzed their expression in primary cultured GnRH neurons, prepared from GnRH-EGFP transgenic rats, by means of perforated patch-clamp recordings. GnRH neurons exhibited delayed-rectifier K+ currents and large conductance voltage- and Ca2+-activated K+ (BK) currents. Moreover, multicell RT-PCR (reverse transcriptase-polymerase chain reaction) experiments revealed the expression of BK channel mRNAs (alpha, beta1, beta2, and beta4). The results show the presence of delayed-rectifier K+ currents and BK currents besides previously reported slow afterhyperpolarization currents. These currents control the action potential repolarization and probably also the firing pattern, thereby regulating the cell excitability of GnRH neurons.

1. Neuroscience Series Ion Channels in Rat Gonadotropin-Releasing Hormone Neurons(1)

To understand neural mechanisms of reproductive physiology, we have generated promoter transgenic rats for gonadotropin-releasing hormone (GnRH) neurons. Using these rats we have analyzed ion channels expressed in rat GnRH neurons by means of patch clamp experiments and reverse transcriptase-polymerase chain reaction. The study revealed that rat GnRH neurons exhibit voltage-gated Na+, K+ and Ca2+ currents. There are delayed rectifier K+ currents, large conductance voltage- and Ca2+ -activated K+ currents (BK currents carried through BK channels) and Ca2+ -activated K+ currents (slow afterhyperpolarization currents carried through SK channels). Ca2+ currents are carried through five different Ca2+ channels, namely L-, N-, P/Q-, R- and T-types. Activation of these ion channels determines excitability of GnRH neurons and their firing pattern of action potentials.