Gonadotropin-releasing Hormone Neurons In Brain Slices Research Articles

Development and prenatal exposure to androgens alter potassium currents in gonadotropin-releasing hormone neurons from female mice.

Pulsatile gonadotropin-releasing hormone (GnRH) release is critical for reproduction. Disruptions to GnRH secretion patterns may contribute to polycystic ovary syndrome (PCOS). Prenatally androgenized (PNA) female mice recapitulate many neuroendocrine abnormalities observed in PCOS patients. PNA and development induce changes in spontaneous GnRH neuron firing rate, response to synaptic input, and the afterhyperpolarization potential of the action potential. We hypothesized potassium currents are altered by PNA treatment and/or development. Whole-cell patch-clamp recordings were made of transient and residual potassium currents of GnRH neurons in brain slices from 3-week-old and adult control and PNA females. At 3 weeks of age, PNA treatment increased transient current density versus controls. Development and PNA altered voltage-dependent activation and inactivation of the transient current. In controls, transient current activation and inactivation were depolarized at 3 weeks of age versus in adulthood. In GnRH neurons from 3-week-old mice, transient current activation and inactivation were more depolarized in control than PNA mice. Development and PNA treatment interacted to shift the time-dependence of inactivation and recovery from inactivation. Notably, in cells from adult PNA females, recovery was prolonged compared to all other groups. Activation of the residual current occurred at more depolarized membrane potentials in 3-week-old than adult controls. PNA depolarized activation of the residual current in adults. These findings demonstrate the properties of GnRH neuron potassium currents change during typical development, potentially contributing to puberty, and further suggest PNA treatment may both alter some typical developmental changes and induce additional modifications, which together may underlie aspects of the PNA phenotype. There was not any clinical trial involved in this work.

Gonadal Feedback Alters the Relationship between Action Potentials and Hormone Release in Gonadotropin-Releasing Hormone Neurons in Male Mice.

In vertebrates, the pulsatile release of gonadotropin-releasing hormone (GnRH) from neurons in the hypothalamus triggers secretion of anterior pituitary gonadotropins, which activate steroidogenesis, and steroids in turn exert typically homeostatic negative feedback on GnRH release. Although long-term episodic firing patterns of GnRH neurons in brain slices resemble the pulsatile release of GnRH and LH in vivo, neither the relationship between GnRH neuron firing and release nor whether this relationship is influenced by gonadal feedback are known. We combined fast-scan cyclic voltammetry and patch-clamp to perform simultaneous measurements of neuropeptide release with either spontaneous action potential firing or in response to neuromodulator or action-potential-spike templates in brain slice preparations from male mice. GnRH release increased with higher frequency spontaneous firing to a point; release reached a plateau after which further increases in firing rate did not elicit further increased release. Kisspeptin, a potent GnRH neuron activator via a Gq-coupled signaling pathway, triggered GnRH release before increasing firing rate, whether globally perfused or locally applied. Increasing the number of spikes in an applied burst template increased release; orchidectomized mice had higher sensitivity to the increased action potential number than sham-operated mice. Similarly, Ca2+ currents triggered by these burst templates were increased in GnRH neurons of orchidectomized mice. These results suggest removal of gonadal feedback increases the efficacy of the stimulus-secretion coupling mechanisms, a phenomenon that may extend to other steroid-sensitive regions of the brain.SIGNIFICANCE STATEMENT Pulsatile secretion of GnRH plays a critical role in fertility. The temporal relationship between GnRH neuron action potential firing and GnRH release remains unknown as does whether this relationship is influenced by gonadal feedback. By combining techniques of fast-scan cyclic voltammetry and patch-clamp we, for the first time, monitored GnRH concentration changes during spontaneous and neuromodulator-induced GnRH neuron firing. We also made the novel observation that gonadal factors exert negative feedback on excitation-secretion coupling to reduce release in response to the same stimulus. This has implications for the control of normal fertility, central causes of infertility, and more broadly for the effects of sex steroids in the brain.

Firing patterns of gonadotropin-releasing hormone neurons are sculpted by their biologic state.

Gonadotropin-releasing hormone (GnRH) neurons form the final pathway for the central neuronal control of fertility. GnRH is released in pulses that vary in frequency in females, helping drive hormonal changes of the reproductive cycle. In the common fertility disorder polycystic ovary syndrome (PCOS), persistent high-frequency hormone release is associated with disrupted cycles. We investigated long- and short-term action potential patterns of GnRH neurons in brain slices before and after puberty in female control and prenatally androgenized (PNA) mice, which mimic aspects of PCOS. A Monte Carlo (MC) approach was used to randomize action potential interval order. Dataset distributions were analysed to assess (i) if organization persists in GnRH neuron activity in vitro, and (ii) to determine if any organization changes with development and/or PNA treatment. GnRH neurons in adult control, but not PNA, mice produce long-term patterns different from MC distributions. Short-term patterns differ from MC distributions before puberty but become absorbed into the distributions with maturation, and the distributions narrow. These maturational changes are blunted by PNA treatment. Firing patterns of GnRH neurons in brain slices thus maintain organization dictated at least in part by the biologic status of the source and are disrupted in models of disease.

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.

Prepubertal Development of GABAergic Transmission to Gonadotropin-Releasing Hormone (GnRH) Neurons and Postsynaptic Response Are Altered by Prenatal Androgenization.

Gonadotropin-releasing hormone (GnRH) neurons regulate reproduction through pulsatile GnRH release. Women with polycystic ovary syndrome (PCOS) have persistently elevated luteinizing hormone release frequency, reflecting GnRH release; this exacerbates hyperandrogenemia and disrupted reproductive cycles that are characteristic of this disorder. Clinical evidence suggests that neuroendocrine features of PCOS may manifest peripubertally. Adult mice prenatally exposed to androgens (PNA) mimic several reproductive features of PCOS. GnRH neurons from these mice have increased firing activity and receive increased GABAergic transmission, which is excitatory. When changes emerge during development is unknown. To study the typical postnatal development of GABAergic transmission and the effects of PNA treatment and sex, whole-cell voltage-clamp recordings were made of GABAergic postsynaptic currents (PSCs) in GnRH neurons in brain slices from prepubertal through adult control and PNA female and male mice. GABAergic transmission was present by 1 week of age in females and males and increased in frequency, reaching adult levels at 3 and 4 weeks, respectively. GABAergic PSC frequency was elevated in 3-week-old PNA versus control females. PSC frequency in both controls and PNA mice was activity independent, suggesting that PNA induces changes in synapse organization. PNA also alters the functional response of GnRH neurons to GABA. GABA induced firing in fewer neurons from 3-week-old PNA than control females; membrane potential depolarization induced by GABA was also reduced in cells from PNA mice at this age. PNA thus induces changes during development in the presynaptic organization of the GABAergic network afferent to GnRH neurons as well as the postsynaptic GnRH neuron response, both of which may contribute to adult reproductive dysfunction.SIGNIFICANCE STATEMENT The central neuronal network that regulates reproduction is overactive in polycystic ovary syndrome (PCOS), a leading cause of infertility. Recent evidence of neuroendocrine dysfunction in midpubertal girls suggests that the pathophysiological mechanisms underlying PCOS may arise before pubertal maturation. Prenatal exposure to androgens (PNA) in mice mimics several neuroendocrine features of PCOS. GABAergic transmission to gonadotropin-releasing hormone (GnRH) neurons is important for reproduction and is increased in adult PNA mice. The typical development of this network and when changes with PNA and sex arise relative to puberty are unknown. These studies provide evidence that PNA alters prepubertal development of the GABAergic network afferent to GnRH neurons, including both the presynaptic organization and postsynaptic response. These changes may contribute to reproductive dysfunction in adults.

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.

Estradiol-Dependent Stimulation and Suppression of Gonadotropin-Releasing Hormone Neuron Firing Activity by Corticotropin-Releasing Hormone in Female Mice

Gonadotropin-releasing hormone (GnRH) neurons are the final central regulators of reproduction, integrating various inputs that modulate fertility. Stress typically inhibits reproduction but can be stimulatory; stress effects can also be modulated by steroid milieu. Corticotropin-releasing hormone (CRH) released during the stress response may suppress reproduction independent of downstream glucocorticoids. We hypothesized CRH suppresses fertility by decreasing GnRH neuron firing activity. To test this, mice were ovariectomized (OVX) and either implanted with an estradiol capsule (OVX+E) or not treated further to examine the influence of estradiol on GnRH neuron response to CRH. Targeted extracellular recordings were used to record firing activity from green fluorescent protein-identified GnRH neurons in brain slices before and during CRH treatment; recordings were done in the afternoon when estradiol has a positive feedback effect to increase GnRH neuron firing. In OVX mice, CRH did not affect the firing rate of GnRH neurons. In contrast, CRH exhibited dose-dependent stimulatory (30 nM) or inhibitory (100 nM) effects on GnRH neuron firing activity in OVX+E mice; both effects were reversible. The dose-dependent effects of CRH appear to result from activation of different receptor populations; a CRH receptor type-1 agonist increased firing activity in GnRH neurons, whereas a CRH receptor type-2 agonist decreased firing activity. CRH and specific agonists also differentially regulated short-term burst frequency and burst properties, including burst duration, spikes/burst, and/or intraburst interval. These results indicate that CRH alters GnRH neuron activity and that estradiol is required for CRH to exert both stimulatory and inhibitory effects on GnRH neurons.

Prepubertal Development of Gonadotropin-Releasing Hormone Neuron Activity Is Altered by Sex, Age, and Prenatal Androgen Exposure.

Gonadotropin-releasing hormone (GnRH) neurons regulate reproduction though pulsatile hormone release. Disruption of GnRH release as measured via luteinizing hormone (LH) pulses occurs in polycystic ovary syndrome (PCOS), and in young hyperandrogenemic girls. In adult prenatally androgenized (PNA) mice, which exhibit many aspects of PCOS, increased LH is associated with increased GnRH neuron action potential firing. How GnRH neuron activity develops over the prepubertal period and whether this is altered by sex or prenatal androgen treatment are unknown. We hypothesized GnRH neurons are active before puberty and that this activity is sexually differentiated and altered by PNA. Dams were injected with dihydrotestosterone (DHT) on days 16 to 18 post copulation to generate PNA mice. Action potential firing of GFP-identified GnRH neurons in brain slices from 1-, 2-, 3-, and 4-week-old and adult mice was monitored. GnRH neurons were active at all ages tested. In control females, activity increased with age through 3 weeks, then decreased to adult levels. In contrast, activity did not change in PNA females and was reduced at 3 weeks. Activity was higher in control females than males from 2 to 3 weeks. PNA did not affect GnRH neuron firing rate in males at any age. Short-term action potential patterns were also affected by age and PNA treatment. GnRH neurons are thus typically more active during the prepubertal period than adulthood, and PNA reduces prepubertal activity in females. Prepubertal activity may play a role in establishing sexually differentiated neuronal networks upstream of GnRH neurons; androgen-induced changes during this time may contribute to the adult PNA, and possibly PCOS, phenotype.

Kisspeptin Inhibits High-Voltage Activated Ca2+ Channels in GnRH Neurons via Multiple Ca2+ Influx and Release Pathways

Kisspeptin plays an important role in puberty and subsequent fertility by activating its receptor, G-protein-coupled receptor 54 (GPR54), and increasing cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) and gonadotropin-releasing hormone (GnRH) secretion in GnRH neurons. Yet the mechanism by which kisspeptin increases [Ca²⁺]_i in GnRH neurons remains to be fully elucidated. In other neurons, voltage-gated Ca²⁺ channel (VGCC) activity has been shown to be inversely related to [Ca²⁺]_i. We used whole-cell patch-clamp recording to examine the effects of kisspeptin-10 (KP-10) on VGCC activity evoked by step depolarizations in GnRH neurons in brain slices from pubertal male GnRH-green fluorescent protein transgenic mice. Prolonged (>30 s) KP-10 application inhibited Ca²⁺ currents. The GPR54 antagonist peptide 234, chelation of intracellular Ca²⁺ by 1,2-bis(2-aminophenoxy)ethane N,N,N′,N′-tetraacetic acid, substitution of Ba²⁺ for Ca²⁺, the calmodulin antagonists calmidazolium and trifluoperazine, the phospholipase C inhibitor edelfosine, the canonical transient receptor potential (TRPC) channel and inositol 1,4,5-trisphosphate receptor (IP₃R) antagonist 2-APB, the TRPC channel antagonist BTP2 and the endoplasmic reticulum Ca²⁺-ATPase blocker cyclopiazonic acid each prevented inhibition. The IP₃R antagonists caffeine (10 µM), heparin and intracellular 2-APB prevented inhibition to a lesser extent. The ryanodine receptor (RyR) antagonists ryanodine and dantrolene prevented inhibition, and the RyR agonist caffeine (30 mM) mimicked the effects of KP-10 on Ca²⁺ currents. Our results suggest that kisspeptin induces Ca²⁺ influx through TRPC channels and Ca²⁺ release via IP₃Rs and RyRs, and that this is followed by Ca²⁺/CaM-dependent inhibition of VGCCs.

GnRH Neurons Directly Listen to the Periphery

GnRH Neurons Directly Listen to the Periphery

Prostaglandin E 2 release from astrocytes triggers gonadotropin-releasing hormone (GnRH) neuron firing via EP2 receptor activation

Astrocytes in the hypothalamus release prostaglandin E(2) (PGE(2)) in response to cell-cell signaling initiated by neurons and glial cells. Upon release, PGE(2) stimulates the secretion of gonadotropin-releasing hormone (GnRH), the neuropeptide that controls reproduction, from hypothalamic neuroendocrine neurons. Whether this effect on GnRH secretion is accompanied by changes in the firing behavior of these neurons is unknown. Using patch-clamp recording we demonstrate that PGE(2) exerts a dose-dependent postsynaptic excitatory effect on GnRH neurons. These effects are mimicked by an EP2 receptor agonist and attenuated by protein kinase A (PKA) inhibitors. The acute blockade of prostaglandin synthesis by indomethacin (INDO) or the selective inhibition of astrocyte metabolism by fluoroacetate (FA) suppresses the spontaneous firing activity of GnRH neurons in brain slices. Similarly, GnRH neuronal activity is reduced in mice with impaired astrocytic PGE(2) release due to defective erbB signaling in astrocytes. These results indicate that astrocyte-to-neuron communication in the hypothalamus is essential for the activity of GnRH neurons and suggest that PGE(2) acts as a gliotransmitter within the GnRH neurosecretory system.

Voltage-Gated Potassium Currents Are Targets of Diurnal Changes in Estradiol Feedback Regulation and Kisspeptin Action on Gonadotropin-Releasing Hormone Neurons in Mice1

Estradiol has both negative and positive feedback actions upon gonadotropin-releasing hormone (GnRH) release; the latter actions trigger the preovulatory GnRH surge. Although neurobiological mechanisms of the transitions between feedback modes are becoming better understood, the roles of voltage-gated potassium currents, major contributors to neuronal excitability, are unknown. Estradiol alters two components of potassium currents in these cells: a transient current, I(A), and a sustained current, I(K). Kisspeptin is a potential mediator between estradiol and GnRH neurons and can act directly on GnRH neurons. We examined how estradiol, time of day, and kisspeptin interact to regulate these conductances in a mouse model exhibiting daily switches between estradiol negative (morning) and positive feedback (evening). Whole-cell voltage clamp recordings were made from GnRH neurons in brain slices from ovariectomized (OVX) mice and from OVX mice treated with estradiol (OVX+E). There were no diurnal changes in either I(A) or I(K) in GnRH neurons from OVX mice. In contrast, in GnRH neurons from OVX+E mice, I(A) and I(K) were greater during the morning when GnRH neuron activity is low and smaller in the evening when GnRH neuron activity is high. Estradiol increased I(A) in the morning and decreased it in the evening, relative to that in cells from OVX mice. Exogenously applied kisspeptin reduced I(A) regardless of time of day or estradiol status. Estradiol, interacting with time of day, and kisspeptin both depolarized I(A) activation. These findings extend our understanding of both the neurobiological mechanisms of estradiol negative vs. positive regulation of GnRH neurons and of kisspeptin action on these cells.

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.

DiurnalIn Vivoand RapidIn VitroEffects of Estradiol on Voltage-Gated Calcium Channels in Gonadotropin-Releasing Hormone Neurons

A robust surge of gonadotropin-releasing hormone (GnRH) release triggers the luteinizing hormone surge that induces ovulation. The GnRH surge is attributable to estradiol feedback, but the mechanisms are incompletely understood. Voltage-gated calcium channels (VGCCs) regulate hormone release and neuronal excitability, and may be part of the surge-generating mechanism. We examined VGCCs of GnRH neurons in brain slices from a model exhibiting daily luteinizing hormone surges. Mice were ovariectomized (OVX), and a subset was treated with estradiol implants (OVX+E). OVX+E mice exhibit negative feedback in the A.M. and positive feedback in the P.M. GnRH neurons express prominent high-voltage-activated (HVA) and small low-voltage-activated (LVA) macroscopic (whole-cell) Ca currents (I(Ca)). LVA-mediated currents were not altered by estradiol or time of day. In contrast, in OVX+E mice, HVA-mediated currents varied with time of day; HVA currents in cells from OVX+E mice were lower than those in cells from OVX mice in the A.M. but were higher in the P.M. These changes were attributable to diurnal alternations in L- and N-type components. There were no diurnal changes in any aspect of HVA-mediated I(Ca) in OVX mice. Acute in vitro treatment of cells from OVX and OVX+E mice with estradiol rapidly increased HVA currents primarily through L- and R-type VGCCs by activating estrogen receptor beta and GPR30, respectively. These results suggest multiple mechanisms contribute to the overall feedback regulation of HVA-mediated I(Ca) by estradiol. In combination with changes in synaptic inputs to GnRH neurons, these intrinsic changes in GnRH neurons may play critical roles in estradiol feedback.

GABAergic Transmission to Gonadotropin-Releasing Hormone (GnRH) Neurons Is Regulated by GnRH in a Concentration-Dependent Manner Engaging Multiple Signaling Pathways

Gonadotropin-releasing hormone (GnRH) neurons are the central regulators of fertility. GnRH stimulates or inhibits GnRH neuronal activity depending on dose. The mechanisms for these actions remain unknown. We hypothesized GnRH acts in part by altering fast synaptic transmission to GnRH neurons. GABAergic and glutamatergic postsynaptic currents (PSCs), both of which can excite these neurons, were recorded from GnRH neurons in brain slices from adult intact and orchidectomized (ORX) males. ORX enhanced the frequency of GABA transmission to GnRH neurons, but had no effect on glutamatergic transmission. Effects of ORX on GABAergic transmission were reversed by estradiol replacement, suggesting GABA is a mediator of steroid feedback in males. GABAergic neurons express type-1 GnRH receptor (GnRHR-1). Low GnRH (20 nm) reduced GABAergic PSC frequency in GnRH neurons from both ORX and intact mice. High GnRH (2 microm) had no effect on either GABAergic or glutamatergic transmission to GnRH neurons. To investigate mechanisms mediating low-dose GnRH suppression of GABAergic transmission, GABAergic PSCs were recorded after arresting G(alphai) activity with pertussis toxin (PTX). PTX abolished the suppressive effect of low GnRH. Moreover, PTX uncovered a stimulatory effect of high GnRH on GABAergic transmission. These data suggest low-dose GnRH suppresses GnRH firing rate in part by decreasing GABAergic transmission to the GnRH neurons, independent of gonadal hormone milieu. Low-dose GnRH appears to exert the suppressive effect by activating GnRHR-I coupled to G(alphai). The concentration-dependent effects of GnRH may be mediated in part by changes in affinity of GnRH to GnRHR-I coupled to different G(alpha) proteins.

Differential Regulation of Gonadotropin-Releasing Hormone Neuron Activity and Membrane Properties by Acutely Applied Estradiol: Dependence on Dose and Estrogen Receptor Subtype

Gonadotropin-releasing hormone (GnRH) neurons are critical to controlling fertility. In vivo, estradiol can inhibit or stimulate GnRH release depending on concentration and physiological state. We examined rapid, nongenomic effects of estradiol. Whole-cell recordings were made of GnRH neurons in brain slices from ovariectomized mice with ionotropic GABA and glutamate receptors blocked. Estradiol was bath applied and measurements completed within 15 min. Estradiol from high physiological (preovulatory) concentrations (100 pm) to 100 nm enhanced action potential firing, reduced afterhyperpolarizing potential (AHP) and increased slow afterdepolarization amplitudes (ADP), and reduced I(AHP) and enhanced I(ADP). The reduction of I(AHP) was occluded by previous blockade of calcium-activated potassium channels. These effects were mimicked by an estrogen receptor (ER) beta-specific agonist and were blocked by the classical receptor antagonist ICI182780. ERalpha or GPR30 agonists had no effect. The acute stimulatory effect of high physiological estradiol on firing rate was dependent on signaling via protein kinase A. In contrast, low physiological levels of estradiol (10 pm) did not affect intrinsic properties. Without blockade of ionotropic GABA and glutamate receptors, however, 10 pm estradiol reduced firing of GnRH neurons; this was mimicked by an ERalpha agonist. ERalpha agonists reduced the frequency of GABA transmission to GnRH neurons; GABA can excite to these cells. In contrast, ERbeta agonists increased GABA transmission and postsynaptic response. These data suggest rapid intrinsic and network modulation of GnRH neurons by estradiol is dependent on both dose and receptor subtype. In cooperation with genomic actions, nongenomic effects may play a role in feedback regulation of GnRH secretion.

Physiologic Regulation of a Tetrodotoxin-Sensitive Sodium Influx That Mediates a Slow Afterdepolarization Potential in Gonadotropin-Releasing Hormone Neurons: Possible Implications for the Central Regulation of Fertility

The brain controls fertility through release of gonadotropin-releasing hormone (GnRH), but the mechanisms underlying action potential patterning and GnRH release are not understood. We investigated whether GnRH neurons exhibit afterdepolarizing potentials (ADPs) and whether these are modified by reproductive state. Whole-cell current-clamp recordings of GnRH neurons in brain slices from ovariectomized mice revealed a slow ADP (sADP) after action potentials generated by brief current injection. Generating two or four spikes enhanced sADP amplitude and duration. sADP amplitude was not affected by blocking selected neurotransmitter/neuromodulator receptors, delayed-rectifier potassium channels, calcium-dependent cation channels, or hyperpolarization-activated cation channels but was halved by the calcium channel blocker cadmium and abolished by tetrodotoxin. Cadmium also reduced peak latency. Intrinsic mechanisms underlying the sADP were investigated using voltage-clamp protocols simulating action potential waveforms. A single action potential produced an inward current, which increased after double and quadruple stimulation. Cadmium did not affect current amplitude but reduced peak latency. Pretreatment with blockers of calcium-activated potassium currents (I(KCa)) reproduced this shift and blocked subsequent cadmium-induced changes, suggesting cadmium changes latency indirectly by blocking I(KCa). Tetrodotoxin abolished the inward current, suggesting that it is carried by sodium. In contrast, I(KCa) blockers increased the inward current, indicating that I(KCa) may oppose generation of the sADP. Strong sADPs were suprathreshold, generating repetitive spontaneous firing. I(ADP), sADP, and excitability were enhanced by in vivo estradiol, which triggers a preovulatory surge of GnRH release. Physiological feedback modification of this inward current and resulting sADP may modulate action potential firing and subsequent GnRH release.

Diurnal and estradiol-dependent changes in gonadotropin-releasing hormone neuron firing activity

A robust gonadotropin-releasing hormone (GnRH) surge is a prerequisite signal for the luteinizing hormone (LH) surge that triggers ovulation. In rodents, the GnRH surge is initiated by elevated estradiol and a diurnal switch in estrogen action from negative to positive feedback. The ability of constant estradiol treatment to induce daily LH surges was tested in adult mice that were ovariectomized (OVX) or OVX and treated with estradiol implants (OVX+E). LH in OVX mice showed no time-of-day difference. In contrast, OVX+E mice showed a large LH surge (8- to 124-fold relative to the a.m.) in p.m. samples on d 2-5 post-OVX+E. Targeted extracellular recordings were used to examine changes in firing activity of GnRH neurons in brain slices. There was no time-of-day difference in cells from OVX mice. In contrast, OVX+E cells recorded in the p.m. showed an increased mean firing rate and instantaneous firing frequency, which could increase GnRH release, and decreased duration of quiescence between bouts of firing, possibly reflecting increased pulse frequency, compared with cells recorded in the a.m. In the a.m., OVX+E cells showed changes in GnRH neuron firing reflecting negative feedback compared with OVX cells, whereas in the p.m., OVX+E cells exhibited changes suggesting positive feedback. These data indicate that differences in pattern and level of individual GnRH neuron firing may reflect the switch in estradiol action and underlie GnRH surge generation. The persistence of altered GnRH neuron activity in slices indicates that this approach can be used to study the neurobiological mechanisms of surge generation.