Richard Anthony DeFazio

The pattern of gonadotropin‐releasing hormone (GnRH) release controls reproduction in vertebrates and is regulated by feedback from the gonads. Evidence is accumulating that steroid‐sensitive hypothalamic arcuate kisspeptin–neurokinin B–dynorphin (KNDy) neurons drive GnRH pulses. KNDy neurons exhibit coordinated, large amplitude calcium signals in vivo that precede LH pulses; coordinated events persist in brain slice recordings of these cells. We used cre‐lox to target GCaMP6f expression to KNDy neurons. Brain slice calcium imaging was done to identify putative unitary calcium events. Newly devised analyses identified two types of calcium events (fast and slow) in intact and gonadectomized male and female adult mice. Neurokinin B and the neurokinin‐3 receptor agonist senktide are strong activators of action potential firing in KNDy neurons. The effect of senktide on calcium events over 5‐min baseline and treatment periods was determined. Gonadectomy unexpectedly increased GCaMP6f fluorescence; illumination levels were adjusted to standardize initial levels. Senktide increased the frequency of fast and slow events; this effect was enhanced in gonadectomized mice for fast events. Senktide increased fast event amplitude only in gonadectomized mice. For slow events, in contrast, senktide increased amplitude in all groups, with the increase being larger in gonadectomized than intact mice. Differential regulation by gonadectomy and senktide modulation of fast and slow events suggests two separate mechanisms for these events, which likely also differ from the large amplitude events observed in vivo in KNDy neurons.

Polyendocrine metabolic ovarian syndrome (PMOS), a common cause of infertility, is marked by persistently high luteinizing hormone (LH) pulse frequency, presumably driven by high‐frequency GnRH pulses. Prenatally androgenized (PNA) mice mimic neuroendocrine PMOS symptoms including high LH‐pulse frequency. GnRH neurons from adult PNA mice have a higher spontaneous firing rate than those from vehicle (VEH) mice; this is reversed in peripubertal mice despite more excitatory inputs at both ages. We hypothesized voltage‐gated Ca ²⁺ currents ( I _Ca ) help set intrinsic excitability of GnRH neurons and are altered by development and/or PNA treatment. Whole‐cell patch‐clamp measurements were made of GnRH neuron I _Ca and excitability in 3 week‐old and adult VEH and PNA mice. PNA treatment increased I _Ca density and depolarized the I _Ca ‐half‐inactivation potential at both ages. In adults, the half‐activation potential of the Ca ²⁺ conductance was depolarized, and the half‐inactivation time of the fast I _Ca component was increased regardless of PNA treatment. There was an age‐related change in firing response to current injections driven primarily by more firing in 3 week‐old VEH mice; 3 week‐old PNA mice had adult‐like responses. Blocking small‐conductance Ca ²⁺ ‐activated K ⁺ current with apamin increased GnRH neuron firing rate except in adult PNA mice. Apamin changed the post‐spike‐train membrane response from hyperpolarization to depolarization; during development, this net effect of apamin was smaller in PNA mice. In summary, GnRH neurons from PNA mice have increased I _Ca and altered I _Ca kinetics. Ca ²⁺ ‐activated K ⁺ currents are less prominent in GnRH neurons from adult PNA mice, perhaps contributing to their increased spontaneous firing activity.

Polycystic ovary syndrome (PCOS), a common cause of infertility, is marked by persistently high luteinizing hormone (LH)-pulse frequency, presumably driven by high-frequency GnRH pulses. Prenatally androgenized (PNA) mice mimic neuroendocrine PCOS symptoms including high LH-pulse frequency. GnRH neurons from adult PNA mice have a higher firing rate than those from vehicle (VEH) mice; this is reversed in prepubertal mice despite more excitatory inputs at both ages. We hypothesized voltage-gated Ca ²⁺ currents (I _Ca ) help set intrinsic excitability of GnRH neurons and are altered by development and/or PNA treatment. Whole-cell patch-clamp recordings were used to measure GnRH neuron I _Ca in 3wk-old and adult VEH and PNA mice. PNA treatment increased I _Ca density and depolarized the I _Ca -half inactivation potential at both ages. In VEH but not PNA mice, the Ca ²⁺ -half activation potential was depolarized in adults versus 3wks. Age decreased the inactivation rate of a fast I _Ca regardless of PNA treatment. GnRH neuron firing rate during current injections was higher at 3wks than in adulthood in VEH mice only. Blocking small-conductance Ca²⁺-activated K⁺ current with apamin increased GnRH neuron firing rate except in adult PNA mice. Apamin changed the post-spike-train membrane response from hyperpolarization to depolarization; during development, this net effect of apamin became smaller in PNA mice. In summary, while GnRH neurons from PNA mice have increased I _Ca , they lack some developmental changes in I _Ca kinetics and intrinsic excitability observed in VEH mice. Ca²⁺-activated K⁺ currents are less prominent in GnRH neurons from adult PNA mice, perhaps contributing to increased spontaneous firing. Significance statement: Hyperactivation of GnRH neurons, which control reproductive endocrine function, can lead to increased LH-pulse frequency and is a hallmark of hyperandrogenemia polycystic ovary syndrome (PCOS). We used a mouse model of prenatal androgenization (PNA) that recapitulates the neuroendocrine aspects of PCOS to test the role of calcium currents (I _Ca ) in the PNA phenotype and the typical pubertal process. PNA treatment increased I _Ca in GnRH neurons both before and after puberty. Calcium plays a crucial role in neurosecretion thus this may enhance GnRH release. Another role of calcium is activation of calcium-sensitive potassium currents, which tend to decrease action potential firing rate. Despite increased I _Ca, calcium-activated potassium currents are less effective in adult PNA mice, perhaps contributing to GnRH neuron hyperactivation.

An upstream network, including glia and arcuate nucleus (ARC) kisspeptin neurons, controls hormone secretion from preoptic area (POA) gonadotropin-releasing hormone (GnRH) neurons, which form the final common pathway for the central control of fertility. In males, chemogenetic activation of Gq-mediated signaling in POA glia activated GnRH neurons and downstream luteinizing hormone (LH) release, while chemogenetic activation of ARC glia had no effect on ARC kisspeptin neurons. We characterized sex differences and time-of-day effects in these critical circuits to understand their effects on reproduction. Chemogenetic activation of glial fibrillary acidic protein (GFAP)-expressing cells increased intracellular calcium concentrations regardless of sex, brain region, or time of day. Activation of POA glia or treatment with the gliotransmitter analog dimethyl prostaglandin E2 (dmPGE2) increased GnRH neuron firing rate, and these responses were dependent upon sex and time of day. In contrast, ARC kisspeptin neuron firing rate was unresponsive to ARC glia activation or dmPGE2 regardless of sex or time of day. POA glial activation increased LH levels in males and females but the response in males was more rapid. ARC glia activation had no effect on LH in females and the response in males was delayed compared to POA glia activation. A similar LH response persisted after ARC kisspeptin neuron ablation, suggesting it is not mediated by those neurons. GnRH neurons, rather than arcuate kisspeptin neurons, are thus the main target of glial regulation of reproductive neuroendocrine output and this regulation is dependent on sex and time of day.

Astrocytes play active roles at synapses and can monitor, respond, and adapt to local synaptic activity. While there is abundant evidence that astrocytes modulate excitatory transmission in the hippocampus, evidence for astrocytic modulation of hippocampal synaptic inhibition remains more limited. Furthermore, to better investigate roles for astrocytes in modulating synaptic transmission, more tools that can selectively activate native G protein signaling pathways in astrocytes with both spatial and temporal precision are needed. Here, we utilized AAV8‐GFAP‐Optoα1AR‐eYFP (Optoα1AR), a viral vector that enables activation of G_q signaling in astrocytes via light‐sensitive α1‐adrenergic receptors. To determine if stimulating astrocytic Optoα1AR modulates hippocampal synaptic transmission, recordings were made in CA1 pyramidal cells with surrounding astrocytes expressing Optoα1AR, channelrhodopsin (ChR2), or GFP. Both high‐frequency (20 Hz, 45‐ms light pulses, 5 mW, 5 min) and low‐frequency (0.5 Hz, 1‐s pulses at increasing 1, 5, and 10 mW intensities, 90 s per intensity) blue light stimulation were tested. 20 Hz Optoα1AR stimulation increased both inhibitory and excitatory postsynaptic current (IPSC and EPSC) frequency, and the effect on miniature IPSCs (mIPSCs) was largely reversible within 20 min. However, low‐frequency stimulation of Optoα1AR did not modulate either IPSCs or EPSCs, suggesting that astrocytic G_q‐dependent modulation of basal synaptic transmission in the hippocampus is stimulation‐dependent. By contrast, low‐frequency stimulation of astrocytic ChR2 was effective in increasing both synaptic excitation and inhibition. Together, these data demonstrate that Optoα1AR activation in astrocytes changes basal GABAergic and glutamatergic transmission, but only following high‐frequency stimulation, highlighting the importance of temporal dynamics when using optical tools to manipulate astrocyte function.

Kisspeptin-expressing neurons in the anteroventral-periventricular nucleus (AVPV) are part of a neural circuit generating the gonadotropin-releasing hormone (GnRH) surge. This process is estradiol-dependent and occurs on the afternoon of proestrus in female mice. On proestrus, AVPV kisspeptin neurons express more kisspeptin and exhibit higher frequency action potentials and burst firing compared with diestrus, which is characterized by a pulsatile rather than a prolonged surge of GnRH secretion. We hypothesized changes in voltage-gated potassium conductances shape activity profiles of these cells in a cycle-dependent manner. Whole-cell voltage-clamp recordings of GFP-identified AVPV kisspeptin neurons in brain slices from diestrous and proestrous mice revealed three subcomponents of the voltage-sensitive K⁺current: fast-transient slow-transient, and residual. During proestrus, the V₅₀of inactivation of the fast-transient current was depolarized and the amplitude of the slow-transient component was reduced compared with diestrus; the residual component was consistent across both stages. Computational models were fit to experimental data, including published estrous-cycle effects on other voltage-gated currents. Computer simulations suggest proestrus-typical K⁺currents are suppressive compared with diestrus. Interestingly, larger T-type, persistent-sodium, and hyperpolarization-activated currents during proestrus compensate for this suppressive effect while also enabling postinhibitory rebound bursting. These findings suggest modulation of voltage-gated K⁺and multiple subthreshold depolarizing currents across the negative to positive feedback transition maintain AVPV kisspeptin neuron excitability in response to depolarizing stimuli. These changes also enable firing in response to hyperpolarization, providing a net increase in neuronal excitability, which may contribute to activation of this population leading up to the preovulatory GnRH surge.

GnRH neurons are the final central neural output regulating fertility. Kisspeptin neurons in the hypothalamic arcuate nucleus (KNDy neurons) are considered the main regulator of GnRH output. GnRH and KNDy neurons are surrounded by astrocytes, which can modulate neuronal activity and communicate over distances. Prostaglandin E2 (PGE2), synthesized primarily by astrocytes, increases GnRH neuron activity and downstream pituitary release of luteinizing hormone (LH). We hypothesized that glial fibrillary acidic protein (GFAP)-expressing astrocytes play a role in regulating GnRH and/or KNDy neuron activity and LH release. We used adeno-associated viruses to target designer receptors exclusively activated by designer drugs (DREADDs) to GFAP-expressing cells to activate Gq- or Gi-mediated signaling. Activating Gq signaling in the preoptic area, near GnRH neurons, but not in the arcuate, increases LH release in vivo and GnRH firing in vitro via a mechanism in part dependent upon PGE2. These data suggest that astrocytes can activate GnRH/LH release in a manner independent of KNDy neurons.

Kisspeptin-expressing neurons in the anteroventral-periventricular nucleus (AVPV) are part of a neural circuit generating the gonadotropin-releasing hormone (GnRH) surge. This process is estradiol-dependent and occurs on the afternoon of proestrus in female mice. On proestrus, AVPV kisspeptin neurons express more kisspeptin and exhibit higher frequency action potentials and burst firing compared to diestrus, which is characterized by a pulsatile rather than a prolonged surge of GnRH secretion. We hypothesized changes in voltage-gated potassium conductances shape activity profiles of these cells in a cycle-dependent manner. Whole-cell voltage-clamp recordings of GFP-identified AVPV kisspeptin neurons in brain slices from diestrous and proestrous mice revealed three subcomponents of the voltage-sensitive K ⁺ current: fast-inactivating, slow-inactivating, and residual. During proestrus, the V50 of inactivation of the fast-inactivating current was depolarized and the amplitude of the slow-inactivating component was reduced compared to diestrus; the residual component was consistent across both stages. Computational models were fit to experimental data, including published estrous-cycle effects on other voltage-gated currents. Computer simulations suggest proestrus-typical K ⁺ currents are suppressive compared to diestrus. Interestingly, larger T-type, persistent-sodium, and hyperpolarization-activated currents during proestrus compensate for this suppressive effect while also enabling post-inhibitory rebound bursting. These findings suggest modulation of voltage-gated K ⁺ and multiple subthreshold depolarizing currents across the negative to positive feedback transition maintain AVPV kisspeptin neuron excitability in response to depolarizing stimuli. These changes also enable firing in response to hyperpolarization, providing a net increase in neuronal excitability, which may contribute to activation of this population leading up to the preovulatory GnRH surge. Significance Statement: GnRH neurons provide the central signal to initiate ovulation by releasing a surge of hormone. GnRH neurons are regulated by other cells including those expressing kisspeptin, a potent stimulator of GnRH secretion. Kisspeptin neurons in the anteroventral-periventricular nucleus (AVPV) express more kisspeptin and become more active during the afternoon of proestrus, the phase of the rodent estrous (reproductive) cycle when the GnRH surge occurs. We found voltage-dependent potassium currents in AVPV kisspeptin neurons change with phase of the estrous cycle. Firing simulations indicated these changes are suppressive if occurring in isolation. But proestrous-typical increases in subthreshold depolarizing currents overcome this suppression and promote greater excitability by increasing rebound firing, possibly contributing to the preovulatory activation of this system.

Gonadotropin-releasing hormone (GnRH) drives pituitary secretion of luteinizing hormone (LH) and follicle-stimulating hormone, which in turn regulate gonadal functions including steroidogenesis. The pattern of GnRH release and thus fertility depend on gonadal steroid feedback. Under homeostatic (negative) feedback conditions, removal of the gonads from either females or males increases the amplitude and frequency of GnRH release and alters the long-term firing pattern of these neurons in brain slices. The neurobiological mechanisms intrinsic to GnRH neurons that are altered by homeostatic feedback are not well studied and have not been compared between sexes. During estradiol positive feedback, which is unique to females, there are correlated changes in voltage-gated potassium currents and neuronal excitability. We thus hypothesized these same mechanisms would be engaged in homeostatic negative feedback. Voltage-gated potassium channels play a direct role in setting excitability and action potential properties. Whole-cell patch-clamp recordings of GFP-identified GnRH neurons in brain slices from sham-operated and castrated adult female and male mice were made to assess fast (I_A) and slow (I_K) inactivating potassium currents as well as action potential properties. Surprisingly, no changes were observed among groups in most potassium current properties, input resistance or capacitance and this was reflected in a lack of differences in excitability and specific action potential properties. These results support the concept that, in contrast to positive feedback, steroid negative feedback regulation of GnRH neurons in both sexes is likely conveyed to GnRH neurons via mechanisms that do not induce major changes in the biophysical properties of these cells. Significance Statement The pattern of activity of gonadotropin-releasing hormone (GnRH) neurons is crucial to reproductive success in both males and females. Direct comparison of GnRH neurons from mice of both sexes during negative feedback and after gonadectomy revealed few differences in potassium currents, excitability, and action potential properties. These results support the hypothesis that neurons presynaptic to GnRH neurons communicate negative feedback to these cells in a manner that does not alter their intrinsic biophysical properties.

Gonadotropin-releasing hormone (GnRH) drives pituitary secretion of luteinizing hormone (LH) and follicle-stimulating hormone, which in turn regulate gonadal functions including steroidogenesis. The pattern of GnRH release and thus fertility depend on gonadal steroid feedback. Under homeostatic (negative) feedback conditions, removal of the gonads from either females or males increases the amplitude and frequency of GnRH release and alters the long-term firing pattern of these neurons in brain slices. The neurobiological mechanisms intrinsic to GnRH neurons that are altered by homeostatic feedback are not well studied and have not been compared between sexes. During estradiol positive feedback, which is unique to females, there are correlated changes in voltage-gated potassium currents and neuronal excitability. We thus hypothesized these same mechanisms would be engaged in homeostatic negative feedback. Voltage-gated potassium channels play a direct role in setting excitability and action potential properties. Whole-cell patch-clamp recordings of GFP-identified GnRH neurons in brain slices from sham-operated and castrated adult female and male mice were made to assess fast (I _A ) and slow (I _K ) inactivating potassium currents as well as action potential properties. Surprisingly, no changes were observed in most potassium current properties, input resistance or capacitance and this was reflected in a lack of differences in excitability and specific action potential properties. These results support the concept that, in contrast to positive feedback, steroid negative feedback regulation of GnRH neurons in both sexes is likely conveyed to GnRH neurons via mechanisms that do not induce major changes in the biophysical properties of these cells. Significance Statement: The pattern of activity of gonadotropin-releasing hormone (GnRH) neurons is crucial to reproductive success in both males and females. Direct comparison of GnRH neurons from mice of both sexes during negative feedback and after gonadectomy revealed few differences in potassium currents, excitability, and action potential properties. These results support the hypothesis that neurons presynaptic to GnRH neurons communicate negative feedback to these cells in a manner that does not alter their intrinsic biophysical properties.