Nitric oxide resets kisspeptin-excited GnRH neurons via PIP2 replenishment.

Fertility relies upon pulsatile release of gonadotropin-releasing hormone (GnRH) that drives pulsatile luteinizing hormone secretion. Kisspeptin (KP) neurons in the arcuate nucleus are at the center of the GnRH pulse generation and the steroid feedback control of GnRH secretion. However, KP evokes a long-lasting response in GnRH neurons that is hard to reconcile with periodic GnRH activity required to drive GnRH pulses. Using calcium imaging, we show that 1) the tetrodotoxin-insensitive calcium response evoked by KP relies upon the ongoing activity of canonical transient receptor potential channels maintaining voltage-gated calcium channels in an activated state, 2) the duration of the calcium response is determined by the rate of resynthesis of phosphatidylinositol 4,5-bisphosphate (PIP2), and 3) nitric oxide terminates the calcium response by facilitating the resynthesis of PIP2 via the canonical pathway guanylyl cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G. In addition, our data indicate that exposure to nitric oxide after KP facilitates the calcium response to a subsequent KP application. This effect was replicated using electrophysiology on GnRH neurons in acute brain slices. The interplay between KP and nitric oxide signaling provides a mechanism for modulation of the refractory period of GnRH neurons after KP exposure and places nitric oxide as an important component for tonic GnRH neuronal pulses.

The astroglial and stem cell functions of adult rat folliculostellate cells.

The mammalian pituitary gland is a complex organ consisting of hormone-producing cells, anterior lobe folliculostellate cells (FSCs), posterior lobe pituicytes, vascular pericytes and endothelial cells, and Sox2-expressing stem cells. We present single-cell RNA sequencing and immunohistofluorescence analyses of pituitary cells of adult female rats with a focus on the transcriptomic profiles of nonhormonal cell types. Samples obtained from whole pituitaries and separated anterior and posterior lobe cells contained all expected pituitary resident cell types and lobe-specific vascular cell subpopulations. FSCs and pituicytes expressed S100B, ALDOC, EAAT1, ALDH1A1, and VIM genes and proteins, as well as other astroglial marker genes, some common and some cell type-specific. We also found that the SOX2 gene and protein were expressed in ~15% of pituitary cells, including FSCs, pituicytes, and a fraction of hormone-producing cells, arguing against its stem cell specificity. FSCs comprised two Sox2-expressing subclusters; FS1 contained more cells but lower genetic diversity, while FS2 contained proliferative cells, shared genes with hormone-producing cells, and expressed genes consistent with stem cell niche formation, regulation of cell proliferation and stem cell pluripotency, including the Hippo and Wnt pathways. FS1 cells were randomly distributed in the anterior and intermediate lobes, while FS2 cells were localized exclusively in the marginal zone between the anterior and intermediate lobes. These data indicate the identity of the FSCs as anterior pituitary-specific astroglia, with FS1 cells representing differentiated cells equipped for classical FSC roles and FS2 cells exhibiting additional stem cell-like features.

In vivo recordings of GnRH neuron firing reveal heterogeneity and dependence upon GABAA receptor signaling.

The gonadotropin-releasing hormone (GnRH) neurons are the key cells regulating fertility in all mammalian species. The scattered distribution of these neurons has made investigation of their properties extremely difficult and the key goal of recording their electrical activity in vivo near impossible. The caudal-most extension of the GnRH neuron continuum brings some cells very close to the base of the brain at the level of the anterior hypothalamic area. Taking insight from this, we developed an experimental procedure in anesthetized GnRH-GFP mice that allows the electrical activity of these GnRH neurons to be recorded in vivo. On-cell recordings revealed that the majority of GnRH neurons (86%) were spontaneously active, exhibiting a range of firing patterns, although only a minority (15%) exhibited burst firing. Mean firing frequencies ranged from 0.06 to 3.65 Hz, with the most common interspike interval being ~500 ms. All GnRH neurons tested were activated by AMPA and kisspeptin. Whereas the GABAA receptor agonist muscimol evoked excitatory, inhibitory, or mixed effects on GnRH neuron firing, the GABAA receptor antagonist picrotoxin resulted in a consistent suppression of firing. These observations represent the first electrical recordings of GnRH neurons in vivo. They reveal that GnRH neurons in vivo exhibit considerable heterogeneity in their firing patterns with both similarities and differences to firing in vitro. These variable patterns of firing in vivo are found to be critically dependent upon ongoing GABAA receptor signaling.

GnRH neurons elaborate a long-range projection with shared axonal and dendritic functions.

Information processing by neurons has been traditionally envisioned to occur in discrete neuronal compartments. Specifically, dendrites receive and integrate synaptic inputs while axons initiate and conduct spikes to distal neuronal targets. We report here in mice, using morphological reconstructions and electrophysiology, that the gonadotropin-releasing hormone (GnRH) neurons that control mammalian fertility do not conform to this stereotype and instead possess a single projection structure that functions simultaneously as an axon and dendrite. Specifically, we show that the GnRH neuron projection to the median eminence to control pituitary hormone secretion possesses a spike initiation site and conducts action potentials while also exhibiting spines and synaptic appositions along its entire length. Classical axonal or dendritic markers are not detectable in the projection process. Activation of ionotropic glutamate and/or GABA receptors along the GnRH neuron projection is capable of depolarizing the membrane potential and initiating action potentials. In addition, focal glutamate application to the projection is able to regulate the width of propagating spikes. These data demonstrate that GnRH neurons elaborate a previously uncharacterized neuronal projection that functions simultaneously as an axon and dendrite. This structure, termed a "dendron," greatly expands the dynamic control of GnRH secretion into the pituitary portal system to regulate fertility.

Vasoactive intestinal peptide excites GnRH neurons via KCa3.1, a potential player in the slow afterhyperpolarization current.

Vasoactive intestinal peptide (VIP) is an important component of the suprachiasmatic nucleus (SCN) which relays circadian information to neuronal populations, including GnRH neurons. Human and animal studies have shown an impact of disrupted daily rhythms (chronic shift work, temporal food restriction, clock gene disruption) on both male and female reproduction and fertility. To date, how VIP modulates GnRH neurons remains unknown. Calcium imaging and electrophysiology on primary GnRH neurons in explants and adult mouse brain slice, respectively, were used to address this question. We found VIP excites GnRH neurons via the VIP receptor, VPAC2. The downstream signaling pathway uses both Gs protein/adenylyl cyclase/protein kinase A (PKA) and phospholipase C/phosphatidylinositol 4,5-bisphosphate (PIP2) depletion. Furthermore, we identified a UCL2077-sensitive target, likely contributing to the slow afterhyperpolarization current (IAHP), as the PKA and PIP2 depletion target, and the KCa3.1 channel as a specific target. Thus, VIP/VPAC2 provides an example of Gs protein-coupled receptor-triggered excitation in GnRH neurons, modulating GnRH neurons likely via the slow IAHP. The possible identification of KCa3.1 in the GnRH neuron slow IAHP may provide a new therapeutical target for fertility treatments.

Protein tyrosine phosphatase receptors N and N2 control pituitary melanotroph development and POMC expressions.

The neuroendocrine marker genes Ptprn and Ptprn2 encode protein tyrosine phosphatase receptors N and N2, two members of protein tyrosine phosphatase receptors void of enzymatic activity, and whose function and mechanism of action have not been elucidated. To explore the role(s) of Ptprn and Ptprn2 on the hypothalamic-pituitary-adrenal axis, we used mice in which both genes were knocked out (DKO). The focus in study was on corticotrophs and melanotrophs from the anterior and intermediate lobes of the pituitary gland, respectively. In both sexes, DKO caused an increase in the expression of the corticotroph/melanotroph genes Pomc and Tbx19 and the melanotroph-specific gene Pax7. We also found in vivo and in vitro increased synthesis and release of beta-endorphin, alpha-MSH, and ACTH in DKO mice, which was associated with increased serum corticosterone levels and adrenal mass. DKO also increased the expression of other melanotroph-specific genes, but not corticotroph-specific genes. The dopaminergic pathway in the hypothalamus and dopaminergic receptors in melanotrophs were not affected in DKO mice. However, hyperplasia of the intermediate lobe was observed in DKO females and males, accompanied by increased POMC immunoreactivity per cell. These results indicate that PTPRNs contribute to hypothalamic-pituitary-adrenal function by being involved in processes governing postnatal melanotroph development and Pomc expression.

Acetylcholine regulation of GnRH neuronal activity: A circuit in the medial septum.

In vertebrates, gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility by regulating gonadotrophs in the anterior pituitary. While it is known that acetylcholine (ACh) influences GnRH secretion, whether the effect is direct or indirect, and the specific ACh receptor (AChR) subtype(s) involved remain unclear. Here, we determined 1) whether ACh can modulate GnRH cellular activity and 2) a source of ACh afferents contacting GnRH neurons. Calcium imaging was used to assay GnRH neuronal activity. With GABAergic and glutamatergic transmission blocked, subtype-specific AChR agonists and antagonists were applied to identify direct regulation of GnRH neurons. ACh and nicotine caused a rise in calcium that declined gradually back to baseline after 5-6 min. This response was mimicked by an alpha3-specific agonist. In contrast, muscarine inhibited GnRH calcium oscillations, and blocking M2 and M4 together prevented this inhibition. Labeling for choline acetyltransferase (ChAT) and GnRH revealed ChAT fibers contacting GnRH neurons, primarily in the medial septum (MS), and in greater number in females than males. ChAT positive cells in the MS are known to express p75NGFRs. Labeling for p75NGFR, ChAT and GnRH indicated that ChAT fibers contacting GnRH cells originate from cholinergic cells within these same rostral areas. Together, these results indicate that cholinergic cells in septal areas can directly regulate GnRH neurons.

Common and female-specific roles of protein tyrosine phosphatase receptors N and N2 in mice reproduction.

Simultaneous knockout of the neuroendocrine marker genes Ptprn and Ptprn2, which encode the protein tyrosine phosphatase receptors N and N2, causes infertility in female mice while males are fertile. To elucidate the mechanism of the sex-specific roles of Ptprn and Ptprn2 in mouse reproduction, we analyzed the effects of their double knockout (DKO) on the hypothalamic-pituitary-gonadal axis. In DKO females, delayed puberty and lack of ovulation were observed, complemented by changes in ovarian gene expression and steroidogenesis. In contrast, testicular gene expression, steroidogenesis, and reproductive organs development were not significantly affected in DKO males. However, in both sexes, pituitary luteinizing hormone (LH) beta gene expression and LH levels were reduced, as well as follicle-stimulating hormone beta gene and gonadotropin-releasing hormone (GnRH) gene, while the calcium-mobilizing and LH secretory actions of GnRH were preserved. Hypothalamic Gnrh1 and Kiss1 gene expression was also reduced in DKO females and males. In parallel, a significant decrease in the density of immunoreactive GnRH and kisspeptin fibers was detected in the hypothalamic arcuate nucleus of DKO females and males. The female-specific kisspeptin immunoreactivity in the rostral periventricular region of the third ventricle was also reduced in DKO females, but not in DKO males. These data indicate a critical role of Ptprn and Ptprn2 in kisspeptin-GnRH neuronal function and sexual dimorphism in the threshold levels of GnRH required to preserve reproductive functions.

Postnatal Development and Maintenance of Functional Pituitary Gonadotrophs Is Dependent on PI4-Kinase A.

Postnatal development of functional pituitary gonadotrophs is necessary for maturation of the hypothalamic-pituitary-gonadal axis, puberty, and reproduction. Here we examined the role of PI4-kinase A, which catalyzes the biosynthesis of PI4P in mouse reproduction by knocking out this enzyme in cells expressing the gonadotropin-releasing hormone (GnRH) receptor. Knockout (KO) mice were infertile, reflecting underdeveloped gonads and reproductive tracts and lack of puberty. The number and distribution of hypothalamic GnRH neurons and Gnrh1 expression in postnatal KOs were not affected, whereas Kiss1/kisspeptin expression was increased. KO of PI4-kinase A also did not alter embryonic establishment and neonatal development and function of the gonadotroph population. However, during the postnatal period, there was a progressive loss of expression of gonadotroph-specific genes, including Fshb, Lhb, and Gnrhr, accompanied by low gonadotropin synthesis. The postnatal gonadotroph population also progressively declined, reaching approximately one-third of that observed in controls at 3 months of age. In these residual gonadotrophs, GnRH-dependent calcium signaling and calcium-dependent membrane potential changes were lost, but intracellular administration of inositol-14,5-trisphosphate rescued this signaling. These results indicate a key role for PI4-kinase A in the postnatal development and maintenance of a functional gonadotroph population.

Targeting KNDy neurons to control GnRH pulses.

Gonadotropin-releasing hormone (GnRH) is the final output of the central nervous system that drives fertility. A characteristic of GnRH secretion is its pulsatility, which is driven by a pulse generator. Each GnRH pulse triggers a luteinizing hormone (LH) pulse. However, the puzzle has been to reconcile the synchronicity of GnRH neurons with the scattered hypothalamic distribution of their cell bodies. A leap toward understanding GnRH pulses was the discovery of kisspeptin neurons near the distal processes of GnRH neurons, which secrete kisspeptins, potent excitatory neuropeptides on GnRH neurons, and equipped with dual, but opposite, self-modulatory neuropeptides, neurokinin B and dynorphin. Over the last decade, this cell-to-cell communication has been dissected in animal models. Today the 50-year quest for the basic mechanism of GnRH pulse generation may be over, but questions about its physiological tuning remain. Here is an overview of recent basic research that frames translational research.

The electrophysiologic properties of gonadotropin-releasing hormone neurons.

For about two decades, recordings of identified gonadotropin-releasing hormone (GnRH) neurons have provided a wealth of information on their properties. We describe areas of consensus and debate the intrinsic electrophysiologic properties of these cells, their response to fast synaptic and neuromodulatory input, Ca2+ imaging correlates of action potential firing, and signaling pathways regulating these aspects. How steroid feedback and development change these properties, functions of GnRH neuron subcompartments and local networks, as revealed by chemo- and optogenetic approaches, are also considered.

Pituitary gonadotroph-specific patterns of gene expression and hormone secretion.

Pituitary gonadotrophs play a key role in reproductive functions by secreting luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The LH secretory activity of gonadotroph is controlled by hypothalamic gonadotropin-releasing hormone (GnRH) via GnRH receptors and is accompanied by only minor effects on high basal Lhb gene expression. The secretory profiles of GnRH and LH are highly synchronized, with the latter reflecting a depletion of prestored LH in secretory vesicles by regulated exocytosis. In contrast, FSH is predominantly released by constitutive exocytosis, and secretory activity reflects the kinetics of Fshb gene expression controlled by GnRH, activin, and inhibin. Here is a review of recent data to improve the understanding of multiple patterns of gonadotroph gene expression and hormone secretion.

The Dopamine D4 Receptor Regulates Gonadotropin-Releasing Hormone Neuron Excitability in Male Mice.

Gonadotropin-releasing hormone (GnRH)-secreting neurons control fertility. The release of GnRH peptide regulates the synthesis and release of both luteinizing hormone (LH) and Follicle stimulation hormone (FSH) from the anterior pituitary. While it is known that dopamine regulates GnRH neurons, the specific dopamine receptor subtype(s) involved remain unclear. Previous studies in adult rodents have reported juxtaposition of fibers containing tyrosine hydroxylase (TH), a marker of catecholaminergic cells, onto GnRH neurons and that exogenous dopamine inhibits GnRH neurons postsynaptically through dopamine D1-like and/or D2-like receptors. Our microarray data from GnRH neurons revealed a high level of Drd4 transcripts [i.e., dopamine D4 receptor (D4R)]. Single-cell RT-PCR and immunocytochemistry confirmed GnRH cells express the Drd4 transcript and protein, respectively. Calcium imaging identified changes in GnRH neuronal activity during application of subtype-specific dopamine receptor agonists and antagonists when GABAergic and glutamatergic transmission was blocked. Dopamine, dopamine with D1/5R-specific or D2/3R-specific antagonists or D4R-specific agonists decreased the frequency of calcium oscillations. In contrast, D1/5R-specific agonists increased the frequency of calcium oscillations. The D4R-mediated inhibition was dependent on Gαi/o protein coupling, while the D1/5R-mediated excitation required Gαs protein coupling. Together, these results indicate that D4R plays an important role in the dopaminergic inhibition of GnRH neurons.

Calcium dynamics in gonadotropin-releasing hormone neurons.

The gonadotropin-releasing hormone (GnRH) neurons represent the key output cells of the neuronal network controlling fertility. Intracellular calcium ion concentration ([Ca(2+)](i)) is likely to be a key signaling tool used by GnRH neurons to regulate and co-ordinate multiple cell processes. This review examines the dynamics and control of [Ca(2+)](i) in GT1 cells, embryonic GnRH neurons in the nasal placode culture, and adult GnRH neurons in the acute brain slice preparation. GnRH neurons at all stages of development display spontaneous [Ca(2+)](i) transients driven, primarily, by their burst firing. However, the intracellular mechanisms generating [Ca(2+)](i) transients, and the control of [Ca(2+)](i) by neurotransmitters, varies markedly across the different developmental stages. The functional roles of [Ca(2+)](i) transients are beginning to be unraveled with one key action being that of regulating the dynamics of GnRH neuron burst firing.

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.

Evaluation of local anesthesia and pain control in dermatological surgery: a prospective study of 120 patients.

Local anaesthesia with lidocaine is widely used in dermatology. The aim of this study was to evaluate pain at different times of dermatological surgery when using local anaesthetic agents. 120 consecutive patients were included during a 3 month period in a dermatological day surgery unit. Pain was estimated by a visual analogue scale, before, during and at the end of the operation. At the end, patients were asked about their satisfaction with local anaesthesia or their preference for general anaesthesia. Fifty five patients had lesions on the face and neck. Other localisations were chest (20 cases), limbs (24 cases), perineum (18 cases) and not recorded in 3 cases. Mean diameter of the lesions was 25.3 mm. Pain occurred during anaesthetic injection in 88.5% of the patients and the score was 5 or more in 42 patients. No pain was recorded during and at the end of the operation in 112 and 118 patients respectively. Fifteen patients would have preferred general to local anaesthesia because of intense pain. Local anaesthesia was judged appropriate by 86% of the patients. However, for lesions of the perineum, general anaesthesia would have been preferred by 38.8% of the patients.

Gonadotropin-releasing hormone-1 neuronal activity is independent of cyclic nucleotide-gated channels.

Pulsatile release of GnRH-1 is essential for secretion of gonadotropin hormones. The frequency of GnRH-1 pulses is regulated during the reproductive cycle by numerous neurotransmitters. Cyclic nucleotide-gated (CNG) channels have been proposed as a mechanism to integrate the cAMP signal evoked by many neurotransmitters. This study reports the expression of the CNGA2 subunit in GnRH-1 neurons obtained from mouse nasal explants and shows the ability of GnRH-1 neurons to increase their activity in response to forskolin (activator of adenylyl cyclases), or 3-isobutyl-1-methylxanthine (inhibitor of phosphodiesterases) even after removal of gamma-aminobutyric acid (A)-ergic input. Next, the endogenous activity of adenylyl cyclases was evaluated as a component of the oscillatory mechanism of GnRH-1 neurons. Inhibition of endogenous activity of adenylyl cyclases did not alter GnRH-1 activity. The potential involvement of CNGA2 subunit in basal or induced activity was tested on GnRH-1 neurons obtained from CNGA2-deficient mice. Without up-regulation of CNGA1 or CNGA3, the absence of functional CNGA2 did not alter either the endogenous GnRH-1 neuronal activity or the response to forskolin, negating CNG channels from cAMP-sensitive mechanisms leading to changes in GnRH-1 neuronal activity. In addition, the potential role of CNGA2 subunit in the synchronization of calcium oscillations previously described was evaluated in GnRH-1 neurons from CNGA2-deficient explants. Synchronized calcium oscillations persisted in CNGA2-deficient GnRH-1 neurons. Taken together, these results indicate that CNGA2 channels are not necessary for either the response of GnRH-1 neurons to cAMP increases or the basal rhythmic activity of GnRH-1 neurons.

Gonadotropin-releasing hormone-1 neuronal activity is independent of hyperpolarization-activated cyclic nucleotide-modulated channels but is sensitive to protein kinase a-dependent phosphorylation.

Pulsatile release of GnRH-1 stimulates the anterior pituitary and induces secretion of gonadotropin hormones. GnRH-1 release is modulated by many neurotransmitters that act via G protein-coupled membrane receptors. cAMP is the most ubiquitous effector for these receptors. GnRH-1 neurons express hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel protein in vivo. HCN channels are involved in neuronal pacemaking and can integrate cAMP signals. cAMP-dependent protein kinase (PKA) is also activated by cAMP signals, and PKA-dependent phosphorylation modulates voltage-activated channels. In this report, these two pathways were examined in GnRH-1 neurons as integrators of forskolin (FSK)-induced stimulation. The HCN3 isoform was detected in GnRH-1 neurons obtained from mouse nasal explants. ZD7288, a HCN channel blocker, significantly reduced the efficiency of FSK to stimulate GnRH-1 neurons, whereas blockade of PKA with Rp-adenosine-3',5'-cyclic monophosphorothioate triethylammonium did not attenuate the FSK-induced stimulation. To ensure that disruption of HCN channels on GnRH-1 neurons was responsible for reduction of FSK stimulation, experiments were performed removing gamma-aminobutyric acid (GABA), the major excitatory input to GnRH-1 neurons in nasal explants. Under these conditions, Rp-adenosine-3',5'-cyclic monophosphorothioate triethylammonium, but not ZD7288, altered the FSK-induced response of GnRH-1 neurons. These studies indicate that PKA-dependent phosphorylation is involved in the FSK-induced stimulation of GnRH-1 neurons rather than HCN channels, and HCN channels integrate the FSK-induced stimulation on GABAergic neurons. In addition, blockade of HCN channels did not modify basal GnRH-1 neuronal activity when GABAergic input was intact or removed, negating a role for these channels in basal GABAergic or GnRH-1 neuronal activity.

Nociceptin/Orphanin-FQ Inhibits Gonadotropin-Releasing Hormone Neurons via G-Protein-Gated Inwardly Rectifying Potassium Channels.

The pulsatile release of gonadotropin-releasing hormone (GnRH) is a key feature of the hypothalamic-pituitary-gonadal axis. Kisspeptin neurons in the arcuate nucleus (ARC) trigger GnRH neuronal activity, but how GnRH neurons return to baseline electrical activity is unknown. Nociceptin/orphanin-FQ (OFQ) is an inhibitory neuromodulator. ARC proopiomelanocortin (POMC) neurons, known to receive inputs from ARC kisspeptin neurons, contact GnRH neurons and coexpress OFQ in the rat. In the present study, the effect of OFQ(1-13) on GnRH neurons was determined in the mouse. We identified transcripts for the OFQ receptor [opioid receptor like 1 (ORL1)] in GnRH neurons, and, using two-model systems (explants and slices), we found that OFQ exerted a potent inhibition on GnRH neurons, with or without excitatory inputs. We confirmed that the inhibition was mediated by ORL1 via Gi/o-protein coupling. The inhibition, occurring independently of levels of intracellular cyclic adenosine monophosphate, was sensitive to inwardly rectifying potassium channels. The only specific blocker of Gi/o-protein-coupled inwardly rectifying potassium (GIRK) channels, tertiapin-Q (TPNQ), was ineffective in the inhibition of OFQ. Two GIRK activators, one sharing the binding site of TPNQ and one active only on GIRK1-containing GIRK channels, failed to trigger an inhibition. In contrast, protein kinase C phosphorylation activation, known to inhibit GIRK2-mediated currents, prevented the OFQ inhibition. These results indicate a specific combination of GIRK subunits, GIRK2/3 in GnRH neurons. In vivo, double-labeled OFQ/POMC fibers were found in the vicinity of GnRH neurons, and OFQ fibers apposed GnRH neurons. Together, this study brings to light a potent neuromodulator of GnRH neurons.