Activation of ionotropic and group I metabotropic glutamate receptors stimulates kisspeptin neuron activity in mice.

Different populations of hypothalamic kisspeptin (KISS1) neurons located in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (ARC) are thought to generate the sex-specific patterns of gonadotropin secretion. These neuronal populations integrate gonadal sex steroid feedback with internal and external cues relayed via the actions of neurotransmitters and neuropeptides. The excitatory amino acid neurotransmitter glutamate, the main excitatory neurotransmitter in the brain, plays a role in regulating gonadotropin secretion, at least partially through engaging KISS1 signaling. The expression and function of individual glutamate receptor subtypes in KISS1 neurons, however, are not well characterized. Here, we used GCaMP-based calcium imaging and patch-clamp electrophysiology to assess the impact of activating individual ionotropic (iGluR) and group I metabotropic (mGluR) glutamate receptors on KISS1 neuron activity in the mouse RP3V and ARC. Our results indicate that activation of all iGluR subtypes and of group I mGluRs, likely mGluR1, consistently drives activity in the majority of KISS1 neurons within the RP3V and ARC of males and females. Our results also revealed, somewhat unexpectedly, sex- and region-specific differences. Indeed, activating (S)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type iGluRs evoked larger responses in female ARCKISS1 neurons than in their male counterparts whereas activating group I mGluRs induced larger responses in RP3VKISS1 neurons than in ARCKISS1 neurons in females. Together, our findings suggest that glutamatergic neurotransmission in KISS1 neurons, and its impact on the activity of these cells, might be sex- and region-dependent in mice.

Serotonin stimulates female preoptic area kisspeptin neurons via activation of type 2 serotonin receptors in mice.

Background: The neuroendocrine control of ovulation is orchestrated by neuronal circuits that ultimately drive the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus to trigger the preovulatory surge in luteinizing hormone (LH) secretion. While estrogen feedback signals are determinant in triggering activation of GnRH neurons, through stimulation of afferent kisspeptin neurons in the rostral periventricular area of the third ventricle (RP3VKISS1 neurons), many neuropeptidergic and classical neurotransmitter systems have been shown to regulate the LH surge. Among these, several lines of evidence indicate that the monoamine neurotransmitter serotonin (5-HT) has an excitatory, permissive, influence over the generation of the surge, via activation of type 2 5-HT (5-HT2) receptors. The mechanisms through which this occurs, however, are not well understood. We hypothesized that 5-HT exerts its influence on the surge by stimulating RP3VKISS1 neurons in a 5-HT2 receptor-dependent manner. Methods: We tested this using kisspeptin neuron-specific calcium imaging and electrophysiology in brain slices obtained from male and female mice. Results: We show that exogenous 5-HT reversibly increases the activity of the majority of RP3VKISS1 neurons. This effect is more prominent in females than in males, is likely mediated directly at RP3VKISS1 neurons and requires activation of 5-HT2 receptors. The functional impact of 5-HT on RP3VKISS1 neurons, however, does not significantly vary during the estrous cycle. Conclusion: Taken together, these data suggest that 5-HT2 receptor-mediated stimulation of RP3VKISS1 neuron activity might be involved in mediating the influence of 5-HT on the preovulatory LH surge.

Circadian and kisspeptin regulation of the preovulatory surge.

Fertility in mammals is ultimately controlled by a small population of neurons - the gonadotropin-releasing hormone (GnRH) neurons - located in the ventral forebrain. GnRH neurons control gonadal function through the release of GnRH, which in turn stimulates the secretion of the anterior pituitary gonadotropins luteinizing hormone (LH) and follicle-stimulating hormone (FSH). In spontaneous ovulators, ovarian follicle maturation eventually stimulates, via sex steroid feedback, the mid-cycle surge in GnRH and LH secretion that causes ovulation. The GnRH/LH surge is initiated in many species just before the onset of activity through processes controlled by the central circadian clock, ensuring that the neuroendocrine control of ovulation and sex behavior are coordinated. This review aims to give an overview of anatomical and functional studies that collectively reveal some of the mechanisms through which the central circadian clock regulates GnRH neurons and their afferent circuits to drive the preovulatory surge.

Prenatal androgen treatment impairs the suprachiasmatic nucleus arginine-vasopressin to kisspeptin neuron circuit in female mice.

Polycystic ovary syndrome (PCOS) is associated with elevated androgen and luteinizing hormone (LH) secretion and with oligo/anovulation. Evidence indicates that elevated androgens impair sex steroid hormone feedback regulation of pulsatile LH secretion. Hyperandrogenemia in PCOS may also disrupt the preovulatory LH surge. The mechanisms through which this might occur, however, are not fully understood. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) convey hormonal cues to gonadotropin-releasing hormone (GnRH) neurons. In rodents, the preovulatory surge is triggered by these hormonal cues and coincident timing signals from the central circadian clock in the suprachiasmatic nucleus (SCN). Timing signals are relayed to GnRH neurons, in part, via projections from SCN arginine-vasopressin (AVP) neurons to RP3VKISS1 neurons. Because rodent SCN cells express androgen receptors (AR), we hypothesized that these circuits are impaired by elevated androgens in a mouse model of PCOS. In prenatally androgen-treated (PNA) female mice, SCN Ar expression was significantly increased compared to that found in prenatally vehicle-treated mice. A similar trend was seen in the number of Avp-positive SCN cells expressing Ar. In the RP3V, the number of kisspeptin neurons was preserved. Anterograde tract-tracing, however, revealed reduced SCNAVP neuron projections to the RP3V and a significantly lower proportion of RP3VKISS1 neurons with close appositions from SCNAVP fibers. Functional assessments showed, on the other hand, that RP3VKISS1 neuron responses to AVP were maintained in PNA mice. These findings indicate that PNA changes some of the neural circuits that regulate the preovulatory surge. These impairments might contribute to ovulatory dysfunction in PNA mice modeling PCOS.

Kisspeptin neuron electrophysiology: Intrinsic properties, hormonal modulation, and regulation of homeostatic circuits.

The obligatory role of kisspeptin (KISS1) and its receptor (KISS1R) in regulating the hypothalamic-pituitary-gonadal axis, puberty and fertility was uncovered in 2003. In the few years that followed, an impressive body of work undertaken in many species established that neurons producing kisspeptin orchestrate gonadotropin-releasing hormone (GnRH) neuron activity and subsequent GnRH and gonadotropin hormone secretory patterns, through kisspeptin-KISS1R signaling, and mediate many aspects of gonadal steroid hormone feedback regulation of GnRH neurons. Here, we review knowledge accrued over the past decade, mainly in genetically modified mouse models, of the electrophysiological properties of kisspeptin neurons and their regulation by hormonal feedback. We also discuss recent progress in our understanding of the role of these cells within neuronal circuits that control GnRH neuron activity and GnRH secretion, energy balance and, potentially, other homeostatic and reproductive functions.

Local kisspeptin excitation of rat oxytocin neurones in late pregnancy.

The hormone, oxytocin, is synthesised by magnocellular neurones of the supraoptic and paraventricular nuclei and is released from the posterior pituitary gland into the circulation to trigger uterine contractions during parturition. Kisspeptin fibre density increases around the supraoptic nucleus over pregnancy and intracerebroventricular kisspeptin excites oxytocin neurones only in late pregnancy. However, the mechanism of this excitation is unknown. Here, we found that microdialysis administration of kisspeptin into the supraoptic nucleus consistently increased the action potential (spike) firing rate of oxytocin neurones in urethane-anaesthetised late-pregnant rats (gestation day 18-21) but not in non-pregnant rats. Hazard analysis of action potential firing showed that kisspeptin specifically increased the probability of another action potential firing immediately after each action potential (post-spike excitability) in late-pregnant rats. Patch-clamp electrophysiology in hypothalamic slices showed that bath application of kisspeptin did not affect action potential frequency or baseline membrane potential in supraoptic nucleus neurones. Moreover, kisspeptin superfusion did not affect the frequency or amplitude of excitatory postsynaptic currents or inhibitory postsynaptic currents in supraoptic nucleus neurones. Taken together, these studies suggest that kisspeptin directly activates oxytocin neurones in late pregnancy, at least in part, via increased post-spike excitability. KEY POINTS: Oxytocin secretion is triggered by action potential firing in magnocellular neurones of the hypothalamic supraoptic and paraventricular nuclei to induce uterine contractions during birth. In late pregnancy, kisspeptin expression increases in rat periventricular nucleus neurones that project to the oxytocin system. Here, we show that intra-supraoptic nucleus administration of kisspeptin increases the action potential firing rate of oxytocin neurones in anaesthetised late-pregnant rats, and that the increased firing rate is associated with increased oxytocin neurone excitability immediately after each action potential. By contrast, kisspeptin superfusion of hypothalamic slices did not affect the activity of supraoptic nucleus neurones or the strength of local synaptic inputs to supraoptic nucleus neurones. Hence, kisspeptin might activate oxytocin neurons in late pregnancy by transiently increasing oxytocin neuron excitability after each action potential.

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.

Estrous Cycle Plasticity in the Central Clock Output to Kisspeptin Neurons: Implications for the Preovulatory Surge.

Coordination of ovulation and behavior is critical to reproductive success in many species. During the female estrous cycle, the preovulatory gonadotropin surge occurs when ovarian follicles reach maturity and, in rodents, it begins just before the daily onset of activity, ensuring that ovulation coincides with sex behavior. Timing of the surge relies on projections from the suprachiasmatic nucleus (SCN), the locus of the central circadian clock, to hypothalamic circuits that regulate gonadotropin secretion. The cellular mechanisms through which the SCN controls these circuits and gates the preovulatory surge to the appropriate estrous cycle stage, however, are poorly understood. We investigated in mice the functional impact of SCN arginine-vasopressin (AVP) neuron projections to kisspeptin (Kiss1) neurons in the rostral periventricular area of the third ventricle (RP3VKiss1), responsible for generating the preovulatory surge. Conditional anterograde tracing revealed that SCNAVP neurons innervate approximately half of the RP3VKiss1 neurons. Optogenetic activation of SCNAVP projections in brain slices caused an AVP-mediated stimulation of RP3VKiss1 action potential firing in proestrus, the cycle stage when the surge is generated. This effect was less prominent in diestrus, the preceding cycle stage, and absent in estrus, following ovulation. Remarkably, in estrus, activation of SCNAVP projections resulted in GABA-mediated inhibition of RP3VKiss1 neuron firing, an effect rarely encountered in other cycle stages. Together, these data reveal functional plasticity in SCNAVP neuron output that drives opposing effects on RP3VKiss1 neuron activity across the ovulatory cycle. This might contribute to gating activation of the preovulatory surge to the appropriate estrous cycle stage.

Sex- and sub region-dependent modulation of arcuate kisspeptin neurones by vasopressin and vasoactive intestinal peptide.

A population of kisspeptin neurones located in the hypothalamic arcuate nucleus (ARN) very likely represent the gonadotrophin-releasing hormone pulse generator responsible for driving pulsatile luteinising hormone secretion in mammals. As such, it has become important to understand the neural inputs that modulate the activity of ARN kisspeptin (ARNKISS ) neurones. Using a transgenic GCaMP6 mouse model allowing the intracellular calcium levels ([Ca2+ ]i ) of individual ARNKISS neurones to be assessed simultaneously, we examined whether the circadian neuropeptides vasoactive intestinal peptide (VIP) and arginine vasopressin (AVP) modulated the activity of ARNKISS neurones directly. To validate this methodology, we initially evaluated the effects of neurokinin B (NKB) on [Ca2+ ]i in kisspeptin neurones residing within the rostral, middle and caudal ARN subregions of adult male and female mice. All experiments were undertaken in the presence of tetrodotoxin and ionotropic amino acid antagonists. NKB was found to evoke an abrupt increase in [Ca2+ ]i in 95%-100% of kisspeptin neurones throughout the ARN of both sexes. By contrast, both VIP and AVP were found to primarily activate kisspeptin neurones located in the caudal ARN of female mice. Although 58% and 59% of caudal ARN kisspeptin neurones responded to AVP and VIP, respectively, in female mice, only 0%-8% of kisspeptin neurones located in other ARN subregions responded in females and 0%-12% of cells in any subregion in males (P < 0.05). These observations demonstrate unexpected sex differences and marked heterogeneity in functional neuropeptide receptor expression amongst ARNKISS neurones organised on a rostro-caudal basis. The functional significance of this unexpected influence of VIP and AVP on ARNKISS neurones remains to be established.

Optical Approaches for Interrogating Neural Circuits Controlling Hormone Secretion.

Developments in optical imaging and optogenetics are transforming the functional investigation of neuronal networks throughout the brain. Recent studies in the neuroendocrine field have used genetic mouse models combined with a variety of light-activated optical tools as well as GCaMP calcium imaging to interrogate the neural circuitry controlling hormone secretion. The present review highlights the benefits and caveats of these approaches for undertaking both acute brain slice and functional studies in vivo. We focus on the use of channelrhodopsin and the inhibitory optogenetic tools, archaerhodopsin and halorhodopsin, in addition to GCaMP imaging of individual cells in vitro and neural populations in vivo using fiber photometry. We also address issues around the use of genetic vs viral delivery of encoded proteins to specific Cre-expressing cell populations, their quantification, and the use of conscious vs anesthetized animal models. To date, optogenetics and GCaMP imaging have proven useful in dissecting functional circuitry within the brain and are likely to become essential investigative tools for deciphering the different neural networks controlling hormone secretion.

Dominant Neuropeptide Cotransmission in Kisspeptin-GABA Regulation of GnRH Neuron Firing Driving Ovulation.

A population of kisspeptin-GABA coexpressing neurons located in the rostral periventricular area of the third ventricle (RP3V) is believed to activate gonadotropin-releasing hormone (GnRH) neurons to generate the luteinizing hormone (LH) surge triggering ovulation. Selective optogenetic activation of RP3V kisspeptin (RP3VKISS) neurons in female mice for >30 s and ≥10 Hz in either a continuous or bursting mode was found to reliably generate a delayed and long-lasting activation of GnRH neuron firing in brain slices. Optogenetic activation of RP3VKISS neurons in vivo at 10 Hz generated substantial increments in LH secretion of similar amplitude to the endogenous LH surge. Studies using GABAA receptor antagonists and optogenetic activation of RP3V GABA (RP3VGABA) neurons in vitro revealed that low-frequency (2 Hz) stimulation generated immediate and transient GABAA receptor-mediated increases in GnRH neuron firing, whereas higher frequencies (10 Hz) recruited the long-lasting activation observed following RP3VKISS neuron stimulation. In vivo, 2 Hz activation of RP3VGABA neurons did not alter LH secretion, whereas 10 Hz stimulation evoked a sustained large increase in LH identical to RP3VKISS neuron activation. Optogenetic activation of RP3VKISS neurons in which kisspeptin had been deleted did not alter LH secretion. These studies demonstrate the presence of parallel transmission streams from RP3V neurons to GnRH neurons that are frequency dependent and temporally distinct. This comprises a rapid and transient GABAA receptor-mediated activation and a slower onset kisspeptin-mediated stimulation of long duration. At the time of the LH surge, GABA release appears to be functionally redundant with the neuropeptide kisspeptin being the dominant cotransmitter influencing GnRH neuron output.SIGNIFICANCE STATEMENT Miscommunication between the brain and ovaries is thought to represent a major cause of infertility in humans. Studies in rodents suggest that a population of neurons located in the rostral periventricular area of the third ventricle (RP3V) are critical for activating the gonadotropin-releasing hormone (GnRH) neurons that trigger ovulation. The present study provides evidence that an RP3V neuron population coexpressing kisspeptin and GABA provides a functionally important excitatory input to GnRH neurons at the time of ovulation. This neural input releases GABA and/or kisspeptin in the classical frequency dependent and temporally distinct nature of amino acid-neuropeptide cotransmission. Unusually, however, the neuropeptide stream is found to be functionally dominant in activating GnRH neurons at the time of ovulation.

Female sexual behavior in mice is controlled by kisspeptin neurons.

Sexual behavior is essential for the survival of many species. In female rodents, mate preference and copulatory behavior depend on pheromones and are synchronized with ovulation to ensure reproductive success. The neural circuits driving this orchestration in the brain have, however, remained elusive. Here, we demonstrate that neurons controlling ovulation in the mammalian brain are at the core of a branching neural circuit governing both mate preference and copulatory behavior. We show that male odors detected in the vomeronasal organ activate kisspeptin neurons in female mice. Classical kisspeptin/Kiss1R signaling subsequently triggers olfactory-driven mate preference. In contrast, copulatory behavior is elicited by kisspeptin neurons in a parallel circuit independent of Kiss1R involving nitric oxide signaling. Consistent with this, we find that kisspeptin neurons impinge onto nitric oxide-synthesizing neurons in the ventromedial hypothalamus. Our data establish kisspeptin neurons as a central regulatory hub orchestrating sexual behavior in the female mouse brain.

Definition of the hypothalamic GnRH pulse generator in mice.

The pulsatile release of luteinizing hormone (LH) is critical for mammalian fertility. However, despite several decades of investigation, the identity of the neuronal network generating pulsatile reproductive hormone secretion remains unproven. We use here a variety of optogenetic approaches in freely behaving mice to evaluate the role of the arcuate nucleus kisspeptin (ARNKISS) neurons in LH pulse generation. Using GCaMP6 fiber photometry, we find that the ARNKISS neuron population exhibits brief (∼1 min) synchronized episodes of calcium activity occurring as frequently as every 9 min in gonadectomized mice. These ARNKISS population events were found to be near-perfectly correlated with pulsatile LH secretion. The selective optogenetic activation of ARNKISS neurons for 1 min generated pulses of LH in freely behaving mice, whereas inhibition with archaerhodopsin for 30 min suppressed LH pulsatility. Experiments aimed at resetting the activity of the ARNKISS neuron population with halorhodopsin were found to reset ongoing LH pulsatility. These observations indicate the ARNKISS neurons as the long-elusive hypothalamic pulse generator driving fertility.

Defining a novel leptin-melanocortin-kisspeptin pathway involved in the metabolic control of puberty.

OBJECTIVE: Puberty is a key developmental phenomenon highly sensitive to metabolic modulation. Worrying trends of changes in the timing of puberty have been reported in humans. These might be linked to the escalating prevalence of childhood obesity and could have deleterious impacts on later (cardio-metabolic) health, but their underlying mechanisms remain unsolved. The neuropeptide α-MSH, made by POMC neurons, plays a key role in energy homeostasis by mediating the actions of leptin and likely participates in the control of reproduction. However, its role in the metabolic regulation of puberty and interplay with kisspeptin, an essential puberty-regulating neuropeptide encoded by Kiss1, remain largely unknown. We aim here to unveil the potential contribution of central α-MSH signaling in the metabolic control of puberty by addressing its role in mediating the pubertal effects of leptin and its potential interaction with kisspeptin. METHODS: Using wild type and genetically modified rodent models, we implemented pharmacological studies, expression analyses, electrophysiological recordings, and virogenetic approaches involving DREADD technology to selectively inhibit Kiss1 neurons, in order to interrogate the physiological role of a putative leptin→α-MSH→kisspeptin pathway in the metabolic control of puberty. RESULTS: Stimulation of central α-MSH signaling robustly activated the reproductive axis in pubertal rats, whereas chronic inhibition of melanocortin receptors MC3/4R, delayed puberty, and prevented the permissive effect of leptin on puberty onset. Central blockade of MC3/4R or genetic elimination of kisspeptin receptors from POMC neurons did not affect kisspeptin effects. Conversely, congenital ablation of kisspeptin receptors or inducible, DREADD-mediated inhibition of arcuate nucleus (ARC) Kiss1 neurons resulted in markedly attenuated gonadotropic responses to MC3/4R activation. Furthermore, close appositions were observed between POMC fibers and ARC Kiss1 neurons while blockade of α-MSH signaling suppressed Kiss1 expression in the ARC of pubertal rats. CONCLUSIONS: Our physiological, virogenetic, and functional genomic studies document a novel α-MSH→kisspeptin→GnRH neuronal signaling pathway involved in transmitting the permissive effects of leptin on pubertal maturation, which is relevant for the metabolic (and, eventually, pharmacological) regulation of puberty onset.

Vasoactive Intestinal Peptide Excites GnRH Neurons in Male and Female Mice.

A variety of external and internal factors modulate the activity of GnRH neurons to control fertility in mammals. A direct, vasoactive intestinal peptide (VIP)-mediated input to GnRH neurons originating from the suprachiasmatic nucleus is thought to relay circadian information within this network. In the present study, we examined the effects of VIP on GnRH neuron activity in male and female mice at different stages of the estrous cycle. We carried out cell-attached recordings in slices from GnRH-green fluorescent protein mice and calcium imaging in slices from a mouse line expressing the genetically encoded calcium indicator GCaMP3 selectively in GnRH neurons. We show that 50%-80% of GnRH neurons increase their firing rate in response to bath-applied VIP (1nM-1000nM) in both male and female mice and that this is accompanied by a robust increase in intracellular calcium concentrations. This effect is mediated directly at the GnRH neuron likely through activation of high-affinity VIP receptors. Because suprachiasmatic nucleus-derived timing cues trigger the preovulatory surge only on the afternoon of proestrus in female mice, we examined the effects of VIP during the estrous cycle at different times of day. VIP responsiveness in GnRH neurons did not vary significantly in diestrous and proestrous mice before or around the time of the expected preovulatory surge. These results indicate that the majority of GnRH neurons in male and female mice express functional VIP receptors and that the effects of VIP on GnRH neurons do not alter across the estrous cycle.

Estrogen permits vasopressin signaling in preoptic kisspeptin neurons in the female mouse.

The cellular mechanisms governing the impact of the central circadian clock on neuronal networks are incompletely understood. We examine here the influence of the suprachiasmatic nucleus output neuropeptide arginine-vasopressin (AVP) on the activity of preoptic area kisspeptin neurons. These cells integrate circadian and hormonal signals within the neuronal network that regulates fertility in females. Electrophysiological recordings in brain slices from kisspeptin-GFP mice showed that AVP dose-dependently increased the firing rate of most kisspeptin neurons. These actions were mediated directly at the kisspeptin neuron. Experiments in mice expressing the calcium indicator GCaMP3 in kisspeptin neurons enabled simultaneous monitoring of intracellular calcium concentrations ([Ca(2+)]i) in multiple cells and revealed that AVP increased [Ca(2+)]i in >80% of diestrous kisspeptin neurons via a mechanism involving voltage-gated calcium channels. We next examined whether AVP signaling in kisspeptin neurons was time and ovarian cycle dependent. AVP exerted the same effects on diestrous and proestrous days of the ovarian cycle, whether hours before [zeitgeber time 4 (ZT4)-ZT6] or just before (ZT10) the expected time of the proestrous preovulatory luteinizing hormone surge. Remarkably, however, AVP signaling was critically dependent on circulating ovarian steroids as AVP no longer excited preoptic kisspeptin neurons in ovariectomized mice, an effect that was fully restored by estradiol treatment. Together, these studies show that AVP exerts a potent and direct stimulatory influence upon the electrical activity and [Ca(2+)]i of most preoptic kisspeptin neurons. Unexpectedly, estrogen is found to permit circadian AVP signaling at preoptic kisspeptin neurons rather than dynamically modulate its activity throughout the estrous cycle.

Electrical properties of kisspeptin neurons and their regulation of GnRH neurons.

Kisspeptin neurons are critical components of the neuronal network controlling the activity of the gonadotropin-releasing hormone (GnRH) neurons. A variety of genetically-manipulated mouse models have recently facilitated the study of the electrical activity of the two principal kisspeptin neuron populations located in the rostral periventricular area of the third ventricle (RP3V) and arcuate nucleus (ARN) in acute brain slices. We discuss here the mechanisms and pathways through which kisspeptin neurons regulate GnRH neuron activity. We then examine the different kisspeptin-green fluorescent protein mouse models being used for kisspeptin electrophysiology and the data obtained to date for RP3V and ARN kisspeptin neurons. In light of these new observations on the spontaneous firing rates, intrinsic membrane properties, and neurotransmitter regulation of kisspeptin neurons, we speculate on the physiological roles of the different kisspeptin populations.

Estrous cycle plasticity in the hyperpolarization-activated current ih is mediated by circulating 17ß-estradiol in preoptic area kisspeptin neurons.

Circulating gonadal steroid hormones are thought to modulate a wide range of brain functions. However, the effects of steroid fluctuations through the ovarian cycle on the intrinsic properties of neurons are not well understood. We examined here whether gonadal steroids modulated the excitability of kisspeptin neurons located in the rostral periventricular region of the third ventricle (RP3V) of female mice. These cells are strongly implicated in sensing the high levels of circulating estradiol on proestrus to activate gonadotropin-releasing hormone (GnRH) neurons that, in turn, trigger ovulation. Electrophysiological studies were undertaken in brain slices from ovariectomized (OVX), diestrous, and proestrous kisspeptin-GFP mice. RP3V kisspeptin neurons exhibited marked changes in the hyperpolarization-evoked depolarizing sag and rebound firing across these groups. The hyperpolarization-activated current Ih was identified to be responsible for the depolarizing sag and was increased across OVX → diestrous → proestrous mice. Experiments in OVX mice given estradiol replacement identified an estradiol-dependent increase in Ih within RP3V kisspeptin neurons. Ih in these cells was found to contribute to their subthreshold membrane properties and the dynamics of rebound firing following hyperpolarizing stimuli in an estrous cycle-dependent manner. Only a minor role was found for Ih in modulating the spontaneous burst firing of RP3V kisspeptin neurons. These observations identify Ih as an ionic current that is regulated in a cyclical manner by circulating estradiol within the female brain, and suggest that such plasticity in the intrinsic properties of RP3V kisspeptin neurons may contribute to the generation of the preovulatory GnRH surge.

Spontaneous kisspeptin neuron firing in the adult mouse reveals marked sex and brain region differences but no support for a direct role in negative feedback.

Kisspeptin-Gpr54 signaling is critical for the GnRH neuronal network controlling fertility. The present study reports on a kisspeptin (Kiss)-green fluorescent protein (GFP) mouse model enabling brain slice electrophysiological recordings to be made from Kiss neurons in the arcuate nucleus (ARN) and rostral periventricular region of the third ventricle (RP3V). Using dual immunofluorescence, approximately 90% of GFP cells in the RP3V of females, and ARN in both sexes, are shown to be authentic Kiss-synthesizing neurons in adult mice. Cell-attached recordings of ARN Kiss-GFP cells revealed a marked sex difference in their mean firing rates; 90% of Kiss-GFP cells in males exhibited slow irregular firing (0.17 ± 0.04 Hz) whereas neurons from diestrous (0.01 ± 0.01 Hz) and ovariectomized (0 Hz) mice were mostly or completely silent. In contrast, RP3V Kiss-GFP cells were all spontaneously active, exhibiting tonic, irregular, and bursting firing patterns. Mean firing rates were significantly (P < 0.05) higher in diestrus (2.1 ± 0.3 Hz) compared with ovariectomized (1.0 ± 0.2 Hz) mice without any changes in firing pattern. Recordings from RP3V Kiss-GFP neurons at the time of the proestrous GnRH surge revealed a significant decline in firing rate after the surge. Together, these observations demonstrate unexpected sex differences in the electrical activity of ARN Kiss neurons and markedly different patterns of firing by Kiss neurons in the ARN and RP3V. Although data supported a positive influence of gonadal steroids on RP3V Kiss neuron firing, no direct evidence was found to support the previously postulated role of ARN Kiss neurons in the estrogen-negative feedback mechanism.