NK3R signalling in the posterodorsal medial amygdala is involved in stress-induced suppression of pulsatile LH secretion in female mice.

Psychosocial stress negatively impacts reproductive function by inhibiting pulsatile luteinizing hormone (LH) secretion. The posterodorsal medial amygdala (MePD) is responsible in part for processing stress and modulating the reproductive axis. Activation of the neurokinin 3 receptor (NK3R) suppresses the gonadotropin-releasing hormone (GnRH) pulse generator, under hypoestrogenic conditions, and NK3R activity in the amygdala has been documented to play a role in stress and anxiety. We investigate whether NK3R activation in the MePD is involved in mediating the inhibitory effect of psychosocial stress on LH pulsatility in ovariectomised female mice. First, we administered senktide, an NK3R agonist, into the MePD and monitored the effect on pulsatile LH secretion. We then delivered SB222200, a selective NK3R antagonist, intra-MePD in the presence of predator odour, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Senktide administration into the MePD dose-dependently suppresses pulsatile LH secretion. Moreover, NK3R signalling in the MePD mediates TMT-induced suppression of the GnRH pulse generator, which we verified using a mathematical model. The model verifies our experimental findings: (i) predator odour exposure inhibits LH pulses, (ii) activation of NK3R in the MePD inhibits LH pulses and (iii) NK3R antagonism in the MePD blocks stressor-induced inhibition of LH pulse frequency in the absence of ovarian steroids. These results demonstrate for the first time that NK3R neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator.

New methods to investigate the GnRH pulse generator.

The exact neural construct underlying the dynamic secretion of gonadotrophin-releasing hormone (GnRH) has only recently been identified despite the detection of multiunit electrical activity volleys associated with pulsatile luteinising hormone (LH) secretion four decades ago. Since the discovery of kisspeptin/neurokinin B/dynorphin neurons in the mammalian hypothalamus, there has been much research into the role of this neuronal network in controlling the oscillatory secretion of gonadotrophin hormones. In this review, we provide an update of the progressive application of cutting-edge techniques combined with mathematical modelling by the neuroendocrine community, which are transforming the functional investigation of the GnRH pulse generator. Understanding the nature and function of the GnRH pulse generator can greatly inform a wide range of clinical studies investigating infertility treatments.

Hypothalamic PVN CRH Neurons Signal Through PVN GABA Neurons to Suppress GnRH Pulse Generator Frequency in Female Mice.

Corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVN) are central to the stress response. Chemogenetic activation of PVN CRH neurons decreases LH pulse frequency but the mechanism is unknown. In the present study, optogenetic stimulation of PVN CRH neurons suppressed LH pulse frequency in estradiol-replaced ovariectomized CRH-cre mice, and this effect was augmented or attenuated by intra-PVN GABAA or GABAB receptor antagonism, respectively. PVN CRH neurons signal to local GABA neurons, which may provide a possible indirect mechanism by which PVN CRH neurons suppress LH pulse frequency. Optogenetic stimulation of potential PVN GABAergic projection terminals in the hypothalamic arcuate nucleus in ovariectomized estradiol-replaced Vgat-cre-tdTomato mice via an optic fiber implanted in the arcuate nucleus suppressed LH pulse frequency. To further determine whether PVN CRH neurons signal through PVN GABA neurons to suppress LH pulsatility, we combined recombinase mice with intersectional vectors to selectively target these neurons. CRH-cre::Vgat-FlpO mice expressing the stimulatory opsin ChRmine in non-GABAergic CRH neurons alone or in combination with the inhibitory opsin NpHR3.3 in non-CRH-expressing GABA neurons in the PVN were used. Optogenetic stimulation of non-GABAergic CRH neurons suppressed pulsatile LH secretion; however, LH pulse frequency was not affected when CRH neurons were stimulated and PVN GABA neurons were simultaneously inhibited. Together, these studies demonstrate that suppression of LH pulse frequency in response to PVN CRH neuronal activation is mediated by GABAergic signalling intrinsic to the PVN and may incorporate PVN GABAergic projection to the hypothalamic GnRH pulse generator.

Optogenetics studies of kisspeptin neurons.

Optical systems and genetic engineering technologies have made it possible to control neurons and unravel neuronal circuit behavior with high temporal and spatial resolution. The application of optogenetic strategies to understand the physiology of kisspeptin neuronal circuits has evolved in recent years among the neuroendocrine community. Kisspeptin neurons are fundamentally involved in controlling mammalian reproduction but also are implicated in numerous other physiological processes, including but not limited to feeding, energy expenditure, core body temperature and behavior. We conducted a review aiming to shed light on the novel findings obtained from in vitro and in vivo optogenetic studies interrogating kisspeptin neuronal circuits to date. Understanding the function of kisspeptin networks in the brain can greatly inform a wide range of clinical studies investigating infertility treatments, gender identity, metabolic disorders, hot flushes and psychosexual disorders.

Chemogenetic activation of PVN CRH neurons disrupts the estrous cycle and LH dynamics in female mice.

Introduction: The impact of stress on reproductive function is significant. Hypothalamic paraventricular nucleus (PVN) corticotrophin-releasing hormone (CRH) plays a major role in regulating the stress response. Understanding how the hypothalamic-pituitary-adrenal (HPA) axis and the hypothalamic-pituitary-gonadal (HPG) axis interact is crucial for comprehending how stress can lead to reproductive dysfunction. However, whether stress influences reproductive function via modulating PVN CRH or HPA sequelae is not fully elucidated. Methods: In this study, we investigated the impact of chemogenetic activation of PVN CRH neurons on reproductive function. We chronically and selectively stimulated PVN CRH neurons in female CRH-Cre mice using excitatory designer receptor exclusively activated by designer drugs (DREADDs) viral constructs, which were bilaterally injected into the PVN. The agonist compound-21 (C21) was delivered through the drinking water. We determined the effects of DREADDs activation of PVN CRH neurons on the estrous cycles, LH pulse frequency in diestrus and metestrus and LH surge in proestrus mice. The effect of long-term C21 administration on basal corticosterone secretion and the response to acute restraint stress during metestrus was also examined. Additionally, computer simulations of a mathematical model were used to determine the effects of DREADDs activation of PVN CRH neurons, simulating chronic stress, on the physiological parameters examined experimentally. Results: As a result, and consistent with our mathematical model predictions, the length of the estrous cycle was extended, with an increase in the time spent in estrus and metestrus, and a decrease in proestrus and diestrus. Additionally, the frequency of LH pulses during metestrus was decreased, but unaffected during diestrus. The occurrence of the preovulatory LH surge during proestrus was disrupted. The basal level of corticosterone during metestrus was not affected, but the response to acute restraint stress was diminished after long-term C21 application. Discussion: These data suggest that PVN CRH neurons play a functional role in disrupting ovarian cyclicity and the preovulatory LH surge, and that the activity of the GnRH pulse generator remains relatively robust during diestrus but not during metestrus under chronic stress exposure in accordance with our mathematical model predictions.

GABA Signaling in the Posterodorsal Medial Amygdala Mediates Stress-induced Suppression of LH Pulsatility in Female Mice.

Psychological stress is linked to infertility by suppressing the hypothalamic GnRH pulse generator. The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream regulator of GnRH pulse generator activity and displays increased neuronal activation during psychological stress. The MePD is primarily a GABAergic nucleus with a strong GABAergic projection to hypothalamic reproductive centers; however, their functional significance has not been determined. We hypothesize that MePD GABAergic signalling mediates psychological stress-induced suppression of pulsatile LH secretion. We selectively inhibited MePD GABA neurons during psychological stress in ovariectomized (OVX) Vgat-cre-tdTomato mice to determine the effect on stress-induced suppression of pulsatile LH secretion. MePD GABA neurons were virally infected with inhibitory hM4DGi-designer receptor exclusively activated by designer drugs (DREADDs) to selectively inhibit MePD GABA neurons. Furthermore, we optogenetically stimulated potential MePD GABAergic projection terminals in the hypothalamic arcuate nucleus (ARC) and determined the effect on pulsatile LH secretion. MePD GABA neurons in OVX female Vgat-cre-tdTomato mice were virally infected to express channelrhodopsin-2 and MePD GABAergic terminals in the ARC were selectively stimulated by blue light via an optic fiber implanted in the ARC. DREADD-mediated inhibition of MePD GABA neurons blocked predator odor and restraint stress-induced suppression of LH pulse frequency. Furthermore, sustained optogenetic stimulation at 10 and 20 Hz of MePD GABAergic terminals in the ARC suppressed pulsatile LH secretion. These results show for the first time that GABAergic signalling in the MePD mediates psychological stress-induced suppression of pulsatile LH secretion and suggest a functionally significant MePD GABAergic projection to the hypothalamic GnRH pulse generator.

Posterodorsal Medial Amygdala Urocortin-3, GABA, and Glutamate Mediate Suppression of LH Pulsatility in Female Mice.

The posterodorsal subnucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic-pituitary-gonadal (HPG) and hypothalamic-pituitary-adrenal (HPA) axes. Inhibition of MePD urocortin-3 (Ucn3) neurons prevents psychological stress-induced suppression of luteinizing hormone (LH) pulsatility while blocking the stress-induced elevations in corticosterone (CORT) secretion in female mice. We explore the neurotransmission and neural circuitry suppressing the gonadotropin-releasing hormone (GnRH) pulse generator by MePD Ucn3 neurons and we further investigate whether MePD Ucn3 efferent projections to the hypothalamic paraventricular nucleus (PVN) control CORT secretion and LH pulsatility. Ucn3-cre-tdTomato female ovariectomized (OVX) mice were unilaterally injected with adeno-associated virus (AAV)-channelrhodopsin 2 (ChR2) and implanted with optofluid cannulae targeting the MePD. We optically activated Ucn3 neurons in the MePD with blue light at 10 Hz and monitored the effect on LH pulses. Next, we combined optogenetic stimulation of MePD Ucn3 neurons with pharmacological antagonism of GABAA or GABAB receptors with bicuculline or CGP-35348, respectively, as well as a combination of NMDA and AMPA receptor antagonists, AP5 and CNQX, respectively, and observed the effect on pulsatile LH secretion. A separate group of Ucn3-cre-tdTomato OVX mice with 17ß-estradiol replacement were unilaterally injected with AAV-ChR2 in the MePD and implanted with fiber-optic cannulae targeting the PVN. We optically stimulated the MePD Ucn3 efferent projections in the PVN with blue light at 20 Hz and monitored the effect on CORT secretion and LH pulses. We reveal for the first time that activation of Ucn3 neurons in the MePD inhibits GnRH pulse generator frequency via GABA and glutamate signaling within the MePD, while MePD Ucn3 projections to the PVN modulate the HPG and HPA axes.

Optogenetic stimulation of Kiss1ARC terminals in the AVPV induces surge-like luteinizing hormone secretion via glutamate release in mice.

Kisspeptin neurons are mainly located in the arcuate (Kiss1ARC, vis-à-vis the GnRH pulse generator) and anteroventral periventricular nucleus (Kiss1AVPV, vis-à-vis the GnRH surge generator). Kiss1ARC send fibre projections that connect with Kiss1AVPV somata. However, studies focused on the role of Kiss1ARC neurons in the LH surge are limited, and the role of Kiss1ARC projections to AVPV (Kiss1ARC→AVPV) in the preovulatory LH surge is still unknown. To investigate its function, this study used optogenetics to selectively stimulate Kiss1ARC→AVPV and measured changes in circulating LH levels. Kiss1ARC in Kiss-Cre-tdTomato mice were virally infected to express channelrhodopsin-2 proteins, and optical stimulation was applied selectively via a fibre optic cannula in the AVPV. Sustained 20 Hz optical stimulation of Kiss1ARC→AVPV from 15:30 to 16:30 h on proestrus effectively induced an immediate increase in LH reaching peak surge-like levels of around 8 ng/ml within 10 min, followed by a gradual decline to baseline over about 40 min. Stimulation at 10 Hz resulted in a non-significant increase in LH levels and 5 Hz stimulation had no effect in proestrous animals. The 20 Hz stimulation induced significantly higher circulating LH levels on proestrus compared with diestrus or estrus, which suggested that the effect of terminal stimulation is modulated by the sex steroid milieu. Additionally, intra-AVPV infusion of glutamate antagonists, AP5+CNQX, completely blocked the increase on LH levels induced by Kiss1ARC→AVPV terminal photostimulation in proestrous animals. These results demonstrate for the first time that optical stimulation of Kiss1ARC→AVPV induces an LH surge-like secretion via glutamatergic mechanisms. In conclusion, Kiss1ARC may participate in LH surge generation by glutamate release from terminal projections in the AVPV.

GnRH pulse generator frequency is modulated by kisspeptin and GABA-glutamate interactions in the posterodorsal medial amygdala in female mice.

Kisspeptin neurons in the arcuate nucleus of the hypothalamus generate gonadotrophin-releasing hormone (GnRH) pulses, and act as critical initiators of functional gonadotrophin secretion and reproductive competency. However, kisspeptin in other brain regions, most notably the posterodorsal subnucleus of the medial amygdala (MePD), plays a significant modulatory role over the hypothalamic kisspeptin population; our recent studies using optogenetics have shown that low-frequency light stimulation of MePD kisspeptin results in increased luteinsing hormone pulse frequency. Nonetheless, the neurochemical pathways that underpin this regulatory function remain unknown. To study this, we have utilised an optofluid technology, precisely combining optogenetic stimulation with intra-nuclear pharmacological receptor antagonism, to investigate the neurotransmission involved in this circuitry. We have shown experimentally and verified using a mathematical model that functional neurotransmission of both GABA and glutamate is a requirement for effective modulation of the GnRH pulse generator by amygdala kisspeptin neurons.

Provocative tests with Kisspeptin-10 and GnRH set the scene for determining social status and environmental impacts on reproductive capacity in male African lions (Panthera leo).

Understanding the hypothalamic factors regulating reproduction facilitates maximising the reproductive success of breeding programmes and in the management and conservation of threatened species, including African lions. To provide insight into the physiology and pathophysiology of the hypothalamic-pituitary-gonadal reproductive axis in lions, we studied the luteinising hormone (LH) and steroid hormone responses to gonadotropin-releasing hormone (GnRH) and its upstream regulator, kisspeptin. Six young (13.3 ± 1.7 months, 56.2 ± 4.3 kg) and four adult (40.2 ± 1.4 months, 174 ± 6 kg) male lions (Ukutula Conservation Centre, South Africa) were used in this study. Lions were immobilised with a combination of medetomidine and ketamine and an intravenous catheter was placed in a jugular, cephalic or medial saphenous vein for blood sampling at 10-min intervals for 220 min. The ten-amino acid kisspeptin which has full intrinsic activity (KP-10, 1 µg/kg) and GnRH (1 µg/kg) were administered intravenously to study their effects on LH and steroid hormone plasma concentrations, measured subsequently by ELISA and liquid chromatography tandem mass spectrometry (LC-MS/MS), respectively. Basal LH levels were similarly low between the age groups, but testosterone and its precursor levels were higher in the adult animals. Adult lions showed a significant LH response to KP-10 (10-fold) and GnRH (11-fold) administration (p < 0.05 and P < 0.001, respectively) whereas in young lions LH increased significantly only in response to GnRH. In adults alone, testosterone and its precursors steadily increased in response to KP-10, with no significant further increase in response to GnRH. Plasma levels of glucocorticoids in response to KP-10 remained unchanged. We suggest that provocative testing of LH and steroid stimulation with kisspeptin provides a new and sensitive tool for determining reproductive status and possibly an index of exposure to stress, environmental insults such as disease, endocrine disruptors and nutritional status. 272 words.

Role of Posterodorsal Medial Amygdala Urocortin-3 in Pubertal Timing in Female Mice.

Post-traumatic stress disorder impedes pubertal development and disrupts pulsatile LH secretion in humans and rodents. The posterodorsal sub-nucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, pubertal timing, as well as emotional processing and anxiety. Psychosocial stress exposure alters neuronal activity within the MePD increasing the expression of Urocortin3 (Ucn3) and its receptor corticotropin-releasing factor type-2 receptor (CRFR2) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. We test the hypothesis that psychosocial stress, processed by the MePD, is relayed to the hypothalamic GnRH pulse generator to delay puberty in female mice. We exposed C57Bl6/J female mice to the predator odor, 2,4,5-Trimethylthiazole (TMT), during pubertal transition and examined the effect on pubertal timing, pre-pubertal LH pulses and anxiety-like behaviour. Subsequently, we virally infected Ucn3-cre-tdTomato female mice with stimulatory DREADDs targeting MePD Ucn3 neurons and determined the effect on pubertal timing and pre-pubertal LH pulse frequency. Exposure to TMT during pubertal development delayed puberty, suppressed pre-pubertal LH pulsatility and enhanced anxiety-like behaviour, while activation of MePD Ucn3 neurons reduced LH pulse frequency and delayed puberty. Early psychosocial stress exposure decreases GnRH pulse generator frequency delaying puberty while inducing anxiety-behaviour in female mice, an effect potentially involving Ucn3 neurons in the MePD.

Mathematical models in GnRH research.

Mathematical modelling is an indispensable tool in modern biosciences, enabling quantitative analysis and integration of biological data, transparent formulation of our understanding of complex biological systems, and efficient experimental design based on model predictions. This review article provides an overview of the impact that mathematical models had on GnRH research. Indeed, over the last 20 years mathematical modelling has been used to describe and explore the physiology of the GnRH neuron, the mechanisms underlying GnRH pulsatile secretion, and GnRH signalling to the pituitary. Importantly, these models have contributed to GnRH research via novel hypotheses and predictions regarding the bursting behaviour of the GnRH neuron, the role of kisspeptin neurons in the emergence of pulsatile GnRH dynamics, and the decoding of GnRH signals by biochemical signalling networks. We envisage that with the advent of novel experimental technologies, mathematical modelling will have an even greater role to play in our endeavour to understand the complex spatiotemporal dynamics underlying the reproductive neuroendocrine system.

Optogenetic Activation of Arcuate Kisspeptin Neurons Generates a Luteinizing Hormone Surge-Like Secretion in an Estradiol-Dependent Manner.

Traditionally, the anteroventral periventricular (AVPV) nucleus has been the brain area associated with luteinizing hormone (LH) surge secretion in rodents. However, the role of the other population of hypothalamic kisspeptin neurons, in the arcuate nucleus (ARC), has been less well characterized with respect to surge generation. Previous experiments have demonstrated ARC kisspeptin knockdown reduced the amplitude of LH surges, indicating that they have a role in surge amplification. The present study used an optogenetic approach to selectively stimulate ARC kisspeptin neurons and examine the effect on LH surges in mice with different hormonal administrations. LH level was monitored from 13:00 to 21:00 h, at 30-minute intervals. Intact Kiss-Cre female mice showed increased LH secretion during the stimulation period in addition to displaying a spontaneous LH surge around the time of lights off. In ovariectomized Kiss-Cre mice, optogenetic stimulation was followed by a surge-like secretion of LH immediately after the stimulation period. Ovariectomized Kiss-Cre mice with a low dose of 17ß-estradiol (OVX+E) replacement displayed a surge-like increase in LH release during period of optic stimulation. No LH response to the optic stimulation was observed in OVX+E mice on the day of estradiol benzoate (EB) treatment (day 1). However, after administration of progesterone (day 2), all OVX+E+EB+P mice exhibited an LH surge during optic stimulation. A spontaneous LH surge also occurred in these mice at the expected time. Taken together, these results help to affirm the fact that ARC kisspeptin may have a novel amplificatory role in LH surge production, which is dependent on the gonadal steroid milieu.

Urocortin3 in the Posterodorsal Medial Amygdala Mediates Stress-induced Suppression of LH Pulsatility in Female Mice.

Psychosocial stress disrupts reproduction and interferes with pulsatile LH secretion. The posterodorsal medial amygdala (MePD) is an upstream modulator of the reproductive axis and stress. Corticotropin-releasing factor type 2 receptors (CRFR2s) are activated in the presence of psychosocial stress together with increased expression of the CRFR2 ligand Urocortin3 (Ucn3) in the MePD of rodents. We investigate whether Ucn3 signalling in the MePD is involved in mediating the suppressive effect of psychosocial stress on LH pulsatility. First, we administered Ucn3 into the MePD and monitored the effect on LH pulses in ovariectomized mice. Next, we delivered Astressin2B, a selective CRFR2 antagonist, intra-MePD in the presence of predator odor, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Subsequently, we virally infected Ucn3-cre-tdTomato mice with inhibitory designer receptor exclusively activated by designer drugs (DREADDs) targeting MePD Ucn3 neurons while exposing mice to TMT or restraint stress and examined the effect on LH pulsatility as well as corticosterone release. Administration of Ucn3 into the MePD dose-dependently inhibited LH pulses and administration of Astressin2B blocked the suppressive effect of TMT on LH pulsatility. Additionally, DREADDs inhibition of MePD Ucn3 neurons blocked TMT and restraint stress-induced inhibition of LH pulses and corticosterone release. These results demonstrate for the first time that Ucn3 neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator and corticosterone secretion. Ucn3 signalling in the MePD plays a role in modulating the hypothalamic-pituitary-gonadal and hypothalamic-pituitary-adrenal axes, and this brain locus may represent a nodal center in the interaction between the reproductive and stress axes.

Dynorphin and GABAA Receptor Signaling Contribute to Progesterone's Inhibition of the LH Surge in Female Mice.

Progesterone can block estrogen-induced luteinising hormone (LH) surge secretion and can be used clinically to prevent premature LH surges. The blocking effect of progesterone on the LH surge is mediated through its receptor in the anteroventral periventricular nucleus (AVPV) of the hypothalamus. However, the underlying mechanisms are unclear. The preovulatory LH surge induced by estrogen is preceded by a significant reduction in hypothalamic dynorphin and gamma-aminobutyric acid (GABA) release. To test the detailed roles of dynorphin and GABA in an LH surge blockade by progesterone, ovariectomized and 17ß-estradiol capsule-implanted (OVX/E2) mice received simultaneous injections of estradiol benzoate (EB) and progesterone (P) or vehicle for 2 consecutive days. The LH level was monitored from 2:30 pm to 8:30 pm at 30-minute intervals. Progesterone coadministration resulted in the LH surge blockade. A continuous microinfusion of the dynorphin receptor antagonist nor-BNI or GABAA receptor antagonist bicuculline into the AVPV from 3:00 pm to 7:00 pm reversed the progesterone-mediated blockade of the LH surge in 7 of 9 and 6 of 10 mice, respectively. In addition, these LH surges started much earlier than the surge induced by estrogen alone. However, 5 of 7 progesterone-treated mice did not show LH surge secretion after microinfusion with the GABAB receptor antagonist CGP-35348. Additionally, peripheral administration of kisspeptin-54 promotes LH surge-like release in progesterone treated mice. These results demonstrated that the progesterone-mediated suppression of the LH surge is mediated by an increase in dynorphin and GABAA receptor signaling acting though kisspeptin neurons in the AVPV of the hypothalamus in female mice.

Optogenetic stimulation of kisspeptin neurones within the posterodorsal medial amygdala increases luteinising hormone pulse frequency in female mice.

Kisspeptin within the arcuate nucleus of the hypothalamus is a critical neuropeptide in the regulation of reproduction. Together with neurokinin B and dynorphin A, arcuate kisspeptin provides the oscillatory activity that drives the pulsatile secretion of gonadotrophin-releasing hormone (GnRH), and therefore luteinising hormone (LH) pulses, and is considered to be a central component of the GnRH pulse generator. It is well established that the amygdala also exerts an influence over gonadotrophic hormone secretion and reproductive physiology. The discovery of kisspeptin and its receptor within the posterodorsal medial amygdala (MePD) and our recent finding showing that intra-MePD administration of kisspeptin or a kisspeptin receptor antagonist results in increased LH secretion and decreased LH pulse frequency, respectively, suggests an important role for amygdala kisspeptin signalling in the regulation of the GnRH pulse generator. To further investigate the function of amygdala kisspeptin, the present study used an optogenetic approach to selectively stimulate MePD kisspeptin neurones and examine the effect on pulsatile LH secretion. MePD kisspeptin neurones in conscious Kiss1-Cre mice were virally infected to express the channelrhodopsin 2 protein and selectively stimulated by light via a chronically implanted fibre optic cannula. Continuous stimulation using 5 Hz resulted in an increased LH pulse frequency, which was not observed at the lower stimulation frequencies of 0.5 and 2 Hz. In wild-type animals, continuous stimulation at 5 Hz did not affect LH pulse frequency. These results demonstrate that selective activation of MePD Kiss1 neurones can modulate hypothalamic GnRH pulse generator frequency.

Kisspeptin as a Behavioral Hormone.

Successful reproduction is dependent not only on hormonal endocrine responses but also on suitable partner selection, copulatory acts, as well as associated emotional, behavioral, and cognitive processes many of which are supported by the limbic system. The reproductive hormone kisspeptin (encoded by the KISS1/kiss1 gene) is now recognized as the key orchestrator of the reproductive axis. In addition to the hypothalamus, prominent kisspeptin neuronal populations have been identified throughout limbic and paralimbic brain regions across an assortment of species. In this review, we detail the emerging roles of kisspeptin signaling in the broader aspects of behavioral, emotional, and cognitive control. Recent studies from zebrafish through humans have provided new molecular and neural insights into the complex role of kisspeptin in interpreting olfactory and auditory cues to govern sexual partner preference, in regulating copulatory behaviors and in influencing mood and emotions. Furthermore, emerging roles for kisspeptin in facilitating memory and learning are also discussed. To this end, these findings shed new light onto the importance of kisspeptin signaling, while informing the pharmacological development of kisspeptin as a potential therapeutic strategy for individuals suffering from associated reproductive, emotional, and cognitive disorders.

The Roles of the Amygdala Kisspeptin System.

The hypothalamic hormone kisspeptin (encoded by the KISS1/kiss1 gene) is the master regulator of the reproductive axis with its role in controlling gonadotrophin hormone secretion now well characterized. However, identification of kisspeptin and its cognate receptor expression within the amygdala, a key limbic brain region whose functions contribute to a broad range of physiological and behavioral processes, has heightened interest concerning kisspeptins' role in the broader aspects of reproductive physiology. In this review, we detail the important developments and key studies examining the emerging functions of this kisspeptin population. These studies provide novel advances in our understanding of the mechanisms controlling reproductive neuroendocrinology by defining the crucial role of the amygdala kisspeptin system in modulating pubertal timing, reproductive hormone secretion, and pulsatility, as well as its influence in governing-related behaviors. To this end, the role of the amygdala kisspeptin system in integrating reproductive hormone secretion with behavior sheds new light onto the potential use of kisspeptin-based therapeutics for reproductive and related psychosexual disorders.

The Origin of GnRH Pulse Generation: An Integrative Mathematical-Experimental Approach.

Fertility critically depends on the gonadotropin-releasing hormone (GnRH) pulse generator, a neural construct comprised of hypothalamic neurons coexpressing kisspeptin, neurokoinin-B and dynorphin. Here, using mathematical modeling and in vivo optogenetics we reveal for the first time how this neural construct initiates and sustains the appropriate ultradian frequency essential for reproduction. Prompted by mathematical modeling, we show experimentally using female estrous mice that robust pulsatile release of luteinizing hormone, a proxy for GnRH, emerges abruptly as we increase the basal activity of the neuronal network using continuous low-frequency optogenetic stimulation. Further increase in basal activity markedly increases pulse frequency and eventually leads to pulse termination. Additional model predictions that pulsatile dynamics emerge from nonlinear positive and negative feedback interactions mediated through neurokinin-B and dynorphin signaling respectively are confirmed neuropharmacologically. Our results shed light on the long-elusive GnRH pulse generator offering new horizons for reproductive health and wellbeing.SIGNIFICANCE STATEMENT The gonadotropin-releasing hormone (GnRH) pulse generator controls the pulsatile secretion of the gonadotropic hormones LH and FSH and is critical for fertility. The hypothalamic arcuate kisspeptin neurons are thought to represent the GnRH pulse generator, since their oscillatory activity is coincident with LH pulses in the blood; a proxy for GnRH pulses. However, the mechanisms underlying GnRH pulse generation remain elusive. We developed a mathematical model of the kisspeptin neuronal network and confirmed its predictions experimentally, showing how LH secretion is frequency-modulated as we increase the basal activity of the arcuate kisspeptin neurons in vivo using continuous optogenetic stimulation. Our model provides a quantitative framework for understanding the reproductive neuroendocrine system and opens new horizons for fertility regulation.

Role of the posterodorsal medial amygdala in predator odour stress-induced puberty delay in female rats.

Puberty onset is influenced by various factors, including psychosocial stress. The present study investigated cat-odour stress on puberty onset and oestrous cyclicity in rats. Female weanling rats were exposed to either soiled cat litter or fresh unused litter for 10 consecutive days. Following vaginal opening (VO), rats were smeared for 14 days to determine oestrous cyclicity. Anxiety-like behaviour was assessed using standard anxiety tests. Brains were collected to determine corticotrophin-releasing factor (CRF), CRF receptor 1 (CRF-R1) and CRF receptor 2 (CRF-R2) mRNA in the paraventricular nucleus (PVN), as well as the central nucleus of the amygdala (CEA) and the medial nucleus of the amygdala (MEA). Cat odour delayed VO and first oestrus, disrupted oestrous cycles and caused anxiogenic responses. Cat odour elicited increased CRF mRNA expression in the PVN but not in the CeA. CRF-R1 and CRF-R2 mRNA levels in the PVN and CeA were unaffected by cat odour; however, CRF-R1 mRNA levels were decreased in the MeA. The role of CRF signalling in the MeA, particularly its posterodorsal subnucleus (MePD), with respect to pubertal timing was directly examined by unilateral intra-MePD administration of CRF (0.2 nmol day-1 for 14 days) via an osmotic mini-pump from postnatal day 24 and was shown to delay VO and first oestrus. These data suggest that CRF signalling in the MePD may be associated with predator odour-induced puberty delay.