Kisspeptin neurones in the posterodorsal medial amygdala modulate sexual partner preference and anxiety in male mice.

The posterodorsal medial amygdala (MePD) is a neural site in the limbic brain involved in regulating emotional and sexual behaviours. There is, however, limited information available on the specific neuronal cell type in the MePD functionally mediating these behaviours in rodents. The recent discovery of a significant kisspeptin neurone population in the MePD has raised interest in the possible role of kisspeptin and its cognate receptor in sexual behaviour. The present study therefore tested the hypothesis that the MePD kisspeptin neurone population is involved in regulating attraction towards opposite sex conspecifics, sexual behaviour, social interaction and the anxiety response by selectively stimulating these neurones using the novel pharmacosynthetic DREADDs (designer receptors exclusively activated by designer drugs) technique. Adult male Kiss-Cre mice received bilateral stereotaxic injections of a stimulatory DREADD viral construct (AAV-hSyn-DIO-hM3 D(Gq)-mCherry) targeted to the MePD, with subsequent activation by i.p. injection of clozapine-N-oxide (CNO). Socio-sexual behaviours were assessed in a counter-balanced fashion after i.p. injection of either saline or CNO (5 mg kg-1 ). Selective activation of MePD kisspeptin neurones by CNO significantly increased the time spent by male mice in investigating an oestrous female, as well as the duration of social interaction. Additionally, after CNO injection, the mice appeared less anxious, as indicated by a longer exploratory time in the open arms of the elevated plus maze. However, levels of copulatory behaviour were comparable between CNO and saline-treated controls. These data indicate that DREADD-induced activation of MePD kisspeptin neurones enhances both sexual partner preference in males and social interaction and also decreases anxiety, suggesting a key role played by MePD kisspeptin in sexual motivation and social behaviour.

Posterodorsal Medial Amygdala Mediates Tail-Pinch Induced Food Intake in Female Rats.

Comfort eating during periods of stress is a common phenomenon observed in both animals and humans. However, the underlying mechanisms of stress-induced food intake remain elusive. The amygdala plays a central role in higher-order emotional processing and the posterodorsal subnucleus of the medial amygdala (MePD), in particular, is involved in food intake. Extra-hypothalamic corticotrophin-releasing factor (CRF) is well recognised for mediating behavioural responses to stress. To explore the possible role of amygdala CRF receptor activation in stress-induced food intake, we evaluated whether a stressor such as tail-pinch, which reliably induces food intake, would fail to do so in animals bearing bilateral neurotoxic lesions of the MePD. Our results showed that ibotenic acid induced lesions of the MePD markedly reduced tail-pinch induced food intake in ovariectomised, 17ß-oestradiol replaced rats. In addition, intra-MePD (right side only) administration of CRF (0.002 or 0.02 ng) via chronically implanted cannulae resulted in a dose-dependent increase in food intake, although higher doses of 0.2 and 2 ng CRF had less effect, producing a bell shaped curve. Furthermore, intra-MePD (bilateral) administration of the CRF receptor antagonist, astressin (0.3 µg per side) effectively blocked tail-pinch induced food intake. These data suggest that the MePD is involved in stress-induced food intake and that the amygdala CRF system may be a mediator of comfort eating.

The Posterodorsal Medial Amygdala Regulates the Timing of Puberty Onset in Female Rats.

Obesity is the major risk factor for early puberty, but emerging evidence indicates other factors including psychosocial stress. One key brain region notable for its role in controlling calorie intake, stress, and behavior is the amygdala. Early studies involving amygdala lesions that included the medial nucleus advanced puberty in rats. More recently it was shown that a critical site for lesion-induced hyperphagia and obesity is the posterodorsal subnucleus of the medial amygdala (MePD), which may explain the advancement of puberty. Glutamatergic activity also increases in the MePD during puberty without a corresponding γ-aminobutyric acid (GABA)ergic change, suggesting an overall activation of this brain region. In the present study, we report that neurotoxic lesioning of the MePD advances puberty and increases weight gain in female rats fed a normal diet. However, MePD lesioned rats fed a 25% nonnutritive bulk diet also showed the dramatic advancement of puberty but without the increase in body weight. In both dietary groups, MePD lesions resulted in an increase in socialization and a decrease in play fighting behavior. Chronic GABAA receptor antagonism in the MePD from postnatal day 21 for 14 days also advanced puberty, increased socialization, and decreased play fighting without altering body weight, whereas glutamate receptor antagonism delayed puberty and decreased socialization without affecting play fighting. In conclusion, our results suggest the MePD regulates the timing of puberty via a novel mechanism independent of change in body weight and caloric intake. MePD glutamatergic systems advance the timing of puberty whereas local GABAergic activation results in a delay.

Relative Importance of the Arcuate and Anteroventral Periventricular Kisspeptin Neurons in Control of Puberty and Reproductive Function in Female Rats.

Kisspeptin plays a critical role in pubertal timing and reproductive function. In rodents, kisspeptin perikarya within the hypothalamic arcuate (ARC) and anteroventral periventricular (AVPV) nuclei are thought to be involved in LH pulse and surge generation, respectively. Using bilateral microinjections of recombinant adeno-associated virus encoding kisspeptin antisense into the ARC or AVPV of female rats at postnatal day 10, we investigated the relative importance of these two kisspeptin populations in the control of pubertal timing, estrous cyclicity, and LH surge and pulse generation. A 37% knockdown of kisspeptin in the AVPV resulted in a significant delay in vaginal opening and first vaginal estrous, abnormal estrous cyclicity, and reduction in the occurrence of spontaneous LH surges, although these retained normal amplitude. This AVPV knockdown had no effect on LH pulse frequency, measured after ovariectomy. A 32% reduction of kisspeptin in the ARC had no effect on the onset of puberty but resulted in abnormal estrous cyclicity and decreased LH pulse frequency. Additionally, the knockdown of kisspeptin in the ARC decreased the amplitude but not the incidence of LH surges. These results might suggest that the role of AVPV kisspeptin in the control of pubertal timing is particularly sensitive to perturbation. In accordance with our previous studies, ARC kisspeptin signaling was critical for normal pulsatile LH secretion in female rats. Despite the widely reported role of AVPV kisspeptin neurons in LH surge generation, this study suggests that both AVPV and ARC populations are essential for normal LH surges and estrous cyclicity.

Overexpression of corticotropin releasing factor in the central nucleus of the amygdala advances puberty and disrupts reproductive cycles in female rats.

Prolonged exposure to environmental stress activates the hypothalamic-pituitary-adrenal (HPA) axis and generally disrupts the hypothalamic-pituitary-gonadal axis. Because CRF expression in the central nucleus of the amygdala (CeA) is a key modulator in adaptation to chronic stress, and central administration of CRF inhibits the hypothalamic GnRH pulse generator, we tested the hypothesis that overexpression of CRF in the CeA of female rats alters anxiety behavior, dysregulates the HPA axis response to stress, changes pubertal timing, and disrupts reproduction. We used a lentiviral vector to increase CRF expression site specifically in the CeA of preweaning (postnatal day 12) female rats. Overexpression of CRF in the CeA increased anxiety-like behavior in peripubertal rats shown by a reduction in time spent in the open arms of the elevated plus maze and a decrease in social interaction. Paradoxically, puberty onset was advanced but followed by irregular estrous cyclicity and an absence of spontaneous preovulatory LH surges associated with proestrous vaginal cytology in rats overexpressing CRF. Despite the absence of change in basal corticosterone secretion or induced by stress (lipopolysaccharide or restraint), overexpression of CRF in the CeA significantly decreased lipopolysaccharide, but not restraint, stress-induced suppression of pulsatile LH secretion in postpubertal ovariectomized rats, indicating a differential stress responsivity of the GnRH pulse generator to immunological stress and a potential adaptation of the HPA axis to chronic activation of amygdaloid CRF. These data suggest that the expression profile of this key limbic brain CRF system might contribute to the complex neural mechanisms underlying the increasing incidence of early onset of puberty on the one hand and infertility on the other attributed to chronic stress in modern human society.

Neurokinin B receptor antagonism decreases luteinising hormone pulse frequency and amplitude and delays puberty onset in the female rat.

The neural mechanisms controlling puberty onset remain enigmatic. Humans with loss of function mutations in TAC3 or TACR3, the genes encoding neurokinin B (NKB) or its receptor, neurokinin-3 receptor (NK3R), respectively, present with severe congenital gonadotrophin deficiency and pubertal failure. Animal studies have shown ambiguous actions of NKB-NK3R signalling with respect to controlling puberty onset. The present study aimed to determine the role of endogenous NKB-NK3R signalling in the control of pulsatile luteinising hormone (LH) secretion and the timing of puberty onset, and also whether precocious pubertal onset as a result of an obesogenic diet is similarly regulated by this neuropeptide system. Prepubertal female rats, chronically implanted with i.c.v. cannulae, were administered SB222200, a NK3R antagonist, or artificial cerebrospinal fluid via an osmotic mini-pump for 14 days. SB222200 significantly delayed the onset of vaginal opening and first oestrus (as markers of puberty) compared to controls in both normal and high-fat diet fed animals. Additionally, serial blood sampling, via chronic indwelling cardiac catheters, revealed that the increase in LH pulse frequency was delayed and that the LH pulse amplitude was reduced in response to NK3R antagonism, regardless of dietary status. These data suggest that endogenous NKB-NK3R signalling plays a role in controlling the timing of puberty and the associated acceleration of gonadotrophin-releasing hormone pulse generator frequency in the female rat.

Neurokinin B signaling in the female rat: a novel link between stress and reproduction.

Acute systemic stress disrupts reproductive function by inhibiting pulsatile gonadotropin secretion. The underlying mechanism involves stress-induced suppression of the GnRH pulse generator, the functional unit of which is considered to be the hypothalamic arcuate nucleus kisspeptin/neurokinin B/dynorphin A neurons. Agonists of the neurokinin B (NKB) receptor (NK3R) have been shown to suppress the GnRH pulse generator, in a dynorphin A (Dyn)-dependent fashion, under hypoestrogenic conditions, and Dyn has been well documented to mediate several stress-related central regulatory functions. We hypothesized that the NKB/Dyn signaling cascade is required for stress-induced suppression of the GnRH pulse generator. To investigate this ovariectomized rats, iv administered with Escherichia coli lipopolysaccharide (LPS) following intracerebroventricular pretreatment with NK3R or κ-opioid receptor (Dyn receptor) antagonists, were subjected to frequent blood sampling for hormone analysis. Antagonism of NK3R, but not κ-opioid receptor, blocked the suppressive effect of LPS challenge on LH pulse frequency. Neither antagonist affected LPS-induced corticosterone secretion. Hypothalamic arcuate nucleus NKB neurons project to the paraventricular nucleus, the major hypothalamic source of the stress-related neuropeptides CRH and arginine vasopressin (AVP), which have been implicated in the stress-induced suppression of the hypothalamic-pituitary-gonadal axis. A separate group of ovariectomized rats was, therefore, used to address the potential involvement of central CRH and/or AVP signaling in the suppression of LH pulsatility induced by intracerebroventricular administration of a selective NK3R agonist, senktide. Neither AVP nor CRH receptor antagonists affected the senktide-induced suppression of the LH pulse; however, antagonism of type 2 CRH receptors attenuated the accompanying elevation of corticosterone levels. These data indicate that the suppression of the GnRH pulse generator by acute systemic stress requires hypothalamic NKB/NK3R signaling and that any involvement of CRH therewith is functionally upstream of NKB.

The physiological role of arcuate kisspeptin neurons in the control of reproductive function in female rats.

Kisspeptin plays a pivotal role in pubertal onset and reproductive function. In rodents, kisspeptin perikarya are located in 2 major populations: the anteroventral periventricular nucleus and the hypothalamic arcuate nucleus (ARC). These nuclei are believed to play functionally distinct roles in the control of reproduction. The anteroventral periventricular nucleus population is thought to be critical in the generation of the LH surge. However, the physiological role played by the ARC kisspeptin neurons remains to be fully elucidated. We used bilateral stereotactic injection of recombinant adeno-associated virus encoding kisspeptin antisense into the ARC of adult female rats to investigate the physiological role of kisspeptin neurons in this nucleus. Female rats with kisspeptin knockdown in the ARC displayed a significantly reduced number of both regular and complete oestrous cycles and significantly longer cycles over the 100-day period of the study. Further, kisspeptin knockdown in the ARC resulted in a decrease in LH pulse frequency. These data suggest that maintenance of ARC-kisspeptin levels is essential for normal pulsatile LH release and oestrous cyclicity.

The role of neurokinin B signalling in reproductive neuroendocrinology.

The KNDy neuropeptides, kisspeptin, neurokinin B (NKB) and dynorphin A (Dyn), have been implicated in regulating pulsatile luteinising hormone (LH) secretion. Studies of the interactions between KNDy signalling systems, however, are currently few. Although the stimulatory effect of kisspeptin and the inhibitory effect of Dyn on the gonadotropin-releasing hormone pulse generator are widely accepted, the effects of NKB in rodents are variable and sometimes controversial. Literature describing increased LH secretion in response to NKB receptor agonism predominates and is in line with human physiology, as well as the pathophysiology of pubertal failure associated with disruption of NKB signalling. However, the robust suppression of the LH pulse, induced by the same treatment under hypoestrogenic conditions, may hold clues as to the mechanisms of reproductive inhibition under pathological conditions. This review discusses the recent evidence for this paradox and outlines a revised working model incorporating the mechanisms by which KNDy neuropeptides modulate the reproductive axis.

Suppression of the GnRH pulse generator by neurokinin B involves a κ-opioid receptor-dependent mechanism.

Neurokinin B (NKB) and its receptor (NK3R) are coexpressed with kisspeptin, Dynorphin A (Dyn), and their receptors [G-protein-coupled receptor-54 (GPR54)] and κ-opioid receptor (KOR), respectively] within kisspeptin/NKB/Dyn (KNDy) neurons in the hypothalamic arcuate nucleus (ARC), the proposed site of the GnRH pulse generator. Much previous research has employed intracerebroventricular (icv) administration of KNDy agonists and antagonists to address the functions of KNDy neurons. We performed a series of in vivo neuropharmacological experiments aiming to determine the role of NKB/NK3R signaling in modulating the GnRH pulse generator and elucidate the interaction between KNDy neuropeptide signaling systems, targeting our interventions to ARC KNDy neurons. First, we investigated the effect of intra-ARC administration of the selective NK3R agonist, senktide, on pulsatile LH secretion using a frequent automated serial sampling method to obtain blood samples from freely moving ovariectomized 17ß-estradiol-replaced rats. Our results show that senktide suppresses LH pulses in a dose-dependent manner. Intra-ARC administration of U50488, a selective KOR agonist, also caused a dose-dependent, albeit more modest, decrease in LH pulse frequency. Thus we tested the hypothesis that Dyn/KOR signaling localized to the ARC mediates the senktide-induced suppression of the LH pulse by profiling pulsatile LH secretion in response to senktide in rats pretreated with nor-binaltorphimine, a selective KOR antagonist. We show that nor-binaltorphimine blocks the senktide-induced suppression of pulsatile LH secretion but does not affect LH pulse frequency per se. In order to address the effects of acute activation of ARC NK3R, we quantified (using quantitative RT-PCR) changes in mRNA levels of KNDy-associated genes in hypothalamic micropunches following intra-ARC administration of senktide. Senktide down-regulated expression of genes encoding GnRH and GPR54 (GNRH1 and Kiss1r, respectively), but did not affect the expression of Kiss1 (which encodes kisspeptin). We conclude that NKB suppresses the GnRH pulse generator in a KOR-dependent fashion and regulates gene expression in GnRH neurons.

The role of GABAergic signalling in stress-induced suppression of gonadotrophin-releasing hormone pulse generator frequency in female rats.

Stress exerts profound inhibitory effects on reproductive function by suppressing the pulsatile release of gonadotrophin-releasing hormone (GnRH) and therefore luteinising hormone (LH). This effect is mediated in part via the corticotrophin-releasing factor (CRF) system, although another potential mechanism is via GABAergic signalling within the medial preoptic area (mPOA) because this has known inhibitory influences on the GnRH pulse generator and shows increased activity during stress. In the present study, we investigated the role of the preoptic endogenous GABAergic system in stress-induced suppression of the GnRH pulse generator. Ovariectomised oestradiol-replaced rats were implanted with bilateral and unilateral cannulae targeting toward the mPOA and lateral cerebral ventricle, respectively; blood samples (25 µl) were taken via chronically implanted cardiac catheters every 5 min for 6 h for the measurement of LH pulses. Intra-mPOA administration of the specific GABA(A) receptor antagonist, bicuculline (0.2 pmol each side, three times at 20-min intervals) markedly attenuated the inhibitory effect of lipopolysaccharide (LPS; 25 µg/kg i.v.) but not restraint (1 h) stress on pulsatile LH secretion. By contrast, restraint but not LPS stress-induced suppression of LH pulse frequency was reversed by application of the selective GABA(B) receptor antagonist, CGP-35348, into the mPOA (1.5 nmol each side, three times at 20-min intervals). However, intra-mPOA application of either bicuculline or CGP-35348 attenuated the inhibitory effect of CRF (1 nmol i.c.v.) on the pulsatile LH secretion. These data indicate a pivotal and differential role of endogenous GABAergic signalling in the mPOA with respect to mediating psychological and immunological stress-induced suppression of the GnRH pulse generator.

The role of the bed nucleus of the stria terminalis in stress-induced inhibition of pulsatile luteinising hormone secretion in the female rat.

The bed nucleus of the stria terminalis (BNST) occupies a central position in the neural circuitry regulating the hypothalamic-pituitary-adrenocortical axis response to stress. The potential role of the BNST in stress-induced suppression of the gondotrophin-releasing hormone (GnRH) pulse generator, the central regulator of the reproductive system, was assessed by examining the effects of micro-infusion of corticotrophin-releasing factor (CRF) or its antagonist into the BNST on pulsatile luteinising hormone (LH) secretion or stress-induced inhibition of LH pulses, respectively. Ovariectomised oestrogen-treated rats were implanted chronically with bilateral cannulae in the dorsolateral BNST and i.v. catheters. CRF (25, 50 or 100 pmol in 200 nl of artificial cerebrospinal fluid) administered bilaterally into the BNST resulted in a dose-dependent decrease in LH pulse frequency, and induced Fos expression in glutamic acid decarboxylase immunostained neurones in the medial preoptic area. These results suggest that the activation of hypothalamic GABAergic neurones in response to intra-BNST administration of CRF may be involved in the suppression of LH pulses. Furthermore, administration of CRF antagonist (280 pmol astressin-B, three times at 20-min intervals) into the BNST effectively blocked the suppression of pulsatile LH secretion in response to restraint (1 h) but not hypoglycaemic (0.25 U insulin/kg, i.v.) stress. These data suggest that CRF innervation of the dorsolateral BNST plays a key, but differential, role in stress-induced suppression of the GnRH pulse generator.

Corticotrophin-releasing factor and stress-induced inhibition of the gonadotrophin-releasing hormone pulse generator in the female.

It is well established that stress activates the hypothalamo-pituitary-adrenal (HPA) axis and suppresses the hypothalamo-pituitary-gonadal (HPG) axis. A large literature dealing with various stressors that regulate gonadotrophin-releasing hormone (GnRH) secretion in a variety of species (including nonhuman primates, sheep, and rats) provides evidence that stress modulates GnRH secretion by activating the corticotrophin-releasing factor (CRF) system and sympathoadrenal pathways, as well as the limbic brain. Different stressors may suppress the HPG axis by activating or inhibiting various pathways in the CNS. In addition to CRF being the principal hypophysiotropic factor driving the HPA axis, it is a potent inhibitor of the GnRH pulse generator. The suppression of the GnRH pulse generator by a variety of stressful stimuli can be blocked by CRF antagonists, suggesting a pivotal role for endogenous CRF. The differential roles for CRF receptor type 1 (CRF-R1) and CRF-R2 in stress-induced suppression of the GnRH pulse generator add to the complexity of CRF regulation of the HPG axis. Although the precise sites and mechanisms of action remain to be elucidated, noradrenergic and gamma-amino-butyric acid (GABA) neurones are implicated in the system's regulation, and opioids and kisspeptin in the medial preoptic area (mPOA) and hypothalamic arcuate nucleus (ARC) may operate downstream of the CRF neuronal system.

Corticotrophin-releasing factor alters the timing of puberty in the female rat.

Puberty is a developmental process that is dependent upon activation of the hypothalamic gonadotrophin-releasing hormone (GnRH) pulse generator. It is well established that the stress neuropeptide, corticotrophin-releasing factor (CRF), has a profound inhibitory action on GnRH pulse generator frequency. Although stress is known to affect the timing of puberty, the role of CRF is unknown. The present study aimed to test the hypothesis that CRF plays a critical role in the timing of puberty. On postnatal day (pnd) 28, female rat pups were chronically implanted with i.c.v. cannulae and received 14 days of administration of either CRF, CRF receptor antagonist (astressin-B) or artificial cerebrospinal fluid via an osmotic mini-pump. A separate group of rats served as nonsurgical controls. As a marker of puberty, rats were monitored for vaginal opening and first vaginal oestrus. Levels of CRF, CRF receptor types 1 and 2 (CRF-R1, CRF-R2) mRNA expression in micropunches of the medial preoptic area (mPOA), hypothalamic paraventricular nucleus (PVN) and arcuate nucleus (ARC) were determined across pubertal development; brain tissue was collected from a naive group of rats on pnd 14, 32, on the day of vaginal opening, and pnd 77 (Adult). Administration of CRF resulted in a delay in the onset of puberty, whereas astressin-B advanced pubertal onset. Additionally, CRF and CRF-R1 mRNA expression was reduced in the mPOA, but not ARC, at puberty. In the PVN, expression of CRF, but not CRF-R1 mRNA, was reduced at the time of puberty. These data support the hypothesis that CRF signalling may play an important role in modulating the timing of puberty in the rat.

Neonatal lipopolysaccharide exposure delays puberty and alters hypothalamic Kiss1 and Kiss1r mRNA expression in the female rat.

Immunological challenge experienced in early life can have long-term programming effects on the hypothalamic-pituitary-adrenal axis that permanently influence the stress response. Similarly, neonatal exposure to immunological stress enhances stress-induced suppression of the hypothalamic-pituitary gonadal (HPG) axis in adulthood, but may also affect earlier development, including the timing of puberty. To investigate the timing of the critical window for this programming of the HPG axis, neonatal female rats were injected with lipopolysaccharide (LPS; 50 microg/kg i.p.) or saline on postnatal days 3 + 5, 7 + 9, or 14 + 16 and monitored for vaginal opening and first vaginal oestrus as markers of puberty. We also investigated the effects of neonatal programming on the development of the expression patterns of kisspeptin (Kiss1) and its receptor (Kiss1r) in hypothalamic sites known to contain kisspeptin-expressing neuronal populations critical to reproductive function: the medial preoptic area (mPOA) and the arcuate nucleus in neonatally-stressed animals. We determined that the critical period for a significant delay in puberty as a result of neonatal LPS exposure is before 7 days of age in the female rat, and demonstrated that Kiss1, but not Kiss1r mRNA, expression in the mPOA is down-regulated in pre-pubertal females. These data suggest that the mPOA population of kisspeptin neurones play a pivotal role in controlling the onset of puberty, and that their function can be affected by neonatal stress.

Kisspeptin stimulation of insulin secretion: mechanisms of action in mouse islets and rats.

AIMS/HYPOTHESIS: Kisspeptin is a novel peptide identified as an endogenous ligand of the G-protein-coupled receptor 54 (GPR-54), which plays a crucial role in puberty and reproductive function. High levels of GPR-54 and kisspeptin have been reported in the pancreas and we have previously shown that kisspeptin potentiates glucose-induced insulin release from isolated islets, although the mechanisms underlying this effect were unclear. METHODS: Insulin secretion from isolated mouse islets was measured to characterise the effects of kisspeptin. The effects of kisspeptin on both p42/44 mitogen-activated protein kinase (MAPK) phosphorylation and intracellular Ca(2+)([Ca(2+)](i)) in mouse islets were also investigated. Furthermore, kisspeptin was administered to rats in vivo and effects on plasma insulin levels measured. RESULTS: In the current study, kisspeptin induced a concentration-dependent potentiation of glucose-induced (20 mmol/l) insulin secretion from mouse islets, with maximal effects at 1 micromol/l, but had no effect on insulin secretion at a substimulatory concentration of glucose (2 mmol/l). Activation of GPR-54 by kisspeptin also caused reversible increases in [Ca(2+)](i) in Fura-2 loaded dispersed islet cells. The kisspeptin-induced potentiation of glucose-induced insulin secretion was completely abolished by inhibitors of phospholipase C and p42/44 MAPK, but not by inhibitors of protein kinase C or p38 MAPK. Intravenous administration of kisspeptin into conscious, unrestrained rats caused an increase in circulating insulin levels, whilst central administration of kisspeptin had no effect, indicating a peripheral site of action. CONCLUSIONS/INTERPRETATION: These observations suggest that neither typical protein kinase C isoforms nor p38 MAPK are involved in the potentiation of glucose-induced insulin release by kisspeptin, but intracellular signalling pathways involving phospholipase C, p42/44 MAPK and increased [Ca(2+)](i) are required for the stimulatory effects on insulin secretion. The observation that kisspeptin is also capable of stimulating insulin release in vivo supports the conclusion that kisspeptin is a regulator of beta cell function.

Down-regulation of hypothalamic kisspeptin and its receptor, Kiss1r, mRNA expression is associated with stress-induced suppression of luteinising hormone secretion in the female rat.

Identification of kisspeptin (Kiss1) and its G protein-coupled receptor 54 (Kiss1r) as an essential component of the hypothalamic-pituitary-gonadal (HPG) axis controlling gonadotrophin secretion raises the possibility that kisspeptin-Kiss1r signalling may play a critical role in the transduction of stress-induced suppression of reproduction. We examined the effects of: (i) three different stressors, known to suppress pulsatile luteinising hormone (LH) secretion; (ii) corticotrophin-releasing factor (CRF); and (iii) corticosterone on Kiss1 and Kiss1r expression in key hypothalamic sites regulating gonadotrophin secretion: the medial preoptic area (mPOA) and arcuate nucleus (ARC). Ovariectomised oestrogen-replaced rats were implanted with i.v., subcutaneous or i.c.v. cannulae. Blood samples were collected at 5-min intervals for 5-6 h for detection of LH. Quantitative reverse transcriptase-polymerase chain reaction was used to determine Kiss1 and Kiss1r mRNA levels in brain punches of the mPOA and ARC collected 6 h after restraint, insulin-induced hypoglycaemia or lipopolysaccharide stress, or after i.c.v. administration of CRF, or acute or chronic subcutaneous administration of corticosterone. We observed down-regulation of at least one component of the kisspeptin-Kiss1r signalling system by each of the stress paradigms within the mPOA and ARC. CRF decreased Kiss1 and Kiss1r expression in both the mPOA and ARC. Both acute and chronic stress levels of corticosterone resulted in a concomitant decrease in Kiss1 and an increase in kiss1r mRNA expression in the mPOA and ARC. This differential regulation of Kiss1 and Kiss1r might account for the lack of effect corticosterone has on pulsatile LH secretion. Considering the pivotal role for kisspeptin-Kiss1r signalling in the control of the HPG axis, these results suggest that the reduced Kiss1-Kiss1r expression may be a contributing factor in stress-related suppression of LH secretion.

A role for the medial preoptic area in CGRP-induced suppression of pulsatile LH secretion in the female rat.

Calcitonin gene-related peptide (CGRP) is involved in a variety of stress responses and plays a pivotal role in stress-induced suppression of the GnRH pulse generator in the rat. Intracerebroventricular administration of CGRP suppresses luteinizing hormone (LH) pulses and increases Fos expression within the medial preoptic area (mPOA) and paraventricular nucleus (PVN). The aims of the present study were to investigate whether the mPOA or PVN are sites of action for CGRP-induced suppression of LH pulses and whether lipopolysaccharide (LPS), restraint or insulin-induced hypoglycaemia, stressors known to suppress LH pulses, affect mRNA expression for CGRP and its receptor subunits (calcitonin receptor-like receptor (CL) and RAMP-1) in the mPOA and PVN. Micro-infusion of CGRP (50, 250 or 500 pmol) into the mPOA, but not the PVN, dose-dependently suppressed LH pulse frequency. LPS, restraint and hypoglycaemia suppressed RAMP-1 mRNA, but not CL or CGRP mRNA expression in the mPOA. In the PVN, all three stressors suppressed CL mRNA expression, but only LPS or restraint suppressed RAMP-1 mRNA, and CGRP mRNA was unaffected. These results provide evidence that, unlike the PVN, the mPOA might play an important role in the inhibitory effect of CGRP on pulsatile LH secretion. Additionally, CGRP receptor function may be involved in this brain region in stress-induced suppression of the GnRH pulse generator.

The role of corticotrophin-releasing hormone receptors in the calcitonin gene-related peptide-induced suppression of pulsatile luteinising hormone secretion in the female rat.

Corticotrophin-releasing hormone (CRH) plays a pivotal role in the suppression of the gonadotrophin-releasing hormone (GRH) pulse generator in response to stress and intracerebroventricular (i.c.v.) administration of calcitonin gene-related peptide (CGRP). We have previously shown both CRH receptor subtypes, CRH-R1 and CRH-R2, are involved in the stress-induced suppression of LH pulses. The aims of the present study were to examine the role of CRH-R1 and CRH-R2 in CGRP-induced suppression of LH pulses, and to investigate the effects of CGRP on CRH expression in the paraventricular nucleus (PVN) and central nucleus of the amygdala (CeA), which have prominent CRH neurone populations that receive dense CGRP innervations. The suppression of LH pulses by CGRP (1.5 microg i.c.v.) was completely prevented by intravenous administration of the CRH-R1 antagonist SSR125543Q (7.5 mg/rat i.v., 30 min before CGRP), but was not affected by the CRH-R2 antagonist, astressin(2)-B (100 microg i.c.v., 10 min before CGRP). CGRP increased the CRH mRNA expression in PVN and CeA. These results provide evidence of a role for CRH-R1 in mediating the suppressive effects of CGRP on pulsatile LH secretion in the female rat, and additionally raise the possibility of an involvement of PVN and CeA CRH neuronal populations in this suppression.

Neonatal lipopolysaccharide exposure exacerbates stress-induced suppression of luteinizing hormone pulse frequency in adulthood.

Early life exposure to immunological challenge has programming effects on the adult hypothalamo-pituitary-adrenocortical axis stress responsivity, and stress is known to suppress GnRH pulse generator activity, especially LH pulses. We investigated the effects of neonatal exposure to endotoxin on stress-induced suppression of pulsatile LH secretion and the involvement of corticotropin-releasing factor (CRF) receptor mechanisms in adult rats. Pups at 3 and 5 d of age were administered lipopolysaccharide (LPS, 50 microg/kg, ip). At 12 wk of age, they were ovariectomized and implanted with sc 17beta-estradiol capsules and i.v. cannulas. Blood samples (25 microl) were collected every 5 min for 5 h for LH measurement. After 2 h of sampling, rats were given LPS (25 microg/kg, iv). CRF and CRF-R1 and CRF-R2 receptor mRNA was determined by RT-PCR in medial preoptic area (mPOA) micropunches collected at 3 h after LPS administration. There was no difference in basal LH pulse frequency between neonatal LPS- and neonatal saline-treated controls. However, neonatal endotoxin-treated rats exhibited a significantly greater LPS stress-induced suppression of LH pulse frequency. Basal mPOA CRF-R1 expression was unchanged in neonatal LPS- and neonatal saline-treated rats. However, CRF-R1 expression was significantly increased in response to LPS stress in neonatal LPS-treated animals but not in neonatal saline-treated controls. CRF and CRF-R2 expression was unchanged in all treatment groups. These data demonstrate that exposure to bacterial endotoxin in early neonatal life programs long-term sensitization of the GnRH pulse generator to the inhibitory influence of stress in adulthood, an effect that might involve up-regulation of CRF-R1 expression in the mPOA.