Co-localisation of kisspeptin with galanin or neurokinin B in afferents to mouse GnRH neurones

The gonadotrophin-releasing hormone (GnRH) secreting neurones, which form the final common pathway for the central regulation of reproduction, are directly targeted by kisspeptin (KP) via the G protein-coupled receptor, GPR54. In these multiple labelling studies, we used ovariectomised mice treated with 17β-oestradiol (OVX + E(2)) or vehicle (OVX + oil) to determine: (i) the ultrastructural characteristics of KP-immunoreactive (IR) afferents to GnRH neurones; (ii) their galanin or neurokinin B (NKB) content; and (iii) the co-expression of galanin or NKB with KP in the two major subpopulations of KP neurones located in the rostral periventricular area of the third ventricle (RP3V) and the arcuate nucleus (Arc). Electron microscopic investigation of the neuronal juxtapositions revealed axosomatic and axodendritic synapses; these showed symmetrical or asymmetrical characteristics, suggesting a phenotypic diversity of KP afferents. Heterogeneity of afferents was also demonstrated by differential co-expression of neuropeptides; in OVX + E(2) mice, KP afferents to GnRH neurones showed galanin-immunoreactivity with an incidence of 22.50 ± 2.41% and NKB-immunoreactivity with an incidence of 5.61 ± 2.57%. In OVX + oil animals, galanin-immunoreactivity in the KP afferents showed a major reduction, appearing in only 5.78 ± 1.57%. Analysis for co-localisation of galanin or NKB with KP was extended to the perikaryal level in animal models, which showed the highest KP incidence; these were OVX + E(2) females for the RP3V and OVX + oil females for the ARC. In the RP3V of colchicine-treated OVX + E(2) animals, 87.84 ± 2.65% of KP-IR neurones were galanin positive. In the Arc of the colchicine-treated OVX + oil animals, galanin immunoreactivity was detected in only 12.50 ± 1.92% of the KP expressing neurones. By contrast, the incidence of co-localisation with NKB in the Arc of those animals was 98.09 ± 1.30%. In situ hybridisation histochemistry of sections from OVX + E(2) animals identified galanin message in more than a third of the KP neurones in the RP3V (38.67 ± 11.57%) and in the Arc (42.50 ± 12.52%). These data suggest that GnRH neurones are innervated by chemically heterogeneous KP cell populations, with a small proportion deriving from the Arc group. The presence of galanin within KP axons innervating GnRH neurones and the oestrogen-dependent regulation of that presence add a new dimension to the roles played by galanin in the central regulation of reproduction.     

Dual phenotype kisspeptin-dopamine neurones of the rostral periventricular area of the third ventricle project to gonadotrophin-releasing hormone neurones

The neuropeptide kisspeptin and its G-protein-coupled receptor, Gpr54, are critical regulators of fertility. Two major populations of kisspeptin neurones exist in the rodent: one in the rostral periventricular area of the third ventricle (RP3V) and another in the arcuate nucleus. The RP3V population of kisspeptin neurones is crucial for the generation of the luteinising hormone surge that drives ovulation in females. The RP3V kisspeptin neurones are sexually dimorphic, with many more neurones in females than males, and they project to gonadotrophin-releasing hormone (GnRH) neurones. Tyrosine hydroxylase (TH) expressing neurones in the RP3V are also sexually dimorphic and are assumed to project to GnRH neurones. In the present study, we examined the coexpression of kisspeptin and TH peptides in the RP3V of dioestrous and pro-oestrous female mice. We also investigated whether kisspeptin and TH peptides colocalised in terminal appositions with GnRH neurones in the rostral preoptic area (rPOA). Approximately half of the kisspeptin neurones in the RP3V were found to also express TH and vice versa, although there was no difference between mice in dioestrus or pro-oestrus. The majority (95%) of GnRH neurones in the rPOA exhibited a close apposition from a kisspeptin fibre, whereas only one quarter exhibited a close apposition from a TH fibre. Many of the TH close appositions with GnRH neurones coexpressed kisspeptin (62-86%), although these dual-labelled appositions comprised <20% of all kisspeptin appositions on GnRH neurones. The percentage of GnRH neurones with kisspeptin, TH and double-labelled appositions did not differ between dioestrous and pro-oestrous mice. These findings indicate that a subpopulation of kisspeptin neurones expressing dopamine innervate GnRH neurones in the rPOA.     

Aromatase knockout mice show normal steroid-induced activation of gonadotrophin-releasing hormone neurones and luteinising hormone surges with a reduced population of kisspeptin neurones in the rostral hypothalamus

We recently reported that female aromatase knockout (ArKO) mice show deficits in sexual behaviour and a decreased population of kisspeptin‐immunoreactive neurones in the rostral periventricular area of the third ventricle (RP3V), resurrecting the question of whether oestradiol actively contributes to female‐typical sexual differentiation. To further address this question, we assessed the capacity of ArKO mice to generate a steroid‐induced luteinising hormone (LH) surge. Adult, gonadectomised wild‐type (WT) and ArKO mice were given silastic oestradiol implants s.c. and, 1 week later, received s.c. injections of either oestradiol benzoate (EB) followed by progesterone, EB alone, or no additional steroids to activate gonadotrophin‐releasing hormone (GnRH) neurones and generate an LH surge. Treatment with EB and progesterone induced significant Fos/GnRH double‐labelling and, consequently, an LH surge in female WT and in ArKO mice of both sexes but not in male WT mice. ArKO mice of both sexes had fewer cells expressing Kiss‐1 mRNA in the RP3V compared to female WT mice but had more Kiss‐1 mRNA‐expressing cells compared to WT males, reflecting an incomplete sexual differentiation of this system. To determine the number of cells expressing kisspeptin, the same experimental design was repeated in Experiment 2 with the addition of groups of WT and ArKO mice that were given EB + progesterone and sacrificed 2 h before the expected LH surge. No differences were observed in the number of kisspeptin‐immunoreactive cells 2 h before and at the time of the LH surge. The finding that ArKO mice of both sexes have a competent LH surge system suggests that oestradiol has predominantly defeminising actions on the GnRH/LH surge system in males and that the steroid‐induced LH surge can occur in females even with a greatly reduced population of kisspeptin neurones in the RP3V.     

Evidence for Changes in Numbers of Synaptic Inputs onto KNDy and GnRH Neurones during the Preovulatory LH Surge in the Ewe

Kisspeptin neurones located in the arcuate nucleus (ARC) and preoptic area (POA) are critical mediators of gonadal steroid feedback onto gonadotrophin‐releasing hormone (GnRH) neurones. ARC kisspeptin cells that co‐localise neurokinin B (NKB) and dynorphin (Dyn), are collectively referred to as KNDy (Kisspeptin/NKB/Dyn) neurones, and have been shown in mice to also co‐express the vesicular glutamate transporter, vGlut2, an established glutamatergic marker. The ARC in rodents has long been known as a site of hormone‐induced neuroplasticity, and changes in synaptic inputs to ARC neurones in rodents occur over the oestrous cycle. Based on this evidence, the the present study aimed to examine possible changes across the ovine oestrous cycle in synaptic inputs onto kisspeptin cells in the ARC (KNDy) and POA, and inputs onto GnRH neurones. Gonadal‐intact breeding season ewes were perfused using 4% paraformaldehyde during either the luteal or follicular phase of the oestrous cycle, with the latter group killed at the time of the luteinising hormone (LH) surge. Hypothalamic sections were processed for triple‐label immunodetection of kisspeptin/vGlut2/synaptophysin or kisspeptin/vGlut2/GnRH. The total numbers of synaptophysin‐ and vGlut2‐positive inputs to ARC KNDy neurones were significantly increased at the time of the LH surge compared to the luteal phase; because these did not contain kisspeptin, they do not arise from KNDy neurones. By contrast to the ARC, the total number of synaptophysin‐positive inputs onto POA kisspeptin neurones did not differ between luteal phase and surge animals. The total number of kisspeptin and vGlut2 inputs onto GnRH neurones in the mediobasal hypothalamus (MBH) was also increased during the LH surge, and could be attributed to an increase in the number of KNDy (double‐labelled kisspeptin + vGlut2) inputs. Taken together, these results provide novel evidence of synaptic plasticity at the level of inputs onto KNDy and GnRH neurones during the ovine oestrous cycle. Such changes may contribute to the generation of the preovulatory GnRH/LH surge.     

Kisspeptin,gonadotrophin‐releasing hormone and oestrogen receptor α colocalise with neuronal nitric oxide synthase neurones in prepubertal female sheep

Puberty is a process that integrates multiple inputs ultimately resulting in an increase in gonadotrophin‐releasing hormone (GnRH) secretion. Although kisspeptin neurones play an integral role in GnRH secretion and puberty onset, other systems are also likely important. One potential component is nitric oxide (NO), a gaseous neurotransmitter synthesised by nitric oxide synthase (NOS). The present study aimed to neuroanatomically characterise neuronal NOS (nNOS) in prepubertal female sheep and determine whether oestradiol exerts effects on this system. Luteinising hormone secretion was reduced by oestradiol treatment in prepubertal ovariectomised ewes. Neurones immunoreactive for nNOS were identified in several areas, with the greatest number present in the ventrolateral portion of the ventromedial hypothalamus, followed by the ventromedial hypothalamus, preoptic area (POA) and arcuate nucleus (ARC). Next, we determined whether nNOS neurones contained oestrogen receptor (ER)α and could potentially communicate oestradiol (E₂) feedback to GnRH neurones. Neuronal NOS neurones contained ERα with the percentage of coexpression (12%‐40%) depending upon the area analysed. We next investigated whether a neuroanatomical relationship existed between nNOS and kisspeptin or nNOS and GnRH neurones. A high percentage of kisspeptin neurones in the POA (79%) and ARC (98%) colocalised with nNOS. Kisspeptin close contacts were also associated with nNOS neurones. A greater number of close contacts were observed in the ARC than the POA. A high percentage of POA GnRH neurones (79%) also expressed nNOS, although no GnRH close contacts were observed onto nNOS neurones. Neither the numbers of nNOS neurones in the POA or hypothalamus, nor the percentage of nNOS coexpression with GnRH, kisspeptin or ERα were influenced by oestradiol. These experiments reveal that a neuroanatomical relationship exists between both nNOS and kisspeptin and nNOS and GnRH in prepubertal ewes. Therefore, nNOS may act both directly and indirectly to influence GnRH secretion in prepubertal sheep.     

Leptin Responsive and GABAergic Projections to the Rostral Preoptic Area in Mice

The adipocyte‐derived hormone leptin plays a critical role in the control of reproduction via signalling in hypothalamic neurones. The drivers of the hypothalamic‐pituitary‐gonadal axis, the gonadotrophin‐releasing hormone (GnRH) neurones, do not have the receptors for leptin. Therefore, intermediate leptin responsive neurones must provide leptin‐to‐GnRH signalling. We investigated the populations of leptin responsive neurones that provide input to the rostral preoptic area (rPOA) where GnRH cell bodies reside. Fluorescent retrograde tracer beads (RetroBeads; Lumafluor Inc., Naples, FL, USA) were injected into the rPOA of transgenic leptin receptor enhanced green fluorescent protein (Lepr‐eGFP) reporter mice. Uptake of the RetroBeads by Lepr‐eGFP neurones was assessed throughout the hypothalamus. RetroBead uptake was most evident in the medial arcuate nucleus (ARC), the dorsomedial nucleus (DMN) and the ventral premammillary nucleus (PMV) of the hypothalamus. The uptake of RetroBeads specifically by Lepr‐eGFP neurones was highest in the medial ARC (18% of tracer‐labelled neurones Lepr‐eGFP‐positive). Because neurones that are both leptin responsive and GABAergic play a critical role in the regulation of fertility by leptin, we next focussed on the location of these populations. To address whether GABAergic neurones in leptin‐responsive hypothalamic regions project to the rPOA, the experiment was repeated in GABA neurone reporter mice (Vgat‐tdTomato). Between 10% and 45% of RetroBead‐labelled neurones in the ARC were GABAergic, whereas uptake of tracer by GABAergic neurones in the DMN and PMV was very low (< 5%). These results show that both leptin responsive and GABAergic neurones from the ARC project to the region of the GnRH cell bodies. Our findings suggest that LEPR‐expressing GABA neurones from the ARC may be mediators of leptin‐to‐GnRH signalling.     

Nutritional Programming of Accelerated Puberty in Heifers: Involvement of Pro‐Opiomelanocortin Neurones in the Arcuate Nucleus

The timing of puberty and subsequent fertility in female mammals are dependent on the integration of metabolic signals by the hypothalamus. Pro‐opiomelanocortin (POMC) neurones in the arcuate nucleus (ARC) comprise a critical metabolic‐sensing pathway controlling the reproductive neuroendocrine axis. α‐Melanocyte‐stimulating hormone (αMSH), a product of the POMC gene, has excitatory effects on gonadotrophin‐releasing hormone (GnRH) neurones and fibres containing αMSH project to GnRH and kisspeptin neurones. Because kisspeptin is a potent stimulator of GnRH release, αMSH may also stimulate GnRH secretion indirectly via kisspeptin neurones. In the present work, we report studies conducted in young female cattle (heifers) aiming to determine whether increased nutrient intake during the juvenile period (4–8 months of age), a strategy previously shown to advance puberty, alters POMC and KISS1 mRNA expression, as well as αMSH close contacts on GnRH and kisspeptin neurones. In Experiment 1, POMC mRNA expression, detected by in situ hybridisation, was greater (P < 0.05) in the ARC in heifers that gained 1 kg/day of body weight (high‐gain, HG; n = 6) compared to heifers that gained 0.5 kg/day (low‐gain, LG; n = 5). The number of KISS1‐expressing cells in the middle ARC was reduced (P < 0.05) in HG compared to LG heifers. In Experiment 2, double‐immunofluorescence showed limited αMSH‐positive close contacts on GnRH neurones, and the magnitude of these inputs was not influenced by nutritional status. Conversely, a large number of kisspeptin‐immunoreactive cells in the ARC were observed in close proximity to αMSH‐containing varicosities. Furthermore, HG heifers (n = 5) exhibited a greater (P < 0.05) percentage of kisspeptin neurones in direct apposition to αMSH fibres and an increased (P < 0.05) number of αMSH close contacts per kisspeptin cell compared to LG heifers (n = 6). These results indicate that the POMC‐kisspeptin pathway may be important in mediating the nutritional acceleration of puberty in heifers.     

Evidence for Suprachiasmatic Vasopressin Neurones Innervating Kisspeptin Neurones in the Rostral Periventricular Area of the Mouse Brain: Regulation by Oestrogen

In rodents, a circadian signal from the suprachiasmatic nucleus (SCN) is essential for the pro‐oestrous surge of gonadotrophin‐releasing hormone (GnRH), which, in turn, induces luteinising hormone (LH) surge and ovulation. We hypothesised that kisspeptin (KP) neurones in the anteroventral periventricular and periventricular preoptic nuclei (AVPV/PeN) form part of the communication pathway between the SCN and GnRH neurones. In anterograde track tracing studies, we first identified vasopressin (VP)‐containing axons of SCN origin in apposition to KP‐immunoreactive (IR) neurones. Studies to quantify this input relied on the observation that VP‐synthesising neurones in the SCN differ from other VP systems in their lack of galanin expression. In ovariectomised mice, 30.79 ± 1.63% of KP‐IR perikarya and proximal dendrites within the AVPV/PeN received galanin‐negative VP‐IR varicosities. Oestrogen‐treatment significantly increased the number of KP‐IR neurones, with their percentage apposed by galanin‐negative VP‐IR varicosities (46.95 ± 1.88%) and the number of VP‐IR appositions on individual KP‐IR neurones. At the ultrastructural level, the VP‐IR terminals formed symmetric synapses with KP‐IR neurones, which was in accordance with the morphology of inhibitory synapses established by SCN neurones. By contrast to VP, vasoactive intestinal polypeptide (VIP), which is synthesised by a distinct subset of SCN neurones, occurred only rarely in axons apposed to KP‐IR neurones. Altogether, our results are consistent with the hypothesis that KP neurones located in the mouse AVPV/PeN receive circadian information from the SCN via a vasopressinergic monosynaptic pathway, which is enhanced by oestrogen.     

Male Effect Pheromone Tickles the Gonadotrophin‐Releasing Hormone Pulse Generator

In sheep and goats, the primer pheromone produced by the male induces out‐of‐seasonal ovulation in anoestrous females, the so‐called ‘male effect.’ Because the initial endocrine event following reception of the pheromone is the stimulation of pulsatile luteinising hormone (LH) secretion, the central target of the pheromone is considered to be the putative gonadotrophin‐releasing hormone (GnRH) pulse generator. Using electrophysiological techniques to record multiple‐unit activity (MUA) in close proximity to kisspeptin neurones in the arcuate nucleus (ARC) of Shiba goats, we found that bursts (volleys) of MUA occur at regular intervals, and repetitive bursts are invariably associated with discrete pulses of LH, suggesting that the ARC kisspeptin neurones may be the intrinsic source of the GnRH pulse generator. A brief exposure of female goats to the pheromone immediately elicited an instantaneous rise in MUA, which is followed by an MUA volley and an accompanying LH pulse, indicating that the pheromone signal is transmitted to a subset of the ARC kisspeptin neurones to activate them. Because it has been suggested that the neurokinin B and dynorphin coexpressed in those neurones play critical roles in generating rhythmic bursts, they may be involved in the intracellular pheromone actions that are responsible for inducing the GnRH pulse.     

Interactions between β-endorphin and kisspeptin neurons of the ewe arcuate nucleus are modulated by photoperiod

Opioid peptides are well-known modulators of the central control of reproduction. Among them, dynorphin coexpressed in kisspeptin (KP) neurons of the arcuate nucleus (ARC) has been thoroughly studied for its autocrine effect on KP release through κ opioid receptors. Other studies have suggested a role for β-endorphin (BEND), a peptide cleaved from the pro-opiomelanocortin precursor, on food intake and central control of reproduction. Similar to KP, BEND content in the ARC of sheep is modulated by day length and BEND modulates food intake in a dose-dependent manner. Because KP levels in the ARC vary with photoperiodic and metabolic status, a photoperiod-driven influence of BEND neurons on neighboring KP neurons is plausible. The present study aimed to investigate a possible modulatory action of BEND on KP neurons located in the ovine ARC. Using confocal microscopy, numerous KP appositions on BEND neurons were found but there was no photoperiodic variation of the number of these interactions in ovariectomized, estradiol-replaced ewes. By contrast, BEND terminals on KP neurons were twice as numerous under short days, in ewes having an activated gonadotropic axis, compared to anestrus ewes under long days. Injection of 5 μg BEND into the third ventricle of short-day ewes induced a significant and specific increase of activated KP neurons (16% vs. 9% in controls), whereas the percentage of overall activated (c-Fos positive) neurons, was similar between both groups. These data suggest a photoperiod-dependent influence of BEND on KP neurons of the ARC, which may influence gonadotropin-releasing hormone pulsatile secretion and inform KP neurons about metabolic status.     

Seasonal changes in the inputs to gonadotropin-releasing hormone neurones in the ewe brain: an assessment by conventional fluorescence and confocal microscopy

The seasonal pattern of breeding in sheep offers an opportunity to examine plasticity of neuronal inputs to gonadotropin-releasing hormone (GnRH) neurones. We used conventional fluorescence microscopy and confocal microscopy to compare the extent of input to GnRH neurones from various neuropeptide/neurotransmitter systems in ewes during the breeding and anestrous seasons. Using double-labelling immunohistochemistry, we counted close appositions between GnRH cells and varicosities that were immunoreactive for either glutamic acid decarboxylase (GAD; for gamma-amino butyric acid-GABA-neurones), dopamine beta hydroxylase (DBH; for noradrenergic neurones), vesicular glutamate transporter-1 (VGluT-1, for glutamatergic neurones), neuropeptide Y (NPY) and tyrosine hydroxylase (TH; for dopaminergic/noradrenergic neurones). The percentage of GnRH cells displaying close appositions to GABA-ergic varicosities was higher (P < 0.02) in anestrus than in the breeding season. The percentage of GnRH cells receiving input from varicosities that were positive for TH, DBH and VGluT-1 was similar in both seasons. Approximately 26-49% of GnRH neurones were seen to receive inputs from NPY, TH, GABAergic or noradrenergic neurones, while a larger number of GnRH cells (72-75%) received input from glutamatergic neurones. Conventional microscopy consistently overestimated the number of close contacts on GnRH neurones compared to confocal microscopy. For TH-immunoreactive varicosities in the preoptic area, only 16-35% were also immunoreactive for DBH, suggesting that the remainder are dopaminergic. Approximately half of the noradrenergic inputs in the preoptic area were also immunoreactive for NPY. In conclusion, we present numerical data on the consensus between light and confocal microscopy and the level of input of various neuronal systems to GnRH cells; the data indicate a seasonal change in the GABAergic input to GnRH neurones.     

Visualisation of Kiss1 Neurone Distribution Using a Kiss1‐CRE Transgenic Mouse

Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE‐GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss‐CRE heterozygous mice with CRE‐activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80‐90% of tdTomato positive neurones in the ARC were co‐labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato‐labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre‐optic areas and to the lateral hypothalamus.     

Gonadotrophin‐Releasing Hormone (GnRH) Exerts Stimulatory Effects on GnRH Neurones in Intact Adult Male and Female Mice

There is substantial evidence for a role of the neuropeptide gonadotrophin‐releasing hormone (GnRH) in the regulation of GnRH neurone secretion but how this is achieved is not understood. We examined here the effects of GnRH on the electrical excitability and intracellular calcium concentration ([Ca²⁺]_i) of GnRH neurones in intact adult male and female mice. Perforated‐patch electrophysiological recordings from GnRH‐green fluorescent protein‐tagged GnRH neurones revealed that 3 nm –3 μm GnRH evoked gradual approximately 3 mV depolarisations in membrane potential from up to 50% of GnRH neurones in male and female mice. The depolarising effect of GnRH was observed on approximately 50% of GnRH neurones throughout the oestrous cycle. However, at pro‐oestrus alone, GnRH was also found to transiently hyperpolarise approximately 30% of GnRH neurones. Both hyperpolarising and depolarising responses were maintained in the presence of tetrodotoxin. Calcium imaging studies undertaken in transgenic GnRH‐pericam mice showed that GnRH suppressed [Ca²⁺]_i in approximately 50% of GnRH neurones in dioestrous and oestrous mice. At pro‐oestrus, 25% of GnRH neurones exhibited a suppressive [Ca²⁺]_i response to GnRH, whereas 17% were stimulated. These results demonstrate that nm to μm concentrations of GnRH exert depolarising actions on approximately 50% of GnRH neurones in males and females throughout the oestrous cycle. This is associated with a reduction in [Ca²⁺]_i. At pro‐oestrus, however, a further population of GnRH neurones exhibit a hyperpolarising response to GnRH. Taken together, these studies indicate that GnRH acts predominantly as a neuromodulator at the level of the GnRH cell bodies to exert a predominant excitatory influence upon GnRH neurones in intact adult male and female mice.     

Evidence of involvement of neurone‐glia/neurone‐neurone communications via gap junctions in synchronised activity of KNDy neurones

Pulsatile secretion of gonadotrophin‐releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so‐called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB‐NK3R signalling. We determined the role of NKB‐NK3R signalling in Ca²⁺ oscillation (an indicator of neuronal activities) of KNDy neurones and its synchronisation mechanism among KNDy neurones. Senktide, a selective agonist for NK3R, increased the frequency of Ca²⁺ oscillations in cultured Kiss1‐GFP cells collected from the mediobasal hypothalamus of the foetal Kiss1‐green fluorescent protein (GFP) mice. The senktide‐induced Ca²⁺ oscillations were synchronised in the Kiss1‐GFP and neighbouring glial cells. Confocal microscopy analysis of these cells, which have shown synchronised Ca²⁺ oscillations, revealed close contacts between Kiss1‐GFP cells, as well as between Kiss1‐GFP cells and glial cells. Dye coupling experiments suggest cell‐to‐cell communication through gap junctions between Kiss1‐GFP cells and neighbouring glial cells. Connexin‐26 and ‐37 mRNA were found in isolated ARC Kiss1 cells taken from adult female Kiss1‐GFP transgenic mice. Furthermore, 18β‐glycyrrhetinic acids and mefloquine, which are gap junction inhibitors, attenuated senktide‐induced Ca²⁺ oscillations in Kiss1‐GFP cells. Taken together, these results suggest that NKB‐NK3R signalling enhances synchronised activities among neighbouring KNDy neurones, and that both neurone‐neurone and neurone‐glia communications via gap junctions possibly contribute to synchronised activities among KNDy neurones.     

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.     

Evidence for Expression of Galanin Receptor Gal-R1 mRNA in Certain Gonadotropin Releasing Hormone Neurones of the Rostral Preoptic Area

Previous studies have shown that galanin plays an important role in the regulation of gonadotropin releasing hormone (GnRH) release. At present, it is not known if this role is exerted by direct or indirect interactions between galanin producing neurones and GnRH neurones. The objective of this study was to determine whether or not GnRH neurones could express galanin receptor Gal-R1 mRNA. Dual in-situ hybridization experiments were carried out with digoxigenin-labelled cRNA probes encoding GnRH in combination with 35S-labelled riboprobe encoding the galanin receptor Gal-R1. In order to detect possible variations in the expression of the Gal-R1 mRNA under different physiological conditions, male rats, intact female rats throughout the phases of the oestrous cycle, ovariectomized (OVX) and steroid-treated rats were analysed. The results show that many cells expressing Gal-R1 mRNA were present throughout the preoptic area. Gal-R1 mRNA-expressing cells were observed in very close proximity with GnRH neurones. In the female rat, some GnRH neurones located in the rostral preoptic area/vascular organ of the lamina terminalis expressed Gal-R1 mRNA. These double-labelled cells were observed at all times of the oestrous cycle, except during diestrus at 08.00 h and pro-oestrus at 18.00 h. Conspicuously, at oestrus 1800 h, we found that 21.6% of rostral GnRH neurones expressed the Gal-R1 mRNA. In addition, dual-labelled GnRH neurones were seen in OVX animals but not in oestrogen plus progesterone-treated ones. In the male rat, colocalization of GnRH mRNA and Gal-R1 receptor mRNA was not observed. In the medial preoptic area, no double-labelled GnRH neurones were detected regardless of the endocrine conditions. These results suggest that, in addition to a possible indirect action of galanin on GnRH cells via neurones located at close proximity, the effects of galanin on GnRH can be mediated by direct activation of galanin receptors in rostral GnRH neurones. This study also shows that expression of Gal-R1 mRNA in GnRH cells is influenced by the levels of circulating gonadal steroids.     

Possible Involvement of Microglia Containing Cyclooxygenase‐1 in the Accumulation of Gonadotrophin‐Releasing Hormone in the Preoptic Area in Female Rats

Prostaglandins (PGs), especially PGE₂, are involved in the hypothalamic control of gonadotrophin‐releasing hormone (GnRH) release, acting at least in part on the terminal of GnRH axons in the median eminence. The present study aimed: (i) to clarify the role of PG(s) in regulating GnRH cell function at the level of the perikarya in the preoptic area; (ii) to determine the cyclooxygenase (COX) isozyme responsible for producing PG(s) that regulates GnRH perikarya; and (iii) to identify cell types that contain the responsible COX isozyme in female rats. A surge of luteinising hormone (LH) secretion was induced by oestrogen and progesterone in ovariectomised rats. Treatment of the rat before the LH surge with indomethacin, a nonselective COX inhibitor, or NS‐398, a selective COX‐2 inhibitor, did not interfere with the surge. However, treatment with indomethacin or flurbiprofen, a selective COX‐1 inhibitor, significantly reduced the number of GnRH‐immunoreactive cells in the preoptic area at the time of peak LH secretion during the surge. NS‐398 did not affect the GnRH immunoreactivity. Double‐labelled immunofluorescent histochemistry revealed COX‐1 immunoreactivity in the vicinity of, but not within, GnRH containing neurones in the preoptic area. COX‐2 immunoreactivity was not found in the same area. The COX‐1 immunoreactivity was almost entirely localised in microglia in the preoptic area, but not in neurones or astrocytes. These results suggest that microglia in the preoptic area containing COX‐1 are responsible for producing PG(s), which, in turn, facilitates the accumulation of GnRH during the gonadotrophin surge in female rats.