Prenatal Testosterone Excess Decreases Neurokinin 3 Receptor Immunoreactivity within the Arcuate Nucleus KNDy Cell Population

Prenatal exposure of the female ovine foetus to excess testosterone leads to neuroendocrine disruptions in adulthood, as demonstrated by defects in responsiveness with respect to the ability of gonadal steroids to regulate gonadotrophin‐releasing hormone (GnRH) secretion. In the ewe, neurones of the arcuate nucleus (ARC), which co‐expresses kisspeptin, neurokinin B (NKB) and dynorphin (termed KNDy cells), play a key role in steroid feedback control of GnRH and show altered peptide expression after prenatal testosterone treatment. KNDy cells also co‐localise NKB receptors (NK3R), and it has been proposed that NKB may act as an autoregulatory transmitter in KNDy cells where it participates in the mechanisms underlying steroid negative‐feedback. In addition, recent evidence suggests that NKB/NK3R signalling may be involved in the positive‐feedback actions of oestradiol leading to the GnRH/luteinising hormone (LH) surge in the ewe. Thus, we hypothesise that decreased expression of NK3R in KNDy cells may be present in the brains of prenatal testosterone‐treated animals, potentially contributing to reproductive defects. Using single‐ and dual‐label immunohistochemistry we found NK3R‐positive cells in diverse areas of the hypothalamus; however, after prenatal testosterone treatment, decreased numbers of NK3R immunoreactive (‐IR) cells were seen only in the ARC. Moreover, dual‐label confocal analyses revealed a significant decrease in the percentage of KNDy cells (using kisspeptin as a marker) that co‐localised NK3R. To investigate how NKB ultimately affects GnRH secretion in the ewe, we examined GnRH neurones in the preoptic area (POA) and mediobasal hypothalamus (MBH) for the presence of NK3R. Although, consistent with earlier findings, we found no instances of NK3R co‐localisation in GnRH neurones in either the POA or MBH; in addition, > 70% GnRH neurones in both areas were contacted by NK3R‐IR presynaptic terminals suggesting that, in addition to its role at KNDy cell bodies, NKB may regulate GnRH neurones by presynaptic actions. In summary, the finding of decreased NK3R within KNDy cells in prenatal testosterone‐treated sheep complements previous observations of decreased NKB and dynorphin in the same population, and may contribute to deficits in the feedback control of GnRH/LH secretion in this animal model.     

The Distribution of Kisspeptin (Kiss)1‐ and Kiss2‐Positive Neurones and Their Connections with Gonadotrophin‐Releasing Hormone‐3 Neurones in the Zebrafish Brain

Kisspeptin is a neuroendocrine hormone with a critical role in the activation of gonadotrophin‐releasing hormone (GnRH) neurones, which is vital for the onset of puberty in mammals. However, the functions of kisspeptin neurones in non‐mammalian vertebrates are not well understood. We have used transgenics to labell kisspeptin neurones (Kiss1 and Kiss2) with mCherry in zebrafish (Danio rerio). In kiss1:mCherry transgenic zebrafish, Kiss1 cells were located in the dorsomedial and ventromedial habenula, with their nerve fibres contributing to the fasciculus retroflexus and projecting to the ventral parts of the interpeduncular and raphe nuclei. In kiss2:mCherry zebrafish, Kiss2 cells were primarily located in the dorsal zone of the periventricular hypothalamus and, to a lesser extent, in the periventricular nucleus of the posterior tuberculum and the preoptic area. Kiss2 fibres formed a wide network projecting into the telencephalon, the mesencephalon, the hypothalamus and the pituitary. To study the relationship of kisspeptin neurones and GnRH3 neurones, these fish were crossed with gnrh3:EGFP zebrafish to obtain kiss1:mCherry/gnrh3:EGFP and kiss2:mCherry/gnrh3:EGFP double transgenic zebrafish. The GnRH3 fibres ascending to the habenula were closely associated with Kiss1 fibres projecting from the ventral habenula. On the other hand, GnRH3 fibres and Kiss2 fibres were adjacent but scarcely in contact with each other in the telencephalon and the hypothalamus. The Kiss2 and GnRH3 fibres in the ventral hypothalamus projected into the pituitary via the pituitary stalk. In the pituitary, Kiss2 fibres were directly in contact with GnRH3 fibres in the pars distalis. These results reveal the pattern of kisspeptin neurones and their connections with GnRH3 neurones in the brain, suggesting distinct mechanisms for Kiss1 and Kiss2 in regulating reproductive events in zebrafish.     

Kisspeptin Neurones do not Directly Signal to RFRP‐3 Neurones but RFRP‐3 may Directly Modulate a Subset of Hypothalamic Kisspeptin Cells in Mice

The neuropeptides kisspeptin (encoded by Kiss1) and RFamide‐related peptide‐3 (also known as GnIH; encoded by Rfrp) are potent stimulators and inhibitors, respectively, of reproduction. Whether kisspeptin or RFRP‐3 might act directly on each other's neuronal populations to indirectly modulate reproductive status is unknown. To examine possible interconnectivity of the kisspeptin and RFRP‐3 systems, we performed double‐label in situ hybridisation (ISH) for the RFRP‐3 receptors, Gpr147 and Gpr74, in hypothalamic Kiss1 neurones of adult male and female mice, as well as double‐label ISH for the kisspeptin receptor, Kiss1r, in Rfrp‐expressing neurones of the hypothalamic dorsal‐medial nucleus (DMN). Only a very small proportion (5‐10%) of Kiss1 neurones of the anteroventral periventricular region expressed Gpr147 or Gpr74 in either sex, whereas higher co‐expression (approximately 25%) existed in Kiss1 neurones in the arcuate nucleus. Thus, RFRP‐3 could signal to a small, primarily arcuate, subset of Kiss1 neurones, a conclusion supported by the finding of approximately 35% of arcuate kisspeptin cells receiving RFRP‐3‐immunoreactive fibre contacts. By contrast to the former situation, no Rfrp neurones co‐expressed Kiss1r in either sex, and Tacr3, the receptor for neurokinin B (NKB; a neuropeptide co‐expressed with arcuate kisspeptin neurones) was found in <10% of Rfrp neurones. Moreover, kisspeptin‐immunoreactive fibres did not readily appose RFRP‐3 cells in either sex, further excluding the likelihood that kisspeptin neurones directly communicate to RFRP‐3 neurones. Lastly, despite abundant NKB in the DMN region where RFRP‐3 soma reside, NKB was not co‐expressed in the majority of Rfrp neurones. Our results suggest that RFRP‐3 may modulate a small proportion of kisspeptin‐producing neurones in mice, particularly in the arcuate nucleus, whereas kisspeptin neurones are unlikely to have any direct reciprocal actions on RFRP‐3 neurones.     

Recent Discoveries on the Control of Gonadotrophin‐Releasing Hormone Neurones in Nonhuman Primates

Since Ernst Knobil proposed the concept of the gonadotrophin‐releasing hormone (GnRH) pulse‐generator in the monkey hypothalamus three decades ago, we have made significant progress in this research area with cellular and molecular approaches. First, an increase in pulsatile GnRH release triggers the onset of puberty. However, the question of what triggers the pubertal increase in GnRH is still unclear. GnRH neurones are already mature before puberty but GnRH release is suppressed by a tonic GABA inhibition. Our recent work indicates that blocking endogenous GABA inhibition with the GABA_A receptor blocker, bicuculline, dramatically increases kisspeptin release, which plays an important role in the pubertal increase in GnRH release. Thus, an interplay between the GABA, kisspeptin, and GnRH neuronal systems appears to trigger puberty. Second, cultured GnRH neurones derived from the olfactory placode of monkey embryos exhibit synchronised intracellular calcium, [Ca²⁺]_i, oscillations and release GnRH in pulses at approximately 60‐min intervals after 14 days in vitro (div). During the first 14 div, GnRH neurones undergo maturational changes from no [Ca²⁺]_i oscillations and little GnRH release to the fully functional state. Recent work also shows GnRH mRNA expression increases during in vitro maturation. This mRNA increase coincides with significant demethylation of a CpG island in the GnRH 5′‐promoter region. This suggests that epigenetic differentiation occurs during GnRH neuronal maturation. Third, oestradiol causes rapid, direct, excitatory action in GnRH neurones and this action of oestradiol appears to be mediated through a membrane receptor, such as G‐protein coupled receptor 30.     

Surge‐Like Luteinising Hormone Secretion Induced by Retrochiasmatic Area NK3R Activation is Mediated Primarily by Arcuate Kisspeptin Neurones in the Ewe

The neuropeptides neurokinin B (NKB) and kisspeptin are potent stimulators of gonadotrophin‐releasing hormone (GnRH)/luteinsing hormone (LH) secretion and are essential for human fertility. We have recently demonstrated that selective activation of NKB receptors (NK3R) within the retrochiasmatic area (RCh) and the preoptic area (POA) triggers surge‐like LH secretion in ovary‐intact ewes, whereas blockade of RCh NK3R suppresses oestradiol‐induced LH surges in ovariectomised ewes. Although these data suggest that NKB signalling within these regions of the hypothalamus mediates the positive‐feedback effects of oestradiol on LH secretion, the pathway through which it stimulates GnRH/LH secretion remains unclear. We proposed that the action of NKB on RCh neurones drives the LH surge by stimulating kisspeptin‐induced GnRH secretion. To test this hypothesis, we quantified the activation of the preoptic/hypothalamic populations of kisspeptin neurones in response to POA or RCh administration of senktide by dual‐label immunohistochemical detection of kisspeptin and c‐Fos (i.e. marker of neuronal activation). We then administered the NK3R agonist, senktide, into the RCh of ewes in the follicular phase of the oestrous cycle and conducted frequent blood sampling during intracerebroventricular infusion of the kisspeptin receptor antagonist Kp‐271 or saline. Our results show that the surge‐like secretion of LH induced by RCh senktide administration coincided with a dramatic increase in c‐Fos expression within arcuate nucleus (ARC) kisspeptin neurones, and was completely blocked by Kp‐271 infusion. We substantiate these data with evidence of direct projections of RCh neurones to ARC kisspeptin neurones. Thus, NKB‐responsive neurones in the RCh act to stimulate GnRH secretion by inducing kisspeptin release from KNDy neurones.     

Chronic Oestradiol Reduces the Dendritic Spine Density of KNDy (Kisspeptin/Neurokinin B/Dynorphin) Neurones in the Arcuate Nucleus of Ovariectomised Tac2‐Enhanced Green Fluorescent Protein Transgenic Mice

Neurones in the arcuate nucleus that express neurokinin B (NKB), kisspeptin and dynorphin (KNDy) play an important role in the reproductive axis. Oestradiol modulates the gene expression and somatic size of these neurones, although there is limited information available about whether their dendritic structure, a correlate of cellular plasticity, is altered by oestrogens. In the present study, we investigated the morphology of KNDy neurones by filling fluorescent neurones in the arcuate nucleus of Tac2‐enhanced green fluorescent protein (EGFP) transgenic mice with biocytin. Filled neurones from ovariectomised (OVX) or OVX plus 17β‐oestradiol (E₂)‐treated mice were visualised with anti‐biotin immunohistochemistry and reconstructed in three dimensions with computer‐assisted microscopy. KNDy neurones exhibited two primary dendrites, each with a few branches confined to the arcuate nucleus. Quantitative analysis revealed that E₂ treatment of OVX mice decreased the cell size and dendritic spine density of KNDy neurones. The axons of KNDy neurones originated from the cell body or proximal dendrite and gave rise to local branches that appeared to terminate within the arcuate nucleus. Numerous terminal boutons were also visualised within the ependymal layer of the third ventricle adjacent to the arcuate nucleus. Axonal branches also projected to the adjacent median eminence and exited the arcuate nucleus. Confocal microscopy revealed close apposition of EGFP and gonadotrophin‐releasing hormone‐immunoreactive fibres within the median eminence and confirmed the presence of KNDy axon terminals in the ependymal layer of the third ventricle. The axonal branching pattern of KNDy neurones suggests that a single KNDy neurone could influence multiple arcuate neurones, tanycytes in the wall of the third ventricle, axon terminals in the median eminence and numerous areas outside of the arcuate nucleus. In parallel with its inhibitory effects on electrical excitability, E₂ treatment of OVX Tac2‐EGFP mice induces structural changes in the somata and dendrites of KNDy neurones.     

Gonadal and Nongonadal Regulation of Sex Differences in Hypothalamic Kiss1 Neurones

The brains of males and females differ anatomically and physiologically, including sex differences in neurone size or number, synapse morphology and specific patterns of gene expression. Brain sex differences may underlie critical sex differences in physiology or behaviour, including several aspects of reproduction, such as the timing of sexual maturation (earlier in females than males) and the ability to generate a preovulatory gonadotrophin surge (in females only). The reproductive axis is controlled by afferent pathways that converge upon forebrain gonadotrophin‐releasing hormone (GnRH) neurones, but GnRH neurones are not sexually dimorphic. Although most reproductive sex differences probably reflect sex differences in the upstream circuits and factors that regulate GnRH secretion, the key sexually‐dimorphic factors that influence reproductive status have remained poorly defined. The recently‐identified neuropeptide kisspeptin, encoded by the Kiss1 gene, is an important regulator of GnRH secretion, and Kiss1 neurones in rodents are sexually dimorphic in specific hypothalamic populations, including the anteroventral periventricular nucleus–periventricular nucleus continuum (AVPV/PeN) and the arcuate nucleus (ARC). In the adult AVPV/PeN, Kiss1 neurones are more abundant in females than males, representing a sex difference that is regulated by oestradiol signalling during critical periods of postnatal and pubertal development. By contrast, Kiss1 neurones in the ARC are not sexually differentiated in adult rodents but, in mice, the regulation of ARC Kiss1 cells by gonadal hormone‐independent factors is sexually dimorphic during prepubertal development. These various sex differences in hypothalamic Kiss1 neurones may relate to known sex differences in reproductive physiology, such as puberty onset and positive feedback.     

Characterisation of Kiss1r (Gpr54)‐Expressing Neurones in the Arcuate Nucleus of the Female Rat Hypothalamus

Kisspeptin is essential in reproduction and acts by stimulating neurones expressing gonadotrophin‐releasing hormone (GnRH). Recent studies suggest that kisspeptin has multiple roles in the modulation of neuronal circuits in systems outside the hypothalamic‐pituitary‐gonadal axis. Our recent research using in situ hybridisation (ISH) clarified the histological distribution of Kiss1r (Gpr54)‐expressing neurones in the rat brain that were presumed to be putative targets of kisspeptin. The arcuate nucleus (ARN) of the hypothalamus is one of the brain regions in which Kiss1r expression in non‐GnRH neurones is prominent. However, the characteristics of Kiss1r‐expressing neurones in the ARN remain unclear. The present study aimed to determine the neurochemical characteristics of Kiss1r‐expressing neurones in the ARN using ISH and immunofluorescence. We revealed that the majority (approximately 63%) of Kiss1r‐expressing neurones in the ARN were pro‐opiomelanocortin (POMC) neurones, which have an anorexic effect in mammals. Additionally, a few Kiss1r‐expressing neurones in the dorsal ARN are tuberoinfundibular dopamine (TIDA) neurones, which control milk production by inhibiting prolactin secretion from the anterior pituitary. TIDA neurones showed a relatively weak Kiss1r ISH signal compared to POMC neurones, as well as low co‐expression of Kiss1r (approximately 15%). We also examined the expression of Kiss1r in neuropeptide Y and kisspeptin neurones, which are reported to arise from POMC‐expressing progenitor cells during development. However, the vast majority of neuropeptide Y and kisspeptin neurones in the ARN did not express Kiss1r. These results suggest that kisspeptin may directly regulate energy homeostasis and milk production by modulating the activity of POMC and TIDA neurones, respectively. Our results provide an insight into the wide variety of roles that kisspeptin plays in homeostatic and neuroendocrine functions.     

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.     

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.     

Photoperiodic Co‐Regulation of Kisspeptin,Neurokinin B and Dynorphin in the Hypothalamus of a Seasonal Rodent

In many species, sexual activity varies on a seasonal basis. Kisspeptin (Kp), a hypothalamic neuropeptide acting as a strong activator of gonadotrophin‐releasing hormone neurones, plays a critical role in this adaptive process. Recent studies report that two other neuropeptides, namely neurokinin B (NKB) and dynorphin (DYN), are co‐expressed with Kp (and therefore termed KNDy neurones) in the arcuate nucleus and that these peptides are also considered to influence GnRH secretion. The present study aimed to establish whether hypothalamic NKB and DYN expression is photoperiod‐dependent in a seasonal rodent, the Syrian hamster, which exhibits robust seasonal rhythms in reproductive activity. The majority of Kp neurones in the arcuate nucleus co‐express NKB and DYN and the expression of all three peptides is decreased under a short (compared to long) photoperiod, leading to a 60% decrease in the number of KNDy neurones under photo‐inhibitory conditions. In seasonal rodents, RFamide‐related peptide (RFRP) neurones of the dorsomedial hypothalamus are also critical for seasonal reproduction. Interestingly, NKB and DYN are also expressed in the dorsomedial hypothalamus but do not co‐localise with RFRP‐immunoreactive neurones, and the expression of both NKB and DYN is higher under a short photoperiod, which is opposite to the short‐day inhibition of RFRP expression. In conclusion, the present study shows that NKB and DYN display different photoperiodic variations in the Syrian hamster hypothalamus. In the arcuate nucleus, NKB and DYN, together with Kp, are down‐regulated under a short photoperiod, whereas, in the dorsomedial hypothalamus, NKB and DYN are up‐regulated under a short photoperiod.     

Characterisation of arcuate nucleus kisspeptin/neurokinin B neuronal projections and regulation during lactation in the rat

Lactation results in negative energy balance in the rat leading to decreased gonadotrophin-releasing hormone (GnRH) release and anoestrus. Inhibited GnRH release may be a result of decreased stimulatory tone from neuropeptides critical for GnRH neuronal activity, such as kisspeptin (Kiss1) and neurokinin B (NKB). The present study aimed to identify neuronal projections from the colocalised population of Kiss1/NKB cells in the arcuate nucleus (ARH) using double-label immunohistochemistry to determine where this population may directly regulate GnRH neuronal activity. Additionally, the present study further examined lactation-induced changes in the Kiss1 system that could play a role in decreased GnRH release. The colocalised ARH Kiss1/NKB fibres projected primarily to the internal zone of the median eminence (ME) where they were in close proximity to GnRH fibres; however, few Kiss1/NKB fibres from the ARH were seen at the level of GnRH neurones in the preoptic area (POA). Arcuate Kiss1/NKB peptide levels were decreased during lactation consistent with previous mRNA data. Surprisingly, anteroventral periventricular (AVPV) Kiss1 peptide levels were increased, whereas Kiss1 mRNA levels were decreased during lactation, suggesting active inhibition of peptide release. These findings indicate ARH Kiss1/NKB and AVPV Kiss1 appear to be inhibited during lactation, which may contribute to decreased GnRH release and subsequent reproductive dysfunction. Furthermore, the absence of a strong ARH Kiss1/NKB projection to the POA suggests regulation of GnRH by this population occurs primarily at the ME level via local projections.     

The Effects of Neurokinin B upon Gonadotrophin Release in Male Rodents

Growing evidence suggests the tachykinin neurokinin B (NKB) may modulate gonadotrophin secretion and play a role in sex‐steroid feedback within the reproductive axis. NKB signalling has recently been identified as being necessary for normal human reproductive function, although the precise mechanisms underpinning this role remain to be established. We have used rodents to explore further the role of NKB within the reproductive axis. In particular, we have studied its interactions with kisspeptin, a neuropeptide essential for reproductive function in rodent and human with close anatomical links to NKB within the hypothalamus. Intraperitoneal administration of NKB (50 nmol) to male mice had no effect on circulating luteinsing hormone (LH) levels and, although i.p. kisspeptin (15 nmol) increased LH five‐fold, co‐administration of NKB and kisspeptin was indistinguishable from kisspeptin alone. Intracerebroventricular administration of NKB (10 nmol) to male mice also had no effect on LH levels, with 1 nmol kisspeptin i.c.v. significantly increasing LH compared to control (0.37 ± 0.18 versus 5.11 ± 0.28 ng/ml, respectively). Interestingly, i.c.v. co‐administration of NKB and kisspeptin caused a significant increase in LH concentrations compared to kisspeptin alone (8.96 ± 1.82 versus 5.11 ± 0.28 ng/ml respectively). We used hypothalamic explants from rats to assess the effect of NKB on gonadotrpohin‐releasing hormone (GnRH) secretion ex vivo. Doses of NKB up to 1000 nm failed to stimulate GnRH secretion, whereas 100 nm kisspeptin robustly increased GnRH secretion. Of note, co‐administration of NKB with kisspeptin abrogated the effect of kisspeptin, producing no GnRH release above basal state. Finally, we analysed the expression of Tac2/Tacr3 (genes encoding NKB and NK3R, respectively) within the arcuate nucleus in different nutritional states. After a 48‐h fast, the expression of both Tac2 and Tacr3 showed a significant increase, in contrast to levels of Kiss1 and Kiss1r mRNA, which remained unchanged. In male rodent models, NKB and kisspeptin have different effects upon gonadotrophin release and appear to interact in a complex manner.     

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.     

Electrophysiological Characteristics of Gonadotrophin‐Releasing Hormone 1–3 Neurones: Insights From a Study of Fish Brains

The vertebrate gonadotrophin‐releasing hormone (GnRH) neurones are considered to consist of one group of hypothalamic neuroendocrine and two groups of extrahypothalamic neuromodulatory GnRH neurones, and each group of neurones expresses different molecular species of GnRH peptide. Different GnRH peptides are produced by one of the three paralogous GnRH genes, gnrh1, gnrh2 and gnrh3, which are considered to have originated from gene duplications. All three GnRH systems are well developed in teleost brains. By taking advantage of this, and especially the use of GnRH‐green fluoresecent protein transgenic fish, the anatomical and electrophysiological properties of all three types of GnRH neurones can now be studied. The hypophysiotropic GnRH1 neurones in the preoptic area show episodic spontaneous electrical activities, whereas the extrahypothalamic GnRH2 neurones in the midbrain and GnRH3 neurones in the terminal nerve show regular intrinsic pacemaker activities. It is suggested that these different electrophysiological properties are related to their different functions (i.e. GnRH1 neurones act as hypophysiotropic neuroendocrine regulators and GnRH2 and GnRH3 neurones act as neuromodulators). The present review focuses on recent electrophysiological analyses of GnRH3 neurones, which have revealed the excitatory GABAergic and the inhibitory FMRFamide‐like peptidergic regulations acting upon them, as well as gap junctional electrotonic coupling.     

Deletion of Stim1 in Hypothalamic Arcuate Nucleus Kiss1 Neurons Potentiates Synchronous GCaMP Activity and Protects against Diet-Induced Obesity

Kisspeptin (Kiss1) neurons are essential for reproduction, but their role in the control of energy balance and other homeostatic functions remains unclear. High-frequency firing of hypothalamic arcuate Kiss1 (Kiss1^ARH) neurons releases kisspeptin into the median eminence, and neurokinin B (NKB) and dynorphin onto neighboring Kiss1^ARH neurons to generate a slow EPSP mediated by TRPC5 channels that entrains intermittent, synchronous firing of Kiss1^ARH neurons. High-frequency optogenetic stimulation of Kiss1^ARH neurons also releases glutamate to excite the anorexigenic proopiomelanocortin (POMC) neurons and inhibit the orexigenic neuropeptide Y/agouti-related peptide (AgRP) neurons via metabotropic glutamate receptors. At the molecular level, the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1) is critically involved in the regulation of neuronal Ca²⁺ signaling and neuronal excitability through its interaction with plasma membrane (PM) calcium (e.g., TRPC) channels. Therefore, we hypothesized that deletion of Stim1 in Kiss1^ARH neurons would increase neuronal excitability and their synchronous firing, which ultimately would affect energy homeostasis. Using optogenetics in combination with whole-cell recording and GCaMP6 imaging in slices, we discovered that deletion of Stim1 in Kiss1 neurons significantly increased the amplitude and duration of the slow EPSP and augmented synchronous [Ca²⁺]i oscillations in Kiss1^ARH neurons. Deletion of Stim1 in Kiss1^ARH neurons amplified the actions of NKB and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD). Therefore, STIM1 appears to play a critical role in regulating synchronous firing of Kiss1^ARH neurons, which ultimately affects the coordination between energy homeostasis and reproduction.SIGNIFICANCE STATEMENT Hypothalamic arcuate kisspeptin (Kiss1^ARH) neurons are essential for stimulating the pulsatile release of gonadotropin-releasing hormone (GnRH) and maintaining fertility. However, Kiss1^ARH neurons appear to be a key player in coordinating energy balance with reproduction. The regulation of calcium channels and hence calcium signaling is critically dependent on the endoplasmic reticulum (ER) calcium-sensing protein stromal interaction molecule 1 (STIM1), which interacts with the plasma membrane (PM) calcium channels. We have conditionally deleted Stim1 in Kiss1^ARH neurons and found that it significantly increased the excitability of Kiss1^ARH neurons and protected ovariectomized female mice from developing obesity and glucose intolerance with high-fat dieting (HFD).     

Roles for Primary Cilia in Gonadotrophin‐Releasing Hormone Neurones in the Mouse

During embryonic development, gonadotrophin‐releasing hormone (GnRH) neurones make an extraordinary migration out of the nose and into the brain where, in adulthood, they drive the pituitary regulation of gonadal function and fertility. Primary cilia are antennae‐like, immotile organelles that project from the surface of nearly all cells, including GnRH neurones. Links between defects in primary cilia and a variety of human pathologies have been discovered that suggest a role for primary cilia in embryogenesis and reproductive function. The present study aimed to investigate whether GnRH neurone primary cilia are critical for their embryonic migration and the adult control of fertility. To achieve this, we used a Cre‐loxP strategy to selectively disrupt primary cilia by deleting Kif3a, an intraflagellar transport protein family member essential for primary cilia assembly and function, specifically in GnRH neurones. Confocal analysis revealed that, in Kif3a^fl/fl (WT‐Kif3a) controls, all GnRH neurones possessed primary cilia, whereas, in GnRH‐Cre^+/?;Kif3a^fl/fl (GnRH‐Kif3aKO) mice, 60% of GnRH neurones lacked any evidence of primary cilia and the remaining 40% possessed only stunted primary cilia (< 2 μm). Despite abolishing normal primary cilia assembly in GnRH neurones from embryogenesis, adult GnRH neurone distribution and reproductive function was remarkably normal. The total number of GnRH neurones was the same in GnRH‐Kif3aKO and WT‐Kif3a controls; however, a significant increase (25%) was identified in the number of GnRH neurones sampled through the midpoint of the rostral pre‐optic area in GnRH‐Kif3aKO mice (P < 0.05). The time to vaginal opening was not different in GnRH‐Kif3aKO mice, although they displayed significantly advanced first oestrus (P < 0.05), and oestrous cycle length was increased (P < 0.05). However, females displayed normal basal levels of luteinising hormone, responded normally to oestrogen‐induced negative‐ and positive‐feedback, and displayed normal fecundity. Taken together, these data suggest that primary cilia and associated signal transduction pathways play a role in the topographical distribution and specific functions of GnRH neurones; however, they are not essential for fertility.     

Redefining the Gonadotrophin‐Releasing Hormone Neurone Dendrite

Gonadotrophin‐releasing hormone (GnRH) neurones are the final output neurones of the complex synaptic network responsible for the central control of fertility. This scattered population of neurones has been shown to have remarkably long dendritic processes by cell‐filling of GnRH neurones in situ with low‐molecular weight dyes. This review focuses on how the functional significance of these long dendritic extensions is being explored through dual somatic–dendritic electrophysiological recordings, computational modelling, immunolabelling for specific channels and multiple modes of microscopy and imaging. Remarkably, recent work has discovered that GnRH neurone dendrites not only actively propagate action potentials, but also comprise the primary site of action potential initiation. These findings, along with the discovery of regionalised expression of active conductances, highlight dendrites of single GnRH neurones as being central sites of signal integration. Moreover, imaging studies have shown that the long dendrites of GnRH neurones intertwine and bundle with one another. The presence of shared synaptic input to bundling dendrites, coupled with their active properties and the increased potency of distally placed synaptic inputs, is suggestive of a novel mechanism of GnRH neurone synchronisation, a feature critical for mammalian reproduction. Together, these discoveries of the GnRH neurone dendrite structure and function are changing the way that we view the central regulation of fertility.