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.     

RhoA in tyrosine hydroxylase neurones regulates food intake and body weight via altered sensitivity to peripheral hormones

Dopamine‐producing tyrosine hydroxylase (TH) neurones in the hypothalamic arcuate nucleus (ARC) have recently been shown to be involved in ghrelin signalling and body weight homeostasis. In the present study, we investigate the role of the intracellular regulator RhoA in hypothalamic TH neurones in response to peripheral hormones. Diet‐induced obesity was found to be associated with increased phosphorylation of TH in ARC, indicating obesity‐associated increased activity of ARC TH neurones. Mice in which RhoA was specifically knocked out in TH neurones (TH‐RhoA^?/? mice) were more sensitive to the orexigenic effect of peripherally administered ghrelin and displayed an abolished response to the anorexigenic hormone leptin. When TH‐RhoA^?/? mice were challenged with a high‐fat high‐sucrose (HFHS) diet, they became hyperphagic and gained more body weight and fat mass compared to wild‐type control mice. Importantly, lack of RhoA prevented development of ghrelin resistance, which is normally observed in wild‐type mice after long‐term HFHS diet feeding. Patch‐clamp electrophysiological analysis demonstrated increased ghrelin‐induced excitability of TH neurones in lean TH‐RhoA^?/? mice compared to lean littermate control animals. Additionally, increased expression of the orexigenic hypothalamic neuropeptides agouti‐related peptide and neuropeptide Y was observed in TH‐RhoA^?/? mice. Overall, our data indicate that TH neurones in ARC are important for the regulation of body weight homeostasis and that RhoA is both a central effector in these neurones and important for the development of obesity‐induced ghrelin resistance. The obese phenotype of TH‐RhoA^?/? mice may be a result of increased sensitivity to ghrelin and decreased sensitivity to leptin, resulting in increased food intake.     

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.     

Microinjection of galanin-like peptide into the medial preoptic area stimulates food intake in adult male rats

Galanin-like peptide (GALP) is a neuropeptide implicated in the regulation of feeding behaviour, metabolism and reproduction. GALP is an endogenous ligand of the galanin receptors, which are widely expressed in the hypothalamus. GALP is predominantly expressed in arcuate nucleus (ARC) neurones, which project to the paraventricular nucleus (PVN) and medial preoptic area (mPOA). Intracerebroventricular or intraparaventricular (iPVN) injection of GALP acutely increases food intake in rats. The effect of GALP injection into the mPOA on feeding behaviour has not previously been studied. In the present study, intra-mPOA (imPOA) injection of GALP potently increased 0-1-h food intake in rats. The dose-response effect of imPOA GALP administration on food intake was similar to that previously observed following iPVN administration. The effects of GALP (1 nmol) or galanin (1 nmol) on food intake were then compared following injection into the PVN, mPOA, ARC, dorsal medial nucleus (DMN), lateral hypothalamus and rostral preoptic area (rPOA). GALP (1 nmol) increased food intake to a similar degree when injected into the imPOA or iPVN, but produced no significant effect when injected into the ARC, DMN, lateral hypothalamus or rPOA. Similarly, galanin (1 nmol) significantly increased food intake following injection imPOA and iPVN. However, the effect was significantly smaller than that following administration of GALP (1 nmol). Galanin also had no significant effect on food intake when administered into the ARC, DMN, lateral hypothalamus and rPOA. These data suggest that the mPOA and the PVN may have specific roles in mediating the orexigenic effect of GALP and galanin.     

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.     

Differential sensitivity of specific neuronal populations of the rat hypothalamus to prolactin action

Prolactin stimulates dopamine release from neuroendocrine dopaminergic (NEDA) neurons in the hypothalamic arcuate nucleus (ARC) to maintain low levels of serum prolactin. Elevated prolactin levels during pregnancy and lactation may mediate actions in other hypothalamic regions such as the paraventricular nucleus (PVN) and rostral preoptic area (rPOA). We predicted that NEDA neurons would be more sensitive prolactin targets than neurons in other regions because they are required to regulate basal prolactin secretion. Moreover, differences in the accessibility of the ARC to prolactin in blood may influence the responsiveness of this population. Therefore, we compared prolactin-induced signaling in different hypothalamic neuronal populations following either systemic or intracerebroventricular (icv) prolactin administration. Phosphorylation of the signal transduction factor, STAT5 (pSTAT5), was used to identify prolactin-responsive neurons. In response to systemic prolactin, pSTAT5-labeled cells were widely observed in the ARC but absent from the rPOA and PVN. Many of these responsive cells in the ARC were identified as NEDA neurons. The lowest icv prolactin dose (10 ng) induced pSTAT5 in the ARC, but with higher doses (>500 ng) pSTAT5 was detected in numerous regions, including the rPOA and PVN. NEDA neurons were maximally labeled with nuclear pSTAT5 in response to 500 ng prolactin and appeared to be more sensitive than dopaminergic neurons in the rPOA. Subpopulations of oxytocin neurons in the hypothalamus were also found to be differentially sensitive to prolactin. These data suggest that differences in the accessibility of the arcuate nucleus to prolactin, together with intrinsic differences in the NEDA neurons, may facilitate homeostatic feedback regulation of prolactin release.     

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.     

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.     

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.     

Neuroanatomical connections between kisspeptin neurones and somatostatin neurones in the female and male rat hypothalamus: A possible involvement of SSTR1 in kisspeptin release

Somatostatin (SST), a neuropeptide involved in the central modulation of several physiological functions, is co‐distributed in the same hypothalamic areas as kisspeptin (KP), the most potent secretagogue of the gonadotrophin‐releasing hormone (GnRH) secretion known to date. Because SST infused i.c.v. evoked a potent inhibition of GnRH release, we explored the neuroanatomical relationships between KP and SST populations in male and female rats. Accordingly, intact males and ovariectomised oestradiol‐replaced females were killed and their brains processed aiming to simultaneously detect KP, SST and synapsin, a marker for synapses. We observed numerous appositions of KP on SST neurones both in the female and male arcuate nucleus (ARC) and ventromedial hypothalamus. A large association between SST terminals and KP neurones at the level of the pre‐optic area was also observed in female rats and in a more limited frame in males. Finally, most KP neurones from the ARC showed SST appositions in both sexes. To determine whether SST could affect KP cell activity, we assessed whether SST receptors (SSTR) were present on KP neurones in the ARC. We also looked for the presence of SSTR1 and SSTR2A in the brain of male rats. Brains were processed using a sequential double immunocytochemistry aiming to detect KP and SSTR1 or KP and SSTR2A. We observed overlapping distributions of immunoreactive neurones for SSTR1 and KP and counted approximately one‐third of KP neurones with SSTR1. By contrast, neurones labelled for SSTR2A or KP were often juxtaposed in the ARC and the occurrence of double‐labelled neurones was sporadic (< 5%). These results suggest that the action of SST on KP neurones would pass mainly through SSTR1 at the level of the ARC.     

Neurokinin 3 Receptor Immunoreactivity in the Septal Region, Preoptic Area and Hypothalamus of the Female Sheep: Colocalisation in Neurokinin B Cells of the Arcuate Nucleus but not in Gonadotrophin-Releasing Hormone Neurones

Recent evidence has implicated neurokinin B (NKB) in the complex neuronal network mediating the effects of gonadal steroids on the regulation of gonadotrophin-releasing hormone (GnRH) secretion. Because the neurokinin 3 receptor (NK3R) is considered to mediate the effects of NKB at the cellular level, we determined the distribution of immunoreactive NK3R in the septal region, preoptic area (POA) and hypothalamus of the ewe. NK3R cells and/or fibres were found in areas including the bed nucleus of the stria terminalis, POA, anterior hypothalamic and perifornical areas, dopaminergic A15 region, dorsomedial and lateral hypothalamus, arcuate nucleus (ARC) and the ventral premammillary nucleus. We also used dual-label immunocytochemistry to determine whether a neuroanatomical basis for direct modulation of GnRH neurones by NKB was evident. No GnRH neurones at any rostral-caudal level were observed to contain NK3R immunoreactivity, although GnRH neurones and fibres were in proximity to NK3R-containing fibres. Because NKB fibres formed close contacts with NKB neurones in the ARC, we determined whether these NKB neurones also contained immunoreactive NK3R. In luteal-phase ewes, 64% ± 11 of NKB neurones colocalised NK3R. In summary, NK3R is distributed in areas of the sheep POA and hypothalamus known to be involved in the control of reproductive neuroendocrine function. Colocalisation of NK3R in NKB neurones of the ARC suggests a potential mechanism for the autoregulation of this subpopulation; however, the lack of NK3R in GnRH neurones suggests that the actions of NKB on GnRH neurosecretory activity in the ewe are mediated indirectly via other neurones and/or neuropeptides.     

Acute Inhibition of Central c‐Jun N‐terminal Kinase Restores Hypothalamic Insulin Signalling and Alleviates Glucose Intolerance in Diabetic Mice

The hypothalamus has been identified as a main insulin target tissue for regulating normal body weight and glucose metabolism. Recent observations suggest that c‐Jun‐N‐terminal kinase (JNK)‐signalling plays a crucial role in the development of obesity and insulin resistance because neuronal JNK‐1 ablation in the mouse prevented high‐fat diet‐induced obesity (DIO) and increased energy expenditure, as well as insulin sensitivity. In the present study, we investigated whether central JNK inhibition is associated with sensitisation of hypothalamic insulin signalling in mice fed a high‐fat diet for 3 weeks and in leptin‐deficient mice. We determined whether i.c.v. injection of a pharmacological JNK‐inhibitor (SP600125) improved impaired glucose homeostasis. By immunohistochemistry, we first observed that JNK activity was increased in the arcuate nucleus (ARC) and the ventromedial hypothalamus (VMH) in both mouse models, relative to normoglycaemic controls. This suggests that up‐regulation of JNK in these regions is associated with glucose intolerance and obesity, independent of leptin levels. Acute i.c.v. injection of SP600125 ameliorated glucose tolerance within 30 min in both leptin‐deficient and DIO mice. Given the acute nature of i.c.v. injections, these effects cannot be attributed to changes in food intake or energy balance. In a hypothalamic cell line, and in the ARC and VMH of leptin‐deficient mice, JNK inhibition by SP600125 consistently improved impaired insulin signalling. This was determined by a reduction of phospho‐insulin receptor substrate‐1 [IRS‐1(Ser612)] protein in a hypothalamic cell line and a decline in the number of pIRS‐1(Ser612) immunoreactive cells in the ARC and VMH. Serine 612 phosphorylation of IRS‐1 is assumed to negatively regulate insulin signalling. In leptin‐deficient mice, in both nuclei, central inhibition of JNK increased the number of cells immunoreactive for phospho‐Akt (Ser473) and phospho‐GSK‐3β (Ser9), which are important markers of insulin signalling. Collectively, our data suggest that the acute inhibition of central JNK improves impaired glucose homeostasis and is associated with sensitisation of hypothalamic insulin signalling.     

Activation of Nesfatin‐1‐Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin‐1 in Rats

Peripheral anorectic hormones, such as glucagon‐like peptide (GLP)‐1, cholecystokinin (CCK)‐8 and leptin, suppress food intake. The newly‐identified anorectic neuropeptide, nesfatin‐1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP‐1 and CCK‐8 and co‐administrations of GLP‐1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin‐1‐immunoreactive (‐IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP‐1 (100 μg/kg) caused significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK‐8 (50 μg/kg) resulted in marked increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP‐1 (33 μg/kg) or leptin. However, co‐administration of GLP‐1 (33 μg/kg) and leptin resulted in significant increases in the number of nesfatin‐1‐IR neurones expressing Fos‐immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP‐1, CCK‐8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin‐1. These results indicate that nesfatin‐1‐expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP‐1 and leptin in addition to CCK‐8, and also suppress food intake in rats.     

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.     

Differential Expression of RFamide‐Related Peptide,a Mammalian Gonadotrophin‐Inhibitory Hormone Orthologue,and Kisspeptin in the Hypothalamus of Abadeh Ecotype Does During Breeding and Anoestrous Seasons

Gonadotrophin‐inhibitory hormone (GnIH) is a novel hypothalamic neuropeptide that was discovered in birds as an inhibitory factor for gonadotrophin release. RFamide‐related peptide (RFRP) is a mammalian GnIH orthologue that inhibits gonadotrophin synthesis and release in mammals through actions on gonadotrophin‐releasing hormone (GnRH) neurones and gonadotrophs, mediated via the GnIH receptor (GnIH‐R), GPR147. On the other hand, hypothalamic kisspeptin provokes the release of GnRH from the hypothalamus. The present study aimed to compare the expression of RFRP in the dorsomedial hypothalamus and paraventricular nucleus (DMH/PVN) and that of kisspeptin in the arcuate nucleus (ARC) of the female goat hypothalamus during anoestrous and breeding seasons. Mature female Abadeh does were used during anoestrus, as well as the follicular and luteal phases of the cycle. The number of RFRP‐immunoreactive (‐IR) neurones in the follicular phase was lower than in the luteal and anoestrous stages. Irrespective of the ovarian stage, the number of RFRP‐IR neurones in the rostral and middle regions of the DMH/PVN was higher than in the caudal region. By contrast, the number of kisspeptin‐IR neurones in the follicular stage was greater than in the luteal stage and during the anoestrous stage. Irrespective of the stage of the ovarian cycle, the number of kisspeptin‐IR neurones in the caudal region of the ARC was greater than in the middle and rostral regions. In conclusion, RFRP‐IR cells were more abundant in the rostral region of the DMH/PVN nuclei of the hypothalamus, with a greater number being found during the luteal and anoestrous stages compared to the follicular stage. On the other hand, kisspeptin‐IR neurones were more abundant in the caudal part of the ARC, with a greater number recorded in the follicular stage compared to the luteal and anoestrous stages.