Recovery of menses after functional hypothalamic amenorrhoea: if, when and why.

BACKGROUND: Prolonged amenorrhoea occurs as a consequence of functional hypothalamic amenorrhoea (FHA) which is most often induced by weight loss, vigorous exercise or emotional stress. Unfortunately, removal of these triggers does not always result in the return of menses. The prevalence and conditions underlying the timing of return of menses vary strongly and some women report amenorrhoea several years after having achieved and maintained normal weight and/or energy balance. A better understanding of these factors would also allow improved counselling in the context of infertility. Although BMI, percentage body fat and hormonal parameters are known to be involved in the initiation of the menstrual cycle, their role in the physiology of return of menses is currently poorly understood. We summarise here the current knowledge on the epidemiology and physiology of return of menses. OBJECTIVE AND RATIONALE: The aim of this review was to provide an overview of (i) factors determining the recovery of menses and its timing, (ii) how such factors may exert their physiological effects and (iii) whether there are useful therapeutic options to induce recovery. SEARCH METHODS: We searched articles published in English, French or German language containing keywords related to return of menses after FHA published in PubMed between 1966 and February 2020. Manuscripts reporting data on either the epidemiology or the physiology of recovery of menses were included and bibliographies were reviewed for further relevant literature. The Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) criteria served to assess quality of observational studies. OUTCOMES: Few studies investigate return of menses and most of them have serious qualitative and methodological limitations. These include (i) the lack of precise definitions for FHA or resumption of menses, (ii) the use of short observation periods with unsatisfactory descriptions and (iii) the inclusion of poorly characterised small study groups. The comparison of studies is further hampered by very inhomogeneous study designs. Consequently, the exact prevalence of resumption of menses after FHA is unknown. Also, the timepoint of return of menses varies strongly and reliable prediction models are lacking. While weight, body fat and energy availability are associated with the return of menses, psychological factors also have a strong impact on the menstrual cycle and on behaviour known to increase the risk of FHA. Drug therapies with metreleptin or naltrexone might represent further opportunities to increase the chances of return of menses, but these require further evaluation. WIDER IMPLICATIONS: Although knowledge on the physiology of return of menses is presently rudimentary, the available data indicate the importance of BMI/weight (gain), energy balance and mental health. The physiological processes and genetics underlying the impact of these factors on the return of menses require further research. Larger prospective studies are necessary to identify clinical parameters for accurate prediction of return of menses as well as reliable therapeutic options.

A synthetic kisspeptin analog that triggers ovulation and advances puberty.

The neuropeptide kisspeptin and its receptor, KiSS1R, govern the reproductive timeline of mammals by triggering puberty onset and promoting ovulation by stimulating gonadotrophin-releasing hormone (GnRH) secretion. To overcome the drawback of kisspeptin short half-life we designed kisspeptin analogs combining original modifications, triazole peptidomimetic and albumin binding motif, to reduce proteolytic degradation and to slow down renal clearance, respectively. These analogs showed improved in vitro potency and dramatically enhanced pharmacodynamics. When injected intramuscularly into ewes (15 nmol/ewe) primed with a progestogen, the best analog (compound 6, C6) induced synchronized ovulations in both breeding and non-breeding seasons. Ovulations were fertile as demonstrated by the delivery of lambs at term. C6 was also fully active in both female and male mice but was completely inactive in KiSS1R KO mice. Electrophysiological recordings of GnRH neurons from brain slices of GnRH-GFP mice indicated that C6 exerted a direct excitatory action on GnRH neurons. Finally, in prepubertal female mice daily injections (0.3 nmol/mouse) for five days significantly advanced puberty. C6 ability to trigger ovulation and advance puberty demonstrates that kisspeptin analogs may find application in the management of livestock reproduction and opens new possibilities for the treatment of reproductive disorders in humans.

Effects of Prolactin and Lactation on A15 Dopamine Neurones in the Rostral Preoptic Area of Female Mice.

There are several distinct populations of dopamine neurones in the hypothalamus. Some of these, such as the A12 tuberoinfundibular dopamine neurones and the A14 periventricular dopamine neurones, are known to be regulated by the anterior pituitary hormone prolactin, whereas others, such as the A13 zona incerta dopaminergic neurones, are not. The present study aimed to investigate the role of prolactin in the regulation of a fourth population of hypothalamic dopamine neurones: the A15 dopamine population in the rostral hypothalamus. These neurones may play a role in the regulation of gonadotrophin-releasing hormone (GnRH) secretion, and we hypothesised that they might contribute to the suppression of GnRH release and infertility caused by hyperprolactinaemia. Under basal (low prolactin) conditions, only 8% of A15 dopamine neurones in the anteroventral periventricular nucleus (AVPV) of vehicle-treated dioestrous mice expressed phosphorylated signal transducer and activator of transcription 5 (pSTAT5), as labelled by immunohistochemistry. We have previously shown that this transcription factor can be used as an index of prolactin-receptor activation. Following acute prolactin administration, 35% of AVPV dopamine neurones co-expressed pSTAT5, whereas, during lactation, when endogenous prolactin levels are chronically elevated, 55% of AVPV dopamine neurones expressed pSTAT5. There was also a significant increase in dopamine turnover in the rostral hypothalamus, both in the diagonal band of Broca at the level of the organum vasculosum of the lamina terminalis and in the rostral preoptic area during lactation, with the 3,4-dihydroxyphenylacetic acid/dopamine ratio increasing from 0.28 ± 0.04 and 0.14 ± 0.01 in dioestrous mice to 0.82 ± 0.06 and 0.38 ± 0.03, respectively, in day 7 lactating mice. It is not yet known whether this change is driven by the hyperprolactinaemia of lactation, or another lactation-specific signal. These data demonstrate that the A15 dopaminergic neurones of the rostral hypothalamus are responsive to exogenous prolactin and may be regulated by endogenous prolactin during lactation.

Prolactin regulation of kisspeptin neurones in the mouse brain and its role in the lactation-induced suppression of kisspeptin expression.

Hyperprolactinaemia is a major cause of infertility in both males and females, although the mechanism by which prolactin inhibits the reproductive axis is not clear. The aim of the present study was to test the hypothesis that elevated prolactin causes suppression of kisspeptin expression in the hypothalamus, resulting in reduced release of gonadotrophin-releasing hormone (GnRH) and consequent infertility. In oestrogen-treated ovariectomised mice, chronic prolactin-treatment prevented the rise in luteinising hormone (LH) seen in vehicle-treated mice. Kiss1 mRNA was significantly suppressed in both the rostral periventricular region of the third ventricle (RP3V) and arcuate nucleus after prolactin treatment. Exogenous prolactin treatment induced phosphorylated signal transducer and activator of transcription 5 (pSTAT5) in kisspeptin neurones, and suppression of endogenous prolactin using bromocriptine reduced levels of pSTAT5 in kisspeptin neurones, suggesting that prolactin acts directly on kisspeptin neurones. By contrast, fewer than 1% of GnRH neurones expressed pSTAT5 in either dioestrous or lactating mice. As reported previously, there was significant suppression of kisspeptin mRNA and protein in the RP3V on day 7 of lactation, although not in the arcuate nucleus. Bromocriptine treatment significantly increased Kiss1 mRNA expression in the RP3V, although not to dioestrous levels. Unilateral thelectomy, aiming to eliminate sensory inputs from nipples on one side of the body, failed to alter the reduction in the number of kisspeptin neurones observed in the RP3V. These data demonstrate that chronic prolactin administration suppressed serum LH, and reduced Kiss1 mRNA levels in both the RP3V and arcuate nucleus, consistent with the hypothesis that prolactin-induced suppression of kisspeptin secretion might mediate the inhibitory effects of prolactin on GnRH secretion. During lactation, however, the suppression of Kiss1 mRNA in the RP3V was only partially reversed by the administration of bromocriptine to block elevated levels of prolactin, suggesting that, although elevated prolactin contributes to lactational anovulation, additional non-neural factors must also contribute to the lactation-induced suppression of kisspeptin neurones.

Lactational anovulation in mice results from a selective loss of kisspeptin input to GnRH neurons.

In mammals, lactation is associated with a period of infertility characterized by the loss of pulsatile secretion of GnRH and cessation of ovulatory cycles. Despite the importance of lactational infertility in determining overall fecundity of a species, the mechanisms by which the suckling stimulus suppresses GnRH secretion remain unclear. Because kisspeptin neurons are critical for fertility, the aim of this study was to test the hypothesis that reduced kisspeptin expression might mediate the lactation-induced suppression of fertility, using mouse models. In the rostral periventricular area of the third ventricle (RP3V), a progressive decrease in RP3V Kiss1 mRNA levels was observed during pregnancy culminating in a 10-fold reduction during lactation compared with diestrous controls. This was associated with approximately 60% reduction in the numbers of kisspeptin-immunoreactive neurons in the RP3V detected during lactation. Similarly, in the arcuate nucleus there was also a significant decrease in Kiss1 mRNA levels during late pregnancy and midlactation, and a notable decrease in kisspeptin fiber density during lactation. The functional characteristics of the RP3V kisspeptin input to GnRH neurons were assessed using electrophysiological approaches in an acute brain slice preparation. Although endogenous RP3V kisspeptin neurons were found to activate GnRH neurons in diestrous mice, this was never observed during lactation. This did not result from an absence of kisspeptin receptors because GnRH neurons responded normally to 100 nM exogenous kisspeptin during lactation. The kisspeptin deficit in lactating mice was selective, because GnRH neurons responded normally to RP3V gamma aminobutryic acid inputs during lactation. These data demonstrate that a selective loss of RP3V kisspeptin inputs to GnRH neurons during lactation is the likely mechanism causing lactational anovulation in the mouse.

Development of a methodology for and assessment of pulsatile luteinizing hormone secretion in juvenile and adult male mice.

Current methodology to monitor pulsatile LH release in mice is limited by inadequate assay sensitivity, resulting in the need for collection of large blood volumes. Thus, assessment of pulsatile LH secretion in mice remains highly challenging, and observations are limited to adult mice. To address this, we developed a highly sensitive ELISA for assessment of mouse LH concentrations in small fractions of whole blood. We demonstrate that this assay is capable of reliably detecting LH down to a theoretical limit of 0.117 ng/mL in a 2-µL fraction of whole blood. Using an established frequent blood collection procedure, we validated the accuracy of this method by determining the pulsatile LH secretion in early-adult (10 weeks old) C57BL6/J male mice. Data demonstrate regular pulsatile release of LH, with peaks in LH secretion rarely exceeding 3 ng/mL. Moreover, assessment of LH release in Gpr54 knockout mice demonstrates the lack of pulsatile LH release after the loss of kisspeptin-mediated pubertal maturation. We next determined age-associated changes in pulsatile LH secretion by assessment of LH secretion in prepubertal (28 days old) C57BL6/J male mice and repeated assessment in the same mice in adulthood (120 days old). Data demonstrate that the rise in total LH secretion in mice after pubertal maturation occurs along with an overall rise in the pulsatile LH secretion rate. This was coupled with a significant increase in the number of LH secretory events (number of pulses). In addition, we observed a decrease in the clearance (increased half-life) and a decrease in the regularity (approximate entropy) of LH release. This method will be of wide general utility within the field of reproductive biology.

Immunohistochemical evidence for the presence of various kisspeptin isoforms in the mammalian brain.

Kisspeptins are small peptides encoded by the Kiss1 gene that have been the focus of intense neuroendocrine research during the last decade. Kisspeptin is now considered to have important roles in the regulation of puberty onset and adult oestrogen-dependent feedback mechanisms on gonadotrophin-releasing hormone secretion. Several kisspeptin antibodies have been generated that have enabled an overall view of kisspeptin peptide distribution in the brain of many mammalian species. However, it remains that the distribution of the different kisspeptin isoforms is unclear in the mammalian brain. In the present study, we report on two new N-terminal-directed kisspeptin antibodies, one against the mouse kisspeptin-52 sequence (AC053) and one against the rat kisspeptin-52 sequence (AC067), and use them to specifically map these long isoforms in the brains of mouse and rat, respectively. Kisspeptin-52 immunoreactivity was detected in the two main kisspeptin neuronal populations of the rostral periventricular area and arcuate nucleus but not in the dorsomedial hypothahamus. A large number of fibres throughout the ventral forebrain were also labelled with these two antibodies. Finally, a comparison with the most commonly used C-terminal-directed kisspeptin antibodies further suggests the presence of shorter kisspeptin fragments in the brain with specific inter- and intracellular expression patterns.

Burst firing in gonadotrophin-releasing hormone neurones does not require ionotrophic GABA or glutamate receptor activation.

Burst firing is a feature of many neuroendocrine cell types, including the hypothalamic gonadotrophin-releasing hormone (GnRH) neurones that control fertility. The role of intrinsic and extrinsic influences in generating GnRH neurone burst firing is presently unclear. In the present study, we investigated the role of fast amino acid transmission in burst firing by examining the effects of receptor antagonists on bursting displayed by green fluorescent protein GnRH neurones in sagittal brain slices prepared from adult male mice. Blockade of AMPA and NMDA glutamate receptors with a cocktail of CNQX and AP5 was found to have no effects on burst firing in GnRH neurones. The frequency of bursts, dynamics of individual bursts, or percentage of firing clustered in bursts was not altered. Similarly, GABA(A) receptor antagonists bicuculline and picrotoxin had no effects upon burst firing in GnRH neurones. To examine the importance of both glutamate and GABA ionotrophic signalling, a cocktail including picrotoxin, CNQX and AP5 was used but, again, this was found to have no effects on GnRH neurone burst firing. To further question the impact of endogenous amino acid release on burst firing, electrical activation of anteroventral periventricular nuclei GABA/glutamate inputs to GnRH neurones was undertaken and found to have no impact on burst firing. Taken together, these observations indicate that bursting in GnRH neurones is not dependent upon acute ionotrophic GABA and glutamate signalling and suggest that extrinsic inputs to GnRH neurones acting through AMPA, NMDA and GABA(A) receptors are unlikely to be required for burst initiation in these cells.

Differential actions of prolactin on electrical activity and intracellular signal transduction in hypothalamic neurons.

In many tissues, including brain, prolactin action is predominantly mediated by the Janus kinase/signal transducer and activator of transcription (STAT) signal transduction pathway, leading to changes in gene transcription. However, prolactin can also exert rapid actions on electrical activity of hypothalamic neurons. Here, we investigate whether both responses occur in a single cell type, focusing on three specific populations known to be influenced by prolactin: GnRH neurons, tuberoinfundibular dopamine (TIDA) neurons, and neurons in the anteroventral-periventricular nucleus in female mice. We performed phosphorylated STAT5 (pSTAT5) immunohistochemistry to identify prolactin-responsive neurons after in vivo prolactin treatment. In addition, we carried out in vitro electrophysiology in slices from transgenic mice expressing green fluorescent protein driven by the GnRH or tyrosine hydroxylase promoters as well as from C57BL/6J mice to assess acute electrical responses to prolactin. Approximately 88% of TIDA neurons expressed pSTAT5 in diestrous mice, rising to 97% after prolactin treatment. All TIDA neurons also showed a rapid increase in firing rate after prolactin treatment. In contrast, very few GnRH neurons (11%) showed pSTAT5 in response to prolactin, and none showed a change in electrical activity. Finally, in the anteroventral-periventricular nucleus, most neurons (69%) responded to prolactin treatment with an increase in pSTAT5, but only 2/38 (∼5%) showed changes in electrical activity in response to prolactin. These observations show that prolactin recruits different combinations of electrical and transcriptional responses in neurons depending upon their anatomical location and phenotype. This may be critical in establishing appropriate responses to prolactin under different physiological conditions.

Gonadal steroid induction of kisspeptin peptide expression in the rostral periventricular area of the third ventricle during postnatal development in the male mouse.

Kisspeptin and its G-protein coupled receptor Gpr54 are essential for the pubertal activation of gonadotrophin-releasing hormone (GnRH) neurones, with Gpr54 mutation or deletion resulting in failed puberty and infertility in humans and mice. The number of kisspeptin-immunoreactive neurones in the rostral periventricular area of the third ventricle (RP3V) increases during pubertal development in concert with the appearance of kisspeptin appositions with GnRH neurones in the mouse rostral preoptic area. We recently demonstrated that the pubertal increase in RP3V kisspeptin neuronal number in females is dependent upon circulating oestradiol levels. The present experiments investigated the potential role of gonadal steroids in the induction of kisspeptin expression in the RP3V during pubertal development in the male mouse. Using immunocytochemistry (ICC), we show that gonadectomy of male pups at postnatal day (P) 20 resulted in a 60-70% reduction in the number of kisspeptin immunoreactive (IR) neurones within the RP3V of P45 mice (P<0.05) compared to sham-treated littermates. We established a profile of circulating testosterone levels during postnatal development in male mice and found that circulating testosterone was low throughout early postnatal development and increased from P35-40 to reach adult levels. Treatment of P20-gonadectomised male mice with 17ß-oestradiol or testosterone from P38-45 restored kisspeptin-IR neurone number in the RP3V to intact control levels (P>0.05). Using double-label ICC, we demonstrate that the majority of RP3V kisspeptin neurones express androgen receptors and oestrogen receptor α, indicating that RP3V kisspeptin neurones in the male mouse are equipped to respond to both androgen and oestrogen signals. These results indicate that, as in females, gonadal steroids are essential for the increase in kisspeptin immunoreactive cell number that occurs in the RP3V during pubertal development in the male mouse.

Roles for oestrogen receptor ß in adult brain function.

Oestradiol exerts a profound influence upon multiple brain circuits. For the most part, these effects are mediated by oestrogen receptor (ER)α. We review here the roles of ERß, the other ER isoform, in mediating rodent oestradiol-regulated anxiety, aggressive and sexual behaviours, the control of gonadotrophin secretion, and adult neurogenesis. Evidence exists for: (i) ERß located in the paraventricular nucleus underpinning the suppressive influence of oestradiol on the stress axis and anxiety-like behaviour; (ii) ERß expressed in gonadotrophin-releasing hormone neurones contributing to oestrogen negative-feedback control of gonadotrophin secretion; (iii) ERß controlling the offset of lordosis behaviour; (iv) ERß suppressing aggressive behaviour in males; (v) ERß modulating responses to social stimuli; and (vi) ERß in controlling adult neurogenesis. This review highlights two major themes; first, ERß and ERα are usually tightly inter-related in the oestradiol-dependent control of a particular brain function. For example, even though oestradiol feedback to control reproduction occurs principally through ERα-dependent mechanisms, modulatory roles for ERß also exist. Second, the roles of ERα and ERß within a particular neural network may be synergistic or antagonistic. Examples of the latter include the role of ERα to enhance, and ERß to suppress, anxiety-like and aggressive behaviours. Splice variants such as ERß2, acting as dominant negative receptors, are of further particular interest because their expression levels may reflect preceeding oestradiol exposure of relevance to oestradiol replacement therapy. Together, this review highlights the predominant modulatory, but nonetheless important, roles of ERß in mediating the many effects of oestradiol upon adult brain function.

Milestones on Steroids and the Nervous System: 10 years of basic and translational research.

During the last 10 years, the conference on 'Steroids and Nervous System' held in Torino (Italy) has been an important international point of discussion for scientists involved in this exciting and expanding research field. The present review aims to recapitulate the main topics that have been presented through the various meetings. Two broad areas have been explored: the impact of gonadal hormones on brain circuits and behaviour, as well as the mechanism of action of neuroactive steroids. Relationships among steroids, brain and behaviour, the sexual differentiation of the brain and the impact of gonadal hormones, the interactions of exogenous steroidal molecules (endocrine disrupters) with neural circuits and behaviour, and how gonadal steroids modulate the behaviour of gonadotrophin-releasing hormone neurones, have been the topics of several lectures and symposia during this series of meetings. At the same time, many contributions have been dedicated to the biosynthetic pathways, the physiopathological relevance of neurosteroids, the demonstration of the cellular localisation of different enzymes involved in neurosteroidogenesis, the mechanisms by which steroids may exert some of their effects, both the classical and nonclassical actions of different steroids, the role of neuroactive steroids on neurodegeneration, neuroprotection, and the response of the neural tissue to injury. In these 10 years, this field has significantly advanced and neuroactive steroids have emerged as new potential therapeutic tools to counteract neurodegenerative events.

Depolarising and hyperpolarising actions of GABA(A) receptor activation on gonadotrophin-releasing hormone neurones: towards an emerging consensus.

The gonadotrophin-releasing hormone (GnRH) neurones represent the final output neurones of a complex neuronal network that controls fertility. It is now appreciated that GABAergic neurones within this network provide an important regulatory influence on GnRH neurones. However, the consequences of direct GABA(A) receptor activation on adult GnRH neurones have been controversial for nearly a decade now, with both hyperpolarising and depolarising effects being reported. This review provides: (i) an overview of GABA(A) receptor function and its investigation using electrophysiological approaches and (ii) re-examines the past and present results relating to GABAergic regulation of the GnRH neurone, with a focus on mouse brain slice data. Although it remains difficult to reconcile the results of the early studies, there is a growing consensus that GABA can act through the GABA(A) receptor to exert both depolarising and hyperpolarising effects on GnRH neurones. The most recent studies examining the effects of endogenous GABA release on GnRH neurones indicate that the predominant action is that of excitation. However, we are still far from a complete understanding of the effects of GABA(A) receptor activation upon GnRH neurones. We argue that this will require not only a better understanding of chloride ion homeostasis in individual GnRH neurones, and within subcellular compartments of the GnRH neurone, but also a more integrative view of how multiple neurotransmitters, neuromodulators and intrinsic conductances act together to regulate the activity of these important cells.

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 changes in responses of hypothalamic and brainstem neuronal populations to prolactin during lactation in the mouse.

During lactation, there are numerous functional adaptations in the maternal brain. There is evidence that the high levels of circulating prolactin present during lactation might contribute to these adaptive changes. The present study aimed to investigate levels of functional prolactin-mediated signal transduction in the brain of lactating mice, using prolactin-induced phosphorylation of signal transducer and activator of transcription 5 (pSTAT5) as a marker, and compare these to the effect of exogenous prolactin during diestrus. On Day 7 of lactation, widespread induction of pSTAT5 was observed in numerous regions of the mouse forebrain and brainstem. In the medial preoptic nucleus, bed nuclei stria terminalis, paraventricular nucleus, and medial amygdala of the forebrain, and in the rostral periaqueductal gray, parabrachial nucleus, dorsal raphe, and the raphe obscurus nucleus of the brainstem, pSTAT5 expression was markedly increased during lactation compared with the response to exogenous prolactin during diestrus. In the anteroventral periventricular nucleus, arcuate nucleus, ventromedial nucleus, and dorsomedial nucleus, responses in lactation were comparable to diestrus. Conversely, in the area postrema of the brainstem, there was a reduction in response to prolactin, with a loss of pSTAT5 expression, during lactation. These differential responses following either acute or chronic elevations in prolactin were not accompanied by any changes in levels of prolactin receptor mRNA, when measured by in situ hybridization. These data are consistent with the hypothesis that prolactin might mediate widespread adaptive responses in the maternal brain.

Enhanced c-Fos expression in superior colliculus, paraventricular thalamus and septum during learning of cue-reward association.

Reward-mediated associative learning is important for recognizing the significance of environmental cues. Such learning involves convergence of multimodal sensory inputs with circuits involved in affective and memory processes. Dopamine-dependent plasticity in the striatum plays a pivotal role, but the wider circuits engaged in cue-reward association are poorly understood. To identify candidate structures that may be of particular interest for further detailed electrophysiological and functional analysis, we quantified c-Fos expression in a selection of brain structures. c-Fos is a well-known marker of cell activation with additional potential importance for synaptic plasticity. We compared c-Fos expression between animals exposed to 100 pairings of a novel conditioned stimulus with a subsequent reward, and control animals exposed to the same number of cues and rewards, but where the cues and rewards occurred at random with respect to each other. We found significant increases in c-Fos expression in the superior colliculus in the group exposed to cue-reward pairing. This is consistent with previous recordings in conscious animals, showing modulation of phasic visual responses of single collicular neurons depending on their association with reward. Further, the data also suggest the possibility that the thalamic paraventricular nucleus and septal nuclei may be selectively activated during cue-reward association learning. Little is known of the neurophysiological responses in these structures during such tasks, so the present results suggest they would be targets of interest for future single-neuron recording experiments, designed to confirm whether the neurons show learning-specific modulation.

Gonadotrophin-releasing hormone (GnRH) exerts stimulatory effects on GnRH neurons 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 mum 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 mum 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.

Distribution of kisspeptin neurones in the adult female mouse brain.

Kisspeptin-GPR54 signalling is essential for normal reproductive functioning. However, the distribution of kisspeptin neuronal cell bodies and their projections is not well established. The present study aimed to provide a detailed account of kisspeptin neuroanatomy in the mouse brain. Using a polyclonal rabbit antibody AC566, directed towards the final ten C-terminal amino acids of murine kisspeptin, three populations of kisspeptin-expressing cell bodies were identified in the adult female mouse brain. One exists as a dense periventricular continuum of cells within the rostral part of the third ventricle, another is found within the arcuate nucleus, and another is identified as a low-density group of scattered cells within the dorsomedial nucleus and posterior hypothalamus. Kisspeptin-immunoreactive fibres were abundant within the ventral aspect of the lateral septum and within the hypothalamus running in periventricular and ventral retrochiasmatic pathways. Notable exclusions from the kisspeptin fibre innervation were the suprachiasmatic and ventromedial nuclei. Outside of the hypothalamus, a small number of kisspeptin fibres were identified in the bed nucleus of the stria terminalis, subfornical organ, medial amygdala, paraventricular thalamus, periaqueductal grey and locus coerulus. All kisspeptin cell body and fibre immunoreactivity was absent in brain tissue from Kiss1 knockout mice. These observations provide a map of kisspeptin neurones in the mouse brain and indicate that a limited number of mostly medial hypothalamic and lateral septal brain regions are innervated by the three hypothalamic kisspeptin cell populations; the functions of these projections remain to be established.

Oestrogen, kisspeptin, GPR54 and the pre-ovulatory luteinising hormone surge.

Ovulation is central to mammalian fertility, yet the precise mechanism through which oestrogen triggers the gonadotrophin-releasing hormone (GnRH) surge that generates the pre-ovulatory luteinising hormone (LH) surge has remained elusive. The recent discovery that kisspeptin-GPR54 signalling is an essential regulator of the neuroendocrine axis at puberty has led investigators to evaluate the role of kisspeptin in the pre-ovulatory GnRH surge mechanism. Kisspeptin neurones are known to express oestrogen and progesterone receptors and have their cell bodies located in brain regions implicated in the positive-feedback mechanism in several mammalian species. In rodents, kisspeptin neurones located in the rostral periventricular area of the third ventricle (RP3V) are positively regulated by oestrogen and most likely are activated by oestrogen at the time of positive feedback. A similar scenario appears to exist for a sub-population of kisspeptin neurones located in the mediobasal hypothalamus of sheep and primates. The majority of GnRH neurones express GPR54, and kisspeptin causes an intense electrical activation of these cells. In concordance with this, kisspeptin administration in vivo results in an abrupt and prolonged release of LH in all mammalian species examined to date. Functional evidence from immunoneutralisation and knockout studies suggests that RP3V kisspeptin neurones projecting to GnRH neurones are an essential component of the surge mechanism in rodents. Taken together, the studies undertaken to date provide substantial evidence in support of a key role of kisspeptin-GPR54 signalling in the generation of the oestrogen-induced pre-ovulatory surge mechanism in mammals.

Genetic dissection of estrogen receptor signaling in vivo.

The multiple actions of estrogen in mammalian physiology are brought about, on a molecular level, by several signaling pathways, and mediated by at least two receptors-estrogen receptor (ER) alpha and beta. Analysis of knock-out mice devoid of either or both receptor isoforms revealed the essential function of estrogen receptor alpha in female reproduction, as ERalpha deficiency leads to a complex endocrine phenotype, severe disturbances in several reproductive organs, and infertility. This reflects the many actions of estrogen in female reproductive endocrinology. To carry the understanding of estrogen action to a cellular resolution, modern genetic technologies can be employed, including artificial chromosome-based transgenesis and conditional gene targeting. The combination of these techniques yields mouse models that lack ERalpha in specific cell types of the body. Using cell-type-specific ERalpha mutants, it could be shown that ERa in neurons is essential for the luteinizing hormone (LH) surge that triggers ovulation. Studies using ERalpha and ERbeta-selective agonists reveal that ERalpha activation is sufficient to induce an ovulatory hormonal stimulus. Thus, genetic analysis and selective pharmacological tools can complement each other in the molecular and cellular dissection of hormone receptor function in vivo.