Journal of Neuroendocrinology

BackgroundKisspeptinergic signaling is well-established as crucial for regulation of reproduction, but its potential broader role in brain function is less understood. This study investigates the distribution and chemotyping of kisspeptin-expressing neurons within the mouse brain. MethodsRNAscope singleplex, duplex and multiplex in situ hybridization methods were used to assess kisspeptin mRNA (Kiss1) expression and its co-expression with other neuropeptides, excitatory and inhibitory neurotransmitter markers, and sex steroid receptors in intact and gonadectomized young adult mice. ResultsSeven distinct kisspeptin neuronal chemotypes were characterized, including within two novel Kiss1-expressing groups described here for the first time: the ventral premammillary nucleus, and the nucleus of the solitary tract. Kiss1 mRNA was also localized in the soma, and within the dendritic compartment, of hypothalamic neurons. Altered Kiss1 expression following gonadectomy suggests a previously unappreciated role for androgen receptors in regulating kisspeptin signaling. ConclusionThis study provides a detailed chemoanatomical map of kisspeptin-expressing neurons in the brain, highlighting their potential functional diversity. The discovery of new kisspeptin-expressing neuronal populations, and gonadectomy-induced changes in Kiss1 expression patterns, provide a basis for further exploration of non-endocrine roles for kisspeptin in brain function.

Study objectivesParadoxical sleep (PS) is a state involving numerous hypothalamic neuronal subpopulations, many remaining neurochemically uncharacterized. Our goal was to compare hypothalamic neurons active during Wakefulness or PS rebound (PSR) and explore their potential overlap, with a focus on melanin-concentrating-hormone (MCH), Orexin (Orx), Lhx6 and a new contingent of Meis2- expressing neurons. MethodIn the same TRAP2-red mouse, neurons activated during Wakefulness (4h) and PSR (2h) express TdTomato and c-Fos, respectively. Double-labelling and triple immunofluorescence with neurochemical markers were performed to characterize and quantify cell populations in hypothalamic structures. ResultsTwelve hypothalamic structures showed distinct activation patterns. The anterior hypothalamic area (AHA), zona incerta (ZI) and tuberal nucleus contained more activated neurons during PSR than Wakefulness, whereas the paraventricular hypothalamic (PVN) and supraoptic (SO) nuclei were predominantly activated during Wakefulness. MCH and Lhx6 neurons were mainly recruited during PSR, whereas Orx neurons were activated during both. A ventral subpopulation of MCH neurons showed higher activation during PSR than the dorsal subpopulation. Additionally, [~]30% of the c-Fos+ neurons in ZI and AHA express Meis2. A similar proportion of TdTomato+ neurons positive for Meis2 were encountered in PVN and SO. Overall, [~]20% of all hypothalamic neurons activated during PSR are now neurochemically identified. ConclusionOur study identifies new neuronal populations activated during PSR in AHA and tuberal nucleus. We further get evidence that Meis2 delineates novel neuronal populations activated during PSR. In summary, our results using TRAP2-red mice characterize new cell populations activated during Wakefulness or PSR, opening experimental paths for determining their function regarding vigilance states. Statement of significanceWakefulness and paradoxical sleep are very similar at the electroencephalographic level. It remains relevant to determine the potential overlap of the neurons active during each vigilance state. We here took advantage of the powerful transgenic TRAP2-red mice to directly compare in the same animal the brain cell activation during both states, with a focus on the hypothalamus. A deeper knowledge of each individual subpopulation of hypothalamic neurons within complex brain circuits underlying the sleep-waking cycle will help the understanding and validation of treatments of sleep disorders, at least those directly linked to demonstrated hypothalamic dysfunction as Narcolepsy (Orx neurons), Amyotrophic Lateral Sclerosis (MCH and Orx signaling) or neurodegenerative diseases (Parkinsons and Alzheimer diseases).

Gonadotropin-releasing hormone (GnRH)-synthesizing neurons orchestrate reproduction centrally. Early studies have proposed the contribution of acetylcholine (ACh) to hypothalamic control of reproduction, although the causal mechanisms have not been clarified. Here, we report that in vivo pharmacogenetic activation of the cholinergic system increased the secretion of luteinizing hormone (LH) in orchidectomized mice. 3DISCO immunocytochemistry and electron microscopy revealed the innervation of GnRH neurons by cholinergic axons. Retrograde viral labeling initiated from GnRH-Cre neurons identified the medial septum and the diagonal band of Broca as exclusive sites of origin for cholinergic afferents of GnRH neurons. In acute brain slices, ACh and the ACh receptor (AChR) agonist carbachol evoked a biphasic effect on the firing rate in GnRH neurons, first increasing and then diminishing it. In the presence of tetrodotoxin, carbachol induced an inward current, followed by a decline in the frequency of mPSCs, indicating a direct influence on GnRH cells. RT-PCR and whole-cell patch-clamp studies revealed that GnRH neurons expressed both nicotinic (4{beta}2, 3{beta}4, and 7) and muscarinic (M1-M5) ACh receptors. The nicotinic AChRs contributed to the nicotine-elicited inward current and the rise in firing rate. Muscarine via M1 and M3 receptors increased, while via M2 and M4 reduced the frequency of both miniature postsynaptic currents (mPSCs) and firing. Optogenetic activation of channelrhodopsin-2-tagged cholinergic axons modified GnRH neuronal activity and evoked co-transmission of ACh and GABA from a subpopulation of boutons. These findings confirm that the central cholinergic system immensely regulates GnRH neurons and activates the HPG-axis via ACh and ACh/GABA neurotransmissions. Significance statementCholinergic drugs influence reproduction centrally, although the exact neuronal targets and regulatory mechanisms remain unsettled. We found that pharmacogenetic activation of the cholinergic system in vivo evoked an augmented LH release. The study also identified cholinergic cell groups in the mouse forebrain that innervate gonadotropin-releasing hormone (GnRH) neurons, the main hypothalamic regulators of reproduction. We also determined the subtypes of nicotinic and muscarinic receptors involved in neuronal information transmission and explored how their ligands affect the electrophysiological activity of GnRH neurons. A subset of cholinergic neurons was found to co-transmit GABA, which excites GnRH cells via GABA-A receptors. The findings suggest a novel, cholinergic regulation of the adult GnRH system in male mice that activates the pituitary-gonadal axis.

Evidence suggests that estradiol-sensing preoptic area GABA neurons are involved in the pre-ovulatory surge mechanism necessary for ovulation. In vivo CRISPR-Cas9 editing was used to achieve a 60-70% knockdown in estrogen receptor alpha (ESR1) expression by GABA neurons located within the region of the rostral periventricular of the third ventricle (RP3V) and medial preoptic nuclei (MPN) in adult female mice. Mice exhibited variable reproductive phenotypes with the only significant finding being those mice with bilateral ESR1 deletion in RP3V GABA neurons that had reduced cFos expression in GnRH neurons at the time of the surge. One sub-population of RP3V GABA neurons expresses kisspeptin. Re-grouping ESR1-edited mice on the basis of their RP3V kisspeptin expression revealed a highly consistent phenotype; mice with a near complete loss of kisspeptin immunoreactivity displayed constant estrus and failed to exhibit surge activation but retained pulsatile LH secretion. These observations demonstrate ESR1-expressing GABA-kisspeptin neurons in the RP3V are essential for the murine preovulatory LH surge mechanism.

In adult mammals, neural stem cells emerge in three neurogenic regions, the subventricular zone of the lateral ventricle (SVZ), the subgranular zone of the dentate gyrus of the hippocampus (SGZ) and the hypothalamus. In the SVZ and the SGZ, neural stem/progenitor cells (NSPCs) express the glial fibrillary acidic protein (GFAP) and selective ablation of these NSPCs drastically decreases cell proliferation in vitro and in vivo. In the hypothalamus, GFAP is expressed by -tanycytes, which are specialized radial glia-like cells in the wall of the third ventricle. To explore the role of these hypothalamic GFAP-positive tanycytes, we used transgenic mice expressing herpes simplex virus thymidine kinase (HSV-Tk) under the control of the mouse Gfap promoter and 4-week intracerebroventricular infusion of the antiviral agent ganciclovir (GCV) that kills dividing cells expressing Tk. While GCV drastically reduced the number and growth of hypothalamus-derived neurospheres from adult transgenic mice in vitro, it caused hypogonadism in vivo. The selective death of dividing tanycytes expressing GFAP indeed caused a marked decrease in testosterone levels and testicular weight, as well as vacuolization of the seminiferous tubules and loss of spermatogenesis. In addition, GCV-treated GFAP-Tk mice showed impaired sexual behavior, but no alteration in food intake or body weight. Our results also show that the selective ablation of GFAP-expressing tanycytes leads to a sharp decrease in the number of gonadotropin-releasing hormone (GnRH)-immunoreactive neurons and blunted LH secretion. Altogether, our data show that GFAP-expressing tanycytes play a central role in the regulation of male reproductive function. Main pointsKilling adult hypothalamic GFAP-expressing cells blunts neurosphere formation in vitro and leads to GnRH deficiency and hypogonadism in vivo. This work pinpoints an unreported role of dividing GFAP-expressing tanycytes in reproductive function.

Post-traumatic stress disorder impedes pubertal development and disrupts pulsatile LH secretion in humans and rodents. The posterodorsal sub-nucleus of the medial amygdala (MePD) is an upstream modulator of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, pubertal timing, as well as emotional processing and anxiety. Psychosocial stress exposure alters neuronal activity within the MePD increasing the expression of Urocortin3 (Ucn3) and its receptor corticotropin-releasing factor type-2 receptor (CRFR2) while enhancing the inhibitory output from the MePD to key hypothalamic reproductive centres. We test the hypothesis that psychosocial stress, processed by the MePD, is relayed to the hypothalamic GnRH pulse generator to delay puberty in female mice. We exposed C57Bl6/J female mice to the predator odor, 2,4,5-Trimethylthiazole (TMT), during pubertal transition and examined the effect on pubertal timing, pre-pubertal LH pulses and anxiety-like behaviour. Subsequently, we virally infected Ucn3-cre-tdTomato female mice with stimulatory DREADDs targeting MePD Ucn3 neurons and determined the effect on pubertal timing and pre-pubertal LH pulse frequency. Exposure to TMT during pubertal development delayed puberty, suppressed pre-pubertal LH pulsatility and enhanced anxiety-like behaviour, while activation of MePD Ucn3 neurons reduced LH pulse frequency and delayed puberty. Early psychosocial stress exposure decreases GnRH pulse generator frequency delaying puberty while inducing anxiety-behaviour in female mice, an effect potentially involving Ucn3 neurons in the MePD.

The signaling pathways that control embryonic development, migration, and differentiation of gonadotropin-releasing hormone (GnRH) neurons, as well as the postnatal fate, function, and survival of differentiated cells, are the subject of ongoing research. Here, we examined the role of phosphoinositides in this complex multistep process by generating GnRH neuron-specific phosphatidylinositol 4-kinase alpha knockout mice. These mice were healthy and indistinguishable from their control littermates in size. However, adult knockout females and males were infertile due to underdeveloped gonads and reproductive organs. Furthermore, hypothalamic GnRH immunoreactivity was absent, and expression of the hypothalamic Gnrh1 gene and pituitary gonadotroph-specific genes was reduced. In contrast, hypothalamic kisspeptin immunoreactivity was preserved, and Kiss1 expression was modified in a nuclei specific-manner, consistent with the loss of circulating sex steroid hormones. Embryonic neurogenesis and migration of GnRH neurons were not impaired, as evidenced by normal Gnrh1 expression in the hypothalamus of neonatal animals and the presence of immunoreactive GnRH neurons in infantile mice in comparable number and distribution to age-matched controls. However, their cellular degeneration was evident, accompanied by reduced Gnrh1 expression. GnRH neuron-specific tdTomato expression confirmed their postnatal degeneration and death, whereas ectopic tdTomato cells located in the lateral septum remained unaffected. Together, these findings indicate that phosphoinositides dependent on phosphatidylinositol 4-kinase alpha activity are not critical for embryonic steps in the development of the GnRH neuronal network, but are essential for the postnatal function and survival of these cells. Significance StatementDifferentiation of neuroendocrine GnRH cells involves neurogenesis in the olfactory placodes, migration to the hypothalamus, projection to the median eminence, and connections with upstream neurons, including kisspeptin neurons. Here we show that knockout of phosphatidylinositol 4-kinase alpha in GnRH neurons does not affect these strps of embryonic development. However, the activity of this enzyme is essential for postnatal survival of GnRH neurons; in the absence of this gene, the neurons die, causing infertility in both female and male mice.

The relaxin-3/relaxin family peptide receptor 3 (RXFP3) neuropeptidergic system is emerging as a potential target for treating various neuropsychiatric diseases, particularly those involving dysregulated stress and arousal. RXFP3 is abundantly expressed in several hypothalamic nuclei, and in the zona incerta (ZI). These regions play a central role in the regulation of stress and arousal, however the function of relaxin-3/RXFP3 within these circuits is unknown. The purpose of this study was to begin characterising this function by describing the distribution and genetic signature of neurons that express RXFP3. We used RNAscope fluorescent in situ hybridisation to characterise the spatial expression pattern and neurochemical phenotype of cells expressing Rxfp3 mRNA throughout the mouse lateral hypothalamus (LH) and ZI. We found that Rxfp3 is expressed across the rostrocaudal extent of both the LH and ZI and follows a parabolic pattern of expression, peaking in more rostral areas of each nucleus. Neurochemical phenotyping of Rxfp3+ cells with Gad1, Slc17a6 (vGlut2), Pvalb, Th, and Sst showed that LH/ZI Rxfp3+ cells co-express each marker to varying extents, generally proportional to their overall abundance within each structure. Furthermore, LH/ZI Rxfp3+ cells overlapped with several known populations involved in various facets of fear learning and defensive behaviour, such as the dopaminergic A13 group, somatostatin-expressing rostral ZI neurons, and glutamatergic LH neurons. The neurochemical diversity of these neurons may reflect the overall role of both the LH and ZI as global regulators of behaviour and the role of relaxin-3/RXFP3 signalling in modulating high-vigilance states.

Ghrelin is a gut-derived hormone that is secreted during conditions of negative caloric balance and acts as a key modulator of feeding, increasing food intake and affecting several physiological systems such as metabolism, behavior and the control of endocrine and autonomic functions. Previous studies showed that ghrelin participates in the stress response, acting on hypothalamic paraventricular nucleus neurons that express corticotropin-releasing hormone (PVNCRH neurons). In the present study, we investigated the effects of ghrelin administration on the behavioral responses to restraint stress in mice. In their homecage, C57Bl6 mice in basal conditions expressed the behaviors of surveying, walking, rearing, grooming and, to a lesser extent, digging, climbing and freezing. Restraint stress increased the time spent in grooming without significant changes in other behaviors. Ghrelin administration did not affect behavior in control mice, but it reversed the effect of restraint stress on grooming. Chemogenetic activation of PVNCRH neurons by clozapine N-Oxide (CNO) administration in hM3Dq DREADD mice increased grooming, while ghrelin mitigated this effect. In addition, CNO administration decreased walking and rearing, both in the presence or absence of ghrelin. Food intake was increased by ghrelin administration, however, it was not affected by stress or CNO. These results indicate that ghrelin decreases the activity of PVNCRH neurons, partially preventing the behavioral effects of restraint stress. The inhibitory input to PVNCRH neurons probably arrives from other nuclei, since GABAergic neurons were not identified in the PVN neurons of these mice.

Early life stress (ELS) is a potent environmental factor that can confer enduring effects on brain structure and function. Exposure to stress during early life has been linked to a wide range of physiopathological consequences later in life. In particular, ELS has been shown to have lasting effects on neurogenesis in the adult brain, suggesting that ELS is a significant regulator of adult neural stem cell function. Here, we investigated the effect of ELS on the numbers and proliferation of neural stem cells in the hypothalamus of adult mice. We show that ELS has long term negative effects on hypothalamic neural stem cell numbers and on their proliferation. Specifically, ELS reduced the total numbers of PCNA+ cells present in hypothalamic areas surrounding the 3rd ventricle; the numbers of PCNA+/Sox2+/Nestin-GFP+ cells present in the medial eminence at the base of the 3rd ventricle; and the number of {beta}-tanycytes around the ventral 3rd ventricle, without affecting the numbers of -tanycytes in more dorsal areas. These results suggest that a reduction of proliferation and tanycyte numbers contributes to the effects of ELS on the hypothalamus and its consequent physiological alterations.

To induce ovulation, neural circuits are sequentially activated by estradiol and progesterone. In female rodents, estradiol-induced neuroprogesterone, synthesized in astrocytes, is essential for the luteinizing hormone (LH) surge and subsequently, ovulation. However, the gonadotropin-releasing hormone (GnRH) neurons do not express the steroid receptors necessary for reproduction: progesterone receptors (PGR) or estrogen receptor- (ER). Steroid information is transduced by rostral periventricular (RP3V) kisspeptin neurons that express ER and PGR and innervate GnRH neurons in the diagonal band of Broca (DBB) and the medial septum. In this study, we tested the hypothesis that estradiol induced neuroprogesterone needed for the LH surge is mediated by kisspeptin. Neuroprogesterone synthesis was inhibited with aminoglutethimide (AGT; s.c.) in 17{beta}-estradiol benzoate (EB)-primed, ovariectomized (ovx) and adrenalectomized (adx) rats. Kisspeptin-10 (20 nmol/{micro}l) was infused into the DBB, trunk blood was collected 53 hours post-EB injection, and serum LH levels were analyzed by ELISA. AGT inhibition of neuroprogesterone synthesis blocked the EB-induced LH surge. Subsequent treatment with either progesterone or DBB kisspeptin-10 infusion restored the LH surge. Kisspeptin restored the LH surge, which was blocked by DBB infusion of kisspeptin receptor (GPR54) antagonist (kisspeptin-234). Finally, knockdown of kisspeptin protein levels in the RP3V with kisspeptin antisense oligodeoxynucleotide (ODN) significantly lowered LH levels in EB-primed rats compared to scrambled ODN, demonstrating the importance of endogenous RP3V kisspeptin for the LH surge. These results support the hypothesis that neuroprogesterone induces both kisspeptin release from RP3V neurons impacting the LH surge.

Oxytocin (OXT), a primitive nonapeptide known to regulate reproduction and social behaviors, is synthesized primarily in the hypothalamus and is secreted via hypophyseal-portal system of the posterior pituitary gland. Given that pituitary hormones, traditionally thought of as regulators of single targets, display an array of central and peripheral actions, OXT also directly affects bone and body composition. Its effects on bone remodeling are physiologically relevant, as elevated OXT levels during pregnancy and lactation could cause calcium mobilization from the maternal skeleton for intergenerational calcium transfer towards fetal bone growth. There is an equally large body of evidence that has established the presence of OXT receptors (OXTRs) in the brain through which central functions, such as social bonding, and peripheral functions, such as the regulation of body composition, can be exerted. To purposefully address the effects of OXT on the brain, we used RNAscope to map OXT and OXTR expression, at the single transcript level, in the whole mouse brain. Identification of brain nuclei with the highest OXT and OXTR transcript density will shed further light on functional OXT nodes that could be further interrogated experimentally to define new physiologic circuitry.

Neural circuits in female rats are exposed to estradiol and sequential progesterone to regulate the luteinizing hormone (LH) surge and thus ovulation. Estradiol induces progesterone receptors (PGRs) in rostral periventricular region of the third ventricle (RP3V) kisspeptin neurons, and positive feedback estradiol concentrations induce neuroprogesterone (neuroP) synthesis in hypothalamic astrocytes that signal to PGRs expressed in kisspeptin neurons to trigger the LH surge. We tested the hypothesis that neuroP-PGR signals through Src family kinase (Src) to trigger the LH surge. As in vitro, PGR and Src are co-expressed in RP3V neurons. Estradiol treatment increased the number of PGR immunopositive cells and PGR and Src colocalization. Infusion of the Src inhibitor (PP2) into the RP3V, attenuated the LH surge measured by ELISA in trunk blood collected 53 hours post-EB injection. While PP2 reduced the LH surge in 50 g EB treated ovariectomized/adrenalectomized (ovx/adx) rats, activation of either PGR or Src in 2g EB primed animals significantly elevated LH concentrations compared with DMSO treated ovx/adx rats. These results support the importance of Src in the estradiol and neuroP triggering of the LH surge.

The regulation of food intake is one of the major research areas in the study of metabolic syndromes such as obesity. Gene targeting studies have clarified the roles of hypothalamic neurons in feeding behaviour. However, our understanding of neural function under physiological conditions is still limited. Immediate early genes, such as activity-regulated cytoskeleton-associated protein (Arc/Arg3.1), are useful markers of neuronal activity. Here, we investigated the role of Arc/Arg3.1 gene-expressing neurons in the hypothalamus after refeeding using the targeted recombination in active populations method. We identified refeeding-responsive prodynorphin/cholecystokinin neurons in the dorsomedial hypothalamus that project to the paraventricular hypothalamic nucleus. Chemogenetic activation of these neurons decreased food intake and promoted positive valence. Our findings provide insight into the role of newly identified hedonic neurons in the process of feeding-induced satiety.

Melanin-concentrating hormone (MCH) is a ubiquitous vertebrate neuropeptide predominantly synthesized by neurons of the diencephalon that can act through two G protein-coupled receptors, called MCHR1 and MCHR2. The expression of Mchr1 has been investigated in both rats and mice, but its synthesis remains poorly described. After identifying an antibody that detects MCHR1 with high specificity, we employed immunohistochemistry to map the distribution of MCHR1 in the CNS of rats and mice. Multiple neurochemical markers were also employed to characterize some of the neuronal populations that synthesize MCHR1. Our results show that MCHR1 is abundantly found in a sensory subcellular structure called the neuronal primary cilium, which has been associated with the detection of free neurochemical agents released to act through volume transmission. Ciliary MCHR1 was found in a wide range of areas, including the olfactory bulb, cortical mantle, striatum, hippocampal formation, amygdala, midline thalamic nuclei, periventricular hypothalamic nuclei, and midbrain areas. No differences were observed between male and female mice, and rats and mice diverged in two key areas: the caudate-putamen nucleus and the subgranular zone of the dentate gyrus. Ciliary MCHR1 was found in close association to several neurochemical markers, including tyrosine hydroxylase, calretinin, kisspeptin, estrogen receptor, oxytocin, vasopressin, and corticotropin-releasing factor. Given the role of neuronal primary cilia in sensing free neurochemical messengers in the extracellular fluid, the widespread distribution of ciliary MCHR1, and the diverse neurochemical populations who synthesize MCHR1, our data indicates that volume transmission may play a prominent role in the normal function of the MCH system.

In nature, species synchronize reproduction and energy metabolism with seasons to optimize survival and growth. While the effects of endocrine-disrupting chemicals (EDCs) exposure on conventional laboratory rodents are increasingly studied, their impacts on mammalian seasonal adaptation remain unexplored. This study investigates the effect of oral exposure to bisphenol A (BPA) on physiological and neuroendocrine seasonal adaptation in Djungarian hamsters. Adult female and male hamsters were orally exposed to BPA (5, 50, or 500 {micro}g/kg/d) or vehicle during a 10-week transition from a long (LP) to short (SP) photoperiod (winter transition) or vice versa (summer transition). Changes in body weight, food intake, and pelage color were monitored weekly and, at the end of the exposure, gene expression of hypothalamic markers of photoperiodic, reproductive and metabolic integration, reproductive organ activity, and glycemia were assessed. Our results revealed sex-specific effects of BPA on acquiring SP and LP phenotypes. During LP to SP transition, females exposed to 500 {micro}g/kg/d BPA exhibited delayed body weight loss and reduced feed efficiency associated with a lower expression of somatostatin in the arcuate nucleus (ARC), while males exposed to 5 {micro}g/kg/d BPA showed an accelerated acquisition of SP-induced metabolic parameters. During SP to LP transition, females exposed to 5 {micro}g/kg/d BPA displayed a faster LP adaptation in reproductive and metabolic parameters, along with quicker ARC kisspeptin downregulation and delayed ARC Pomc upregulation, while males exposed to BPA exhibited decreased expression of central photoperiodic integrators without changes in the physiological LP acquisition. This pioneering study investigating EDC impacts on mammalian seasonal physiology shows that BPA alters the dynamic of metabolic adaptation to both SP and LP transitions with marked sex dimorphism, causing temporal discordance in seasonal adaptation between males and females. These findings emphasize the importance of investigating EDCs impact on non-conventional animal models, providing insights into wildlife physiology. HighlightsO_LIDjungarian hamsters seasonal adaptation is disrupted by BPA oral exposure C_LIO_LIBPA delays in females and accelerates in males the metabolic adaptation to short days C_LIO_LIBPA accelerates in females, not in males, metabolic/reproductive adaptation to long days C_LIO_LIBPA affects the photoperiodic expression of central reproductive and metabolic genes C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=89 SRC="FIGDIR/small/580037v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@3ddec8org.highwire.dtl.DTLVardef@1ed3536org.highwire.dtl.DTLVardef@4083c8org.highwire.dtl.DTLVardef@18bd837_HPS_FORMAT_FIGEXP M_FIG Graphical abstract C_FIG

Vasopressin (AVP), a nonapeptide synthesized predominantly by magnocellular hypothalamic neurons, is conveyed to the posterior pituitary via the pituitary stalk, where AVP is secreted into the circulation. Known to regulate blood pressure and water homeostasis, it also modulates diverse social behaviors, such as pair-bonding, social recognition and cognition in mammals including humans. Importantly, AVP modulates social behaviors in a sex-specific manner, perhaps, due to sex differences in the distribution in the brain of AVP and its main receptor AVPR1a. There is a corpus of integrative studies for the expression of AVP and AVPR1a in various brain regions, and their functions in modulating central and peripheral actions. In order to purposefully address sexually dimorphic and novel roles of AVP on central and peripheral functions through its AVPR1a, we utilized RNAscope to map Avp and Avpr1a single transcript expression in the mouse brain. As the most comprehensive atlas of AVP and AVPR1a in the mouse brain, this compendium highlights the importance of newly identified AVP/AVPR1a neuronal nodes that may stimulate further functional studies.

The pituitary is the vertebrate endocrine gland responsible for the production and secretion of several essential peptide hormones. These, in turn, control many aspects of an animals physiology and development, including growth, reproduction, homeostasis, metabolism and stress responses. In teleost fish, each hormone is presumably produced by a specific cell type. However, key details on the regulation of, and communication between these cell types remain to be resolved. We have therefore used single-cell sequencing to generate gene expression profiles for 2592 and 3804 individual cells from the pituitaries of female and male adult medaka (Oryzias latipes), respectively. Based on expression profile clustering, we define 15 and 16 distinct cell types in the female and male pituitary, respectively, of which ten are involved in the production of a single peptide hormone. Collectively, our data provide a high-quality reference for studies on pituitary biology and the regulation of hormone production, both in fish and in vertebrates in general.

Gonadotropin-releasing hormone (GnRH) neurons are the final common target of a complex network of cells cooperating for the central control of reproduction. The balance between excitatory and inhibitory transsynaptic and non-synaptic inputs is crucial for the maintenance of the GnRH rhythms: the pulse and the surge. The precise mechanisms behind this remain under debate. In this work, we challenge the hypothesis that excitatory and inhibitory inputs from kisspeptin and neuronal nitric oxide (NO) synthase (nNOS)-expressing neurons orchestrates GnRH release, in a microcircuit that we call the Kisspeptin/nNOS/GnRH (KiNG) neuronal network. Our work specifically focuses on the role of nNOS neurons located in the organum vasculosum laminae terminalis (OV) and the median preoptic nucleus (MePO). nNOS and kisspeptin neurons interact anatomically and functionally, with the kisspeptin receptor (Kiss1r) being differentially regulated in nNOS-expressing neurons across the female estrous cycle. Using a novel viral tool allowing for the measurement of NO/cGMP levels with exquisite sensitivity, we demonstrate that kisspeptin is able to induce NO-dependent cGMP production in the OV/MePO, including in GnRH neurons in vivo. Using electrophysiological, genetic, chemogenetic and pharmacologic approaches, we reveal that NO production from nNOS neurons in the OV/MePO is needed to fine-tune the GnRH/LH response to kisspeptin, and specifically to turn off GnRH release, thus generating pulses. Our findings provide valuable insights into the tripartite KiNG neuronal network governing the regulation of the GnRH/LH pulse and surge.

Puberty is a critical transition period to achieve fertility and reproductive capacity in all mammalian species. At puberty, the hypothalamic-pituitary-gonadal (HPG) is activated by neuroendocrine changes in the brain. Central to this are Kiss1 neurons that produce kisspeptin, a neuropeptide which is a potent stimulator of gonadotropin releasing hormone (GnRH) secretion. Kiss1 neurons in the arcuate region of the hypothalamus (Kiss1ARC) increase pulsatile secretion of GnRH at puberty. Other developmental maturational changes in the brain are often accompanied by neuronal plasticity changes but this has not been studied in Kiss1 neurons. Electrophysiological characterisation of Kiss1ARC neurons from female mice shows that these neurons undergo profound intrinsic plasticity at puberty with a critical window between 3 and 4 weeks. Immature Kiss1ARC neurons cannot sustain depolarisation-evoked firing for even 500 ms and instead fire a brief burst of high frequency spikes before falling silent. This would make them unsuitable for the sustained activity that is needed to activate GnRH neurons and trigger LH secretion in the HPG axis. After puberty, sustained firing can be maintained, which endows post-puberty Kiss1ARC neurons with a mature physiological phenotype that is amenable to neuropeptide modulation for generation of burst firing and pulsatile release of kisspeptin. There is a corresponding decrease in the threshold for action potential initiation, a more hyperpolarised post-spike trough and a larger medium after-hyperpolarisation (mAHP). Gene expression analysis showed a significant decrease in Scn2a (Nav1.2 channel), Kcnq2 (Kv7.2 channel) and Lrrc55 (BK channel auxiliary {gamma}3-subunit) expression and an increase in Hcn1 (hyperpolarization activated cyclic nucleotide-gated potassium channel) expression which may contribute to the observed electrophysiological changes. Ovariectomy and {beta}-estradiol replacement defined a window of estrogen-dependent plasticity of action potential firing at puberty, such that post-puberty Kiss1ARC neurons achieve a mature physiological phenotype for activation of the HPG axis.