In vivo imaging of the GnRH pulse generator reveals a temporal order of neuronal activation and synchronization during each pulse.

A hypothalamic pulse generator located in the arcuate nucleus controls episodic release of gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) and is essential for reproduction. Recent evidence suggests this generator is composed of arcuate "KNDy" cells, the abbreviation based on coexpression of kisspeptin, neurokinin B, and dynorphin. However, direct visual evidence of KNDy neuron activity at a single-cell level during a pulse is lacking. Here, we use in vivo calcium imaging in freely moving female mice to show that individual KNDy neurons are synchronously activated in an episodic manner, and these synchronized episodes always precede LH pulses. Furthermore, synchronization among KNDy cells occurs in a temporal order, with some subsets of KNDy cells serving as "leaders" and others as "followers" during each synchronized episode. These results reveal an unsuspected temporal organization of activation and synchronization within the GnRH pulse generator, suggesting that different subsets of KNDy neurons are activated at pulse onset than afterward during maintenance and eventual termination of each pulse. Further studies to distinguish KNDy "leader" from "follower" cells is likely to have important clinical significance, since regulation of pulsatile GnRH secretion is essential for normal reproduction and disrupted in pathological conditions such as polycystic ovary syndrome and hypothalamic amenorrhea.

Localization of kisspeptin, NKB, and NK3R in the hypothalamus of gilts treated with the progestin altrenogest.

Mechanisms in the brain controlling secretion of gonadotropin hormones in pigs, particularly luteinizing hormone (LH), are poorly understood. Kisspeptin is a potent LH stimulant that is essential for fertility in many species, including pigs. Neurokinin B (NKB) acting through neurokinin 3 receptor (NK3R) is involved in kisspeptin-stimulated LH release, but organization of NKB and NK3R within the porcine hypothalamus is unknown. Hypothalamic tissue from ovariectomized (OVX) gilts was used to determine the distribution of immunoreactive kisspeptin, NKB, and NK3R cells in the arcuate nucleus (ARC). Almost all kisspeptin neurons coexpressed NKB in the porcine ARC. Immunostaining for NK3R was distributed throughout the preoptic area (POA) and in several hypothalamic areas including the periventricular and retrochiasmatic areas but was not detected within the ARC. There was no colocalization of NK3R with gonadotropin-releasing hormone (GnRH), but NK3R-positive fibers in the POA were in close apposition to GnRH neurons. Treating OVX gilts with the progestin altrenogest decreased LH pulse frequency and reduced mean circulating concentrations of LH compared with OVX control gilts (P < 0.01), but the number of kisspeptin and NKB cells in the ARC did not differ between treatments. The neuroanatomical arrangement of kisspeptin, NKB, and NK3R within the porcine hypothalamus confirms they are positioned to stimulate GnRH and LH secretion in gilts, though differences with other species exist. Altrenogest suppression of LH secretion in the OVX gilt does not appear to involve decreased peptide expression of kisspeptin or NKB.

Comparison of Magnetic Resonance Angiography and Computed Tomography Angiography Stereoscopic Cerebral Vascular Models.

In this paper, we will discuss and compare the stereoscopic models developed from two types of radiographic data, Magnetic Resonance Angiography (MRA) images and Computed Tomography Angiography (CTA) images. Stereoscopic models were created using surface or volume segmentation and semi-auto combined segmentation techniques. Although, the CTA data were found to improve the speed and quality of constructing virtual vascular models compared to conventional CT data, small blood vessels were difficult to capture during the imaging and reconstruction process thereby limiting the fidelity of the stereoscopic models. Thus, high contrast Magnetic Resonance Angiography (MRA) images offer better resolution to visualize and capture the smaller branches of the cerebral vasculature than CTA images.

Regulation of GnRH pulsatility in ewes.

Early work in ewes provided a wealth of information on the physiological regulation of pulsatile gonadotropin-releasing hormone (GnRH) secretion by internal and external inputs. Identification of the neural systems involved, however, was limited by the lack of information on neural mechanisms underlying generation of GnRH pulses. Over the last decade, considerable evidence supported the hypothesis that a group of neurons in the arcuate nucleus that contain kisspeptin, neurokinin B and dynorphin (KNDy neurons) are responsible for synchronizing secretion of GnRH during each pulse in ewes. In this review, we describe our current understanding of the neural systems mediating the actions of ovarian steroids and three external inputs on GnRH pulsatility in light of the hypothesis that KNDy neurons play a key role in GnRH pulse generation. In breeding season adults, estradiol (E2) and progesterone decrease GnRH pulse amplitude and frequency, respectively, by actions on KNDy neurons, with E2 decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E2 inhibits GnRH pulse frequency by decreasing kisspeptin and increasing dynorphin release, actions that wane as the lamb matures to allow increased pulsatile GnRH secretion at puberty. Less is known about mediators of undernutrition and stress, although some evidence implicates kisspeptin and dynorphin, respectively, in the inhibition of GnRH pulse frequency by these factors. During the anoestrus, inhibitory photoperiod acting via melatonin activates A15 dopaminergic neurons that innervate KNDy neurons; E2 increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release.

Neural mechanisms controlling seasonal reproduction: principles derived from the sheep model and its comparison with hamsters.

Seasonal reproduction is a common adaptive strategy among mammals that allows for breeding to occur at times of the year when it is most advantageous for the subsequent survival and growth of offspring. A major mechanism responsible for seasonal reproduction is a striking increase in the responsiveness of gonadotropin-releasing hormone (GnRH) neurons to the negative feedback effects of estradiol. The neural and neuroendocrine circuitry responsible for mammalian seasonal reproduction has been primarily studied in three animal models: the sheep, and two species of hamsters. In this review, we first describe the afferent signals, neural circuitry and transmitters/peptides responsible for seasonal reproductive transitions in sheep, and then compare these mechanisms with those derived from studies in hamsters. The results suggest common principles as well as differences in the role of specific brain nuclei and neuropeptides, including that of kisspeptin cells of the hypothalamic arcuate nucleus, in regulating seasonal reproduction among mammals.

Prenatal testosterone exposure decreases colocalization of insulin receptors in kisspeptin/neurokinin B/dynorphin and agouti-related peptide neurons of the adult ewe.

Insulin serves as a link between the metabolic and reproductive systems, communicating energy availability to the hypothalamus and enabling reproductive mechanisms. Adult Suffolk ewes prenatally exposed to testosterone (T) display an array of reproductive and metabolic dysfunctions similar to those seen in women with polycystic ovarian syndrome (PCOS), including insulin resistance. Moreover, prenatal T treatment alters neuropeptide expression in KNDy (co-expressing kisspeptin, neurokinin B/dynorphin) and agouti-related peptide (AgRP) neurons in the arcuate nucleus, two populations that play key roles in the control of reproduction and metabolism, respectively. In this study, we determined whether prenatal T treatment also altered insulin receptors in KNDy and AgRP neurons, as well as in preoptic area (POA) kisspeptin, pro-opiomelanocortin (POMC), and gonadotropin-releasing hormone (GnRH) neurons of the adult sheep brain. Immunofluorescent detection of the beta subunit of insulin receptor (IRß) revealed that KNDy, AgRP and POMC neurons, but not GnRH or POA kisspeptin neurons, colocalize IRß in control females. Moreover, prenatal T treatment decreased the percentage of KNDy and AgRP neurons that colocalized IRß, consistent with reduced insulin sensitivity. Administration of the anti-androgen drug, Flutamide, during prenatal T treatment, prevented the reduction in IRß colocalization in AgRP, but not in KNDy neurons, suggesting that these effects are programmed by androgenic and oestrogenic actions, respectively. These findings provide novel insight into the effects of prenatal T treatment on hypothalamic insulin sensitivity and raise the possibility that decreased insulin receptors, specifically within KNDy and AgRP neurons, may contribute to the PCOS-like phenotype of this animal model.

Endogenous opioid-induced neuroplasticity of dopaminergic neurons in the ventral tegmental area influences natural and opiate reward.

Natural reward and drugs of abuse converge on the mesolimbic pathway and activate common mechanism of neural plasticity in the nucleus accumbens. Chronic exposure to opiates induces plasticity in dopaminergic neurons of the ventral tegmental area (VTA), which regulates morphine reward tolerance. Here, we test the hypotheses that mating-induced release of endogenous opioids in the VTA causes morphological changes of VTA dopamine cells in male rats, which in-turn regulate the long-term expression of experience-induced reinforcement of sexual behavior. First, sexual experience decreased VTA dopamine soma size 1 and 7 days, but not 30 days after the last mating session. This effect was blocked with naloxone before each mating session; thus, VTA dopamine cell plasticity was dependent on action of endogenous opioids. In turn, VTA plasticity was associated with altered opiate reward, as sexually experienced males did not form conditioned place preference for 0.5 mg/kg morphine. Next, it was determined whether endogenous opioid action mediates sexual reward and memory in male rats treated with naloxone during mating experience, either systemically or intra-VTA. Naloxone did not prevent the initial experience-induced facilitation of sexual behavior over repeated mating sessions, or conditioned place preference for mating. However, naloxone treatment attenuated the longer-term expression of experience-induced facilitation of sexual behavior and neural activation in mesolimbic areas induced by mating-associated conditioned cues. Together, these data demonstrate that endogenous opioids during mating induce neural plasticity in VTA dopamine neurons that appear critical for morphine reward and long-term memory for natural reward behavior.

Sex differences and effects of prenatal exposure to excess testosterone on ventral tegmental area dopamine neurons in adult sheep.

Prenatal testosterone (T) excess in sheep results in a wide array of reproductive neuroendocrine deficits and alterations in motivated behavior. The ventral tegmental area (VTA) plays a critical role in reward and motivated behaviors and is hypothesised to be targeted by prenatal T. Here we report a sex difference in the number VTA dopamine cells in the adult sheep, with higher numbers of tyrosine hydroxylase (TH)-immunoreactive (-ir) cells in males than females. Moreover, prenatal exposure to excess T during either gestational days 30-90 or 60-90 resulted in increased numbers of VTA TH-ir cells in adult ewes compared to control females. Stereological analysis confirmed significantly greater numbers of neurons in the VTA of males and prenatal T-treated ewes, which was primarily accounted for by greater numbers of TH-ir cells. In addition, immunoreactivity for TH in the cells was denser in males and prenatal T-treated females, suggesting that sex differences and prenatal exposure to excess T affects both numbers of cells expressing TH and the protein levels within dopamine cells. Sex differences were also noted in numbers of TH-ir cells in the substantia nigra, with more cells in males than females. However, prenatal exposure to excess T did not affect numbers of TH-ir cells in the substantia nigra, suggesting that this sex difference is organised independently of prenatal actions of T. Together, these results demonstrate sex differences in the sheep VTA dopamine system which are mimicked by prenatal treatment with excess T.

Natural and drug rewards act on common neural plasticity mechanisms with ΔFosB as a key mediator.

Drugs of abuse induce neuroplasticity in the natural reward pathway, specifically the nucleus accumbens (NAc), thereby causing development and expression of addictive behavior. Recent evidence suggests that natural rewards may cause similar changes in the NAc, suggesting that drugs may activate mechanisms of plasticity shared with natural rewards, and allowing for unique interplay between natural and drug rewards. In this study, we demonstrate that sexual experience in male rats when followed by short or prolonged periods of loss of sex reward causes enhanced amphetamine reward, indicated by sensitized conditioned place preference for low-dose (0.5 mg/kg) amphetamine. Moreover, the onset, but not the longer-term expression, of enhanced amphetamine reward was correlated with a transient increase in dendritic spines in the NAc. Next, a critical role for the transcription factor ΔFosB in sex experience-induced enhanced amphetamine reward and associated increases in dendritic spines on NAc neurons was established using viral vector gene transfer of the dominant-negative binding partner ΔJunD. Moreover, it was demonstrated that sexual experience-induced enhanced drug reward, ΔFosB, and spinogenesis are dependent on mating-induced dopamine D1 receptor activation in the NAc. Pharmacological blockade of D1 receptor, but not D2 receptor, in the NAc during sexual behavior attenuated ΔFosB induction and prevented increased spinogenesis and sensitized amphetamine reward. Together, these findings demonstrate that drugs of abuse and natural reward behaviors act on common molecular and cellular mechanisms of plasticity that control vulnerability to drug addiction, and that this increased vulnerability is mediated by ΔFosB and its downstream transcriptional targets.

A role for neurokinin B in pulsatile GnRH secretion in the ewe.

The recent description of infertility in humans with loss-of-function mutations in genes for neurokinin B (NKB) or its receptor (NK3R) has focused attention on the importance of this tachykinin in the control of GnRH secretion. In a number of species, NKB neurons in the arcuate nucleus also produce two other neuropeptides implicated in the control of GnRH secretion: (1) kisspeptin, which is also essential for fertility in humans, and (2) dynorphin, an inhibitory endogenous opioid peptide. A number of characteristics of this neuronal population led to the hypothesis that they may be responsible for driving synchronous release of GnRH during episodic secretion of this hormone, and there is now considerable evidence to support this hypothesis in sheep and goats. In this article, we briefly review the history of work on the NKB system in sheep and then review the anatomy of NKB signaling in the ewe. We next describe evidence from a number of species that led to development of a model for the role of these neurons in episodic GnRH secretion. Finally, we discuss recent experiments in sheep and goats that tested this hypothesis and led to a modified version of the model, and then broaden our focus to briefly consider the possible roles of NKB in other species and systems.

Neuroanatomy of the kisspeptin signaling system in mammals: comparative and developmental aspects.

Our understanding of kisspeptin and its actions depends, in part, on a detailed knowledge of the neuroanatomy of the kisspeptin signaling system in the brain. In this chapter, we will review our current knowledge of the distribution of kisspeptin cells, fibers, and receptors in the mammalian brain, including the development, phenotype, and projections of different kisspeptin subpopulations. A fairly consistent picture emerges from this analysis. There are two major groups of kisspeptin cell bodies: a large number in the arcuate nucleus (ARC) and a smaller collection in the rostral periventricular area of the third ventricle (RP3V) of rodents and preoptic area (POA) of non-rodents. Both sets of neurons project to GnRH cell bodies, which contain Kiss1r, and the ARC kisspeptin population also projects to GnRH axons in the median eminence. ARC kisspeptin neurons contain neurokinin B and dynorphin, while a variable percentage of those cells in the RP3V of rodents contain galanin and/or dopamine. Neurokinin B and dynorphin have been postulated to contribute to the control of GnRH pulses and sex steroid negative feedback, while the role of galanin and dopamine in rostral kisspeptin neurons is not entirely clear. Kisspeptin neurons, fibers, and Kiss1r are found in other areas, including widespread areas outside the hypothalamus, but their physiological role(s) in these regions remains to be determined.

KNDy Neurons of the Hypothalamus and Their Role in GnRH Pulse Generation: an Update.

There is considerable evidence that synchronized activity within a reciprocally connected population of cells in the arcuate nucleus (ARC) coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy cells) is crucial for the generation of gonadotrophin-releasing hormone (GnRH) pulses in mammals. The initial "KNDy hypothesis" proposed that pulsatile GnRH secretion is elicited by episodic kisspeptin release from KNDy cells following synchronized activation and termination of the population by NKB and dynorphin, respectively. Since then, the role of KNDy cells as a critical component of the pulse generator has been further supported by studies at the single-cell level, demonstrating that the population is both necessary and sufficient for pulsatility. In addition, there have been considerable modifications and expansion of the original hypothesis, including work demonstrating the critical role of glutamate in synchronization of the KNDy cell network, functional interactions with other ARC subpopulations, and the existence of species differences in the role of dynorphin in pulse generation. Here we review these recent changes and discuss how the translation of these findings has led to the development of new therapies for disorders related to pulse generation. We also outline critical gaps in knowledge that are currently limiting the application of KNDy research in the clinic, particularly regarding the role of dynorphin in pulse generation in primates.

KNDy neurons as the GnRH pulse generator: Recent studies in ruminants.

This review considers three aspects of recent work on the role of KNDy neurons in GnRH pulse generation in ruminants. First, work on basic mechanisms of pulse generation includes several tests of this hypothesis, all of which support it, and evidence that Kiss1r-containing neurons form a positive feedback circuit with the KNDy neural network that strengthen the activity of this network. The second section on pathways mediating external inputs focuses on the influence of nutrition and photoperiod, and describes the evidence supporting roles for proopiomelanocortin (POMC) and agouti-related peptide (AgRP) afferents to KNDy cells in each of these. Finally, we review studies exploring the potential applications of manipulating signaling by kisspeptin, and the other KNDy peptides, to control reproductive function in domestic animals and conclude that, although these approaches show some promise, they do not have major advantages over current practices at this time.

Lesions of KNDy and Kiss1R Neurons in the Arcuate Nucleus Produce Different Effects on LH Pulse Patterns in Female Sheep.

The current model for the synchronization of GnRH neural activity driving GnRH and LH pulses proposes that a set of arcuate (ARC) neurons that contain kisspeptin, neurokinin B, and dynorphin (KNDy neurons) is the GnRH pulse generator. This study tested the functional role of ovine KNDy neurons in pulse generation and explored the roles of nearby Kiss1 receptor (Kiss1R)-containing cells using lesions produced with saporin (SAP) conjugates. Injection of NK3-SAP ablated over 90% of the KNDy cells, while Kiss-SAP (saporin conjugated to kisspeptin-54) lesioned about two-thirds of the Kiss1R population without affecting KNDy or GnRH cell number. Both lesions produced a dramatic decrease in LH pulse amplitude but had different effects on LH pulse patterns. NK3-SAP increased interpulse interval, but Kiss-SAP did not. In contrast, Kiss-SAP disrupted the regular hourly occurrence of LH pulses, but NK3-SAP did not. Because Kiss1R is not expressed in KNDy cells, HiPlex RNAScope was used to assess the colocalization of 8 neurotransmitters and 3 receptors in ARC Kiss1R-containing cells. Kiss1R cells primarily contained transcript markers for GABA (68%), glutamate (28%), ESR1 (estrogen receptor-α) mRNA, and OPRK1 (kappa opioid receptor) mRNA. These data support the conclusion that KNDy neurons are essential for GnRH pulses in ewes, whereas ARC Kiss1R cells are not but do maintain the amplitude and regularity of GnRH pulses. We thus propose that in sheep, ARC Kiss1R neurons form part of a positive feedback circuit that reinforces the activity of the KNDy neural network, with GABA or glutamate likely being involved.

Concurrent exposure to methamphetamine and sexual behavior enhances subsequent drug reward and causes compulsive sexual behavior in male rats.

Methamphetamine (Meth) users report having heightened sexual pleasure, numerous sexual partners, and engaging in unprotected sex due to loss of inhibitory control. This compulsive sexual behavior contributes to increased prevalence of sexually transmitted infections, but the neural basis for this is unknown. We previously established a paradigm for compulsive sexual behavior in male rats in which visceral illness induced by lithium chloride was paired with sexual behavior (Davis et al., 2010; Frohmader et al., 2010a). The current study examined the effects of repeated Meth administration on sexual performance, compulsive sexual behavior, and sex or Meth reward. First, results demonstrated that seven daily administrations of 2 mg/kg, but not 1 mg/kg, Meth increased latencies to initiate mating. This impairment was evident 30 min after last Meth administration, but dissipated after 1 or 7 d of subsequent drug abstinence. Repeated 1 mg/kg Meth exposure resulted in compulsive sex-seeking behavior 2 weeks following last Meth administration. This effect was dependent on Meth administration being concurrent with sexual experience and was not observed in sexually experienced animals that received Meth alone. Moreover, concurrent Meth and sexual experience enhanced conditioned place preference (CPP) for Meth, and for concurrent Meth and mating compared with Meth or mating alone. In contrast, CPP for mating alone was decreased. Together, these data indicate that the association between drug use and mating may be required for expression of compulsive sexual behavior and is correlated with increased reward seeking for concurrent Meth exposure and mating.

Activation of gastrin-releasing peptide receptors in the lumbosacral spinal cord is required for ejaculation in male rats.

INTRODUCTION: Ejaculation is a complex reflex mediated by a spinal ejaculation generator located in the lumbosacral spinal cord and consisting of a population of lumbar spinothalamic (LSt) neurons. LSt neurons and their intraspinal axonal projections contain several neuropeptides, including gastrin-releasing peptide (GRP). AIM: To test the hypothesis that GRP is critically involved in mediating ejaculation by acting in autonomic and motor areas of the lumbosacral spinal cord, utilizing a physiological paradigm to investigate ejaculatory reflexes in isolation of supraspinal inputs. METHODS: Dual immunohistochemistry for GRP and galanin was performed to investigate co-expression of GRP in LSt cells of control male rats. Next, anesthetized, spinalized male rats received intrathecal infusions of either GRP antagonist RC-3095 (0, 10, or 20 nmol/10 µL) or GRP (0, 0.2, 0.5 nmol/10 µL). Ejaculatory reflexes were induced by electrical stimulation of the dorsal penile nerve (DPN) which reliably triggers rhythmic increases in seminal vesicle pressure (SVP) and contractions of the bulbocavernosus muscle (BCM), indicative of the emission and expulsion phases of ejaculation, respectively. MAIN OUTCOME MEASURES: GRP in LSt cells was expressed as percentages of co-expression. SVP and electromyographic recording (EMG) of BCM activity following drug treatment and DPN stimulation were recorded and analyzed for numbers of SVP increases, BCM events and bursts. RESULTS: GRP was exclusively expressed in LSt cells and axons. Intrathecal infusion of RC-3095, but not saline, blocked SVP increases and BCM bursting induced by DPN stimulation. Intrathecal infusions of GRP, but not saline, triggered SVP increases and BCM bursting in 43-66% of animals and facilitated SVP increases and BCM bursting induced by subthreshold DPN stimulation in all animals. CONCLUSION: These data support a critical role for GRP for control of the emission and expulsion phases of ejaculation in male rats by acting in LSt target areas in the lumbosacral spinal cord.

A pivotal role of lumbar spinothalamic cells in the regulation of ejaculation via intraspinal connections.

INTRODUCTION: A population of lumbar spinothalamic cells (LSt cells) has been demonstrated to play a pivotal role in ejaculatory behavior and comprise a critical component of the spinal ejaculation generator. LSt cells are hypothesized to regulate ejaculation via their projections to autonomic and motor neurons in the lumbosacral spinal cord. AIM: The current study tested the hypothesis that ejaculatory reflexes are dependent on LSt cells via projections within the lumbosacral spinal cord. METHODS: Male rats received intraspinal injections of neurotoxin saporin conjugated to substance P analog, previously shown to selectively lesion LSt cells. Two weeks later, males were anesthetized and spinal cords were transected. Subsequently, males were subjected to ejaculatory reflex paradigms, including stimulation of the dorsal penile nerve (DPN), urethrogenital stimulation or administration of D3 agonist 7-OH-DPAT. Electromyographic recordings of the bulbocavernosus muscle (BCM) were analyzed for rhythmic bursting characteristic of the expulsion phase of ejaculation. In addition, a fourth commonly used paradigm for ejaculation and erections in unanesthetized, spinal-intact male rats was utilized: the ex copula reflex paradigm. MAIN OUTCOME MEASURES: LSt cell lesions were predicted to prevent rhythmic bursting of BCM following DPN, urethral, or pharmacological stimulation, and emissions in the ex copula paradigm. In contrast, LSt cell lesions were not expected to abolish erectile function as measured in the ex copula paradigm. RESULTS: LSt cell lesions prevented rhythmic contractions of the BCM induced by any of the ejaculatory reflex paradigms in spinalized rats. However, LSt cell lesions did not affect erectile function nor emissions determined in the ex copula reflex paradigm. CONCLUSIONS: These data demonstrate that LSt cells are essential for ejaculatory, but not erectile reflexes, as previously reported for mating animals. Moreover, LSt cells mediate ejaculation via projections within the spinal cord, presumably to autonomic and motor neurons.

Neuroendocrine control of gonadotropin-releasing hormone: Pulsatile and surge modes of secretion.

The concept that different systems control episodic and surge secretion of gonadotropin-releasing hormone (GnRH) was well established by the time that GnRH was identified and formed the framework for studies of the physiological roles of GnRH, and later kisspeptin. Here, we focus on recent studies identifying the neural mechanisms underlying these two modes of secretion, with an emphasis on their core components. There is now compelling data that kisspeptin neurons in the arcuate nucleus that also contain neurokinin B (NKB) and dynorphin (i.e., KNDy cells) and their projections to GnRH dendrons constitute the GnRH pulse generator in mice and rats. There is also strong evidence for a similar role for KNDy neurons in sheep and goats, and weaker data in monkeys and humans. However, whether KNDy neurons act on GnRH dendrons and/or GnRH soma and dendrites that are found in the mediobasal hypothalamus (MBH) of these species remains unclear. The core components of the GnRH/luteinising hormone surge consist of an endocrine signal that initiates the process and a neural trigger that drives GnRH secretion during the surge. In all spontaneous ovulators, the core endocrine signal is a rise in estradiol secretion from the maturing follicle(s), with the site of estrogen positive feedback being the rostral periventricular kisspeptin neurons in rodents and neurons in the MBH of sheep and primates. There is considerable species variations in the neural trigger, with three major classes. First, in reflex ovulators, this trigger is initiated by coitus and carried to the hypothalamus by neural or vascular pathways. Second, in rodents, there is a time of day signal that originates in the suprachiasmatic nucleus and activates rostral periventricular kisspeptin neurons and GnRH soma and dendrites. Finally, in sheep nitric oxide-producing neurons in the ventromedial nucleus, KNDy neurons and rostral kisspeptin neurons all appear to participate in driving GnRH release during the surge.

Prenatal androgen treatment impairs the suprachiasmatic nucleus arginine-vasopressin to kisspeptin neuron circuit in female mice.

Polycystic ovary syndrome (PCOS) is associated with elevated androgen and luteinizing hormone (LH) secretion and with oligo/anovulation. Evidence indicates that elevated androgens impair sex steroid hormone feedback regulation of pulsatile LH secretion. Hyperandrogenemia in PCOS may also disrupt the preovulatory LH surge. The mechanisms through which this might occur, however, are not fully understood. Kisspeptin (KISS1) neurons of the rostral periventricular area of the third ventricle (RP3V) convey hormonal cues to gonadotropin-releasing hormone (GnRH) neurons. In rodents, the preovulatory surge is triggered by these hormonal cues and coincident timing signals from the central circadian clock in the suprachiasmatic nucleus (SCN). Timing signals are relayed to GnRH neurons, in part, via projections from SCN arginine-vasopressin (AVP) neurons to RP3VKISS1 neurons. Because rodent SCN cells express androgen receptors (AR), we hypothesized that these circuits are impaired by elevated androgens in a mouse model of PCOS. In prenatally androgen-treated (PNA) female mice, SCN Ar expression was significantly increased compared to that found in prenatally vehicle-treated mice. A similar trend was seen in the number of Avp-positive SCN cells expressing Ar. In the RP3V, the number of kisspeptin neurons was preserved. Anterograde tract-tracing, however, revealed reduced SCNAVP neuron projections to the RP3V and a significantly lower proportion of RP3VKISS1 neurons with close appositions from SCNAVP fibers. Functional assessments showed, on the other hand, that RP3VKISS1 neuron responses to AVP were maintained in PNA mice. These findings indicate that PNA changes some of the neural circuits that regulate the preovulatory surge. These impairments might contribute to ovulatory dysfunction in PNA mice modeling PCOS.

Artificial feeding synchronizes behavioral, hormonal, metabolic and neural parameters in mother-deprived neonatal rabbit pups.

Nursing in the rabbit is under circadian control, and pups have a daily anticipatory behavioral arousal synchronized to this unique event, but it is not known which signal is the main entraining cue. In the present study, we hypothesized that food is the main entraining signal. Using mother-deprived pups, we tested the effects of artificial feeding on the synchronization of locomotor behavior, plasma glucose, corticosterone, c-Fos (FOS) and PERIOD1 (PER1) rhythms in suprachiasmatic, supraoptic, paraventricular and tuberomammillary nuclei. At postnatal day 1, an intragastric tube was placed by gastrostomy. The next day and for the rest of the experiment, pups were fed with a milk formula through the cannula at either 02:00 h or 10:00 h [feeding time = zeitgeber time (ZT)0]. At postnatal days 5-7, pups exhibited behavioral arousal, with a significant increase in locomotor behavior 60 min before feeding. Glucose levels increased after feeding, peaking at ZT4-ZT12 and then declining. Corticosterone levels were highest around the time of feeding, and then decreased to trough concentrations at ZT12-ZT16, increasing again in anticipation of the next feeding bout. In the brain, the suprachiasmatic nucleus had a rhythm of FOS and PER1 that was not significantly affected by the feeding schedule. Conversely, the supraoptic, paraventricular and tuberomammillary nuclei had rhythms of both FOS and PER1 induced by the time of scheduled feeding. We conclude that the nursing rabbit pup is a natural model of food entrainment, as food, in this case milk formula, is a strong synchronizing signal for behavioral, hormonal, metabolic and neural parameters.