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.

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.

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.

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.

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.

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.

Prenatal Androgen Exposure Alters KNDy Neurons and Their Afferent Network in a Model of Polycystic Ovarian Syndrome.

Polycystic ovarian syndrome (PCOS), the most common endocrinopathy affecting women worldwide, is characterized by elevated luteinizing hormone (LH) pulse frequency due to the impaired suppression of gonadotrophin-releasing hormone (GnRH) release by steroid hormone negative feedback. Although neurons that co-express kisspeptin, neurokinin B, and dynorphin (KNDy cells) were recently defined as the GnRH/LH pulse generator, little is understood about their role in the pathogenesis of PCOS. We used a prenatal androgen-treated (PNA) mouse model of PCOS to determine whether changes in KNDy neurons or their afferent network underlie altered negative feedback. First, we identified elevated androgen receptor gene expression in KNDy cells of PNA mice, whereas progesterone receptor and dynorphin gene expression was significantly reduced, suggesting elevated androgens in PCOS disrupt progesterone negative feedback via direct actions upon KNDy cells. Second, we discovered GABAergic and glutamatergic synaptic input to KNDy neurons was reduced in PNA mice. Retrograde monosynaptic tract-tracing revealed a dramatic reduction in input originates from sexually dimorphic afferents in the preoptic area, anteroventral periventricular nucleus, anterior hypothalamic area and lateral hypothalamus. These results reveal 2 sites of neuronal alterations potentially responsible for defects in negative feedback in PCOS: changes in gene expression within KNDy neurons, and changes in synaptic inputs from steroid hormone-responsive hypothalamic regions. How each of these changes contribute to the neuroendocrine phenotype seen in in PCOS, and the role of specific sets of upstream KNDy afferents in the process, remains to be determined.

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.

Food Entrainment, Arousal, and Motivation in the Neonatal Rabbit Pup.

In the newborn rabbit, the light entrainable circadian system is immature and once a day nursing provides the primary timing cue for entrainment. In advance of the mother's arrival, pups display food anticipatory activity (FAA), and metabolic and physiological parameters are synchronized to this daily event. Central structures in the brain are also entrained as indicated by expression of Fos and Per1 proteins, GFAP, a glial marker, and cytochrome oxidase activity. Under fasting conditions, several of these rhythmic parameters persist in the periphery and brain, including rhythms in the olfactory bulb (OB). Here we provide an overview of these physiological and neurobiological changes and focus on three issues, just beginning to be examined in the rabbit. First, we review evidence supporting roles for the organum vasculosum of lamina terminalis (OVLT) and median preoptic nucleus (MnPO) in homeostasis of fluid ingestion and the neural basis of arousal, the latter which also includes the role of the orexigenic system. Second, since FAA in association with the daily visit of the mother is an example of conditioned learning, we review evidence for changes in the corticolimbic system and identified nuclei in the amygdala and extended amygdala as part of the neural substrate responsible for FAA. Third, we review recent evidence supporting the role of oxytocinergic cells of the paraventricular hypothalamic nucleus (PVN) as a link to the autonomic system that underlies physiological events, which occur in preparation for the upcoming next daily meal. We conclude that the rabbit model has contributed to an overall understanding of food entrainment.

Evidence that synaptic plasticity of glutamatergic inputs onto KNDy neurones during the ovine follicular phase is dependent on increasing levels of oestradiol.

Neurones in the arcuate nucleus co-expressing kisspeptin, neurokinin B (NKB) and dynorphin (KNDy) play a critical role in the control of gonadotrophin-releasing hormone (GnRH) and luteinising hormone (LH) secretion. In sheep, KNDy neurones mediate both steroid-negative- and -positive-feedback during pulsatile and preovulatory surge secretions of GnRH/LH, respectively. In addition, KNDy neurones receive glutamatergic inputs expressing vGlut2, a glutamate transporter that serves as a marker for those terminals, from both KNDy neurones and other populations of glutamatergic neurones. Previous work reported higher numbers of vGlut2-positive axonal inputs onto KNDy neurones during the LH surge than in luteal phase ewes. In the present study, we further examined the effects of the ovarian steroids progesterone (P) and oestradiol (E2 ) on glutamatergic inputs to KNDy neurones. Ovariectomised (OVX) ewes received either no further treatment (OVX) or steroid treatments that mimicked the luteal phase (low E2  + P), and early (low E2 ) or late follicular (high E2 ) phases of the oestrous cycle (n = 4 or 5 per group). Brain sections were processed for triple-label immunofluorescent detection of NKB/vGlut2/synaptophysin and analysed using confocal microscopy. We found higher numbers of vGlut2 inputs onto KNDy neurones in high E2 compared to the other three treatment groups. These results suggest that synaptic plasticity of glutamatergic inputs onto KNDy neurones during the ovine follicular phase depend on increasing levels of E2 required for the preovulatory GnRH/surge. These synaptic changes likely contribute to the positive-feedback action of oestrogen on GnRH/LH secretion and thus the generation of the preovulatory surge in the sheep.

Morphological and functional evidence for sexual dimorphism in neurokinin B signalling in the retrochiasmatic area of sheep.

Neurokinin B (NKB) is critical for fertility in humans and stimulates gonadotrophin-releasing hormone/luteinising hormone (LH) secretion in several species, including sheep. There is increasing evidence that the actions of NKB in the retrochiasmatic area (RCh) contribute to the induction of the preovulatory LH surge in sheep. In the present study, we determined whether there are sex differences in the response to RCh administration of senktide, an agonist to the NKB receptor (neurokinin receptor-3 [NK3R]), and in NKB and NK3R expression in the RCh of sheep. To normalise endogenous hormone concentrations, animals were gonadectomised and given implants to mimic the pattern of ovarian steroids seen in the oestrous cycle. In females, senktide microimplants in the RCh produced an increase in LH concentrations that lasted for at least 8 hours after the start of treatment, whereas a much shorter increment (approximately 2 hours) was seen in males. We next collected tissue from gonadectomised lambs 18 hours after the insertion of oestradiol implants that produce an LH surge in female, but not male, sheep for immunohistochemical analysis of NKB and NK3R expression. As expected, there were more NKB-containing neurones in the arcuate nucleus of females than males. Interestingly, there was a similar sexual dimorphism in NK3R-containing neurones in the RCh, NKB-containing close contacts onto these RCh NK3R neurones, and overall NKB-positive fibres in this region. These data demonstrate that there are both functional and morphological sex differences in NKB-NK3R signalling in the RCh and raise the possibility that this dimorphism contributes to the sex-dependent ability of oestradiol to induce an LH surge in female sheep.

Evidence that Nitric Oxide Is Critical for LH Surge Generation in Female Sheep.

Elevated and sustained estradiol concentrations cause a gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) surge that is necessary for ovulation. In sheep, several different neural systems have been implicated in this stimulatory action of estradiol and this study focused on somatostatin (SST) neurons in the ventral lateral region of the ventral medial nucleus (vlVMN) which express c-Fos during the surge. First, we determined if increased activity of SST neurons could be related to elevated GnRH secretion by assessing SST synapses onto GnRH neurons and neurons coexpressing kisspeptin, neurokinin B, dynorphin (KNDy). We found that the percentage of preoptic area GnRH neurons that receive SST input increased during the surge compared with other phases of the cycle. However, since SST is generally inhibitory, and pharmacological manipulation of SST signaling did not alter the LH surge in sheep, we hypothesized that nitric oxide (NO) was also produced by these neurons to account for their activation during the surge. In support of this hypothesis we found that (1) the majority of SST cells in the vlVMN (>80%) contained neuronal nitric oxide synthase (nNOS); (2) the expression of c-Fos in dual-labeled SST-nNOS cells, but not in single-labeled cells, increased during the surge compared with other phases of the cycle; and (3) intracerebroventricular (ICV) infusion of the nitric oxide synthase inhibitor, N(G)-nitro-L-arginine methyl ester, completely blocked the estrogen-induced LH surge. These data support the hypothesis that the population of SST-nNOS cells in the vlVMN are a source of NO that is critical for the LH surge, and we propose that they are an important site of estradiol positive feedback in sheep.

Role for Kisspeptin and Neurokinin B in Regulation of Luteinizing Hormone and Testosterone Secretion in the Fetal Sheep.

Evidence suggests that the hypothalamic-pituitary-gonadal (HPG) axis is active during the critical period for sexual differentiation of the ovine sexually dimorphic nucleus, which occurs between gestational day (GD) 60 and 90. Two possible neuropeptides that could activate the fetal HPG axis are kisspeptin and neurokinin B (NKB). We used GD85 fetal lambs to determine whether intravenous administration of kisspeptin-10 (KP-10) or senktide (NKB agonist) could elicit luteinizing hormone (LH) release. Immunohistochemistry and fluorescent in situ hybridization (FISH) were employed to localize these peptides in brains of GD60 and GD85 lamb fetuses. In anesthetized fetuses, KP-10 elicited robust release of LH that was accompanied by a delayed rise in serum testosterone in males. Pretreatment with the GnRH receptor antagonist (acyline) abolished the LH response to KP-10, confirming a hypothalamic site of action. In unanesthetized fetuses, senktide, as well as KP-10, elicited LH release. The senktide response of females was greater than that of males, indicating a difference in NKB sensitivity between sexes. Gonadotropin-releasing hormone also induced a greater LH discharge in females than in males, indicating that testosterone negative feedback is mediated through pituitary gonadotrophs. Kisspeptin and NKB immunoreactive cells in the arcuate nucleus were more abundant in females than in males. Greater than 85% of arcuate kisspeptin cells costained for NKB. FISH revealed that the majority of these were kisspeptin/NKB/dynorphin (KNDy) neurons. These results support the hypothesis that kisspeptin-GnRH signaling regulates the reproductive axis of the ovine fetus during the prenatal critical period acting to maintain a stable androgen milieu necessary for brain masculinization.

Does the KNDy Model for the Control of Gonadotropin-Releasing Hormone Pulses Apply to Monkeys and Humans?

There is now considerable evidence supporting the role of a subpopulation of neurons in the arcuate nucleus of the hypothalamus that coexpress kisspeptin, neurokinin B, and dynorphin (abbreviated as KNDy neurons) as the long sought-after gonadotropin-releasing hormone (GnRH) pulse generator. The "KNDy hypothesis" of pulse generation has largely been based on findings in rodents and ruminants, and there is considerably less information about the anatomical and functional organization of the KNDy subpopulation in the primate hypothalamus. In this review, we focus on the applicability of this hypothesis, and the roles of kisspeptin, neurokinin B, and dynorphin in reproduction, to humans and nonhuman primates, reviewing available data and pointing out important gaps in our current knowledge. With recent application of drugs that target KNDy peptides and their receptors to therapeutic treatments for reproductive disorders, it is imperative we fully understand the primate KNDy network and its role in the control of GnRH secretion, as well as species differences in this system that may exist between humans, nonhuman primates, and other mammals.

Evidence That the LH Surge in Ewes Involves Both Neurokinin B-Dependent and -Independent Actions of Kisspeptin.

Recent evidence has implicated neurokinin B (NKB) signaling in the retrochiasmatic area (RCh) of the ewe in the LH surge. To test this hypothesis, we first lesioned NK3R neurons in this area by using a saporin conjugate (NK3-SAP). Three weeks after bilateral injection of NK3-SAP or a blank control (BLK-SAP) into the RCh, an LH surge was induced by using an artificial follicular-phase model in ovariectomized ewes. NK3-SAP lesioned approximately 88% of RCh NK3R-containing neurons and reduced the amplitude of the estrogen-induced LH surge by 58%, an inhibition similar to that seen previously with intracerebroventricular (icv) infusion of a KISS1R antagonist (p271). We next tested the hypothesis that NKB signaling in the RCh acts via kisspeptin by determining whether the combined effects of NK3R-SAP lesions and icv infusion of p271 were additive. Experiment 1 was replicated except that ewes received two sequential artificial follicular phases with infusions of p271 or vehicle using a crossover design. The combination of the two treatments decreased the peak of the LH surge by 59%, which was similar to that seen with NK3-SAP (52%) or p271 (54%) alone. In contrast, p271 infusion delayed the onset and peak of the LH surge in both NK3-SAP- and BLK-SAP-injected ewes. Based on these data, we propose that NKB signaling in the RCh increases kisspeptin levels critical for the full amplitude of the LH surge in the ewe but that kisspeptin release occurs independently of RCh input at the onset of the surge to initiate GnRH secretion.

Kisspeptin/Neurokinin B/Dynorphin (KNDy) cells as integrators of diverse internal and external cues: evidence from viral-based monosynaptic tract-tracing in mice.

Neurons in the hypothalamic arcuate nucleus (ARC) that co-express kisspeptin, neurokinin B and dynorphin (KNDy cells) are essential for mammalian reproduction as key regulators of gonadotropin-releasing hormone (GnRH) secretion. Although multiple endogenous and exogenous signals act indirectly via KNDy neurons to regulate GnRH, the identity of upstream neurons that provide synaptic input to this subpopulation is unclear. We used rabies-mediated tract-tracing in transgenic Kiss1-Cre mice combined with whole-brain optical clearing and multiple-label immunofluorescence to create a comprehensive and quantitative brain-wide map of neurons providing monosynaptic input to KNDy cells, as well as identify the estrogen receptor content and peptidergic phenotype of afferents. Over 90% of monosynaptic input to KNDy neurons originated from hypothalamic nuclei in both male and female mice. The greatest input arose from non-KNDy ARC neurons, including proopiomelanocortin-expressing cells. Significant female-dominant sex differences in afferent input were detected from estrogen-sensitive hypothalamic nuclei critical for reproductive endocrine function and sexual behavior in mice, indicating KNDy cells may provide a unique site for the coordination of sex-specific behavior and gonadotropin release. These data provide key insight into the structural framework underlying the ability of KNDy neurons to integrate endogenous and environmental signals important for the regulation of reproductive function.

Prenatal Testosterone Exposure Alters GABAergic Synaptic Inputs to GnRH and KNDy Neurons in a Sheep Model of Polycystic Ovarian Syndrome.

Prenatal testosterone (T)-treated female sheep display reproductive deficits similar to women with polycystic ovarian syndrome (PCOS), including an increase in LH pulse frequency due to actions of the central GnRH pulse generator. In this study, we used multiple-label immunocytochemistry to investigate the possibility of changes in the γ-aminobutyric acid (GABA) neurotransmitter system at two key components of the GnRH pulse generator in prenatal T-treated sheep: kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus, and GnRH neurons in the preoptic area (POA) and mediobasal hypothalamus (MBH). We observed a significant decrease and increase, respectively, in the number of GABAergic synapses onto POA and MBH GnRH neurons in prenatal T-treated ewes; additionally, there was a significant increase in the number of GABAergic inputs onto KNDy neurons. To determine the actions of GABA on GnRH and KNDy neurons, we examined colocalization with the chloride transporters NKCC1 and KCC2, which indicate stimulatory or inhibitory activation of neurons by GABA, respectively. Most GnRH neurons in both POA and MBH colocalized NKCC1 cotransporter whereas none contained the KCC2 cotransporter. Most KNDy neurons colocalized either NKCC1 or KCC2, and 28% of the KNDy population contained NKCC1 alone. Therefore, we suggest that, as in the mouse, GABA in the sheep is stimulatory to GnRH neurons, as well as to a subset of KNDy neurons. Increased numbers of stimulatory GABAergic inputs to both MBH GnRH and KNDy neurons in prenatal T-treated animals may contribute to alterations in steroid feedback control and increased GnRH/LH pulse frequency seen in this animal model of PCOS.