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.

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.

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.

KNDy Cells Revisited.

In the past decade since kisspeptin/neurokinin B/dynorphin (KNDy) cells were first identified in the mammalian hypothalamus, a plethora of new research has emerged adding insights into the role of this neuronal population in reproductive neuroendocrine function, including the basis for GnRH pulse generation and the mechanisms underlying the steroid feedback control of GnRH secretion. In this mini-review, we provide an update of evidence regarding the roles of KNDy peptides and their postsynaptic receptors in producing episodic GnRH release and assess the relative contribution of KNDy neurons to the "GnRH pulse generator." In addition, we examine recent work investigating the role of KNDy neurons as mediators of steroid hormone negative feedback and review evidence for their involvement in the preovulatory GnRH/LH surge, taking into account species differences that exist among rodents, ruminants, and primates. Finally, we summarize emerging roles of KNDy neurons in other aspects of reproductive function and in nonreproductive functions and discuss critical unresolved questions in our understanding of KNDy neurobiology.

Evidence That Dynorphin Acts Upon KNDy and GnRH Neurons During GnRH Pulse Termination in the Ewe.

A subpopulation of neurons located within the arcuate nucleus, colocalizing kisspeptin, neurokinin B, and dynorphin (Dyn; termed KNDy neurons), represents key mediators of pulsatile GnRH secretion. The KNDy model of GnRH pulse generation proposes that Dyn terminates each pulse. However, it is unknown where and when during a pulse that Dyn is released to inhibit GnRH secretion. Dyn acts via the κ opioid receptor (KOR), and KOR is present in KNDy and GnRH neurons in sheep. KOR, similar to other G protein-coupled receptors, are internalized after exposure to ligand, and thus internalization can be used as a marker of endogenous Dyn release. Thus, we hypothesized that KOR will be internalized at pulse termination in both KNDy and GnRH neurons. To test this hypothesis, GnRH pulses were induced in gonad-intact anestrous ewes by injection of neurokinin B (NKB) into the third ventricle and animals were euthanized at times of either pulse onset or termination. NKB injections produced increased internalization of KOR within KNDy neurons during both pulse onset and termination. In contrast, KOR internalization into GnRH neurons was seen only during pulse termination, and only in GnRH neurons within the mediobasal hypothalamus (MBH). Overall, our results indicate that Dyn is released onto KNDy cells at the time of pulse onset, and continues to be released during the duration of the pulse. In contrast, Dyn is released onto MBH GnRH neurons only at pulse termination and thus actions of Dyn upon KNDy and GnRH cell bodies may be critical for pulse termination.

Do Substance P and Neurokinin A Play Important Roles in the Control of LH Secretion in Ewes?

There is now general agreement that neurokinin B (NKB) acts via neurokinin-3-receptor (NK3R) to stimulate secretion of GnRH and LH in several species, including rats, mice, sheep, and humans. However, the roles of two other tachykinins, substance P (SP) and neurokinin A, which act primarily via NK1R and NK2R, respectively, are less clear. In rodents, these signaling pathways can stimulate LH release and substitute for NKB signaling; in humans, SP is colocalized with kisspeptin and NKB in the mediobasal hypothalamus. In this study, we examined the possible role of these tachykinins in control of the reproductive axis in sheep. Immunohistochemistry was used to describe the expression of SP and NK1R in the ovine diencephalon and determine whether these proteins are colocalized in kisspeptin or GnRH neurons. SP-containing cell bodies were largely confined to the arcuate nucleus, but NK1R-immunoreactivity was more widespread. However, there was very low coexpression of SP or NK1R in kisspeptin cells and none in GnRH neurons. We next determined the minimal effective dose of these three tachykinins that would stimulate LH secretion when administered into the third ventricle of ovary-intact anestrous sheep. A much lower dose of NKB (0.2 nmol) than of neurokinin A (2 nmol) or SP (10 nmol) consistently stimulated LH secretion. Moreover, the relative potency of these three neuropeptides parallels the relative selectivity of NK3R. Based on these anatomical and pharmacological data, we conclude that NKB-NK3R signaling is the primary pathway for the control of GnRH secretion by tachykinins in ewes.

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.

κ-Opioid Receptor Is Colocalized in GnRH and KNDy Cells in the Female Ovine and Rat Brain.

Kisspeptin-neurokinin B-dynorphin (KNDy) cells of the hypothalamus are a key component in the neuroendocrine regulation of GnRH secretion. Evidence in sheep and other species suggests that dynorphin released by KNDy cells inhibits pulsatile GnRH secretion by acting upon κ-opioid receptors (KOR). However, the precise anatomical location and neurochemical phenotype of KOR-expressing cells in sheep remain unknown. To this end, we determined the distribution of KOR mRNA and protein in the brains of luteal phase ewes, using an ovine specific KOR mRNA probe for in situ hybridization and an antibody whose specificity we confirmed by Western blot analyses and blocking peptide controls. KOR cells were observed in a number of regions, including the preoptic area (POA); anterior hypothalamic area; supraoptic and paraventricular nuclei; ventromedial, dorsomedial, and lateral hypothalamus; and arcuate nucleus. Next, we determined whether KOR is colocalized in KNDy and/or GnRH cells. Dual-label immunofluorescence and confocal analysis of the KNDy population showed a high degree of colocalization, with greater than 90% of these neurons containing KOR. Surprisingly, GnRH cells also showed high levels of colocalization in sheep, ranging from 74.4% to 95.4% for GnRH cells in the POA and medial basal hypothalamus, respectively. Similarly, 97.4% of GnRH neurons in the POA of ovariectomized, steroid-primed female rats also contained immunoreactive KOR protein. These findings suggest that the inhibitory effects of dynorphin on pulsatile GnRH secretion may occur either indirectly by actions upon KOR within the KNDy population and/or directly via the activation of KOR on GnRH cells.

Evidence that orphanin FQ mediates progesterone negative feedback in the ewe.

Orphanin FQ (OFQ), a member of the opioid family, is found in many areas of the hypothalamus and, when given centrally OFQ inhibits episodic LH secretion in rodents and sheep. Because GnRH neurons are devoid of the appropriate receptors to mediate steroid negative feedback directly, neurons that release OFQ may be involved. Using immunocytochemistry, we first determined that most OFQ neurons in the arcuate nucleus (ARC) and other hypothalamic regions of luteal phase ewes contained both estrogen receptor α and progesterone (P) receptor. Given a similar high degree of steroid receptor colocalization in other ARC subpopulations, we examined whether OFQ neurons of the ARC contained those other neuropeptides and neurotransmitters. OFQ did not colocalize with kisspeptin, tyrosine hydroxylase, or agouti-related peptide, but all ARC OFQ neurons coexpressed proopiomelanocortin. To test for a role for endogenous OFQ, we examined the effects of an OFQ receptor antagonist, [Nphe1,Arg14,Lys15]Nociceptin-NH2 (UFP-101) (30 nmol intracerebroventricular/h), on LH secretion in steroid-treated ewes in the breeding season and ovary-intact ewes in anestrus. Ovariectomized ewes with luteal phase concentrations of P and estradiol showed a significant increase in LH pulse frequency during infusion of UFP-101 (4.5 ± 0.5 pulses/6 h) compared with saline infusion (2.6 ± 0.4 pulses/6 h), whereas ewes implanted with only estradiol did not. Ovary-intact anestrous ewes displayed no significant differences in LH pulse amplitude or frequency during infusion of UFP-101. Therefore, we conclude that OFQ mediates, at least in part, the negative feedback action of P on GnRH/LH pulse frequency in sheep.

Kisspeptin neurons from mice to men: similarities and differences.

The discovery that kisspeptin was critical for normal fertility in humans ushered in a new chapter in our understanding of the control of GnRH secretion. In this paper, we will review recent data on the similarities and differences across several mammalian species in the role of kisspeptin in reproductive neuroendocrinology. In all mammals examined to date, there is strong evidence that kisspeptin plays a key role in the onset of puberty and is necessary for both tonic and surge secretion of GnRH in adults, although kisspeptin-independent systems are also apparent in these studies. Similarly, two groups of kisspeptin neurons, one in the arcuate nucleus (ARC) and the other more rostrally, have been identified in all mammals, although the latter is concentrated in a limited area in rodents and more scattered in other species. Estrogen has divergent actions on kisspeptin expression in these two regions across these species, stimulating it the latter and inhibiting expression in the former. There is also strong evidence that the rostral population participates in the GnRH surge, whereas the ARC population contributes to steroid-negative feedback. There may be species differences in the role of these two populations in puberty, with the ARC cells important in rats, sheep, and monkeys, whereas both have been implicated in mice. ARC kisspeptin neurons also appear to participate in the GnRH surge in sheep and guinea pigs, whereas the data on this possibility in rodents are contradictory. Similarly, both populations are sexually dimorphic in sheep and humans, whereas most data in rodents indicate that this occurs only in the rostral population. The functional consequences of these species differences remain to be fully elucidated but are likely to have significance for understanding normal neuroendocrine control of reproduction as well as for use of kisspeptin agonists/antagonists as a therapeutic tool.

KNDy (kisspeptin/neurokinin B/dynorphin) neurons are activated during both pulsatile and surge secretion of LH in the ewe.

KNDy (kisspeptin/neurokinin B/dynorphin) neurons of the arcuate nucleus (ARC) appear to mediate the negative feedback actions of estradiol and are thought to be key regulators of pulsatile LH secretion. In the ewe, KNDy neurons may also be involved with the positive feedback actions of estradiol (E(2)) to induce the LH surge, but the role of kisspeptin neurons in the preoptic area (POA) remains unclear. The goal of this study was to identify which population(s) of kisspeptin neurons is (are) activated during the LH surge and in response to the removal of E(2)-negative feedback, using Fos as an index of neuronal activation. Dual-label immunocytochemistry for kisspeptin and Fos was performed on sections containing the ARC and POA from ewes during the luteal phase of the estrous cycle, or before or after the onset of the LH surge (experiment 1), and from ovary-intact, short-term (24 h) and long-term (>30 d) ovariectomized (OVX) ewes in anestrus (experiment 2). The percentage of kisspeptin neurons expressing Fos in both the ARC and POA was significantly higher during the LH surge. In contrast, the percentage of kisspeptin/Fos colocalization was significantly increased in the ARC, but not POA, after both short- and long-term E(2) withdrawal. Thus, POA kisspeptin neurons in the sheep are activated during, and appear to contribute to, E(2)-positive feedback, whereas ARC kisspeptin (KNDy) neurons are activated during both surge and pulsatile modes of secretion and likely play a role in mediating both positive and negative feedback actions of E(2) on GnRH secretion in the ewe.

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.

Lesions of orexin neurons block conditioned place preference for sexual behavior in male rats.

The hypothalamic neuropeptide orexin (hypocretin) mediates reward related to drugs of abuse and food intake. However, a role for orexin in sexual reward has yet to be investigated. Orexin neurons are activated by sexual behavior, but endogenous orexin does not appear to be essential for sexual performance and motivation in male rats. Therefore, the goal of the current study was to test the hypothesis that orexin is critically involved in processing of sexual reward in male rats. First, it was demonstrated following exposure to conditioned contextual cues associated with sexual behavior in a conditioned place preference paradigm that cFos expression is induced in orexin neurons, indicating activation of orexin neurons by cues predicting sexual reward. Next, orexin-cell specific lesions were utilized to determine the functional role of orexin in sexual reward processing. Hypothalami of adult male rats were infused with orexin-B-conjugated saporin, resulting in greater than 80% loss of orexin neurons in the perifornical-dorsomedial and lateral hypothalamus. Orexin lesions did not affect expression of sexual behavior, but prevented formation of conditioned place preference for a sexual behavior paired chamber. In contrast, intact sham-treated males or males with partial lesions developed a conditioned place preference for mating. Orexin lesioned males maintained the ability to form a conditioned place aversion to lithium chloride-induced visceral illness, indicating that orexin lesions did not disrupt associative contextual memory. Overall, these findings suggest that orexin is not essential for sexual performance or motivation, but is critical for reward processing and conditioned cue-induced seeking of sexual behavior.

Anatomy of the kisspeptin neural network in mammals.

Kisspeptin has been recognized as a key regulator of GnRH secretion during puberty and adulthood, conveying the feedback influence of endogenous gonadal steroids onto the GnRH system. Understanding the functional roles of this peptide depends on knowledge of the anatomical framework in which it acts, including the location of kisspeptin-expressing cells in the brain and their connections. In this paper, we review current data on the anatomy of the kisspeptin neuronal network, including its colocalization with gonadal steroid hormone receptors, anatomical sites of interaction with the GnRH system, and recent evidence of neurochemical heterogeneity among different kisspeptin neuronal populations. Evidence to date suggests that kisspeptin cells in mammals comprise an interconnected network, with reciprocal connections both within and between separate cell populations, and with GnRH neurons. At the same time, there is more functional and anatomical heterogeneity in this system than originally thought, and many unanswered questions remain concerning anatomical relationships of kisspeptin neurons with other neuroendocrine and neural systems in the brain.

Orexin mediates initiation of sexual behavior in sexually naive male rats, but is not critical for sexual performance.

The hypothalamic neuropeptide orexin mediates arousal, sleep, and naturally rewarding behaviors, including food intake. Male sexual behavior is altered by orexin receptor-1 agonists or antagonists, suggesting a role for orexin-A in this naturally rewarding behavior. However, the specific role of endogenous orexin-A or B in different elements of male sexual behavior is currently unclear. Therefore, the current studies utilized markers for neural activation and orexin cell-specific lesions to test the hypothesis that orexin is critical for sexual motivation and performance in male rats. First, cFos expression in orexin neurons was demonstrated following presentation of a receptive or non-receptive female without further activation by different elements of mating. Next, the functional role of orexin was tested utilizing orexin-B conjugated saporin, resulting in orexin cell body lesions in the hypothalamus. Lesions were conducted in sexually naive males and subsequent sexual behavior was recorded during four mating trials. Lesion males showed shortened latencies to mount and intromit during the first, but not subsequent mating trials, suggesting lesions facilitated initiation of sexual behavior in sexually naive, but not experienced males. Likewise, lesions did not affect sexual motivation in experienced males, determined by runway tests. Finally, elevated plus maze tests demonstrated reduced anxiety-like behaviors in lesioned males, supporting a role for orexin in anxiety associated with initial exposure to the female in naive animals. Overall, these findings show that orexin is not critical for male sexual performance or motivation, but may play a role in arousal and anxiety related to sexual behavior in naive animals.

Bidirectional interactions between the circadian and reward systems: is restricted food access a unique zeitgeber?

Reward is mediated by a distributed series of midbrain and basal forebrain structures collectively referred to as the brain reward system. Recent evidence indicates that an additional regulatory system, the circadian system, can modulate reward-related learning. Diurnal or circadian changes in drug self-administration, responsiveness to drugs of abuse and reward to natural stimuli have been reported. These variations are associated with daily rhythms in mesolimbic electrical activity, dopamine synthesis and metabolism, and local clock gene oscillations. Conversely, the presentation of rewards appears capable of influencing circadian timing. Rodents can anticipate a daily mealtime by the entrainment of a series of oscillators that are anatomically distinct from the suprachiasmatic nucleus. Other work has indicated that restricted access to non-nutritive reinforcers (e.g. drugs of abuse, sex) or to palatable food in the absence of an energy deficit is capable of inducing relatively weak anticipatory activity, suggesting that reward alone is sufficient to induce anticipation. Recent attempts to elucidate the neural correlates of anticipation have revealed that both restricted feeding and restricted palatable food access can entrain clock gene expression in many reward-related corticolimbic structures. By contrast, restricted feeding alone can induce or entrain clock gene expression in hypothalamic nuclei involved in energy homeostasis. Thus, under ad libitum feeding conditions, the weak anticipatory activity induced by restricted reward presentation may result from the entrainment of reward-associated corticolimbic structures. The additional induction or entrainment of oscillators in hypothalamic regulatory areas may contribute to the more robust anticipatory activity associated with restricted feeding schedules.

Dynorphin immunoreactive fibers contact GnRH neurons in the human hypothalamus.

Dynorphin, an endogenous opioid peptide, mediates progesterone-negative feedback on gonadotropin-releasing hormone (GnRH) neurons in other species. The role of dynorphin in humans is unclear. The objective of this study was to determine if dynorphin fibers have close contacts with GnRH neurons in humans. Dual-label immunocytochemistry was performed on postmortem human hypothalamic tissue. The majority of GnRH neurons, 87.5%, had close contacts with dynorphin fibers and multiple close contacts were common, 62.5%. There were no regional differences between the hypothalamus and preoptic area in the distribution of close contacts. More close contacts were identified on the GnRH dendrites compared to the cell bodies (P < .001), but this difference was not significant when corrected for length. In conclusion, dynorphin fibers form close contacts with GnRH neurons in humans. This neuroanatomical evidence may suggest that dynorphin has effects on GnRH regulation in humans as seen in other species.