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Puberty is the process of achieving the capacity for reproduction and depends upon increased gonadotropin-releasing hormone (GnRH) secretion. Neurons in the arcuate nucleus of the hypothalamus (ARC) coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (i.e. KNDy cells) are critical for puberty onset. In humans and livestock, mistimed puberty has negative consequences for health and production, respectively. Puberty is largely impacted by genetics and genes associated with its timing include those for kisspeptin and NKB. Epigenetic modification of genes and transcriptional activity also influence puberty onset. Adequate nutrition is required for normal puberty onset and the hormone leptin links metabolic status and puberty. Leptin acts through ARC neurons reciprocally connected with KNDy neurons that transduce nutritional signals into inputs influencing GnRH neurons. Further work using multiple animal models, including agriculturally important species, will be necessary for understanding the neural mechanisms underlying puberty onset and the problems associated with mistimed puberty.

The anatomy and morphology of gonadotropin-releasing hormone (GnRH) neurons makes them both a joy and a challenge to investigate. They are a highly unique population of neurons given their developmental migration into the brain from the olfactory placode, their relatively small number, their largely scattered distribution within the rostral forebrain, and, in some species, their highly varied individual anatomical characteristics. These unique features have posed technological hurdles to overcome and promoted fertile ground for the establishment and use of creative approaches. Historical and more contemporary discoveries defining GnRH neuron anatomy remain critical in shaping and challenging our views of GnRH neuron function in the regulation of reproductive function. We begin this review with a historical overview of anatomical discoveries and developing methodologies that have shaped our understanding of the reproductive axis. We then highlight significant discoveries across specific groups of mammalian species to address some of the important comparative aspects of GnRH neuroanatomy. Lastly, we touch on unresolved questions and opportunities for future neuroanatomical research on this fascinating and important population of neurons.

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.

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 comprised of arcuate “KNDy” cells, the abbreviation based on co-expression 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.

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.

The discovery of episodic LH secretion in 1970 initiated considerable effort by reproductive neuroendocrinologists to understand the neural mechanisms underlying the synchronous release of GnRH that drives pulsatile LH secretion. Our previous chapter reviewed the history of these efforts and considered a new model for the GnRH pulse generator based on KNDy neurons in the arcuate nucleus that contain k isspeptin, n eurokinin B, and d ynorphin. In this update of that chapter, we retain the original historical material and discuss recent experimental tests of the roles of these peptides. Based on these studies, we propose a modified model for the GnRH pulse generator in ruminants and rodents, the core of which is comprised of KNDy neurons, and discuss whether this model is applicable to humans and other primates.

20-HETE is a potent vasoconstrictor that is implicated in the regulation of blood pressure, cerebral blood flow and neuronal death following ischemia. Numerous human genetic studies have shown that inactivating variants in the cytochrome P450 enzymes that produce 20-HETE are associated with hypertension, stroke and cerebrovascular disease. However, little is known about the expression and cellular distribution of the cytochrome P450A enzymes (CYP4A) that produce 20-HETE or the newly discovered 20-HETE receptor (GPR75) in the brain. The present study examined the cell types and regions in the rat forebrain that express CYP4A and GPR75. Brain tissue slices from Sprague Dawley (SD), Dahl Salt-Sensitive (SS) and CYP4A1 transgenic rat strains, as well as cultured human cerebral pericytes and cerebral vascular smooth muscle cells, were analyzed by fluorescent immunostaining. Tissue homogenates from these strains and cultured cells were examined by Western blot. In the cerebral vasculature, CYP4A and GPR75 were expressed in endothelial cells, vascular smooth muscle cells and the glial limiting membrane of pial arteries and penetrating arterioles but not in the endothelium of capillaries. CYP4A, but not GPR75, was expressed in astrocytes. CYP4A and GPR75 were both expressed in a subpopulation of pericytes on capillaries. The diameters of capillaries were significantly decreased at the sites of first and second-order pericytes that expressed CYP4A. Capillary diameters were unaffected at the sites of other pericytes that did not express CYP4A. These findings implicate 20-HETE as a paracrine mediator in various components of the neurovascular unit and are consistent with 20-HETE's emerging role in the regulation of cerebral blood flow, blood-brain barrier integrity, the pathogenesis of stroke and the vascular contributions to cognitive impairment and dementia. Moreover, this study highlights GPR75 as a potential therapeutic target for the treatment of these devastating conditions.

The vision of a scientist as a lone investigator reaching an epiphany is a widely cherished narrative. Consistent with this ideal, single-author papers were frequent 50 years ago, when the Society for Neuroscience started. However, the basic and translational questions and the public health

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 E2decreasing kisspeptin and progesterone increasing dynorphin release onto GnRH neurons. In pre-pubertal lambs, E2inhibits 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; E2increases dopamine release from these neurons to inhibit KNDy neurons and suppress the frequency of kisspeptin and GnRH release.

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.

Compulsive sexual behavior (CSB) is widely regarded as a “behavioral addiction,” and is a major threat to quality of life and both physical and mental health. However, CSB has been slow to be recognized clinically as a diagnosable disorder. CSB is co-morbid with affective disorders as well as substance use disorders, and recent neuroimaging studies have demonstrated shared or overlapping neural pathologies disorders, especially in brain regions controlling motivational salience and inhibitory control. Clinical neuroimaging studies are reviewed that have identified structural and/or function changes in prefrontal cortex, amygdala, striatum, and thalamus in individuals suffering from CSB. A preclinical model to study the neural underpinnings of CSB in male rats is discussed consisting of a conditioned aversion procedure to examine seeking of sexual behavior despite known negative consequences. Using this preclinical model, a role of the medial prefrontal cortex was identified, including neural plasticity during comorbidity of CSB and psychostimulant abuse. This review summarizes recent human behavioral and neuroimaging studies, in addition to preclinical models that can be used to study the underlying neurobiology of CSB.

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.

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.

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.

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.

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.

Spinal cord injury (SCI) causes sexual dysfunction, including anejaculation in men. Likewise, chronic mid-thoracic contusion injury impairs ejaculatory reflexes in male rats. Ejaculation is controlled by a spinal ejaculation generator (SEG) comprised of a population of lumbar spinothalamic (LSt) neurons. LSt neurons co-express four neuropeptides, including gastrin-releasing peptide (GRP) and galanin and control ejaculation via release of these peptides in lumbar and sacral autonomic and motor nuclei. Here, we tested the hypothesis that contusion injury causes a disruption of the neuropeptides that are expressed in LSt cell bodies and axon terminals, thereby causing ejaculatory dysfunction. Male Sprague Dawley rats received contusion or sham surgery at spinal levels T6-7. Five to six weeks later, animals were perfused and spinal cords were immunoprocessed for galanin and GRP. Results showed that numbers of cells immunoreactive for galanin were not altered by SCI, suggesting that LSt cells are not ablated by SCI. In contrast, GRP immunoreactivity was decreased in LSt cells following SCI, evidenced by fewer GRP and galanin/GRP dual labeled cells. However, SCI did not affect efferent connections of LSt, cells as axon terminals containing galanin or GRP in contact with autonomic cells were not reduced following SCI. Finally, no changes in testosterone plasma levels or androgen receptor expression were noted after SCI. In conclusion, chronic contusion injury decreased immunoreactivity for GRP in LSt cell soma, but did not affect LSt neurons per se or LSt connections within the SEG. Since GRP is essential for triggering ejaculation, such loss may contribute to ejaculatory dysfunction following SCI.

Sheep have been a useful preclinical model for the exploration of the role of kisspeptin and its receptor, KISS1R, in the control of pulsatile section of gonadotropin releasing hormone (GnRH). However, lack of reagents to interrogate KISS1R neurons in sheep has limited progress in this species. To address this limitation, we tested the feasibility of using fluorescent in situ hybridization to monitor kiss1R mRNA and a kisspeptin-saporin conjugate to ablate KISS1R-containing neurons in a cell specific manner. Using custom-designed probes for RNAscope to detect ovine kiss1R and GnRH, a high degree of colocalization was observed in the preoptic area. In future work, we can use this technique to identify mRNAs for several different possible neurotransmitters in other kiss1R-containing neurons. The effects of kiss-saporin injections was tested in mice to determine optimal concentrations and survival times. Male and female mice of two strains received infusions of 200nL of 100ng/uL, 200ng/uL and 400ng/uL concentrations of kiss-sap and blank-sap into the preoptic area, which contains the greatest density of GnRH neuron cell bodies, and, at the level of the GnRH neuron dendron prior to termination in the median eminence. Mice were perfused two or three weeks after infusion. Two adult Suffolk ewes received unilateral kiss-saporin injections (1 ul of 700 ng/ul concentration) into the preoptic area, and one ewe received kiss-sap on one side and blank-sap on the other; brains were perfused three weeks later. Mouse and sheep brains were sectioned and immunostained for GnRH. Analysis of tissue from ewes revealed that GnRH neurons were severely reduced on the side ipsilateral to the kiss-sap injection compared to the contralateral side. In contrast, GnRH neurons were found to be intact in mice, independent of injection site, dosage, survival time, strain and sex of the mice. These results indicate that kiss-sap can be used in sheep to test the physiological role of different KISS1R-containing neuronal populations. They also demonstrate a striking difference between mice and sheep in the ability to lesion GnRH cells using kiss-saporin. This raises the possibility of differences in cell surface localization and/or internalization of KISS1R between these two species, as both are needed for saporin lesions. In conclusion, these results demonstrate the feasibility of detecting kiss1R expression in sheep GnRH neurons and using GnRH cell-specific lesions, while indicating potential differences in KISS1R cellular localization between sheep and mice.

Estradiol controls reproductive status and sexual behavior by acting upon a variety of neurochemical systems in the brain to mediate the sexually dimorphic release of gonadotrophins. However, the neural pathways that transmit steroid hormone information to gonadotropin-releasing hormone (GnRH) neurons are unclear. Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons of the arcuate nucleus (ARC) are critical for reproductive status and implicated to relay a number of peripheral signals, including gonadal steroids, to regulate GnRH secretion. We recently reported the distribution of primary afferents to KNDy neurons (Moore et al, International Congress for Neuroendocrinology, 2018), however, it is not known if KNDy neurons receive synaptic input from steroid hormone-sensitive cells. To address this, we utilized rabies-mediated tract-tracing to unequivocally determine the distribution of estradiol-sensitive monosynaptic inputs to KNDy neurons. Adult male (n=6) and female (n=4) Kiss1-Cre mice were stereotaxically injected in the ARC with Cre-dependent adenoassociated viruses (AAVs) to express avian receptor protein with GFP (TVA-GFP) and rabies glycoprotein (G) in ARC kisspeptin (KNDy) neurons. After three weeks, the glycoprotein-deleted rabies virus (RVdG) containing mCherry was injected in the same location. RVdG exclusively infects KNDy cells expressing TVA, and, as G is required for rabies to cross transynaptically, RVdG crosses into primary afferents of KNDy neurons to express mCherry. As RVdG induces toxicity in cells, a time-point trial determined mCherry-positive cells retain protein expression after 5 days. Mice were perfused and coronal sections immunolabeled for GFP to identify KNDy cells, mCherry to visualize KNDy afferents and estrogen receptor alpha (ERα). Immunoreactivity was mapped using fluorescent microscopy and slide scanning software (MBFbioscience) and dual labeled mCherry- and ERα- positive cells analyzed using confocal microscopy. Male and female mice were statistically compared using students t tests. Dual-labeled cells were detected within the bed nucleus of the stria terminalis and the hypothalamic rostral periventricular area, medial preoptic nucleus (MPON), ventromedial and anterior hypothalamus, ARC and premammillary nucleus. Compared to male mice, females exhibited a significantly higher percentage of dual-labeled cells in the periventricular nucleus (PeN, 46.7±16.4% in females, 7.8±5.1% in males, p

The spinal ejaculation generator is comprised of lumbar spinothalamic (LSt) cells and their axonal projections to autonomic and motor neurons in the lumbosacral spinal cord. LSt cells regulate ejaculatory reflexes by release of neuropeptides that are co-expressed in their axons, as previously demonstrated for gastrin-releasing peptide and enkephalin. Here, the role of two other neuropeptides co-expressed in LSt cells for ejaculatory reflexes is demonstrated: galanin and cholecystokinin (CCK). Adult male rats were anesthetized, spinalized, and received intrathecal infusions of galanin receptor antagonist Galantide (1 or 10 nmol) or CCK receptor antagonist proglumide (71 or 714 nmol). The dorsal penile nerve (DPN) was electrically stimulated to trigger ejaculatory reflexes and seminal vesicle pressure (SVP) and rhythmic contractions of the bulbocavernosus muscle (BCM) were analyzed as parameters of emission and expulsion respectively. Treatment with galanin or CCK antagonists significantly reduced SVP increases and BCM bursting, demonstrating that galanin and CCK are required for ejaculation. Next, anesthetized, spinalized males received intrathecal infusions of galanin (0.15 or 0.3 nmol) or CCK(26-33) (4.35 nmol) and effects on subthreshold DPN stimulations were determined. Intrathecal infusions of galanin or CCK facilitated ejaculatory reflexes induced by subthreshold DPN stimulation in all animals, but did not trigger ejaculatory reflexes in the absence of DPN stimulation. Together, these results demonstrate that galanin and CCK both act in the spinal ejaculation generator to regulate ejaculation. However, effects of galanin and CCK were dependent on DPN stimulation, suggesting that these neuropeptides may act in concert with other LSt co-expressed neuropeptides.

Seasonal reproduction in sheep is primarily due to a dramatic increase in the ability of estradiol (E2) to inhibit the pulsatile secretion of gonadotropin-releasing hormone (GnRH) during the nonbreeding season [anestrus (ANS)]. Recent findings suggest that kisspeptin/neurokinin B/dynorphin (KNDy) neurons of the arcuate nucleus (ARC) play a key role in conveying this negative feedback influence, with dopaminergic projections from the retrochiasmatic area acting upon KNDy cells to decrease kisspeptin release and thus inhibit GnRH pulses. However, several questions remain unanswered: (1) Are the coexpressed KNDy peptides, neurokinin B (NKB) and dynorphin, under seasonal regulation similar to kisspeptin? (2) Are seasonal changes in these peptides and their colocalization of D2 dopamine receptors (D2Rs) steroid dependent? and (3) Do KNDy neurons receive direct input from dopaminergic terminals? We used dual- and triple-label immunofluorescence to analyze brain sections through the ARC of ovariectomized (OVX) and OVX plus E2 ewes perfused during either the breeding season or ANS. Results showed (1) steroid-dependent and steroid-independent seasonal changes in kisspeptin and NKB, but not dynorphin, immunoreactivity; (2) increased D2R coexpression during ANS that was dependent on the presence of E2; and (3) evidence that KNDy cells receive direct contact from dopaminergic terminals and that this input increases during ANS. These results support the hypothesis that dopamine acts to inhibit GnRH secretion in ANS by directly suppressing the activity of ARC KNDy neurons, and implicate NKB as well as kisspeptin in seasonal shifts in E2-negative feedback in the sheep.

The use of psychostimulants is often associated with hypersexuality, and psychostimulant users have identified the effects of drug on sexual behavior as a reason for further use. It was previously demonstrated in male rats that methamphetamine (Meth), when administered concurrently with sexual behavior results in impairment of inhibition of sexual behavior in a conditioned sex aversion (CSA) paradigm where mating is paired with illness. This is indicative of maladaptive sex behavior following Meth and sex experience. The present study examined the neural pathways activated during inhibition of sexual behavior in male rats and the effects of concurrent Meth and sexual behavior on neural activity, using ERK phosphorylation (pERK). First, exposure to conditioned aversive stimuli in males trained to inhibit sexual behavior in the CSA paradigm increased pERK expression in medial prefrontal (mPFC), orbitofrontal cortex (OFC) and areas in striatum and amygdala. Second, effects of concurrent Meth and sex experience were tested in males that were exposed to four daily sessions of concurrent Meth (1 mg/kg) or saline and mating and subsequently exposed to CSA one week after last treatment. Meth and mating-treated males showed significant impairment of inhibition of mating, higher pERK expression under baseline conditions, and disrupted pERK induction by exposure to the conditioned aversive stimuli in mPFC and OFC. These alterations of pERK occurred in CaMKII-expressing neurons, suggesting changes in efferent projections of these areas. Altogether, these data show that concurrent Meth and mating experience causes maladapative sexual behavior that is associated with alterations in neural activation in mPFC and OFC.

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.

Work during the last decade has led to a novel hypothesis for a question that is half a century old: how is the secretory activity of GnRH neurons synchronized to produce episodic GnRH secretion. This hypothesis posits that a group of neurons in the arcuate nucleus (ARC) that contain kisspeptin, neurokinin B (NKB), and dynorphin (known as KNDy neurons) fire simultaneously to drive each GnRH pulse. Kisspeptin is proposed to be the output signal to GnRH neurons with NKB and dynorphin acting within the KNDy network to initiate and terminate each pulse, respectively. This review will focus on the importance of neuroanatomical studies in general and, more specifically, on the work of Dr Marcel Amstalden during his postdoctoral fellowship with the authors, to the development and testing of this hypothesis. Critical studies in sheep that laid the foundation for much of the KNDy hypothesis included the report that a group of neurons in the ARC contain both NKB and dynorphin and appear to form an interconnected network capable of firing synchronously, and Marcel's observations that the NKB receptor is found in most KNDy neurons, but not in any GnRH neurons. Moreover, reports that almost all dynorphin-NKB neurons and kisspeptin neurons in the ARC contained steroid receptors led directly to their common identification as "KNDy" neurons. Subsequent anatomical work demonstrating that KNDy neurons project to GnRH somas and terminals, and that kisspeptin receptors are found in GnRH, but not KNDy neurons, provided important tests of this hypothesis. Recent work has explored the time course of dynorphin release onto KNDy neurons and has begun to apply new approaches to the issue, such as RNAscope in situ hybridization and the use of whole tissue optical clearing with light-sheet microscopy. Together with other approaches, these anatomical techniques will allow continued exploration of the functions of the KNDy population and the possible role of other ARC neurons in generation of GnRH pulses.

Many factors, including social elements, influence drug addiction in humans and can be modeled in laboratory rodents. In general, the presence of social reward is protective against drug abuse and the absence or removal of social reward in both humans and rodents increases vulnerability to drug addiction. The current review chapter is focused on studies from our lab that have examined the effects of sociosexual behavior in male rats on drug-induced behaviors, including changes in both psychostimulant and opiate behavior. Furthermore, we review the underlying neural mechanisms by which these effects occur. Together, these results may help elucidate the neural mechanisms underlying the interaction between social and drug rewards and the mechanisms by which a loss of social rewards increase the vulnerability to drug addiction development.

Vulnerability to develop addiction is influenced by numerous factors, including social behavior. Specifically, in human users, drug taking in a socio-sexual context appears to enhance further drug-seeking behavior. Users report heightened sexual pleasure as a motivation for further drug use and display risk behaviors even when tested in drug-free state. Here, using a preclinical model of limited voluntary drug use in rats, the hypothesis was tested that methamphetamine (Meth)-taking concurrently with socio-sexual experience increases vulnerability to addiction. Male Sprague Dawley rats were socially housed and underwent limited-access Meth self-administration (maximum 1 mg/kg/session). Meth-taking was either concurrent or non-concurrent with sexual behavior: concurrent animals were mated with a receptive female immediately after each session, while non-concurrent animals gained equivalent sexual experience the week prior. Next, drug-seeking behaviors were measured during cue reactivity, extinction, and reinstatement sessions using different extinction and reinstatement protocols in 4 separate studies. Both groups equally acquired Meth self-administration and did not differ in total Meth intake. However, drug-seeking behavior was significantly higher in concurrent animals during cue reactivity tasks, extinction sessions, and cue- or Meth-induced reinstatement tests. In addition, sexual behavior in the absence of Meth triggered reinstatement of drug-seeking in concurrent animals. These results indicate that Meth-taking in a socio-sexual context significantly enhances vulnerability for drug addiction in male rats. This preclinical paradigm of drug self-administration concurrent with socio-sexual behavior provides a useful model for studying the underlying neurobiology of socially driven vulnerability to drug addiction.

The 3(rd) World Conference on Kisspeptin, “Kisspeptin 2017: Brain and Beyond” was held March 30–31 at the Rosen Centre Hotel in Orlando, Florida, providing an international forum for multidisciplinary scientists to meet and share cutting-edge research on kisspeptin biology and its relevance to human health and disease. The meeting built upon previous world conferences focused on the role of kisspeptin and associated peptides in the control of gonadotropin-releasing hormone (GnRH) secretion and reproduction. Based on recent discoveries, the scope of this meeting was expanded to include functions of kisspeptin and related peptides in other physiological systems including energy homeostasis, pregnancy, ovarian and uterine function, and thermoregulation. In addition, discussions addressed the translation of basic knowledge of kisspeptin biology to the treatment of disease, with the goal of seeking consensus about the best approaches to improve human health. The two-day meeting featured a non-traditional structure, with each day starting with poster sessions followed by lunch discussions and facilitated large-group sessions with short presentations to maximize the exchange of new, unpublished data. Topics were identified by a survey prior to the meeting, and focused on major unresolved questions, important controversies, and future directions in the field. Finally, career development activities provided mentoring for trainees and junior investigators, and networking opportunities for those individuals with established researchers in the field. Overall, the meeting was rated as a success by attendees and covered a wide range of lively and provocative discussion topics on the changing nature of the field of “kisspeptinology” and its future.

The 3rd World Conference on Kisspeptin, "Kisspeptin 2017: Brain and Beyond" was held March 30-31 at the Rosen Centre Hotel in Orlando, Florida, providing an international forum for multidisciplinary scientists to meet and share cutting-edge research on kisspeptin biology and its relevance to human health and disease. The meeting built upon previous world conferences focused on the role of kisspeptin and associated peptides in the control of gonadotropin-releasing hormone (GnRH) secretion and reproduction. Based on recent discoveries, the scope of this meeting was expanded to include functions of kisspeptin and related peptides in other physiological systems including energy homeostasis, pregnancy, ovarian and uterine function, and thermoregulation. In addition, discussions addressed the translation of basic knowledge of kisspeptin biology to the treatment of disease, with the goal of seeking consensus about the best approaches to improve human health. The two-day meeting featured a non-traditional structure, with each day starting with poster sessions followed by lunch discussions and facilitated large-group sessions with short presentations to maximize the exchange of new, unpublished data. Topics were identified by a survey prior to the meeting, and focused on major unresolved questions, important controversies, and future directions in the field. Finally, career development activities provided mentoring for trainees and junior investigators, and networking opportunities for those individuals with established researchers in the field. Overall, the meeting was rated as a success by attendees and covered a wide range of lively and provocative discussion topics on the changing nature of the field of "kisspeptinology" and its future. This article is protected by copyright. All rights reserved.

Work over the last 15 years on the control of pulsatile LH secretion has focused largely on a set of neurons in the arcuate nucleus (ARC) that contains two stimulatory neuropeptides, critical for fertility in humans (kisspeptin and neurokinin B (NKB)) and the inhibitory endogenous opioid peptide (EOP), dynorphin, and are now known as KNDy (kisspeptin-NKB-dynorphin) neurons. In this review, we consider the role of each of the KNDy peptides in the generation of GnRH pulses and the negative feedback actions of ovarian steroids, with an emphasis on NKB and dynorphin. With regard to negative feedback, there appear to be important species differences. In sheep, progesterone inhibits GnRH pulse frequency by stimulating dynorphin release, and estradiol inhibits pulse amplitude by suppressing kisspeptin. In rodents, the role of KNDy neurons in estrogen negative feedback remains controversial, progesterone may inhibit GnRH via dynorphin, but the physiological significance of this action is unclear. In primates, an EOP, probably dynorphin, mediates progesterone negative feedback, and estrogen inhibits kisspeptin expression. In contrast, there is now compelling evidence from several species that kisspeptin is the output signal from KNDy neurons that drives GnRH release during a pulse and may also act within the KNDy network to affect pulse frequency. NKB is thought to act within this network to initiate each pulse, although there is some redundancy in tachykinin signaling in rodents. In ruminants, dynorphin terminates GnRH secretion at the end of pulse, most likely acting on both KNDy and GnRH neurons, but the data on the role of this EOP in rodents are conflicting.

Many factors, including social elements, influence drug addiction in humans and can be modeled in laboratory rodents. In general, the presence of social reward is protective against drug abuse and the absence or removal of social reward in both humans and rodents increases vulnerability to drug addiction. The current review chapter is focused on studies from our lab that have examined the effects of sociosexual behavior in male rats on drug-induced behaviors, including changes in both psychostimulant and opiate behavior. Furthermore, we review the underlying neural mechanisms by which these effects occur. Together, these results may help elucidate the neural mechanisms underlying the interaction between social and drug rewards and the mechanisms by which a loss of social rewards increase the vulnerability to drug addiction development.

Work over the last 15 years on the control of pulsatile LH secretion has focused largely on a set of neurons in the arcuate nucleus (ARC) that contains two stimulatory neuropeptides, critical for fertility in humans (kisspeptin and neurokinin B (NKB)) and the inhibitory endogenous opioid peptide (EOP), dynorphin, and are now known as KNDy (kisspeptin-NKB-dynorphin) neurons. In this review, we consider the role of each of the KNDy peptides in the generation of GnRH pulses and the negative feedback actions of ovarian steroids, with an emphasis on NKB and dynorphin. With regard to negative feedback, there appear to be important species differences. In sheep, progesterone inhibits GnRH pulse frequency by stimulating dynorphin release, and estradiol inhibits pulse amplitude by suppressing kisspeptin. In rodents, the role of KNDy neurons in estrogen negative feedback remains controversial, progesterone may inhibit GnRH via dynorphin, but the physiological significance of this action is unclear. In primates, an EOP, probably dynorphin, mediates progesterone negative feedback, and estrogen inhibits kisspeptin expression. In contrast, there is now compelling evidence from several species that kisspeptin is the output signal from KNDy neurons that drives GnRH release during a pulse and may also act within the KNDy network to affect pulse frequency. NKB is thought to act within this network to initiate each pulse, although there is some redundancy in tachykinin signaling in rodents. In ruminants, dynorphin terminates GnRH secretion at the end of pulse, most likely acting on both KNDy and GnRH neurons, but the data on the role of this EOP in rodents are conflicting.

Contains fulltext : 133843.pdf (Publisher’s version ) (Closed access) Sexual behavior of the male rat can be described as a 'sequence': a series of behavioral transitions eventually leading to a consummatory act: ejaculation. A 'funnel-model' is presented to describe the behavioral progression during the sexual sequence. The ejaculation itself is extensively controlled by the 'spinal ejaculation generator', consisting of several elements with afferent sources of genitosensory information, with ascending projection fibers to inform the brainstem and forebrain as well as with descending afferent fibers providing the supraspinal control mechanisms with the opportunity to restrict ejaculations to the optimal moments and circumstances. The messages ascending from the spinal cord reach several interconnected thalamic, hypothalamic and limbic brain areas and are integrated with olfactory information. These brain areas play a role in mechanisms like 'sexual satiety' or a temporary interruption of sexual activities (post-ejaculatory interval), but the exact facilitatory and inhibitory mechanisms involved have not been elucidated yet. In the 'downward' mechanisms controlling the spinal 'release' of an ejaculation, the medial preoptic nucleus plays an important role in cooperation with a number of brainstem areas. This nucleus is also explicitly involved in the rewarding experiences coming with an ejaculation. Finally, the role of several neurotransmitters and-peptides on male sexual behavior are discussed shortly, because sometimes they show remarkable effects on specific aspects of the behavioral sequence. We conclude that, despite our increased knowledge about the brain mechanisms involved in the control of ejaculation, we are still far away from a complete understanding and quite a few questions remain to be resolved. 01 juni 2014

Contains fulltext : 133842.pdf (Publisher’s version ) (Closed access) The sequential organization of sexual behavior of the female rat is described, eventually leading to the lordotic posture, shown during mating. A complex set of signals: olfactory, cutaneous sensory as well as genitosensory, is guiding the female to this specific posture, eventually. Genitosensory signals converge in the lumbosacral levels of the spinal cord, from where they are dispersed to a series of supraspinal brain areas, in the brainstem, thalamus, hypothalamus and limbic system. The similarity with the neural activation patterns observed in the male rat is remarkable. In a number of brain areas, however: the midbrain periaqueductal gray, the ventrolateral part of the ventromedial hypothalamic nucleus (VMHvl) and the medial preoptic-lateral septum regions, specific male-female differences have been observed. Especially the VMHvl is an intriguing area, as it has been shown that the same neurons may be involved in 'opposite behavior' like aggression and the induction of lordosis. The motor mechanisms controlling the lordosis posture in the rat as well as in some other mammals are discussed, as well as some aspects of the reward mechanisms contributing to female sex. We conclude that we have collected a great amount of neurophysiological knowledge over the last 20 years, but that the unresolved questions are still numerous. In this field, there is still much to explore. 01 juni 2014

Psychostimulant reward, as assessed via the conditioned place preference (CPP) paradigm, exhibits a daily rhythm with peaks in the late dark and early light periods, and a nadir near the light-to-dark transition. While this diurnal rhythm is correlated with neural activity in several corticolimbic structures, the brain regions mediating this behavioral rhythm remain unknown. Here, we examine the role of the ventral medial prefrontal cortex (mPFC). The effects of excitotoxic mPFC lesions on daily rhythms in amphetamine CPP were examined at previously observed peak (zeitgeber time [ZT] 23) and nadir times (ZT11). mPFC lesions encompassing the prelimbic and infralimbic subregions increased the CPP for amphetamine at the nadir time, thereby eliminating the daily rhythm in amphetamine reward. To examine the effects of transient mPFC inactivation, rats received intra-mPFC infusions of GABA receptor agonists during the acquisition or expression phases of CPP testing. Inactivation of the ventral mPFC at either of these phases also eliminated the daily rhythm in amphetamine-induced CPP via an increase in drug-paired chamber dwell time at the baseline nadir. Together, these results indicate that the ventral mPFC plays a critical role in mediating the diurnal rhythm in amphetamine CPP during both the acquisition and expression of learned reward-context associations. Moreover, as the loss of rhythmicity occurs via an increase at the nadir point, these results suggest that excitatory output from the ventral mPFC normally inhibits context-elicited reward seeking prior to the light-to-dark transition.

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.

Experience with sexual behavior causes cross-sensitization of amphetamine reward, an effect dependent on a period of sexual reward abstinence. We previously showed that ΔFosB in the nucleus accumbens (NAc) is a key mediator of this cross-sensitization, potentially via dopamine receptor activation. However, the role of mesolimbic dopamine for sexual behavior or cross-sensitization between natural and drug reward is unknown. This was tested using inhibitory designer receptors exclusively activated by designer drugs in ventral tegmental area (VTA) dopamine cells. rAAV5/hSvn-DIO-hm4D-mCherry was injected into the VTA ofTH::Creadult male rats. Males received clozapine N-oxide (CNO) or vehicle injections before each of 5 consecutive days of mating or handling. Following an abstinence period of 7 d, males were tested for amphetamine conditioned place preference (CPP). Next, males were injected with CNO or vehicle before mating or handling for analysis of mating-induced cFos, sex experience-induced ΔFosB, and reduction of VTA dopamine soma size. Results showed that CNO did not affect mating behavior. Instead, CNO prevented sexual experience-induced cross-sensitization of amphetamine CPP, ΔFosB in the NAc and medial prefrontal cortex, and decreases in VTA dopamine soma size. Expression of hm4D-mCherry was specific to VTA dopamine cells and CNO blocked excitation and mating-induced cFos expression in VTA dopamine cells. These findings provide direct evidence that VTA dopamine activation is not required for initiation or performance of sexual behavior. Instead, VTA dopamine directly contributes to increased vulnerability for drug use following loss of natural reward by causing neuroplasticity in the mesolimbic pathway during the natural reward experience.SIGNIFICANCE STATEMENTDrugs of abuse act on the neural pathways that mediate natural reward learning and memory. Exposure to natural reward behaviors can alter subsequent drug-related reward. Specifically, experience with sexual behavior, followed by a period of abstinence from sexual behavior, causes increased reward for amphetamine in male rats. This study demonstrates that activation of ventral tegmental area dopamine neurons during sexual experience regulates cross-sensitization of amphetamine reward. Finally, ventral tegmental area dopamine cell activation is essential for experience-induced neural adaptations in the nucleus accumbens, prefrontal cortex, and ventral tegmental area. These findings demonstrate a role of mesolimbic dopamine in the interaction between natural and drug rewards, and identify mesolimbic dopamine as a key mediator of changes in vulnerability for drug use after loss of natural reward.

Different factors influence the development of drug addiction in humans, including social reward experiences. In animals, experience with social rewards, such as sexual behavior, pair bonding, social and environmental enrichment, can be protective. However, loss or lack of social rewards can lead to a vulnerability to drug-seeking behavior. The effects of social reward experience on drug-seeking behavior are associated with changes in the neural pathways that control drug-related behavior. This review will provide an introduction and overview of the mesolimbic pathway and the influence of social reward experience on drug-seeking behavior in rodents. Moreover, the research from our laboratory on effects of sexual experience and loss of sex reward on psychostimulant and opiate reward will be reviewed. Finally, we will review current knowledge of the neural mechanisms that underlie these interactions. Investigations of the neural underpinnings by which social and drug rewards interact contribute to improved understanding of the neural basis of vulnerability for drug addiction and reward-related behaviors in general.

The neuropeptides neurokinin B (NKB) and kisspeptin are potent stimulators of gonadotrophin-releasing hormone (GnRH)/luteinsing hormone (LH) secretion and are essential for human fertility. We have recently demonstrated that selective activation of NKB receptors (NK3R) within the retrochiasmatic area (RCh) and the preoptic area (POA) triggers surge-like LH secretion in ovary-intact ewes, whereas blockade of RCh NK3R suppresses oestradiol-induced LH surges in ovariectomised ewes. Although these data suggest that NKB signalling within these regions of the hypothalamus mediates the positive-feedback effects of oestradiol on LH secretion, the pathway through which it stimulates GnRH/LH secretion remains unclear. We proposed that the action of NKB on RCh neurones drives the LH surge by stimulating kisspeptin-induced GnRH secretion. To test this hypothesis, we quantified the activation of the preoptic/hypothalamic populations of kisspeptin neurones in response to POA or RCh administration of senktide by dual-label immunohistochemical detection of kisspeptin and c-Fos (i.e. marker of neuronal activation). We then administered the NK3R agonist, senktide, into the RCh of ewes in the follicular phase of the oestrous cycle and conducted frequent blood sampling during intracerebroventricular infusion of the kisspeptin receptor antagonist Kp-271 or saline. Our results show that the surge-like secretion of LH induced by RCh senktide administration coincided with a dramatic increase in c-Fos expression within arcuate nucleus (ARC) kisspeptin neurones, and was completely blocked by Kp-271 infusion. We substantiate these data with evidence of direct projections of RCh neurones to ARC kisspeptin neurones. Thus, NKB-responsive neurones in the RCh act to stimulate GnRH secretion by inducing kisspeptin release from KNDy neurones.

Puberty onset involves increased gonadotrophin-release (GnRH) release as a result of decreased sensitivity to oestrogen (E2 )-negative feedback. Because GnRH neurones lack E2 receptor α, this pathway must contain interneurones. One likely candidate is KNDy neurones (kisspeptin, neurokinin B, dynorphin). The overarching hypothesis of the present study was that the prepubertal hiatus in luteinising hormone (LH) release involves reduced kisspeptin and/or heightened dynorphin input. We first tested the specific hypothesis that E2 would reduce kisspeptin-immunopositive cell numbers and increase dynorphin-immunopositive cell numbers. We found that kisspeptin cell numbers were higher in ovariectomised (OVX) lambs than OVX lambs treated with E2 (OVX+ E2 ) or those left ovary-intact. Very few arcuate dynorphin cells were identified in any group. Next, we hypothesised that central blockade of κ-opioid receptor (KOR) would increase LH secretion at a prepubertal (6 months) but not postpubertal (10 months) age. Luteinising hormone pulse frequency and mean LH increased during infusion of a KOR antagonist, norbinaltorphimine, in OVX + E2 lambs at the prepubertal age but not in the same lambs at the postpubertal age. We next hypothesised that E2 would increase KOR expression in GnRH neurones or alter synaptic input to KNDy neurones in prepubertal ewes. Oestrogen treatment decreased the percentage of GnRH neurones coexpressing KOR (approximately 68%) compared to OVX alone (approximately 78%). No significant differences in synaptic contacts per cell between OVX and OVX + E2 groups were observed. Although these initial data are consistent with dynorphin inhibiting pulsatile LH release prepubertally, additional work will be necessary to define the source and mechanisms of this inhibition.

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. Staudt MD, Truitt WA, McKenna KE, de Oliveira CVR, Lehman MN, and Coolen LM. A pivotal role of lumbar spinothalamic cells in the regulation of ejaculation via intraspinal connections. J Sex Med 2012;9:2256–2265.