Hypothalamic kisspeptin (Kiss), neurokinin B (NKB), and dynorphin‐containing (KNDy) neurons in the arcuate nucleus (ARC) have consistently been shown to be the central generator of gonadotropin‐releasing hormone (GnRH) and corresponding luteinizing hormone (LH) pulses in mammals and possibly contribute to surge secretion as well. Additionally, recent evidence from experiments in sheep suggests that ARC Kiss1R‐containing neurons play an important role in regulating the timing and amplitude of LH pulses. In this study, we examined the functional role of ARC KNDy and Kiss1R‐containing neurons in ovine LH surge secretion via injection of saporin–ligand conjugates (SAP) to ablate these neural populations. NKB‐SAP injections significantly reduced the percentage of ARC Kiss1 (~65% decrease) cells compared to control animals, and a surge‐like increase of LH was prevented in ewes with the greatest degree of Kiss1 cell ablation. Kiss‐SAP injections had no effect on Kiss1 cell percentage or ARC Kiss1R cell number compared to controls, the latter perhaps due to Kiss1R suppression in control animals from elevated estradiol concentrations during the LH surge. However, Kiss‐SAP injections consistently and robustly decreased LH surge amplitude, with 80% of Kiss‐SAP‐treated ewes failing to generate a surge. While the exact identity of these ARC Kiss1R neurons has yet to be fully elucidated, they likely act downstream or in concert with KNDy neurons and possibly integrate other surge‐centric signaling pathways to generate the ovine LH surge. These results support the conclusion that KNDy neurons contribute significantly to the ovine LH surge, while ARC Kiss1R neurons appear to be necessary for a functional surge to occur in sheep.

Kisspeptin neurons play a critical role in the estradiol feedback effects on gonadotropin-releasing hormone (GnRH) neurons and luteinizing hormone (LH) secretion. Endogenous opioid peptides regulate LH secretion, but the neuroendocrine mechanisms involved remain elusive. We used RNAscope to characterize the expression of kappa (Oprk1)-, mu (Oprm1)-, and delta (Oprd1)-opioid receptors in GnRH (Gnrh1) neurons and kisspeptin neurons of the rostral periventricular area of the third ventricle (Kiss1RP3V) and arcuate nucleus (Kiss1ARC) in cycling mice and rats with physiological low (metestrus) and high (proestrus) levels of ovarian steroids. In mice, all opioid receptors were colocalized with Gnrh1, with increased coexpression of Oprk1 on proestrus compared with metestrus. Most Kiss1RP3V neurons expressed Oprk1, Oprm1, or Oprd1, with no changes seen during the estrous cycle. The three opioid receptors were also expressed in Kiss1ARC neurons, and the expression of Oprk1 in Kiss1ARC neurons was reduced on proestrus compared with metestrus. When investigated in cycling rats, Kiss1ARC neurons displayed the same pattern of Oprk1 variation as in mice. However, whereas the mouse Kiss1ARC neurons displayed a predominance of Oprk1 expression, all three opioid receptors were similarly expressed in the rat. Our results show that Oprk1 is the main opioid receptor present in Kiss1ARC neurons of mice but not rats, whereas Oprk1, Oprm1, and Oprd1 are abundantly expressed in mouse Kiss1RP3V and GnRH neurons. Fluctuations in ovarian steroids are likely to modulate Oprk1 levels in GnRH and Kiss1ARC neurons during the ovarian cycle, implicating this opioid receptor in the feedback control of LH secretion in female rodents.

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

Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p < 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.

Abstract Neurons within the arcuate nucleus (ARC) of the hypothalamus containing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) have an important role in regulating the pulsatile secretion of gonadotropin releasing hormone (GnRH) and luteinizing hormone (LH). In sheep, kisspeptin neurons also contribute to the LH surge, as kisspeptin receptor (Kiss1r) antagonist administration reduces surge amplitude by 50% and KNDy neurons are likely involved, based on increased Fos expression at the time of the surge. However, the extent to which kisspeptin acts within the ARC regulate the GnRH/LH surge remains unclear. Thus, herein we tested the hypothesis that deletion of KNDy or ARC Kiss1r-containing neurons would impair the LH surge. Adult female sheep received bilateral injections targeting the ARC of NKB-saporin (NKB-SAP, n = 8), kisspeptin-saporin (Kiss-SAP, n = 10), or blank-saporin (Blank-SAP, n = 7) as a control. In other work, NKB-SAP lesioned over 90% of ovine KNDy neurons, while Kiss-SAP lesioned 67% of Kiss1r-containing cells without affecting KNDy or GnRH cell number. Ewes were also ovariectomized and a subcutaneous silastic estradiol (E2) implant was inserted at the time of neurosurgery. Two artificial luteal phases were simulated with progesterone-containing CIDRs, immediately followed by E2 treatment via implants to induce an LH surge. Blood samples were collected every two to four hours over two days and analyzed for LH via radioimmunoassay. LH surge amplitude in six of eight NKB-SAP ewes (49.5 ± 11.7 ng/mL) was significantly reduced compared with Blank-SAP control ewes (156.7 ± 20.2 ng/mL, p = 0.0001), a reduction similar to that produced by treatment with a Kiss1r antagonist. Nine of ten Kiss-SAP treated ewes displayed little to no increase of LH at the time of the expected surge (16.6 ± 5.3 ng/mL, p < 0.0001). Lesion effectiveness is currently being assessed by RNAscope, however all Kiss-SAP animals examined to date have significantly reduced ARC Kiss1r cell numbers except a single ewe which exhibited a normal LH surge. Based on these data, we propose that in ewes, KNDy neurons contribute to, but are not required for, the LH surge. In contrast, ARC Kiss1r-containing cells are essential for a functional LH surge. Given these results, we are currently assessing the role of ARC Kiss1r neurons in ovine puberty using a similar approach. Our data to date shows that time to puberty onset is similar for Kiss-SAP, Blank-SAP, and non-surgical control animals as measured by an increase in progesterone (p = 0.35). Blood samples to detect LH pulses and the LH surge are currently being analyzed, as are ARC Kiss1r cell numbers.

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

Significance Hypothalamic oscillators that generate pulsatile patterns of hormone secretion represent a fundamental physiological feature regulating homeostatic systems. How individual cells within these neural ensembles generate and coordinate episodic activity and resultant pulse secretion is unknown. Recently, arcuate KNDy (kisspeptin/neurokinin B/dynorphin) cells were identified as a critical component of the gonadotrophin-releasing hormone (GnRH) pulse generator required for reproduction. Using in vivo calcium imaging of KNDy neurons in freely moving mice, we reveal that, prior to each GnRH pulse, individual KNDy cells demonstrate synchronized activity with striking temporal order, with subsets of cells behaving as “leaders” or “followers.” Future work to distinguish these novel subpopulations and define mechanisms underlying the temporal ordering of cellular synchronization may provide avenues to regulate pulse secretion.

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.

Spinal cord injury (SCI) in men is commonly associated with sexual dysfunction, including anejaculation, and chronic mid-thoracic contusion injury in male rats also impairs ejaculatory reflexes. Ejaculation is controlled by a spinal ejaculation generator consisting of a population of lumbar spinothalamic (LSt) neurons that control ejaculation through release of four neuropeptides including galanin and gastrin releasing peptide (GRP) onto lumbar and sacral autonomic and motor nuclei. It was recently demonstrated that spinal contusion injury in male rats caused reduction of GRP-immunoreactivity, but not galanin-immunoreactivity in LSt cells, indicative of reduced GRP peptide levels, but inconclusive results for galanin. The current study further tests the hypothesis that contusion injury causes a disruption of GRP and galanin mRNA in LSt cells. Male rats received mid-thoracic contusion injury and galanin and GRP mRNA were visualized 8 weeks later in the lumbar spinal cord using fluorescent in situ hybridization. Spinal cord injury significantly reduced GRP and galanin mRNA in LSt cells. Galanin expression was higher in LSt cells compared to GRP. However, expression of the two transcripts were positively correlated in LSt cells in both sham and SCI animals, suggesting that expression for the two neuropeptides may be co-regulated. Immunofluorescent visualization of galanin and GRP peptides demonstrated a significant reduction in GRP-immunoreactivity, but not galanin in LSt cells, confirming the previous observations. In conclusion, SCI reduced GRP and galanin expression in LSt cells with an apparent greater impact on GRP peptide levels. GRP and galanin are both essential for triggering ejaculation and thus such reduction may contribute to ejaculatory dysfunction following SCI in rats.

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 kisspeptin, neurokinin B, and dynorphin. 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.

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 contained 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.

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.

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.

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. (C) 2020 Elsevier Inc. All rights reserved.