NK3R signalling in the posterodorsal medial amygdala is involved in stress-induced suppression of pulsatile LH secretion in female mice.

Psychosocial stress negatively impacts reproductive function by inhibiting pulsatile luteinizing hormone (LH) secretion. The posterodorsal medial amygdala (MePD) is responsible in part for processing stress and modulating the reproductive axis. Activation of the neurokinin 3 receptor (NK3R) suppresses the gonadotropin-releasing hormone (GnRH) pulse generator, under hypoestrogenic conditions, and NK3R activity in the amygdala has been documented to play a role in stress and anxiety. We investigate whether NK3R activation in the MePD is involved in mediating the inhibitory effect of psychosocial stress on LH pulsatility in ovariectomised female mice. First, we administered senktide, an NK3R agonist, into the MePD and monitored the effect on pulsatile LH secretion. We then delivered SB222200, a selective NK3R antagonist, intra-MePD in the presence of predator odour, 2,4,5-trimethylthiazole (TMT) and examined the effect on LH pulses. Senktide administration into the MePD dose-dependently suppresses pulsatile LH secretion. Moreover, NK3R signalling in the MePD mediates TMT-induced suppression of the GnRH pulse generator, which we verified using a mathematical model. The model verifies our experimental findings: (i) predator odour exposure inhibits LH pulses, (ii) activation of NK3R in the MePD inhibits LH pulses and (iii) NK3R antagonism in the MePD blocks stressor-induced inhibition of LH pulse frequency in the absence of ovarian steroids. These results demonstrate for the first time that NK3R neurons in the MePD mediate psychosocial stress-induced suppression of the GnRH pulse generator.

Continuous acceleration of neural activity of the GnRH pulse generator during chronic peripheral infusion of neurokinin 3 receptor agonist in goats.

Secretion of pulsatile gonadotropin-releasing hormone (GnRH) is essential for reproduction. Kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B (NKB) and its receptor (NK3R), are believed to be components of the GnRH pulse generator that regulates pulsatile GnRH secretion. We examined the effects of peripheral infusion of senktide, an NK3R selective agonist, on GnRH pulse generator activity by monitoring multiple unit activity (MUA) in the goat ARC. Previous studies have shown that characteristic increases in MUA (MUA volleys) reflect GnRH pulse generator activity. Senktide was infused intravenously or intravaginally for 2 h while recording MUA. Both infusions significantly increased the MUA volley frequency compared with the control. These results demonstrate that peripherally administered senktide acts centrally to sustainably accelerate the neural activity of the GnRH pulse generator throughout the infusion period. This suggests the possibility of practical applications of NK3R agonists for improving reproductive activity in farm animals.

Effect of a neurokinin 3 receptor-selective agonist administration on the embryos recovered from superovulated cows.

Superovulation (SOV) treatment of cows results in unovulated follicles and inconsistent quality of the recovered embryos. It has been demonstrated that luteinizing hormone (LH) secretion is suppressed during SOV treatment of cows, which may cause insufficient follicle development and variation in the development of recovered embryos and unovulated follicles. Pulsatile gonadotropin-releasing hormone/LH secretion is controlled by the activity of kisspeptin, neurokinin B and dynorphin (KNDy) neurons in the arcuate nucleus in many mammals. As neurokinin B promotes the activity of KNDy neurons, we hypothesized that senktide, a neurokinin B receptor agonist, has the potential as a therapeutic drug to improve the ovulation rate and quality of recovered embryos in SOV-treated cows via stimulation of LH secretion. Senktide was administered intravenously (30 or 300 nmol/min) for 2 h, beginning from 72 h after the start of SOV treatment. LH secretion was examined before and after administration, and embryos were collected 7 d after estrus. Senktide administration increased LH secretion in SOV-treated cows. The ratios of code 1, code 1 and 2, and blastocyst stage embryos to recovered embryos were increased by senktide (300 nmol/min) administration. Moreover, the mRNA levels of MTCO1, COX7C, and MTATP6 were upregulated in recovered embryos of senktide (300 nmol/min)-administered animals. These results indicate that the administration of senktide to SOV-treated cows enhances LH secretion and upregulates the expression of genes involved in mitochondrial metabolism in embryos, thereby improving embryo development and embryo quality.

Neurokinin 3 receptor-selective agonist, senktide, decreases core temperature in Japanese Black cattle.

Heat stress disrupts reproductive function in cattle. In summer, high ambient temperature and humidity elevate core body temperature, which is considered to be detrimental to reproductive abilities in cattle. Neurokinin B (NKB) is a factor that generates pulsatile GnRH and subsequent LH secretion in mammals. Recent studies have reported that NKB-neurokinin 3 receptor (NK3R) signaling is associated with heat-defense responses in rodents. The present study aimed to clarify the role of NKB-NK3R signaling in thermoregulation in cattle. We examined the effects of an NK3R-selective agonist, senktide, on vaginal temperature as an indicator of core body temperature in winter and summer. In both seasons, continuous infusion of senktide for 4 h immediately decreased vaginal temperature, and the mean temperature change in the senktide-treated group was significantly lower than that of both vehicle- and GnRH-treated groups. Administration of GnRH induced LH elevation, but there was no significant difference in vaginal temperature change between GnRH- and vehicle-treated groups. Moreover, we investigated the effects of senktide on ovarian temperature. Senktide treatment seemed to suppress the increase in ovarian temperature from 2 h after the beginning of administration, although the difference between groups was not statistically significant. Taken together, these results suggest that senktide infusion caused a decline in the vaginal temperature of cattle, in both winter and summer seasons, and this effect was not due to the gonadotropin-releasing action of senktide. These findings provide new therapeutic options for senktide to support both heat-defense responses and GnRH/LH pulse generation.

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.

The efficacy of a newly developed neurokinin 3 receptor agonist B21-750 on luteinizing hormone secretion in cycling goats.

This study aimed to investigate the efficacy of a newly developed NK3 receptor agonist (B21-750) on the secretion of luteinizing hormone (LH) in association with ovarian steroid hormones during the follicular phase (FP, n = 5) and luteal phase (LP, n = 5) of Shiba goats. The FP group was treated with both prostaglandin F2α and progesterone-controlled internal drug release (CIDR) inserts for 10 d, and B21-750 (200 nmol) was injected 12 h after removing the CIDR. Meanwhile, the LP group received B21-750 injections on a day during the mid-luteal phase. LH secretion increased at 1 h after B21-750 injection in both groups. The percent changes in the area under the curve of LH was higher during the hour after injection than during the hour before injection in both groups. Thus, this study demonstrated that B21-750 induces rapid LH secretion for a short period during both the follicular and luteal phases.

Kisspeptin and Neurokinin B Signaling Network Underlies the Pubertal Increase in GnRH Release in Female Rhesus Monkeys.

Loss-of-function or inactivating mutations in the genes coding for kisspeptin and its receptor (KISS1R) or neurokinin B (NKB) and the NKB receptor (NK3R) in humans result in a delay in or the absence of puberty. However, precise mechanisms of kisspeptin and NKB signaling in the regulation of the pubertal increase in gonadotropin-releasing hormone (GnRH) release in primates are unknown. In this study, we conducted a series of experiments infusing agonists and antagonists of kisspeptin and NKB into the stalk-median eminence, where GnRH, kisspeptin, and NKB neuroterminal fibers are concentrated, and measuring GnRH release in prepubertal and pubertal female rhesus monkeys. Results indicate that (1) similar to those previously reported for GnRH stimulation by the KISS1R agonist (i.e., human kisspeptin-10), the NK3R agonist senktide stimulated GnRH release in a dose-responsive manner in both prepubertal and pubertal monkeys; (2) the senktide-induced GnRH release was blocked in the presence of the KISS1R antagonist peptide 234 in pubertal but not prepubertal monkeys; and (3) the kisspeptin-induced GnRH release was blocked in the presence of the NK3R antagonist SB222200 in the pubertal but not prepubertal monkeys. These results are interpreted to mean that although, in prepubertal female monkeys, kisspeptin and NKB signaling to GnRH release is independent, in pubertal female monkeys, a reciprocal signaling mechanism between kisspeptin and NKB neurons is established. We speculate that this cooperative mechanism by the kisspeptin and NKB network underlies the pubertal increase in GnRH release in female monkeys.

Targeting neurokinin-3 receptor: a novel anti-angiogenesis strategy for cancer treatment.

Angiogenesis is essential for tumor growth and metastasis, controlling angiogenesis is a promising strategy in cancer treatment. However, thus farther severe side effects of anti-angiogenic drugs have been rather demonstrated, stimulating interest in seeking novel targets of anti-angiogenesis. Neurokinin receptors, also known as tachykinin receptors, are usually considered as drug targets due to diverse physiological functions and their tractability. Although Neurokinin B, the selective natural agonist of neurokinin-3 receptor, have been shown to exhibit anti-angiogenesis activity, the effect and mechanism of neurokinin-3 receptor-mediated angiogenesis still remains unclear. In the present study, we demonstrated that [Mephe7]NKB, an analogue of NKB, possess significant anti-angiogenic effect on CAM. Furthermore, by introducing the tumor angiogenesis homing sequence (NGR), we designed and synthesized two novel agonist analogues of NK3R, NK3R-A1 and NK3R-A2. Both of the two analogues exhibit more efficient anti-migration effect on HUVECs by activating NK3R in vitro, and showed potent antitumor activities with no significant side effects in vivo. Taken together, our results illuminated that NK3R might be a potential novel target for the anti-angiogenesis therapy. Notably, NK3R-A1 might be used as a template for the development of the anti-tumor drugs on the basis of the anti-angiogenesis strategy.

A Comprehensive Method To Quantify Adaptations by Male and Female Mice With Hot Flashes Induced by the Neurokinin B Receptor Agonist Senktide.

Vasomotor symptoms (VMS; or hot flashes) plague millions of reproductive-aged men and women who have natural or iatrogenic loss of sex steroid production. Many affected individuals are left without treatment options because of contraindications to hormone replacement therapy and the lack of equally effective nonhormonal alternatives. Moreover, development of safer, more effective therapies has been stymied by the lack of an animal model that recapitulates the hot-flash phenomenon and enables direct testing of hypotheses regarding the pathophysiology underlying hot flashes. To address these problems, we developed a murine model for hot flashes and a comprehensive method for measuring autonomic and behavioral thermoregulation in mice. We designed and constructed an instrument called a thermocline that produces a thermal gradient along which mice behaviorally adapt to a thermal challenge to their core body temperature set point while their thermal preference over time is tracked and recorded. We tested and validated this murine model for VMS by administration of a TRPV1 agonist and a neurokinin B receptor agonist, capsaicin and senktide, respectively, to unrestrained mice and observed their autonomic and behavioral responses. Following both treatments, the mice exhibited a VMS-like response characterized by a drop in core body temperature and cold-seeking behavior on the thermocline. Senktide also caused a rise in tail skin temperature and increased Fos expression in the median preoptic area, a hypothalamic temperature control center. This dynamic model may be used to fully explore the cellular and molecular bases for VMS and to develop and test new therapeutic options.

Both Estrogen and Androgen Modify the Response to Activation of Neurokinin-3 and κ-Opioid Receptors in Arcuate Kisspeptin Neurons From Male Mice.

Gonadal steroids regulate the pattern of GnRH secretion. Arcuate kisspeptin (kisspeptin, neurokinin B, and dynorphin [KNDy]) neurons may convey steroid feedback to GnRH neurons. KNDy neurons increase action potential firing upon the activation of neurokinin B receptors (neurokinin-3 receptor [NK3R]) and decrease firing upon the activation of dynorphin receptors (κ-opioid receptor [KOR]). In KNDy neurons from intact vs castrated male mice, NK3R-mediated stimulation is attenuated and KOR-mediated inhibition enhanced, suggesting gonadal secretions are involved. Estradiol suppresses spontaneous GnRH neuron firing in male mice, but the mediators of the effects on firing in KNDy neurons are unknown. We hypothesized the same gonadal steroids affecting GnRH firing pattern would regulate KNDy neuron response to NK3R and KOR agonists. To test this possibility, extracellular recordings were made from KNDy neurons in brain slices from intact, untreated castrated or castrated adult male mice treated in vivo with steroid receptor agonists. As observed previously, the stimulation of KNDy neurons by the NK3R agonist senktide was attenuated in intact vs castrated mice and suppression by dynorphin was enhanced. In contrast to observations of steroid effects on the GnRH neuron firing pattern, both estradiol and DHT suppressed senktide-induced KNDy neuron firing and enhanced the inhibition caused by dynorphin. An estrogen receptor-α agonist but not an estrogen receptor-ß agonist mimicked the effects of estradiol on NK3R activation. These observations suggest the steroid modulation of responses to activation of NK3R and KOR as mechanisms for negative feedback in KNDy neurons and support the contribution of these neurons to steroid-sensitive elements of a GnRH pulse generator.

A Direct Neurokinin B Projection from the Arcuate Nucleus Regulates Magnocellular Vasopressin Cells of the Supraoptic Nucleus.

Central administration of neurokinin B (NKB) agonists stimulates immediate early gene expression in the hypothalamus and increases the secretion of vasopressin from the posterior pituitary through a mechanism that depends on the activation of neurokinin receptor 3 receptors (NK3R). The present study reports that, in the rat, immunoreactivity for NK3R is expressed in magnocellular vasopressin and oxytocin neurones in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus, and that NKB immunoreactivity is expressed in fibres in close juxtaposition with vasopressin neurones at both of these sites. Retrograde tracing in the rat shows that some NKB-expressing neurones in the arcuate nucleus project to the SON and, in mice, using an anterograde tracing approach, it is found that kisspeptin-expressing neurones of the arcuate nucleus, which are known to co-express NKB, project to the SON and PVN. Finally, i.c.v. injection of the NK3R agonist senktide is shown to potently increase the electrical activity of vasopressin neurones in the SON in vivo with no significant effect detected on oxytocin neurones. The results suggest that NKB-containing neurones in the arcuate nucleus regulate the secretion of vasopressin from magnocellular neurones in rodents, and the possible significance of this is discussed.

Neurokinin 3 Receptor-Expressing Neurons in the Median Preoptic Nucleus Modulate Heat-Dissipation Effectors in the Female Rat.

KNDy neurons facilitate tail skin vasodilation and modulate the effects of estradiol on thermoregulation. We hypothesize that KNDy neurons influence cutaneous vasodilation via projections to neurons in the median preoptic nucleus (MnPO) that express the neurokinin 3 receptor (NK3R). In support of this hypothesis, focal microinjections of senktide, an NK3R agonist, into the MnPO lowers core temperature (TCORE) in the female rat. To further study the role of MnPO NK3R neurons in thermoregulation, these neurons were specifically ablated using a conjugate of a selective NK3R agonist and saporin (NK3-SAP). NK3-SAP or blank-SAP (control) was injected into the MnPO/medial septum. Tail skin temperature (TSKIN) and TCORE were measured in ovariectomized rats exposed to 3 ambient temperatures (TAMBIENT) before and after estradiol-17ß (E2) treatment. Before killing, we injected senktide (sc), monitored TCORE for 70 minutes, and harvested brains for Fos immunohistochemistry. Ablation of MnPO NK3R neurons lowered TSKIN at neutral and subneutral TAMBIENT regardless of E2 treatment. However, ablation did not prevent the effects of E2 on TCORE and TSKIN. In control rats, senktide injections induced hypothermia with numerous Fos-immunoreactive cells in the MnPO. In contrast, in NK3-SAP rats, senktide did not alter TCORE and minimal Fos-immunoreactive neurons were identified in the MnPO. These data show that NK3R neurons in the MnPO are required for the hypothermic effects of senktide but not for the E2 modulation of thermoregulation. The lower TSKIN in NK3-SAP-injected rats suggests that MnPO NK3R neurons, like KNDy neurons, facilitate cutaneous vasodilation, an important heat-dissipation effector.

Evidence that Neurokinin B Controls Basal Gonadotropin-Releasing Hormone Secretion but Is Not Critical for Estrogen-Positive Feedback in Sheep.

BACKGROUND: Loss-of-function mutations in genes encoding kisspeptin or neurokinin B (NKB) or their receptors cause infertility. NKB is coproduced in kisspeptin neurons in the arcuate nucleus (ARC), and these neurons also produce the NKB receptor (NK3R), allowing autosynaptic function. We tested the hypothesis that NKB action in ARC kisspeptin neurons is aligned with increased pulsatile secretion of luteinizing hormone (LH) and/or activation of the estrogen-induced LH surge in ewes. METHODS: Using in situ hybridization and immunohistochemistry, we examined NKB expression in kisspeptin neurons during the ovine estrous cycle. We infused kisspeptin, senktide (an NK3R agonist), or dynorphin into the lateral ventricle during the luteal phase of the estrous cycle to determine effects on pulsatile LH secretion. Finally, we examined the effect of an NK3R antagonist (MRK-08) in ovariectomized ewes. RESULTS: NKB (Tac3) mRNA expression in mid-ARC kisspeptin neurons was elevated during the mid-to-late follicular phase of the estrous cycle. The number of NKB-immunoreactive cells and NKB/kisspeptin terminals in the median eminence was similar during the estrous cycle. Kisspeptin and senktide increased LH pulse frequency and mean LH levels. Central MRK-08 infusion eliminated the LH pulses but did not prevent an estrogen-positive feedback on LH secretion. CONCLUSIONS: NKB expression in ARC kisspeptin neurons is upregulated during the late follicular phase of the estrous cycle, when the pulsatile secretion of gonadotropin-releasing hormone (GnRH)/LH is maximal. When GnRH/LH secretion is minimal, central senktide infusion induces LH secretion, similar to the response to kisspeptin. Although the increase in LH in response to senktide appeared surge-like, we did not observe any change in the surge following NK3R antagonist treatment. We conclude that NKB plays a role in increasing basal GnRH/LH pulsatility in the follicular phase of the cycle but is not essential for estrogen-induced positive feedback.

Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats.

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.

Effects of senktide, a neurokinin 3 receptor agonist, on luteinizing hormone secretion and follicular development in anestrous Shiba goats: a pilot study.

BACKGROUND: Recent studies suggest that neurokinin B and its receptor, neurokinin 3 receptor, have an essential role in the regulation of gonadotropin-releasing hormone and luteinizing hormone (LH) release in several mammalian species. As the first trial, this pilot study reports the effect of intravenous treatment with senktide, a selective agonist of neurokinin 3 receptor, on LH secretion, follicular development in female goats that were clinically diagnosed with anestrus. FINDINGS: Anestrous goats were intravenously administered 200 nmol senktide at 4-h intervals for 24 h. Most of them examined (5/6 cases) showed a pulsatile increase in LH secretion after each injection of senktide, whereas the remaining one case showed a surge-like increase of LH secretion. Ovulation was confirmed in 5/6 cases at the range of 48-96 h after the beginning of treatment. CONCLUSIONS: This pilot study demonstrated that intravenous treatment with senktide has therapeutic action in goats with anestrus by inducing LH release, which could promote follicular development and ovulation.

The effects of chronic subcutaneous administration of an investigational kisspeptin analog, TAK-683, on gonadotropin-releasing hormone pulse generator activity in goats.

The continuous activation of the kisspeptin receptor by its agonists causes the abrogation of kisspeptin signaling, leading to decreased pulsatile luteinizing hormone (LH) secretion. Employing this phenomenon as a tool for probing kisspeptin action, this study aimed to clarify the role of kisspeptin in gonadotropin-releasing hormone (GnRH) pulse generation in goats. We examined the effects of chronic administration of TAK-683, an investigational kisspeptin analog, on LH secretion, GnRH immunostaining, pituitary responses to exogenous GnRH, and GnRH pulse generator activity, reflected by a characteristic increase in multiple-unit activity (MUA volley). An osmotic pump containing TAK-683 was subcutaneously implanted on day 0. TAK-683 treatment dose-dependently suppressed pulsatile LH secretion on day 1. Higher doses of chronic TAK-683 profoundly suppressed pulsatile LH secretion but had little effect on GnRH immunostaining patterns and pituitary responses to GnRH on day 5. In ovariectomized goats, MUA volleys occurred at approximately every 30 min on day -1. On day 5 of chronic TAK-683 administration, pulsatile LH secretion was markedly suppressed, whereas MUA volleys were similar to those observed on day -1. Male pheromones and senktide (neurokinin B receptor agonist) induced an MUA volley but had no effect on LH secretion during chronic TAK-683 administration. The results indicate that the chronic administration of a kisspeptin analog profoundly suppresses pulsatile LH secretion without affecting GnRH content, pituitary function or GnRH pulse generator activity, and they suggest an indispensable role for kisspeptin signaling in the cascade driving GnRH/LH pulses by the GnRH pulse generator.

Neurokinin B signaling in the female rat: a novel link between stress and reproduction.

Acute systemic stress disrupts reproductive function by inhibiting pulsatile gonadotropin secretion. The underlying mechanism involves stress-induced suppression of the GnRH pulse generator, the functional unit of which is considered to be the hypothalamic arcuate nucleus kisspeptin/neurokinin B/dynorphin A neurons. Agonists of the neurokinin B (NKB) receptor (NK3R) have been shown to suppress the GnRH pulse generator, in a dynorphin A (Dyn)-dependent fashion, under hypoestrogenic conditions, and Dyn has been well documented to mediate several stress-related central regulatory functions. We hypothesized that the NKB/Dyn signaling cascade is required for stress-induced suppression of the GnRH pulse generator. To investigate this ovariectomized rats, iv administered with Escherichia coli lipopolysaccharide (LPS) following intracerebroventricular pretreatment with NK3R or κ-opioid receptor (Dyn receptor) antagonists, were subjected to frequent blood sampling for hormone analysis. Antagonism of NK3R, but not κ-opioid receptor, blocked the suppressive effect of LPS challenge on LH pulse frequency. Neither antagonist affected LPS-induced corticosterone secretion. Hypothalamic arcuate nucleus NKB neurons project to the paraventricular nucleus, the major hypothalamic source of the stress-related neuropeptides CRH and arginine vasopressin (AVP), which have been implicated in the stress-induced suppression of the hypothalamic-pituitary-gonadal axis. A separate group of ovariectomized rats was, therefore, used to address the potential involvement of central CRH and/or AVP signaling in the suppression of LH pulsatility induced by intracerebroventricular administration of a selective NK3R agonist, senktide. Neither AVP nor CRH receptor antagonists affected the senktide-induced suppression of the LH pulse; however, antagonism of type 2 CRH receptors attenuated the accompanying elevation of corticosterone levels. These data indicate that the suppression of the GnRH pulse generator by acute systemic stress requires hypothalamic NKB/NK3R signaling and that any involvement of CRH therewith is functionally upstream of NKB.

Molecular recognition of tachykinin receptor selective agonists: insights from structural studies.

This Review deals essentially with the elucidation of structural features of Tachykinin family of neuropeptides, which are known to interact through three distinct GPCR subtypes, namely NK1 (Neurokinin 1), NK2 (Neurokinin 2) and NK3 (Neurokinin 3) receptors. In mammals, Tachykinins have been shown to elicit a wide array of activities such as powerful vasodilatation, hypertensive action and stimulation of extravascular smooth muscle and are known to be involved in a variety of clinical conditions including chronic pain, Parkinson's disease, Alzheimer's disease, depression, rheumatoid arthritis, irritable bowel syndrome and asthma. This broad spectrum of action of Tachykinins is attributed to the lack of selectivity of tachykinins to their receptors. All tachykinins interact with all the three-receptor subtypes with SP preferring NK1, NKA preferring NK2 and NKB preferring NK3. This lack of specificity can be accounted for by the conformational flexibility of these short, linear peptides. Hence, identification of structural features of the agonists important for receptor binding and biological activity is of great significance in unraveling the molecular mechanisms involved in tachykinin receptor activation and also in rational design of novel therapeutic agents. Understanding structure of the ligand-receptor complex and analysis of topography of the binding pocket of the tachykinin receptor is also crucial in rational design of drugs.

Activation of neurokinin 3 receptors stimulates GnRH release in a location-dependent but kisspeptin-independent manner in adult mice.

GnRH neurons form the final common pathway for the central control of reproduction. GnRH release occurs from terminals in the external layer of the median eminence (ME) for neuroendocrine control of the pituitary, and near GnRH-GnRH fiber appositions within the preoptic area (POA). Whether or not control of GnRH secretion by neuromodulators is different in these 2 areas is unknown. Mutations in neurokinin B (NKB) or the neurokinin-3 receptor (NK3R) are linked to hypogonadotropic hypogonadism in humans, suggesting that NKB may regulate GnRH secretion. Using fast scan cyclic voltammetry through carbon-fiber microelectrodes, we examined real-time GnRH release in response to the NK3R agonist senktide in the ME and POA. Coronal brain slices were acutely prepared from adult gonad-intact GnRH-green fluorescent protein male mice, and carbon-fiber microelectrodes were placed either within green fluorescent protein-positive terminal fields of the ME or near GnRH-GnRH fiber appositions in the POA. Senktide induced GnRH release consistently in the ME but not the POA, indicating that GnRH release is differentially regulated by NKB in a location-dependent manner. Senktide also induced GnRH secretion in the ME of kisspeptin-knockout (Kiss1 knockout) mice. Interestingly, release amplitude was lower compared with wild-type mice. These data indicate regulation of GnRH release by NK3R agonists is site specific and suggest that kisspeptin is not a required mediator between NK3R activation and GnRH secretion in the ME. This information will be useful for informing future models of afferent regulation of GnRH release.

Neurokinin B activates arcuate kisspeptin neurons through multiple tachykinin receptors in the male mouse.

Kisspeptin neurons located in the arcuate nucleus (ARN) coexpress dynorphin and neurokinin B (NKB) and may interact to influence gonadotropin secretion. Using a kisspeptin-green fluorescent protein mouse model, the present study examined whether the neuropeptides kisspeptin, dynorphin, and NKB modulate the electrical activity of ARN kisspeptin neurons in the adult male mouse. Cell-attached recordings showed that kisspeptin itself had no effect on kisspeptin neuron firing. Dynorphin and the κ-opioid receptor agonist U50-488 evoked a potent suppression of all ARN kisspeptin neuron firing that was blocked completely by the κ-opioid receptor antagonist nor-Binaltorphimine. Both NKB and Senktide, a neurokinin 3 receptor agonist, exerted a potent stimulatory action on ∼95% of ARN kisspeptin neurons. Although the selective neurokinin 3 receptor antagonists SB222200 and SR142801 blocked the effects of Senktide on kisspeptin neurons, they surprisingly had no effect on NKB activation of firing. Studies with selective neurokinin 1 receptor (SDZ-NKT343) and neurokinin 2 receptor (GR94800) antagonists revealed that the activation of kisspeptin neurons by NKB was only blocked completely by a cocktail of antagonists against all 3 tachykinin receptors. Whole-cell recordings revealed that individual kisspeptin neurons were activated directly by all 3 tachykinins substance, P, neurokinin A, and NKB. These experiments show that dynorphin and NKB have opposing actions on the electrical activity of kisspeptin neurons supporting the existence of an interconnected network of kisspeptin neurons in the ARN. However, the effects of NKB result from an unexpected activation of multiple tachykinin receptors.