dynorphins and senktide

The onset of puberty is the result of an increase in secretion of hypothalamic gonadotrophin-releasing hormone (GnRH). This action is a result of not only the development of stimulatory inputs to its release, but also the gradual decrease in inhibitory inputs that restrain release of the peptide prior to pubertal onset. Dynorphin (DYN) is one of the inhibitory inputs produced in the medial basal hypothalamus (MBH); however, little is known about what substance(s) control its prepubertal synthesis and release. Because neurokinin B (NKB) increases in the hypothalamus as puberty approaches, we considered it a candidate for such a role. An initial study investigated the acute effects of an NKB agonist, senktide, on the secretion of DYN from MBH tissues incubated in vitro. In other experiments, central injections of senktide were administered to animals for 4 days then MBHs were collected for assessment of DYN synthesis or for the in vitro secretion of both DYN and GnRH. Because insulin-like growth factor (IGF)-1 has been shown to play an important role at puberty, additional animals received central injections of this peptide for 4 days to assess NKB and DYN synthesis or the in vitro secretion of NKB. The results obtained show that senktide administration up-regulates the NKB receptor protein, at the same time as suppressing the DYN and its receptor. Senktide consistently suppressed DYN and elevated GnRH secretion in the same tissue incubates from both the acute and chronic studies. IGF-1 administration caused an increase in NKB protein, at the same time as decreasing DYN protein. Furthermore, the central administration of IGF-1 caused an increase in NKB release, an action blocked by the IGF-1 receptor blocker, JB-1. These results indicate that the IGF-1/NKB pathway contributes to suppressing the DYN inhibitory tone on prepubertal GnRH secretion and thus facilitates the puberty-related increase in the release of GnRH to accelerate the onset of puberty.

Topics: Animals; Dynorphins; Female; Gonadotropin-Releasing Hormone; Hypothalamus, Middle; Insulin-Like Growth Factor I; Microinjections; Neurokinin B; Peptide Fragments; Peptides; Rats; Receptor, IGF Type 1; Receptors, Neurokinin-3; Receptors, Opioid; Sexual Maturation; Substance P; Up-Regulation

Pulsatile secretion of gonadotrophin-releasing hormone (GnRH)/luteinising hormone is indispensable for the onset of puberty and reproductive activities at adulthood in mammalian species. A cohort of neurones expressing three neuropeptides, namely kisspeptin, encoded by the Kiss1 gene, neurokinin B (NKB) and dynorphin A, localised in the hypothalamic arcuate nucleus (ARC), so-called KNDy neurones, comprises a putative intrinsic source of the GnRH pulse generator. Synchronous activity among KNDy neurones is considered to be required for pulsatile GnRH secretion. It has been reported that gap junctions play a key role in synchronising electrical activity in the central nervous system. Thus, we hypothesised that gap junctions are involved in the synchronised activities of KNDy neurones, which is induced by NKB-NK3R signalling. We determined the role of NKB-NK3R signalling in Ca

Topics: Animals; Cells, Cultured; Connexins; Dynorphins; Gap Junctions; Glycyrrhetinic Acid; Kisspeptins; Medulla Oblongata; Mefloquine; Mice, Transgenic; Neuroglia; Neurokinin B; Neurons; Peptide Fragments; Substance P

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-β 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.

Topics: Androgens; Animals; Arcuate Nucleus of Hypothalamus; Dihydrotestosterone; Dynorphins; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Estrogens; Gonadotropin-Releasing Hormone; Kisspeptins; Male; Mice; Neurokinin B; Neurons; Orchiectomy; Patch-Clamp Techniques; Peptide Fragments; Receptors, Neurokinin-3; Receptors, Opioid, kappa; Receptors, Steroid; Substance P

Kisspeptin/neurokinin B/dynorphin (KNDy) neurons, which coexpress kisspeptins (Kps), neurokinin B (NKB), and dynorphin (Dyn), regulate gonadotropin secretion. The KNDy model proposes that NKB (a stimulator, through NK3R) and Dyn (an inhibitor, through κ-opioid receptor) shape Kp secretion onto GnRH neurons. However, some aspects of this paradigm remain ill defined. Here we aimed to characterize the following: 1) the effects of NKB signaling on FSH secretion and 2) the role of Dyn in gonadotropin secretion after NK3R activation; 3) additionally, we explored the roles of other tachykinin receptors, NK1R and NK2R, on gonadotropin release. Thus, the effects of the NK3R agonist, senktide, on FSH release were explored across postnatal development in male and female rats; gonadotropin responses to agonists of NK1R substance P and NK2R [neurokinin A (NKA)] were also monitored. Moreover, the effects of senktide on gonadotropin secretion were assessed after antagonizing Dyn actions by nor-binaltorphimine didydrochloride. Before puberty, rats of both sexes showed increased FSH secretion to senktide (and Kp-10). Conversely, adult female rats were irresponsive to senktide in terms of FSH, despite proven LH responses, whereas the adult males did not display FSH or LH responses to senktide, even at high doses. In turn, substance P and NKA stimulated gonadotropin secretion in prepubertal rats, whereas in adults modest gonadotropin responses to NKA were detected. By pretreatment with a Dyn antagonist, adult males became responsive to senktide in terms of LH secretion and displayed elevated basal LH and FSH levels; nor-binaltorphimine didydrochloride treatment uncovered FSH responses to senktide in adult females. Furthermore, the expression of Pdyn and Opkr1 (encoding Dyn and κ-opioid receptor, respectively) in the mediobasal hypothalamus was greater in males than in females at prepubertal ages. Overall, our data contribute to refining our understanding on how the elements of the KNDy node and related factors (ie, other tachykinins) differentially participate in the control of gonadotropins at different stages of rat postnatal maturation.

Topics: Aging; Animals; Dynorphins; Enkephalins; Female; Follicle Stimulating Hormone; Hypothalamus; Kisspeptins; Luteinizing Hormone; Male; Neurokinin B; Peptide Fragments; Protein Precursors; Rats, Wistar; Receptors, Neurokinin-1; Receptors, Neurokinin-2; Substance P

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.. 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.. 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.. 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.

Topics: Animals; Arcuate Nucleus of Hypothalamus; Dynorphins; Estrogens; Estrous Cycle; Female; Gonadotropin-Releasing Hormone; Immunohistochemistry; In Situ Hybridization; Kisspeptins; Luteinizing Hormone; Models, Animal; Neurokinin B; Neurons; Neurotransmitter Agents; Ovariectomy; Peptide Fragments; Receptors, Neurokinin-3; RNA, Messenger; Sheep; Substance P

Reproductive function is tightly regulated by an intricate network of central and peripheral factors; however, the precise mechanism triggering critical reproductive events, such as puberty onset, remains largely unknown. Recently, the neuropeptides kisspeptin (encoded by Kiss1) and neurokinin B (NKB, encoded by TAC3 in humans and Tac2 in rodents) have been placed as essential gatekeepers of puberty. Studies in humans and rodents have revealed that loss-of-function mutations in the genes encoding either kisspeptin and NKB or their receptors, Kiss1r and neurokinin 3 receptor (NK3R), lead to impaired sexual maturation and infertility. Kisspeptin, NKB, and dynorphin A are co-expressed in neurons of the arcuate nucleus (ARC), so-called Kisspeptin/NKB/Dyn (KNDy) neurons. Importantly, these neurons also co-express NK3R. Compelling evidence suggests a stimulatory role of NKB (or the NK3R agonist, senktide) on LH release in a number of species. This effect is likely mediated by autosynaptic inputs of NKB on KNDy neurons to induce the secretion of gonadotropin-releasing hormone (GnRH) in a kisspeptin--dependent manner, with the coordinated actions of other neuroendocrine factors, such as dynorphin, glutamate, or GABA. Thus, we have proposed a model in which NKB feeds back to the KNDy neuron to shape the pulsatile release of kisspeptin, and hence GnRH, in a mechanism also dependent on the sex steroid level. Additionally, NKB may contribute to the regulation of the reproductive function by metabolic cues. Investigating how NKB and kisspeptin interact to regulate the gonadotropic axis will offer new insights into the control of GnRH release during puberty onset and the maintenance of the reproductive function in adulthood, offering a platform for the understanding and treatment of a number of reproductive disorders.

Topics: Animals; Dynorphins; gamma-Aminobutyric Acid; Glutamic Acid; Gonadal Steroid Hormones; Humans; Kisspeptins; Luteinizing Hormone; Mutation; Neurokinin B; Neurons; Peptide Fragments; Puberty; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; Receptors, Neurokinin-3; Rodentia; Sexual Maturation; Substance P

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.

Topics: 3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; Analgesics, Non-Narcotic; Animals; Arcuate Nucleus of Hypothalamus; Dynorphins; Green Fluorescent Proteins; Kisspeptins; Male; Membrane Potentials; Mice; Mice, Transgenic; Neurokinin B; Neurons; Oligopeptides; Patch-Clamp Techniques; Peptide Fragments; Piperidines; Quinolines; Receptors, Neurokinin-2; Receptors, Neurokinin-3; Receptors, Opioid, kappa; Receptors, Tachykinin; Substance P

Pulsatile GnRH release is essential to fertility and is modulated by gonadal steroids, most likely via steroid-sensitive afferents. Arcuate neurons coexpressing kisspeptin, neurokinin B (NKB), and dynorphin (KNDy neurons) are steroid-sensitive and have been postulated to both generate GnRH pulses and mediate steroid feedback on pulse frequency. KNDy neurons are proposed to interact with one another via NKB and dynorphin to activate and inhibit the KNDy network, respectively, and thus alter kisspeptin output to GnRH neurons. To test the roles of NKB and dynorphin on KNDy neurons and the steroid sensitivity of these actions, targeted extracellular recordings were made of Tac2(NKB)-GFP-identified neurons from castrate and intact male mice. Single-cell PCR confirmed most of these cells had a KNDy phenotype. The neurokinin 3 receptor (NK3R) agonist senktide increased action potential firing activity of KNDy neurons. Dynorphin reduced spontaneous KNDy neuron activity, but antagonism of κ-opioid receptors (KOR) failed to induce firing activity in quiescent KNDy neurons. Senktide-induced activation was greater in KNDy neurons from castrate mice, whereas dynorphin-induced suppression was greater in KNDy neurons from intact mice. Interactions of dynorphin with senktide-induced activity were more complex; dynorphin treatment after senktide had no consistent inhibitory effect, whereas pretreatment with dynorphin decreased senktide-induced activity only in KNDy neurons from intact but not castrate mice. These data suggest dynorphin-mediated inhibition of senktide-induced activity requires gonadal steroid feedback. Together, these observations support the hypotheses that activation of NK3R and KOR, respectively, excites and inhibits KNDy neurons and that gonadal steroids modulate these effects.

Topics: Animals; Arcuate Nucleus of Hypothalamus; Benzeneacetamides; Dynorphins; Gonadotropin-Releasing Hormone; Green Fluorescent Proteins; Kisspeptins; Male; Membrane Potentials; Mice; Mice, Transgenic; Neurokinin B; Neurons; Orchiectomy; Peptide Fragments; Pyrrolidines; Receptors, Neurokinin-3; Receptors, Opioid, kappa; Substance P; Time Factors

Puberty in mammals is timed by an increase in gonadotropin-releasing hormone (GnRH) secretion. Previous studies have shown involvement of the two neuropeptides, kisspeptin and neurokinin B (NKB), in controlling puberty onset. Little is known about the role of the other key neuropeptide, dynorphin, in controlling puberty onset, although these three neuropeptides colocalize in the arcuate kisspeptin neurons. The arcuate kisspeptin neuron, which is also referred to as the KNDy neuron, has recently been considered to play a role as an intrinsic source of the GnRH pulse generator. The present study aimed to determine if attenuation of inhibitory dynorphin-kappa-opioid receptor (KOR) signaling triggers the initiation of puberty in normal developing female rats. The present study also determined if stimulatory NKB-neurokinin 3 receptor (NK3R) signaling advances puberty onset. Female Wistar-Imamichi rats were weaned and intraperitoneally implanted with osmotic minipumps filled with nor-binaltorphimine (nor-BNI), a KOR antagonist, or senktide, a NK3R agonist, at 20 days of age. Fourteen days of intraperitoneal infusion of nor-BNI or senktide advanced puberty onset, manifested as vaginal opening and the first vaginal estrus in female rats. Frequent blood sampling showed that nor-BNI significantly increased luteinizing hormone (LH) pulse frequency at 29 days of age compared with vehicle-treated controls. Senktide tended to increase this frequency, but its effect was not statistically significant. The present results suggest that the inhibitory input of dynorphin-KOR signaling plays a role in the prepubertal restraint of GnRH/LH secretion in normal developing female rats and that attenuation of dynorphin-KOR signaling and increase in NKB-NK3R signaling trigger the onset of puberty in female rats.

Topics: Animals; Drug Implants; Dynorphins; Female; Luteinizing Hormone; Naltrexone; Narcotic Antagonists; Nerve Tissue Proteins; Neurokinin B; Neurons; Ovary; Peptide Fragments; Puberty, Precocious; Random Allocation; Rats; Rats, Wistar; Receptors, Neurokinin-3; Receptors, Opioid, kappa; Sexual Maturation; Signal Transduction; Substance P; Weaning

Neurons in the arcuate nucleus (ARC) that concomitantly express kisspeptin, neurokinin B (NKB) and dynorphin A are termed KNDy neurons and are likely candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. Our hypothesis is that KNDy neurons are functionally and anatomically interconnected to generate discrete neural signals that govern pulsatile GnRH secretion. Our goal was to address this hypothesis using electrophysiological and anatomical experiments in goats. Bilateral electrodes targeting KNDy neurons were implanted into ovariectomized goats, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys), was measured. Spontaneous and pheromone- or senktide (an NKB receptor agonist)-induced MUA volleys were simultaneously recorded from both sides of the ARC. An anterograde tracer, biotinylated dextran amine (BDA), was also injected unilaterally into the ARC of castrated male goats, and the distribution of fibers containing both BDA and NKB was examined using dual-labeling histochemistry. The results showed that MUA volleys, regardless of origin (spontaneous or experimentally induced), occur simultaneously between the right and left sides of the ARC. Tract tracing indicated that axons projecting from NKB neurons in the ARC were directly apposed to other NKB neuronal cells located bilaterally in the ARC. These results demonstrate that GnRH pulse generator activity occurs synchronously between both sides of the ARC in goats and that KNDy neurons are bilaterally interconnected in the ARC via NKB-containing fibers. Taken together, the results suggest that KNDy neurons form a neuronal circuit to synchronize burst activity among KNDy neurons and thereby generate discrete neural signals that govern pulsatile GnRH secretion.

Topics: Animals; Arcuate Nucleus of Hypothalamus; Axons; Dynorphins; Electrodes; Electrophysiological Phenomena; Female; Gene Expression Regulation, Developmental; Goats; Gonadotropin-Releasing Hormone; Kisspeptins; Male; Neurokinin B; Neurons; Peptide Fragments; Pheromones; Substance P

Neurokinin B (NKB) and its receptor (neurokinin-3 receptor) are coexpressed with kisspeptin and dynorphin A (Dyn) within neurons of the hypothalamic arcuate nucleus, the suggested site of the GnRH pulse generator. It is thought that these neuropeptides interact to regulate gonadotropin secretion. Using the ovariectomized (OVX) and OVX 17β-estradiol-replaced rat models, we have carried out a series of in vivo neuropharmacological and electrophysiological experiments to elucidate the hierarchy between the kisspeptin, NKB, and Dyn signaling systems. Rats were implanted with intracerebroventricular cannulae and cardiac catheters for frequent (every 5 min) automated serial blood sampling. Freely moving rats were bled for 6 h, with intracerebroventricular injections taking place after a 2-h control bleeding period. A further group of OVX rats was implanted with intra-arcuate electrodes for the recording of multiunit activity volleys, which coincide invariably with LH pulses. Intracerebroventricular administration of the selective neurokinin-3 receptor agonist, senktide (100-600 pmol), caused a dose-dependent suppression of LH pulses and multiunit activity volleys. The effects of senktide did not differ between OVX and 17β-estradiol-replaced OVX animals. Pretreatment with a selective Dyn receptor (κ opioid receptor) antagonist, norbinaltorphimine (6.8 nmol), blocked the senktide-induced inhibition of pulsatile LH secretion. Intracerebroventricular injection of senktide did not affect the rise in LH concentrations after administration of kisspeptin (1 nmol), and neither did kisspeptin preclude the senktide-induced suppression of LH pulses. These data show that NKB suppresses the frequency of the GnRH pulse generator in a Dyn/κ opioid receptor-dependent fashion.

Topics: Animals; Arcuate Nucleus of Hypothalamus; Dynorphins; Female; Gonadotropin-Releasing Hormone; Kisspeptins; Luteinizing Hormone; Narcotic Antagonists; Neurokinin B; Ovariectomy; Peptide Fragments; Rats; Rats, Sprague-Dawley; Receptors, G-Protein-Coupled; Receptors, Kisspeptin-1; Receptors, Neurokinin-3; Receptors, Opioid; Receptors, Opioid, kappa; Signal Transduction; Substance P