Multiple-unit Activity Volleys Research Articles

Peripheral administration of a κ-opioid receptor agonist nalfurafine inactivates gonadotropin-releasing hormone pulse generator activity in goats

Neurons co-expressing kisspeptin, neurokinin B, and dynorphin A (KNDy neurons), located in the arcuate nucleus (ARC) of the hypothalamus, are indicated to be the gonadotropin-releasing hormone (GnRH) pulse generator. Dynorphin A is reported to suppress GnRH pulse generator activity. Nalfurafine is a selective agonist of the κ-opioid receptor (KOR), a receptor for dynorphin A, clinically used as an anti-pruritic drug. This study aimed to evaluate the effects of nalfurafine on GnRH pulse generator activity and luteinizing hormone (LH) pulses using female goats. Nalfurafine (0, 2, 4, 8, or 16 μg/head) was intravenously injected into ovariectomized Shiba goats. The multiple unit activity (MUA) in the ARC area was recorded, and plasma LH concentrations were measured 2 and 48 h before and after injection, respectively. The MUA volley interval during 0–2 h after injection was significantly increased in the nalfurafine 8 and 16 μg groups compared with the vehicle group. In 0–2 h after injection, the number of LH pulses was significantly decreased in the nalfurafine 8 and 16 μg groups, and the mean and baseline LH were significantly decreased in all nalfurafine-treated groups (2, 4, 8, and 16 μg) compared with the vehicle group. These results suggest that nalfurafine inhibits the activity of the GnRH pulse generator in the ARC, thus suppressing pulsatile LH secretion. Therefore, nalfurafine could be used as a reproductive inhibitor in mammals.

Facilitatory and inhibitory role of central amylin administration in the regulation of the gonadotropin-releasing hormone pulse generator activity in goats

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is essential for regulating reproductive functions in mammals. GnRH pulses are governed by a neural mechanism that is termed the GnRH pulse generator. In the present study, we investigated the role of central calcitonin receptor (CTR) signaling in the regulation of the GnRH pulse generator activity in ovariectomized goats by administering amylin, an endogenous ligand for CTR, into the lateral ventricle. GnRH pulse generator activity was measured using multiple unit activity (MUA) recordings in the mediobasal hypothalamus. We analyzed changes in the interval of characteristic increases in MUA (MUA volleys). The MUA volley interval shortened immediately after amylin administration, followed by prolonged intervals. Double in situ hybridization for KISS1 (kisspeptin gene) and CALCR (CTR gene) revealed that low expression levels of CALCR were found in the arcuate kisspeptin neurons, which is suggested as the main population of neurons, involved in GnRH pulse generator activity. These results suggest that central amylin-CTR signaling has a biphasic role in the regulation of GnRH pulse generator activity by acting on cells other than the arcuate kisspeptin neurons in goats.

Electrophysiological approach to the hypothalamic GnRH pulse generator

The hypothalamic GnRH pulse generator that regulates intermittent GnRH discharge into the pituitary portal circulation and thereby modulates pulsatile secretion of LH has been recognized as a key determinant of the reproductive function in mammals. Thus, various internal, as well as external, factors first modify the electrical activity of the GnRH pulse generator, which then alters the pulsatile pattern of gonadotrophin secretion and eventually influences reproductive function. Here, we describe a procedure that has permitted long-term recording of electrophysiological manifestation of the GnRH pulse generator activity and its application to our research which uses ovariectomized and cyclic female goats as experimental models. We have successfully recorded characteristic increases in neuronal activity associated with pulsatile LH secretion from conscious goats by means of a multiple unit activity (MUA) recording technique, which is an adaptation of that developed originally for use in rhesus monkeys. The unitary relationship between periodical increases in MUA (MUA volleys) and LH pulses is well maintained under a variety of experimental conditions, providing evidence that these MUA volleys are the consequence of GnRH pulse generator activity.

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.

A Population of Kisspeptin/Neurokinin B Neurons in the Arcuate Nucleus May Be the Central Target of the Male Effect Phenomenon in Goats

Exposure of females to a male pheromone accelerates pulsatile gonadotropin-releasing hormone (GnRH) secretion in goats. Recent evidence has suggested that neurons in the arcuate nucleus (ARC) containing kisspeptin and neurokinin B (NKB) play a pivotal role in the control of GnRH secretion. Therefore, we hypothesized that these neurons may be the central target of the male pheromone. To test this hypothesis, we examined whether NKB signaling is involved in the pheromone action, and whether ARC kisspeptin/NKB neurons receive input from the medial nucleus of the amygdala (MeA)—the nucleus suggested to relay pheromone signals. Ovariectomized goats were implanted with a recording electrode aimed at a population of ARC kisspeptin/NKB neurons, and GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA) volleys, was measured. Pheromone exposure induced an MUA volley and luteinizing hormone (LH) pulse in control animals, whereas the MUA and LH responses to the pheromone were completely suppressed by the treatment with an NKB receptor antagonist. These results indicate that NKB signaling is a prerequisite for pheromone action. In ovariectomized goats, an anterograde tracer was injected into the MeA, and possible connections between the MeA and ARC kisspeptin/NKB neurons were examined. Histochemical observations demonstrated that a subset of ARC kisspeptin/NKB neurons receive efferent projections from the MeA. These results suggest that the male pheromone signal is conveyed via the MeA to ARC kisspeptin neurons, wherein the signal stimulates GnRH pulse generator activity through an NKB signaling-mediated mechanism in goats.

Effects of Brief Exposure of Male Pheromone on Multiple-Unit Activity at Close Proximity to Kisspeptin Neurons in the Goat Arcuate Nucleus

Exposure of females to the male pheromone induces pulsatile release of gonadotropin-releasing hormone (GnRH) in goats. Recently, kisspeptin neurons in the arcuate nucleus (ARC) have been suggested to represent the proximate source of the GnRH pulse generator. In this study, we examined the effects of the pheromone on multiple-unit activity (MUA) in female goats fitted with recording electrodes aimed at the ARC kisspeptin neurons. In all eight goats, periodic bursts in MUA (MUA volleys), which were considered to be electrophysiological manifestations of the GnRH pulse generator, were observed. The mean intervolley interval (T) during the control period was calculated in each goat that was then exposed to the male pheromone for 1 sec at timings of 1/4 T, 1/2 T or 3/4 T after one regularly occurring MUA volley. An instantaneous rise in MUA was observed immediately after the exposure regardless of timing. Exposure at a timing of 3/4 T resulted in an MUA volley within 60 sec following the instantaneous rise in all goats. In contrast, an MUA volley was induced in only 2 goats by exposure at 1/2 T, while exposure at 1/4 T failed to induce an MUA volley in any goats. These results suggest that transmission of the pheromone signal to the ARC, represented by an instantaneous rise, activates the GnRH pulse generator. Moreover, the timing-dependent pheromone action in inducing an MUA volley indicates that the GnRH pulse generator has a refractory period for the pheromone signal after the burst.

Male Effect Pheromone Tickles the Gonadotrophin‐Releasing Hormone Pulse Generator

In sheep and goats, the primer pheromone produced by the male induces out-of-seasonal ovulation in anoestrous females, the so-called 'male effect.' Because the initial endocrine event following reception of the pheromone is the stimulation of pulsatile luteinising hormone (LH) secretion, the central target of the pheromone is considered to be the putative gonadotrophin-releasing hormone (GnRH) pulse generator. Using electrophysiological techniques to record multiple-unit activity (MUA) in close proximity to kisspeptin neurones in the arcuate nucleus (ARC) of Shiba goats, we found that bursts (volleys) of MUA occur at regular intervals, and repetitive bursts are invariably associated with discrete pulses of LH, suggesting that the ARC kisspeptin neurones may be the intrinsic source of the GnRH pulse generator. A brief exposure of female goats to the pheromone immediately elicited an instantaneous rise in MUA, which is followed by an MUA volley and an accompanying LH pulse, indicating that the pheromone signal is transmitted to a subset of the ARC kisspeptin neurones to activate them. Because it has been suggested that the neurokinin B and dynorphin coexpressed in those neurones play critical roles in generating rhythmic bursts, they may be involved in the intracellular pheromone actions that are responsible for inducing the GnRH pulse.

Neurokinin B and Dynorphin A in Kisspeptin Neurons of the Arcuate Nucleus Participate in Generation of Periodic Oscillation of Neural Activity Driving Pulsatile Gonadotropin-Releasing Hormone Secretion in the Goat

Gonadotropin-releasing hormone (GnRH) neurons in the basal forebrain are the final common pathway through which the brain regulates reproduction. GnRH secretion occurs in a pulsatile manner, and indirect evidence suggests the kisspeptin neurons in the arcuate nucleus (ARC) serve as the central pacemaker that drives pulsatile GnRH secretion. The purpose of this study was to investigate the possible coexpression of kisspeptin, neurokinin B (NKB), and dynorphin A (Dyn) in neurons of the ARC of the goat and evaluate their potential roles in generating GnRH pulses. Using double and triple labeling, we confirmed that all three neuropeptides are coexpressed in the same population of neurons. Using electrophysiological techniques to record multiple-unit activity (MUA) in the medial basal hypothalamus, we found that bursts of MUA occurred at regular intervals in ovariectomized animals and that these repetitive bursts (volleys) were invariably associated with discrete pulses of luteinizing hormone (LH) (and by inference GnRH). Moreover, the frequency of MUA volleys was reduced by gonadal steroids, suggesting that the volleys reflect the rhythmic discharge of steroid-sensitive neurons that regulate GnRH secretion. Finally, we observed that central administration of Dyn-inhibit MUA volleys and pulsatile LH secretion, whereas NKB induced MUA volleys. These observations are consistent with the hypothesis that kisspeptin neurons in the ARC drive pulsatile GnRH and LH secretion, and suggest that NKB and Dyn expressed in those neurons are involved in the process of generating the rhythmic discharge of kisspeptin.

Twelve-hour pair housing attenuates stress-induced hyperthermia in response to conditioned stimulus

The hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator regulates intermittent GnRH discharge into the pituitary portal circulation, and thereby modulates the pattern of pulsatile secretion of luteinizing hormone (LH). Various internal and external factors first modify the GnRH pulse generator activity, which then alters the pulsatile pattern of gonadotrophin secretion, and eventually influence reproductive function. We have successfully recorded characteristic increases in neuronal activity associated with pulsatile LH secretion from the ovariectomized and cycling female goat by means of a multiple unit activity (MUA) recording technique. The unitary relationship between periodic increases in MUA volleys and LH pulses is well maintained under a variety of experimental conditions, providing evidence that these MUA volleys are the consequence of GnRH pulse generator activity. A procedure that has permitted long-term recording of electrophysiological manifestation of the GnRH pulse generator activity and its research application are described.

Further Evidence for the Role of Glucose as a Metabolic Regulator of Hypothalamic Gonadotropin-Releasing Hormone Pulse Generator Activity in Goats

The present study examined the relative importance of blood glucose vs. free fatty acids as a metabolic signal regulating GnRH release as measured electrophysiologically by multiple-unit activity (MUA) in the arcuate nucleus/median eminence region in ovariectomized, estradiol-treated goats. MUA was recorded before, during, and after: 1) cellular glucoprivation by peripheral infusion of 2-deoxy-d-glucose (2DG; 25, 50, and 75 mg/kg.h, iv); 2) peripheral hypoglycemia in response to various doses (15-195 mU/kg.h, iv) of insulin infusion; and 3) cellular lipoprivation induced by peripheral infusion of sodium mercaptoacetate (MA; 2.4 mg/kg.h alone or combined with 25 mg/kg.h of 2DG, iv), and effects on the interval of characteristic increases in MUA (MUA volleys) were examined. Infusion of the highest dose of 2DG increased the mean interval between MUA volleys, whereas the lower doses of 2DG had no effect on volley interval. The MUA volley intervals lengthened as insulin-induced hypoglycemia became profound. There was a negative correlation between MUA volley intervals and blood glucose concentrations during insulin infusion, and coinfusion of glucose with insulin returned the MUA volley interval to a normal frequency. Infusion of MA alone or MA with 2DG did not increase MUA volley intervals. These findings demonstrate that glucose availability, but not fatty acids, regulates the GnRH pulse generator activity in the ruminant. Glucose is considered a key metabolic regulator that fine-tunes pulsatile GnRH release.

Lipopolysaccharide-induced suppression of the hypothalamic gonadotropin-releasing hormone pulse generator in ovariectomized goats.

Ovariectomized goats were implanted with the electrode arrays for monitoring the electrophysiological manifestation of the activity of the hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator, namely multiple-unit activity (MUA) volleys associated with pulsatile luteinizing hormone secretion. They were then subjected to i.v. challenges of lipopolysaccharide (LPS) at the dose of 200 or 400 ng/kg. The interval between the MUA volleys was significantly prolonged by higher dose of LPS whereas neither amplitude nor duration of the MUA volleys was altered. These results suggest that immunological disturbance as evoked by LPS administration directly affects the hypothalamic GnRH pulse generator by slowing down the pulse frequency, and thereby lowers gonadotropin secretion from the anterior pituitary gland, which would culminate in gonadal suppression.

Neural activity in the VMH associated with suppression of the circulatory system in rats

Spontaneous neural activity within the ventromedial nucleus of the hypothalamus (VMH) was monitored in rats to search for neurons regulating the autonomic nervous system. By means of multiple unit activity (MUA) recording method, unique explosive rises in neural activity (MUA volleys), 1 to 4 min in duration, were recorded in conscious freely moving animals. Heart rate was monitored as an autonomic parameter and found to decrease when MUA volleys appeared. These MUA volleys also occurred under urethane anesthesia, and blood pressure and heart rate decreased simultaneously with the volleys, but body temperature remained constant. This fall in blood pressure (but not heart rate) was replicated by electrical stimulation through the electrodes that recorded MUA volleys, suggesting that the neurons responsible for MUA volleys can suppress the circulatory system. The frequency of MUA volleys exhibited a clear diurnal variation: they appeared every 15 or 30 min in the light phase but only seldom in the dark. This diurnal variation seems to be an endogenous circadian rhythm because it was indicated to freerun after blinding the animals. These results suggest that there is a discrete population of neurons in the VMH that fires predominantly during the light phase in an episodic manner and suppresses the circulatory system.

Electrophysiological correlates of GnRH secretion

Abstract The hypothalamic gonadotropin-releasing hormone (GnRH) pulse generator regulates intermittent GnRH discharge into the pituitary portal circulation, and thereby modulates the pattern of pulsatile secretion of luteinizing hormone (LH). Various internal and external factors first modify the GnRH pulse generator activity, which then alters the pulsatile pattern of gonadotrophin secretion, and eventually influence reproductive function. We have successfully recorded characteristic increases in neuronal activity associated with pulsatile LH secretion from the ovariectomized and cycling female goat by means of a multiple unit activity (MUA) recording technique. The unitary relationship between periodic increases in MUA volleys and LH pulses is well maintained under a variety of experimental conditions, providing evidence that these MUA volleys are the consequence of GnRH pulse generator activity. A procedure that has permitted long-term recording of electrophysiological manifestation of the GnRH pulse generator activity and its research application are described.

Changes in the gonadotropin-releasing hormone pulse generator activity during the estrous cycle in the goat.

The hypothalamic GnRH pulse generator activity, which was assessed by the characteristic increases in the multiple unit activity (MUA) specifically associated with the pulsatile LH secretion (MUA volleys), was monitored throughout the estrous cycle of the female goat. Blood samples were collected daily for analysis of progesterone, estradiol and LH levels to monitor ovarian cyclicity. The frequency of the MUA volleys showed a reciprocal relationship with the plasma progesterone profile; being low during the luteal phase, whereas increased during the follicular phase until the onset of the subsequent luteal phase. On the other hand, the duration of the MUA volleys changed in parallel with plasma progesterone levels showing considerable prolongation in the luteal phase. More frequent blood sampling was carried out at 2-hour intervals for timing the LH surge during the follicular phase induced by prostaglandin F2alpha (PGF2alpha) administration. During the PGF2alpha-induced follicular phase, the MUA volley frequency was abruptly increased from 4 h after PGF2alpha injection, and this high frequency was maintained throughout the follicular phase except for the period around the preovulatory LH surge when the MUA volley frequency was lowered temporarily. These results were in accordance with our previous observation that the recurrence of MUA volleys continued with decreased frequency during the estradiol-induced LH surge in ovariectomized goats. The present study revealed that GnRH pulse generator activity changes dramatically during the estrous cycle in the goat, and it is suggested that GnRH pulse generator activity becomes fully potentiated following luteolysis to induce the final follicular development and to stimulate estradiol secretion, which eventually provokes another neural mechanism responsible for the induction of the LH surge.

Electrophysiological Recording of Hypothalamic GnRH Pulse Generator Activity in Castrated Male Goats.

Electrophysiological recording of the hypothalamic GnRH pulse generator activity was attempted in 3 long-term castrated male goats in which electrodes had been implanted in the medial basal hypothalamus (MBH). While multiple unit activity (MUA) signals from the electrode were continuously recorded, each animal received subcutaneous implants containing testosterone for 7 days (Experiment 1). They were similarly treated with estradiol alone, estradiol plus testosterone, and testosterone alone for 7 days each consecutively (Experiment 2). These steroidal treatments enabled to maintain the plasma levels of testosterone and estradiol within physiological ranges at 2-4 ng/ml and 3-5 pg/ml, respectively. Blood samples for the determination of pulsatile LH secretion were collected at 6-min intervals for 4 h on the last day of each treatment period. The short-lived sudden increase in MUA spikes (MUA volley) was detected being always preceding the occurrence of the LH pulse in the peripheral blood, and this synchrony of MUA volley with the LH pulse was constant in all the animals regardless of steroidal treatments. Testosterone treatment resulted in the increase of the MUA volley intervals from 27 min to 43 min (Experiment 1). Treatment with estradiol in Experiment 2 also resulted in prolonged MUA volley intervals similar to those of testosterone treatment and there was no additive or synergistic effect between these two steroids. The present study demonstrated that the electrophysiological manifestation of the hypothalamic GnRH pulse generator could be successfully monitored in male goats as reported in females, and it is suggested that estradiol may exert a negative feedback effect on the GnRH pulse generator activity in a similar manner to that of testosterone but at much lower doses in male goats.

Electrophysiological studies on the hypothalamic GnRH pulse generator

Gonadal function in mammals depends on gonadotropins secreted from the pituitary gland in a pulsatile manner. This pulsatility is governed by the periodic activation of the hypothalamic GnRH pulse generator. By means of multiple unit activity (MUA) recording techniques, characteristics increases in the neuronal activity, each of which is associated with the initiation of pulsatile LH secretion, have been recorded in the medial basal hypothalamus of the monkey, rat and goat. An unambiguous unitary relationship between the increased electrical activity (volley) and the LH pulse under a variety of physiological and experimental conditions indicates that the MUA volleys represent the electrical activity of the GnRH pulse generator. Hypothalamic MUA recordings provide direct access to the central component of the neuroendocrine control system which governs reproductive function.