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Orexin A/hypocretin-1 inhibits pulsatile luteinizing hormone (LH) secretion in female rats. In this study, we investigated whether this inhibition was tied to the fasting state, as suggested by our previous study. We first examined whether orexin A inhibited pulsatile LH secretion when food was available ad libitumduring blood sampling. Next, we investigated the effect of intravenous administration of glucose (400 mg/kg) or lactic acid (negative control; 400 mg/kg) on orexin A-induced inhibition of pulsatile LH secretion. We found that orexin A did not affect pulsatile LH secretion in the presence of food, although it increased feeding behavior. Injection of orexin A significantly inhibited pulsatile LH secretion when food was withheld during blood sampling (p < 0.05); this inhibitory effect was rapidly reversed by intravenous injection of glucose but not lactic acid. Because orexin A did not seem to affect pulsatile LH secretion when food was available ad libitum, we speculate that orexin A has an effect on LH secretion when orexin A-induced hunger is accompanied by stress, such as the absence of food. Furthermore, glucose as well as food may act as a satiety factor in gonadotropin-releasing hormone pulse generation.

p-Nonylphenol (NP) and 4-tert-octylphenol (OP) are known to mimic the action of estrogens as endocrine disruptors. However, their acute effects on the pituitary and the hypothalamus functions in vivo have been uncertain. We therefore determined their effects on the anterior pituitary, in particular, gonadotropin secretion. Two weeks after ovariectomy, the rats were given a subcutaneous injection of 10 mg NP, 10 mg OP, 10 mg bisphenol A, 1 µg 17β-estradiol, or sesame oil alone as control. Twenty-four hours after the treatment, the expression of progesterone receptor mRNA in the anterior pituitary and the level of luteinizing hormone (LH), follicle-stimulating hormone, and prolactin were determined. The expression of progesterone receptor mRNA in the anterior pituitary was significantly increased by either NP, OP, bisphenol A, or estradiol, but bisphenol A was less effective. The level of LH was significantly decreased by either NP or OP, but not by bisphenol A and estradiol. Only estradiol significantly increased the level of prolactin. The level of follicle-stimulating hormone was unchanged by any of the treatments. To check the effects of NP and OP on pulsatile LH secretion, blood samplings were done at 6-min intervals for 3 h. Twenty-four hours after treatment in ovariectomized adult rats, we found that the injection of NP significantly decreased the amplitude of LH pulses and the mean LH concentrations, but not the frequency of LH pulses. The injection of OP significantly decreased the mean LH concentrations without affecting the frequency and amplitude of the LH pulses. Finally, the rats given an injection of NP or sesame oil were intravenously injected with 50 ng of gonadotropin-releasing hormone (GnRH) to check whether NP affected the LH secretory responsiveness of the anterior pituitary to GnRH. We found that the responsiveness to GnRH in NP-injected rats was significantly attenuated compared to the sesame oil-injected rats. The present study suggests that NP, even with a single injection, suppresses the pulsatile LH secretion in adult ovariectomized rats, probably by affecting the anterior pituitary level.

We have suggested that gamma-aminobutyric acid (GABA) in the hypothalamus plays a tonic inhibitory role in the control of the luteinizing hormone (LH) release in intact male rats. To assess whether feedback from the testis alters the inhibitory GABAergic tone in the medial preoptic area (MPO) of male rats, an in vivo microdialysis study was performed in gonadally intact (n = 10), castrated (n = 12) and castrated testosterone-primed (n = 10) male rats. The microdialysis samples were collected and sequential blood samples were also obtained at 1-hour intervals. GABA in the dialysate was determined by high-performance liquid chromatography system and serum LH concentration was determined by radioimmunoassay. Episodic GABA release in the MPO was observed in all three groups of male rats, although castrated male rats showed lower GABA release (2.3 ± 0.3 ng/h) than intact and castrated testosterone-primed male rats (4.0 ± 0.5 and 4.6 ± 1.0 ng/h, respectively). Conversely, castrated male rats showed higher serum LH concentration (7.31 ± 0.46 ng/ml) than intact and castrated testosterone-primed male rats (0.71 ± 0.04 and 0.53 ± 0.07 ng/ml, respectively). In addition, intravenous infusion of bicuculline significantly increased serum LH in intact male rats, whereas bicuculline did not alter serum LH concentrations in castrated male rats. These results are consistent with the hypothesis that the feedback of testosterone stimulates GABA release in the region of the GnRH cell bodies and dendrites in male rats.

Butyl benzyl phthalate (BBP) and bisphenol A (BPA), so-called endocrine disrupters, are known to mimic the action of estrogens: they are thus liable to influence reproductive functions. Since little is known about their action on gene expression in the adult hypothalamus, we examined the effects of these chemicals on the expression of estrogen-regulated mRNAs, i.e., progesterone receptor (PR) mRNA, preproenkephalin (PPE) mRNA and neurotensin (NT) mRNA, in the hypothalamus and pituitary of adult female rats. Two weeks after ovariectomy, rats were subcutaneously injected with 10 mg BBP, 10 mg BPA, or 10 µg 17β-estradiol (E2) in sesame oil, or with sesame oil alone as a control. Twenty-four hours after the injection, tissues including the preoptic area (POA), mediobasal hypothalamus (MBH) and anterior pituitary were collected. Northern blot revealed that injection of E2 resulted in expected changes, i.e., significant increases in PR mRNA in the POA, MBH and anterior pituitary, and in PPE mRNA in the MBH. We also found that injection of BPA significantly increased PR mRNA in the POA and anterior pituitary, while injection of BBP increased PR mRNA in the POA and anterior pituitary, although the increase in the anterior pituitary was not significant. No significant effect of E2, BPA, or BBP on NT mRNA in the POA was detected. The present study demonstrates that the two endocrine disrupters BPA and BBP can increase the expression of PR mRNA in the POA of adult ovariectomized rats.

To determine whether gonadotropin-releasing hormone (GnRH) neurons in culture without the hypothalamus secrete GnRH in a pulsatile fashion, the nasal placode (NAP) was obtained at day 13.5 of gestation and cultured by a roller tube method. If the GnRH release occurs in a pulsatile fashion, it can be said that the pulse generator of GnRH exists inherently in each cell or community of cells in the culture. The concentration of GnRH in the NAP culture medium collected at 8-min intervals for 160 min after 2- to 4-week cultures showed that GnRH release occurred in a pulsatile fashion with a mean interpulse interval of 29.8 ± 2.3 min (n = 9). When the NAP was cultured with tissues of the forebrain vesicle (n = 3) or the hypothalamus (n = 4), GnRH was also released in a pulsatile fashion with similar intervals (27.3 ± 1.0 min for the NAP+forebrain vesicle culture and 36.0 ± 6.3 min for the NAP+hypothalamus culture) as those in cultures without brain tissues. It is concluded that pulsatile GnRH release is an inherent function of GnRH neurons.

The effects of N-methyl-D-aspartate (NMDA) and luteinizing hormone releasing hormone (LH-RH) on luteinizing hormone (LH) secretion were examined in ovariectomized estrogen-primed rats under nonstressed and acutely stressed conditions. The basal LH levels were significantly elevated 15 min after the onset of acute immobilization stress, but were not altered in emotionally stressed or nonstressed rats. Intravenous injections of 10 and 40 mg/kg NMDA significantly elevated serum LH levels by 161 and 212%, respectively, from baseline within 10 min in nonstressed animals. However, the NMDA-induced LH release was significantly reduced when tested 30 min after the onset of acute immobilization stress. Acute emotional stress, which did not affect the baseline LH, also suppressed the LH release response to NMDA, suggesting that the reduced LH responses to NMDA in stressed animals was not due to the elevated baseline level. Pituitary LH release responses to LH-RH were not affected by acute immobilization. We conclude from these results: (1) acute immobilization stress exerts both stimulatory and inhibitory effects on LH release, while acute emotional stress has only an inhibitory effect in estrogen-primed ovariectomized rats; (2) this inhibition occurs at the suprapituitary level, and (3) it involves a suppression of the responsiveness of the hypothalamic LH-RH neuronal system to the excitatory amino acid input.

Neuronal activity in the mediobasal hypothalamus was recorded during the steroid-induced surge of luteinizing hormone (LH) in ovariectomized female rats by means of multiunit activity (MUA) recording techniques. The day of subcutaneous implantation of estradiol-containing silastic tubing, which was done at 12.00 h, was designated as day 1, and progesterone (1 mg) dissolved in sesame oil was injected at 12.00 h on day 5. The hypothalamic MUA and serum LH level were monitored between 10.00 and 20.00 h on days 1, 4 and 5. All 5 animals examined showed characteristic increases (volleys) in MUA associated with pulsatile LH secretion. After implantation of estradiol on day 1, gradual and significant decreases in the serum LH level and in MUA volley frequency were observed. In the afternoon of both days 4 and 5, an LH surge occurred, though it was much greater on day 5 than day 4 due to progesterone injection on day 5. However, neither MUA volleys nor considerable changes in baseline MUA were discernible throughout the recording period on days 4 and 5. More than 5 days after the removal of estradiol-containing tubing, MUA volleys were found to reappear in all the animals. These results suggest that estradiol has an inhibitory effect on gonadotropin-releasing hormone (GnRH) pulse generator activity, and that the GnRH pulse generator is not involved in inducing the LH surge in the rat.

Effects of selective γ-aminobutyric acida (GABAa) and GABAb receptor agonists on noradrenaline (NA)-induced and N-methyl-D-aspartate (NMDA)-induced luteinizing-hormone (LH) secretion were examined in ovariectomized estrogen-primed rats. Experiments were performed in unanesthetized animals bearing chronic intracerebroventricular (i.c.v.) cannulae. Lev. injections of NA or NMD A stimulated LH secretion in animals that had received prior i.c.v. injections of saline. The effects of NA and NMDA were significantly attenuated by a prior i.c.v. injection of phentolamine (a selective α-adrenergic-receptor blocker) and 2-amino-5-phosphonovaleric acid (a selective NMDA receptor antagonist), respectively. NA-induced LH release was also inhibited by i.c.v. injections of either muscimol (a selective GABAa receptor agonist) or baclofen (a selective GABAb receptor agonist). On the other hand, although muscimol inhibited the effect of NMDA, baclofen did not inhibit but slightly augmented the NMDA-induced release of LH. These results support the hypothesis that both GABAa and GABAb receptors are involved in the GABAergic inhibition of LH secretion, and further suggest that they are probably located at different sites in the neural mechanism regulating LH secretion in the female rat.

The effect of activation of central gamma-aminobutyric acidB (GABAB) receptors on pulsatile luteinizing hormone (LH) secretion was examined in unanesthetized ovariectomized rats. Intraventricular injections of 2 or 10 micrograms baclofen, a selective GABAB receptor agonist, usually arrested the existing pulsatile LH secretion for a short period, then LH pulses resumed with reduced frequencies and increased amplitudes. The increase in the pulse amplitude was not necessarily accompanied by a reduction in the pulse frequency, but was observed even after nonelongated interpulse intervals in animals receiving the GABA agonist. Mean LH levels showed complex bi- and triphasic fluctuations after the central administration of high and low doses of baclofen, respectively. It was concluded that central GABAB receptors are involved in the modulatory mechanism affecting the LH pulse frequency and amplitude in the ovariectomized rat.

The effects of noradrenergic and opioid peptidergic receptor blockade, either alone or in combination, on the electrical activity of luteinizing hormone-releasing hormone (LHRH) pulse generator were studied in ovariectomized rats fitted with chronically implanted electrode arrays in the medial basal hypothalamus. Both alpha- and beta-adrenergic receptor antagonists, i.e. phenoxybenzamine (5 mg/kg i.v.) and propranolol (5 mg/kg i.v.), respectively, significantly increased the intervals between characteristic increases (volleys) in hypothalamic multiunit activity (MUA), which were associated with the initiation of LH pulses. In contrast to this, an opioid receptor antagonist naloxone (2 mg/kg i.v.) significantly decreased the intervals between the MUA volleys. Naloxone given after the injection of propranolol induced MUA volleys with a latency of a few minutes. However, when given after the injection of phenoxybenzamine, naloxone failed to induce such immediate MUA volleys. These changes in the intervals between the MUA volleys were faithfully reflected by the pulsatile LH secretion. These results suggest that norepinephrine facilitates LHRH pulse generator activity through both alpha- and beta-adrenergic-receptors, and that the action of opioid peptides on it requires an alpha-adrenergic receptor-mediated mechanism.

The relationship between noradrenergic (NA) stimulatory and gamma-aminobutyric acid (GABA)-mediated inhibitory systems in the control of luteinizing hormone (LH) secretion was examined in ovariectomized rats. Stimulation of the GABAA receptor by an intraventricular (i.c.v.) administration of a GABAA agonist, muscimol, significantly attenuated the LH secretory response to the subsequent i.c.v. injection of NA in estrogen-primed rats. On the other hand, blockade of alpha-adrenergic receptors by phenoxybenzamine did not interfere with the stimulatory effect of an i.c.v. injection of a GABAA antagonist bicuculline. In a different series of experiments, ovariectomized animals had been treated with i.c.v. injections of 6-hydroxydopamine (6-OHDA) or its vehicle. An i.c.v. injection of muscimol or phentolamine significantly inhibited the estrogen-induced surge of LH secretion in vehicle-treated rats. Muscimol also inhibited the existing pulsatile LH secretion in vehicle-treated, estrogen-unprimed animals. In animals in which hypothalamic NA terminals were presumed to be destroyed by 6-OHDA, the inhibitory effect of phentolamine was significantly diminished while that of muscimol was unaltered. These results permit the following conclusions: (1) the central GABAergic system inhibits LH secretion via GABAA receptors; (2) this inhibitory GABA system operates without mediation by the NA system, and (3) the GABAergic non-NA-mediated system can affect physiological patterns of pituitary LH secretion in female rats.

The functions of tuberoinfundibular dopaminergic (TIDA) neurons after long-term estradiol treatment were investigated in Fischer 344 (F344) rats which have high susceptibility to estradiol-induced prolactin (PRL)-secreting pituitary tumors. Dopamine synthesis in and release from TIDA neurons were determined in vitro by 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence following incubation with a DOPA decarboxylase inhibitor and endogenous dopamine release from the median eminence, respectively. The concentration of serum PRL and the weight of the anterior pituitary in ovariectomized F344 rats were markedly increased 3 weeks after a single injection of 2 mg estradiol valerate (EV) and decreased thereafter, but still showed higher levels at 15 and 24 weeks than control ovariectomized rats. Dopamine contents in the median eminence were decreased 3 weeks and unchanged 24 weeks after EV treatment. DOPA accumulation and basal dopamine release in the median eminence of F344 rats were decreased 3 weeks and increased 15 and 24 weeks after EV treatment, similarly to those of Wistar rats as reported previously. However, EV treatment, which caused similar increases in the concentrations of serum PRL and estradiol in F344 and Wistar rats, decreased KCl-induced dopamine release in Wistar rats at 3 weeks, but failed to do so in F344 rats. KCl-induced dopamine release 24 weeks after a single EV injection in F344 rats was greater than that in control rats, whereas the dopamine release 24 weeks after the last treatment of 4 injections at 3-week intervals was not different from that in control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

p-Nonylphenol (NP) and 4-tert-octylphenol (OP) are known to mimic the action of estrogens as endocrine disruptors. However, their acute effects on the pituitary and the hypothalamus functions in vivo have been uncertain. We therefore determined their effects on the anterior pituitary, in particular, gonadotropin secretion. Two weeks after ovariectomy, the rats were given a subcutaneous injection of 10 mg NP, 10 mg OP, 10 mg bisphenol A, 1 microg 17beta-estradiol, or sesame oil alone as control. Twenty-four hours after the treatment, the expression of progesterone receptor mRNA in the anterior pituitary and the level of luteinizing hormone (LH), follicle-stimulating hormone, and prolactin were determined. The expression of progesterone receptor mRNA in the anterior pituitary was significantly increased by either NP, OP, bisphenol A, or estradiol, but bisphenol A was less effective. The level of LH was significantly decreased by either NP or OP, but not by bisphenol A and estradiol. Only estradiol significantly increased the level of prolactin. The level of follicle-stimulating hormone was unchanged by any of the treatments. To check the effects of NP and OP on pulsatile LH secretion, blood samplings were done at 6-min intervals for 3 h. Twenty-four hours after treatment in ovariectomized adult rats, we found that the injection of NP significantly decreased the amplitude of LH pulses and the mean LH concentrations, but not the frequency of LH pulses. The injection of OP significantly decreased the mean LH concentrations without affecting the frequency and amplitude of the LH pulses. Finally, the rats given an injection of NP or sesame oil were intravenously injected with 50 ng of gonadotropin-releasing hormone (GnRH) to check whether NP affected the LH secretory responsiveness of the anterior pituitary to GnRH. We found that the responsiveness to GnRH in NP-injected rats was significantly attenuated compared to the sesame oil-injected rats. The present study suggests that NP, even with a single injection, suppresses the pulsatile LH secretion in adult ovariectomized rats, probably by affecting the anterior pituitary level.

To assess sex differences in stress-induced acetylcholine (ACh) release in the hippocampus and corticosterone (CS) release from the adrenal cortex, an in vivo microdialysis study was performed in intact male (n = 6) and cycling female (diestrus 1, n = 5; proestrus, n = 6) rats. Dialysates and blood samples were taken from 11.00 to 16.00 h in freely moving rats, but restraint stress was applied from 12.00 to 13.00 h. Basal ACh release in the hippocampus was low without significant differences between groups. Although ACh release promptly increased with the onset of restraint stress in both sexes, the magnitude of the increase in males was significantly greater than in female rats during diestrus and proestrus (p < 0.01). Basal serum CS concentrations were significantly greater in proestrus than in diestrus (p < 0.01) or in male rats (p < 0.01). Serum CS concentrations significantly increased at the onset of restraint stress in both sexes (p < 0.01), but the magnitude of the increase was significantly greater in female than in male rats (p < 0.01). These results indicate that sex and/or gonadal steroid environment can affect the stress-induced ACh release in the hippocampus and CS release from the adrenal cortex.

We recently established a primary cell culture system of gonadotropin-releasing hormone (GnRH) neurons originating from olfactory placodes of rat embryos at E13.5 and showed that cultured olfactory placodes released GnRH into the medium in a pulsatile fashion with an interpulse interval of about 30 min. Since the reported presence of γ-aminobutyric acid (GABA) neurons in the culture of rat olfactory placode raises questions as to the role played by these GABA neurons in the GnRH pulse generation, we immunostained GnRH neurons and GABA neurons in this culture system to examine the interrelationship between both types of neurons, and determined the effects of GABA and the GABAA receptor antagonist, bicuculline, on GnRH release. The immunohistochemical study showed that GnRH neurons received fiber terminals from GABA neurons. GnRH neurons in culture released GnRH into the medium at intervals of 30–40 min, confirming our previous study. Treatment with 20 µM GABA prolonged the interpulse interval and decreased the amplitude of GnRH pulses. Bicuculline administered at 20 µM did not affect either parameter, but 50 µM bicuculline elevated the mean GnRH level, although it did not affect either the interpulse interval or the amplitude of GnRH pulses. In addition, 50 µM bicuculline increased the mean trough levels of GnRH pulses, although 20 µM bicuculline did not. In light of the in vivo studies performed previously, we suggest that the GnRH pulse generator, which probably consists of a small population of GnRH neurons in the culture, does not involve GABA neurons to generate the pulsatile GnRH release, although it may be responsive to the inhibitory transmitter GABA. We also found that there may be another population of GnRH neurons in the culture whose activity is strongly suppressed by the tonic inhibition of GABA neurons. Although it is speculative, these latter GnRH neurons may be responsible for the surge of GnRH release.

Orexins are hypothalamic neuropeptides which stimulate luteinizing hormone (LH) secretion in estrogen- and progesterone-treated ovariectomized (OVX) rats and suppress it in OVX rats not treated with estrogen, suggesting a modulation by estrogen of the response to orexins. We examined the effects of orexin A on pulsatile LH secretion in OVX rats treated with a very small dose of estrogen so as to maintain the pulsatile secretion of LH. The estrogen treatment was done 24 h before the blood sampling by subcutaneously implanting a silicone tube (id = 1.5 mm, od = 2.5 mm, length = 25 mm) containing 17β-estradiol (E2) dissolved in sesame oil at 20 µg/ml. In OVX rats treated with sesame oil as a control, the intracerebroventricular (icv) injection of orexin A (0.3 nmol, dissolved in 3 µl artificial cerebrospinal fluid) had no significant effect on the parameters of pulsatile LH secretion, i.e., pulse frequency and pulse amplitude, although it caused a small but statistically significant decrease in overall mean LH concentrations within 1 h. In OVX rats treated with E2, the icv injection of orexin A significantly suppressed the pulsatile LH secretion; the frequency decreased for more than 2 h, inducing a rapid decline in overall mean LH concentrations. In view of the finding that a much higher dose of orexin A suppresses pulsatile LH secretion in OVX rats not treated with E2, we suggest that the suppressive action of orexin A on pulsatile LH secretion is potentiated by estrogen.

Although it has been assumed that synchronized firing of gonadotropin-releasing hormone (GnRH) neurons is necessary for pulsatile GnRH secretion, there is no clear evidence for this. In the present study we simultaneously recorded spontaneous action potentials from multiple cells. Immortalized GnRH neurons (GT1-7 cells) were cultured on a multi-electrode dish (MED) and action potentials recorded by an extracellular recording method. One to two weeks after the beginning of culture, spontaneous action potentials appeared, exhibiting bursts composed of 5–10 action potentials. Burst activity was intermittent and periodic with mean burst intervals of 13.3 s. Furthermore, burst activity was recorded almost simultaneously from several micro-electrodes, suggesting that electrical activities of GT1-7 cells were synchronized with each other. Periodic bursts were completely and reversibly blocked by 1–5 µM tetrodotoxin, indicating that voltage-dependent Na+ channels are involved in their generation. γ-Aminobutyric acid (GABA) given at a 10-µM concentration shortened inter-burst intervals, whereas 10 µM bicuculline lengthened them. Finally, the gap junctional blockers n-octyl alcohol (1 mM) and carbenoxolone (100 µM) reversibly blocked periodic burst activity. The present study provides direct evidence that the electrical activity of GT1-7 cells exhibits synchronous and periodic bursts composed of action potentials. In addition, endogenous GABA is involved in GT1-7 cells in determining burst frequency. Although the precise mechanism of synchronized burst activities needs to be clarified, gap junctional communications among GT1-7 cells are at least partially involved.

To know whether age-related changes occur in the activity of the pulse generator of gonadotropin-releasing hormone (GnRH), old (26 months) and young (3 months) female rats were examined by recording multiunit activity (MUA) in the median eminence region of the hypothalamus, concurrently with blood samplings through an intra-atrial cannula at 6-min intervals to determine serum luteinizing hormone (LH) concentrations. We have regarded the MUA showing intermittent increases (volleys) at 20–30 min intervals, followed by LH pulses, as the electrical activity of the GnRH pulse generator. We were successful in recording MUA in 18 (26%) of 69 old ovariectomized rats and in 8 (32%) of 25 young ovariectomized rats. The overall mean (±SE) of the interval between MUA volleys in old ovariectomized rats was 35.1 ± 2.0 min (n = 18), which was significantly longer than that of 22.5 ± 1.5 min (n = 8) in young ovariectomized rats. The mean interval between LH pulses in old ovariectomized rats was 32.2 ± 3.6 (n = 10), also being significantly longer than that of 23.3 ± 1.0 (n = 8) in young ovariectomized rats. Further, the LH pulse amplitude in old rats (0.95 ± 0.07 ng/ml) was significantly smaller than in young rats (3.40 ± 0.36 ng/ml). The present study also confirmed that the increase in serum LH after intravenous injection of 50 ng GnRH was much smaller in old ovariectomized rats. These results show that the electrical activity of the GnRH pulse generator is certainly reduced with age. Taken together with findings suggesting an age-dependent decrease in stimulated transmitter release, attenuation in both frequency and amplitude of GnRH pulses as well as in pituitary responsiveness to GnRH pulses may account for the decreased pulsatile LH secretion observed in aging rats.

A possible functional relationship between endogenous opioid peptides (EOPs), corticotropin-releasing hormone (CRH) and excitatory amino acids (EAAs) in the control of LH secretion was investigated in ovariectomized estrogen-primed rats. An intraventricular (icv) injection of an EAA agonist, N-methyl-D-aspartate (NMDA), or an EOP antagonist, naloxone, produced an abrupt increase in the serum LH level. While icv pretreatment of the animals with 2-amino-5-phosphonovaleric acid, a specific NMDA receptor antagonist, did not affect by itself basal LH levels, it significantly suppressed the NMDA-induced and also the naloxone-induced LH release. An icv injection of CRH also interfered with the naloxone-induced LH release. However, the NMDA-induced LH release was not affected by an icv injection of CRH or of beta-endorphin. These results suggest that the sites of EOP and CRH inhibition may be located upstream of the site of NMDA stimulation on the GnRH neuronal pathway, and that CRH can inhibit LH secretion without mediation by EOP neurons.

Pulsatile release of luteinizing hormone (LH) is governed by pulsatile release of LH-releasing hormone (LHRH), and each LHRH pulse is produced by synchronized firing of LHRH neurons. However, the mechanism of this synchronized firing is unknown. An increase (volley) in the multiunit activity (MUA), at 20- to 30-min intervals, accompanied by an LH pulse, is found in the median eminence (ME) of ovariectomized rats, suggesting that the MUA reflects the activity of LHRH neurons directly or indirectly. On the hypothesis that LHRH released from LHRH neurons locally in the hypothalamus was involved in the mechanism responsible for the synchronized firings of LHRH neurons, we examined the effect of exogenously administered LHRH on the MUA showing volleys in association with pulsatile LH release in ovariectomized rats. Intravenous injection of LHRH (50 micrograms/kg) evoked a premature volley (average 5.0 min after the injection). Microinjection of LHRH (1 ng) into the ME also evoked a premature volley (average 4.6 min after the injection), accompanied by an LH pulse. Microinjection of LHRH (1 ng) into the medial preoptic area did not cause these effects. The results suggest that LHRH released from LHRH neurons locally in the hypothalamus somehow activates its own and neighboring LHRH neurons, causing synchronized firings of LHRH neurons, and that this mechanism exists at least in and around the ME.

Substance P (SP) which is synthesized and secreted by anterior pituitary cells has been suggested to alter pituitary functions as a local modulator. We determined which cell type(s) of rat anterior pituitary gland secretes SP. A new technique, named the sandwich cell immunoblot assay (CIBA), was developed to identify two protein substances that are secreted simultaneously from the same cells. Monodispersed anterior pituitary cells were sandwiched with pairs of transfer membranes and incubated to absorb secretory substances on the membranes. Small reference points for identifying the location of cell blots were simultaneously put on the pairs of transfer membranes after the incubation. The validity of the sandwich CIBA was revealed by the following: (1) cell blots were detected at the same locations on the pairs of transfer membranes that were immunostained with the same antisera against the anterior pituitary hormones, and (2) the areas of these cell blots detected at the same locations on different membranes correlated well. When one of the pair of transfer membranes was immunostained for SP and the remaining immunostained for one of the anterior pituitary hormones, it was found that 78 and 27% of SP-immunoreactive cell blots showed also GH and TSH immunoreactivity, respectively. None of the SP-immunoreactive cell blots showed immunoreactivity for PRL, ACTH, LH or FSH. These results suggest that the sandwich CIBA is useful to identify two substances cosecreted from a cell and that a subpopulation of rat somatotropes or thyrotropes cosecretes SP.

Whether estrogen has the brain as a site of its negative feedback action was investigated by checking the acute effect of estradiol-17 beta (E2) on the electrical activity of the luteinizing hormone-releasing hormone (LHRH) pulse generator in ovariectomized rats fitted with chronically implanted electrode arrays in the medial basal hypothalamus. Before subcutaneous E2 implantation, the hypothalamic multiunit activity (MUA) exhibited, at an average frequency of 2.43/h, characteristic increases (volleys), each of which was associated with the initiation of an LH pulse. Within 2 h after E2 implantation, the frequency of MUA volleys was reduced significantly, probably associated with decreases in the frequency of LH pulses. The decrease in the amplitude of LH pulses occurred later than 2 h, but this effect was considered not to be mediated by the brain since the duration of MUA volleys was not changed. Since the mean serum LH concentration started to decrease within 2 h after E2 implantation, it was concluded that the acute inhibitory E2 effect on LH release is mediated by the brain. On the day after E2 implantation, intravenous infusion of naloxone (2 mg/kg/h) promptly elicited intermittent MUA volleys each of which was associated with an LH pulse. This suggests that operation of the LHRH pulse generator in the ovariectomized condition is restrained by the action of E2 with the mediation of endogenous opioid peptide neurons.

The role of GABA neurons in the control of pulsatile release of LHRH was investigated by checking the effect of the GABAa receptor agonist, muscimol, and antagonist, bicuculline, on the electrical activity of the luteinizing hormone-releasing hormone (LHRH) pulse generator in the ovariectomized rat fitted with chronically implanted electrode arrays in the medial basal hypothalamus. In untreated control animals, the hypothalamic multiunit activity (MUA) exhibited, at an average of 20.5-min intervals, characteristic increases (volleys), each of which was associated with the initiation of an LH pulse. A bolus i.v. injection of muscimol (2 mg/kg) significantly increased the interval between MUA volleys and LH pulses without affecting the pulse amplitude. Continuous i.v. infusion of saline increased the interval between MUA volleys to an average of 24.1 min without affecting the LH pulse amplitudes. Bicuculline infusion (10 mg/kg/h) altered neither the interval between MUA volleys nor the pulsatile release of LH. This was further checked in the condition where presynaptic inhibition by opioid peptides on the noradrenergic system was presumably decreased by naloxone. Naloxone infusion (0.5 or 0.7 mg/ kg/h) caused the MUA volleys to occur markedly frequently, an average of 13.8-min intervals. The interval during combined infusion of bicuculline with naloxone was an average of 16.2 min, suggesting that bicuculline could not decrease the interval that was set by naloxone. The results show that, although exogenous GABAa receptor agonist is capable of inhibiting the activity of LHRH pulse generator, the reduction in the endogenous GABAa receptor activity does not cause a significant effect, suggesting a minor role of inhibitory GABA neurons in the control of pulsatile release of LHRH. Further, together with the well-known fact that activation of GABAa receptor hyperpolarizes neurons postsynaptically, it is assumed that the GABAergic system, unlike the opioidergic one, is not involved in the presynaptic inhibition of the adrenergic receptor system which is probably implicated in the frequency control of LHRH pulse generator.

Characteristic increases in neuronal activity accompanied by the initiation of each luteinizing hormone (LH) pulse have been successfully recorded in freely moving ovariectomized rats by means of multiunit activity (MUA) recording techniques. In the present study, the effect of the opioid receptor antagonist naloxone on this neuronal activity was examined in rats ovariectomized for 6-10 weeks. Electrodes were chronically implanted into the medial basal hypothalamus (n = 78) or the medial preoptic area (n = 29). During the MUA recording, blood samples were taken through an indwelling atrial cannula at 3- or 6-min intervals to determine the serum LH concentration. Naloxone was administered intravenously for 1 h by either intermittent injection every 6 min (0.05 mg/kg, 10 times) or continuous infusion (0.1, 0.2 and 0.5 mg/kg/h). Explosive rises in the MUA (volleys) associated with the initiation of LH pulse were recorded in 7 animals in which the tips of electrodes were located in the arcuate nucleus-median eminence region. In 5 animals studied, the mean (+/- SE) duration and interval of the MUA volleys were 2.1 +/- 0.1 and 22.1 +/- 0.9 min, respectively. Naloxone given every 6 min significantly increased the duration to 2.6 +/- 0.1 min, and decreased the interval to 9.5 +/- 0.9 min. This naloxone treatment disturbed the clear pulsatility of LH secretion observed in the pretreatment control period. Analysis of the effects of naloxone infused at 3 different doses revealed that the facilitatory effects of naloxone on the duration and frequency of the MUA volleys were dose-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of naloxone, an opiate receptor antagonist, on the secretory pattern of luteinizing hormone (LH) was evaluated in 4-, 16- and 32-week ovariectomized (OVX) rats. Freely moving rats bearing cardiac cannulae were bled every 6 min for 3 h. During the first 90 min of bleeding, an equal volume of saline was injected after withdrawal of each blood sample, while during the second 90 min the replacement was done with saline containing naloxone at a dose of 0.5 (lower dose) or 1.0 (higher dose) mg/kg/h; statistical comparison was done between both treatment periods. In 4-week OVX rats, a significant increase in the mean LH level was observed through the 90-min period of lower-dose naloxone treatment, without significant changes in the pulse frequency and amplitude. In 4-week OVX rats treated with the higher dose and in 16-week OVX rats treated with either the lower or the higher dose, a significant elevation in mean LH levels was observed only during the first 30 min of treatment, which was probably related to the first, large LH peak in the naloxone treatment period. However, together with pulses appearing later in the treatment period, no significant changes were found in the LH pulsatility except for a decrease in frequency in 4-week OVX rats. In 32-week OVX rats, even the lower-dose naloxone did not induce an elevation in mean LH levels. The results demonstrate that naloxone can increase the LH secretion in the absence of ovarian hormones, and further that the effect on LH changes depending upon the dose of naloxone and the time after ovariectomy.

To clarify the regulatory mechanisms for the secretion of somatostatin (SRIF) from the hypothalamus, the effects of intracerebroventricular (i.c.v.) administration of growth hormone-releasing factor (GRF) and corticotropin-releasing factor (CRF) on SRIF secretion into hypophysial portal blood were examined in pentobarbital-anesthetized male rats. Neither the concentration of SRIF in portal plasma nor the secretion rate of SRIF was changed after i.c.v. administration of 0.9% saline. Administration of 10 ng or 5 micrograms human GRF i.c.v. produced a significant increase in the portal plasma concentration and secretion rate of SRIF. Likewise, 5 micrograms CRF significantly increased the portal plasma concentration and secretion rate of SRIF. These results suggest that the neuropeptides GRF and CRF centrally influence SRIF secretion into hypophysial portal blood.

Diurnal variations in concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), corticosterone, estradiol-17 beta and estrone in serum were investigated in 27- to 35-day-old female rats maintained on a 14-hour light and 10-hour dark schedule. Groups of 6-8 rats were decapitated at 3-hour intervals during a 24-hour period. Hormones were measured by radioimmunoassay. LH levels were highly variable over a 24-hour period in rats younger than 32-33 days of age, while in 34- to 35-day-old animals the levels were less variable and a moderate LH peak was seen at 24.00 h. A distinct FSH diurnal rhythm with the peak at 15.00 h was demonstrated in rats older than 32 days of age. The 27- to 28-day-old rat had a diurnal TSH rhythm with the peak at 12.00 h, but older rats had a rhythm with two peaks at 24.00 and 9.00 h. The diurnal corticosterone rhythm analogous to the adult's was established at 32 days of age, although a significant variation having a peak during the dark period appeared earlier. Similarly, a distinct estradiol rhythm appeared at 29 days of age with the peak at 12.00 h, followed by a change in the peak phase afterwards. Serum estrone levels showed a manifest variation in the 27- to 28-day-old rat, but no variation was observed in the older rats. These results not only confirm the previous reports on the existence of pulsatile LH secretion, and corticosterone and estradiol diurnal rhythms, but also demonstrate the existence of diurnal rhythms in FSH, TSH and estrone levels in the immature female rat. Furthermore, it is possible that the rhythm once developed may change in phase, or even disappear as puberty approaches.

The present study was undertaken to determine the role of the hypothalamic periventricular (PV) region in the regulation of the secretory pattern of growth hormone (GH) in adult male rats. The PV regions were destroyed stereotaxically by means of a modified Halasz's knife which was lowered through the midline and rotated several times at the level of the region. In sham-operated controls, the knife was lowered to the same region but no rotation was performed. Serial blood samplings were performed at 10-min intervals for 10 h (10.00-20.00 h), via an intracardiac cannula, at 2, 4 and 6 weeks postoperatively. In 7 sham-operated controls, GH was secreted with a surge period of about 3.5 h, with peak levels of 120-170 ng/ml whole blood and basal levels of about 10 ng/ml whole blood. In 11 PV-lesioned animals, the basal levels of GH were unaltered but the surge period decreased to about 3.0 h and peak levels increased to about twice as high as in sham-operated controls. Somatostatin concentrations in the median eminence of PV-lesioned rats were significantly decreased to about 32% of that of sham-operated rats. The results suggest that, via somatostatin neurons, the PV region plays an inhibitory role in the regulation of periodicity and peak levels of GH secretory bursts.

To study the secretory pattern of growth hormone (GH) in the adult male rat in a continuous sleep state, the GH concentration was measured in blood sampled at 10-min intervals through an intracardiac cannula for 9-10 h long-term anesthesia with thiopental sodium. The cortical EEG was monitored to maintain stable EEG sleep throughout the sampling period. In 41 animals subjected to anesthesia, 32 showed an apparent fluctuation in GH levels. The analysis of the time series of the GH concentration in blood by the power spectrum and least-squares method revealed that 27 animals had periodicities within the mean +/- 2SD range obtained for the unanesthetized, but similarly sampled, animals from 2.40 to 3.85 h. The mean (+/- SE) of the periodicities for 27 animals was 3.01 +/- 0.06 h, similar to that obtained for the unanesthetized animals, approximately 3.0 h. The results indicate that, although the GH-controlling mechanism in the rat is facilitated under sleep, it has a 3.0-hour period ultradian rhythm mechanism that is independent from that for sleep.

The temporal correlation of the secretion of corticosterone (CS) and growth hormone (GH) with the sleep-wakefulness cycle in adult male rats was studied by serial blood sampling at 10-min intervals over a 11-hour period from 11.00 to 22.00 h. Cortical EEGs recorded continuously during the blood sampling were scored into wakefulness and sleep, and the amounts of sleep for every 10 min was plotted against the CS and GH values. All 11-hour time series of CS, GH and amount of sleep were found to have three major ultradian rhythms with periodicities of 1.5 h and its multiples. For CS, there was a significantly negative cross-correlation between the amounts of sleep. Although the most prominent CS rhythm was the 1.5-hour period, CS secretion occurring with a 3.0-hour period had such a phase relationship with the 3.0-hour period sleep rhythm that the secretion began in the late stage of sleep cycle and reached its peak around the time of wakefulness between sleep cycles. In contrast, a GH secretory burst occurred in the early stage of the sleep cycle occurring with a 3.0-hour period, with a definite time lag after the onset of the sleep cycle. The present study demonstrates that three different functions, CS, GH secretions and sleep-wakefulness, have common ultradian rhythms with similar periods, and manifest their rhythms based on their own pacemakers. It is assumed further that each pacemaker is fixed in specific phase relations with others.

To determine if luteinizing hormone releasing hormone (LHRH) might act within the central nervous system to modulate its own secretion, microinjection of LHRH into the medial preoptic area (MPO) was performed at 13:00 h in both the ovariectomized estrogen-primed and proestrous rat, and the effects on the afternoon rise in the serum concentration of luteinizing hormone (LH) were studied. Microinjection of 5 ng LHRH into the MPO in the ovariectomized estrogen-primed rat resulted in a significant increase in serum LH from 16:00 to 19:00 h compared to the value in the saline-injected rat, whereas the injection at a dose of 2 ng did not induce a significant change. The injection of 5 ng LHRH into the third ventricle in the ovariectomized estrogen-primed rat induced a transient increase in serum LH immediately after the injection, followed by recovery to the preinjection concentration by 1 h, indicating that the potentiating effect of 5 ng LHRH in the MPO was not due to the LHRH leaked into the third ventricle. Finally, microinjection of 5 ng LHRH in the proestrous rat induced an early increase in serum LH, resulting in an advancement of the peak of the afternoon rise by 1 h. It is suggested that LHRH secreted in the MPO may, directly or indirectly, stimulate its own secretion in the median eminence to produce the surge of LH secretion.

A new method for estimation of in vitro neurosecretory activity of tuberoinfundibular dopaminergic (TIDA) neurons was developed by measuring the rate of synthesis of dihydroxyphenylalanine (DOPA) in the median eminence of hypothalamic slices. Sagittal hypothalamic slices of ovariectomized rats were incubated in a medium containing 3-hydroxybenzylhydrazine (NSD 1015), an inhibitor of DOPA decarboxylase. DOPA accumulated in the median eminence following incubation with NSD 1015 was determined by high-performance liquid chromatography with electro-chemical detection. The amount of DOPA accumulated in vitro in the median eminence was maximal in a medium containing 10 mM NSD 1015 and linear up to 120 min at 37 degrees C. Increasing the concentration of tyrosine in medium stimulated the synthesis of DOPA in the median eminence. The synthesis of DOPA was blocked by 1 mM alpha-methyltyrosine, an inhibitor of tyrosine hydroxylase. The rate of in vitro synthesis of DOPA in the median eminence was 33% of that of in vivo synthesis. Incubation in a medium containing 50 mM K+ to depolarize neurons caused a 2.4-fold increase in DOPA synthesis in the median eminence. The high K+-induced increase in DOPA synthesis was blocked by omission of Ca2+ and addition of 1 mM EGTA into the medium, suggesting Ca2+ dependency of depolarization-activated DOPA synthesis. These results indicate that this in vitro assay is a useful means to study the regulatory mechanisms of TIDA neurons.

Experiments were carried out to elucidate the influence of stress on the diurnal change in adrenal biosynthetic activity during controlled 24 h light-dark cycles. stress Groups of 10 rabbits were deca

We have shown that the implantation of cholecystokinin octapeptide sulfated form (CCK-8-S) or dopamine (DA) in the medial preoptic area (MPO) in ovariectomized estrogen-primed rats induced an enhancement of the afternoon rise in LH secretion. The present study was done to examine the possible functional interaction of both substances in the brain. Two groups of ovariectomized estradiol (5 micrograms, s.c.)-primed rats were administered intraperitoneally either pimozide (1 mg/kg BW; pimozide-treated group) or 0.1 M tartaric acid as the control solution (pimozide-untreated group). CCK-8-S or DA was implanted in the MPO through chronically implanted guide cannulae and the effects on serum LH and FSH concentrations were compared between both groups. An empty cannula was used as the control. Implantation of either CCK-8-S or DA in the pimozide-untreated group induced marked enhancements of afternoon rises in LH and FSH levels, which normally occurred, but with a lesser magnitude in the animals with the empty cannula. In the pimozide-treated group, however, neither CCK-8-S nor DA induced changes in the LH and FSH levels. It was evident that both effects of CCK-8-S and DA on LH and FSH secretion were blocked by pretreatment with the DA receptor blocker. These data indicate that DA receptors are required to be intact when CCK-8-S implants manifest their action in stimulating gonadotropin secretion.

The effects of 17 beta-estradiol (E2) and prolactin (PRL) on the excitability of neurons in the arcuate nucleus were examined using hypothalamic slices obtained from ovariectomized rats. Neither E2 nor PRL affected the excitability of neurons having antidromic responses due to median eminence stimulation. On the other hand, E2 and PRL altered the spontaneous activity of unidentified neurons, which were not activated by median eminence stimulation, by 6 and 11%, respectively. Both excitatory and inhibitory responses were recorded, which were not abolished in the low Ca2+/high Mg2+ medium. These results suggest that E2 and PRL can rapidly modulate the excitability of some arcuate, but presumably not tuberoinfundibular, neurons by acting directly on the postsynaptic membrane.

Pulsatile secretion of luteinizing hormone (LH) in ovariectomized rats was evaluated before and after local implantation of crystalline estradiol benzoate (EB) into various regions within the forebrain. Serum concentrations of LH were measured by radioimmunoassay in samples collected at 6-min intervals through indwelling cardiac catheters. In rats with EB implanted in the preoptic suprachiasmatic area (POSC), and in the nucleus of the tractus diagonalis and the diagonal band of Broca (DBB) to a lesser extent, the mean LH concentration and LH pulse frequency decreased rapidly while the pulse amplitude did not change for 3 h after implantation. Rats with the EB implant in the bed nucleus of stria terminalis, the medial preoptic area, the medial septal nucleus, the anterior hypothalamic area or the third ventricle showed unchanged frequencies of LH secretory pulses. Implantation of progesterone into the POSC of ovariectomized rats produced no significant change in LH secretory profiles. It was suggested that the sites of action of estrogen in decreasing the LH pulse frequency are not widespread but rather restricted within a small part of the brain, including the POSC and DBB, in the ovariectomized rat.

To study the temporal correlation of secretory profiles of corticosterone (CS) and growth hormone (GH) in adult male rats in the continuous-sleep state, CS and GH concentrations were measured in blood sampled at 10-min intervals through an intracardiac cannula for 11-12 h under long-term thiopental anesthesia. The cortical electroencephalogram (EEG) was monitored to maintain stable sleep during the sampling period. Sixteen rats were subjected to the experiment. By analysis of power spectrum and least-squares methods all the time series of CS and GH concentrations in blood were revealed to have three major ultradian rhythms with periodicities of 1.5 h and its multiples. Although there were no significant positive or negative cross-correlation coefficients between the periodicities of CS and GH, a tendency was evident for GH concentrations to be high when CS concentrations were low, and CS concentrations to be high when GH concentrations were low. The results demonstrate that CS and GH secretions are able to manifest the same ultradian rhythms as those in the unanesthetized, freely moving condition even in the continuous-sleep state due to thiopental anesthesia. Combined with our previous findings, the CS, GH and sleep-wakefulness rhythms appear to be governed by separate pacemakers, although sharing the same basic ultradian rhythm.

Separate regions of the anterior periventricular area (PVA), median eminence (ME) and frontal cortex from brains of hypophysectomized female and intact male rats were extracted and homogenized. The LH-RH content of these homogenates was determined by a specific double antibody radioimmunoassay. LH-RH was found in the PVA as well as in the ME of both hypophysectomized female and intact male rats; it was not detected in the frontal cortex. Extracellular recordings were made from neurons in the PVA and 55 neurons were activated antidromically by stimulating the ME. This suggests that PVA neurons exist with axons terminating in the ME. It is assumed that these axons are unmyelinated since their conduction velocity was approximately 0.3–0.8 m/sec. In addition to the antidromically-activated neurons, 26 neurons with characteristics of transsynaptic activation were recorded in the PVA following stimulation of the ME. Some of the units recorded from the ME-arcuate (ARC) region were also activated either antidromically or transsynaptically by stimulating the PVA. The effect of PVA stimulation on LH release was tested in ovariectomized, estrogen-treated rats with or without anterior deafferentation of the hypothalamus. Increased LH concentration in serum was elicited by PVA stimulation of rats in which deafferentation was either not performed or not completed; no increase in serum LH level was observed following PVA stimulation in rats with complete deafferentation. These data suggest that PVA neurons synthesize LH-RH, which is released at the ME.

Effects of microinjection of neurotensin (NT) in the medial preoptic area on luteinizing hormone (LH) release were examined in unanesthetized ovariectomized rats with or without estrogen priming. Microinjection of NT (100 ng) into the medial preoptic area of ovariectomized estrogen-primed rats significantly facilitated the circadian afternoon rise of LH secretion as compared to the hormone values in control animals microinjected with physiological saline. The timing of the afternoon LH rise was not altered by the peptide. In ovariectomized estrogen-unprimed rats, on the other hand, NT did not affect the existing pulsatile LH secretion nor induced an afternoon rise of the hormone secretion. These results show that NT can stimulate the preoptic LH release mechanism which undergoes the circadian clock under the influence of estrogen.

Variations in serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), prolactin (PRL), growth hormone (GH), and corticosterone in the states of arousal, slow wave sleep and paradoxical sleep were studied as a function of time from the onset of arousal and sleep which were determined polygraphically in 27- to 30-day-old female rats. Markedly high LH values occurred more frequently during slow wave sleep than during other stages, resulting in a tendency to greater mean LH levels in the state of slow wave sleep. Variations in PRL values at various states of the sleep-waking cycle were not very large, but a statistically significant peak occurred shortly after the sleep onset. The mean PRL values obtained after 5 min of paradoxical sleep were lower than those obtained during slow wave sleep. Mean GH values were higher during sleep than arousal state, and the mean during paradoxical sleep was highest. GH levels peaked at 30 min after sleep onset. Corticosterone values were highest in the arousal state, and decreased abruptly after 20 min of sleep. There were no significant variations in FSH and TSH levels in serum during various sleep-waking states. These secretory patterns of anterior pituitary hormones during a sleep-waking cycle are quite similar to those in man.