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The effect of gamma-aminobutyric acid (GABA) on antidromically identified tubero-infundibular (TI) neurons was examined in hypothalamic slices of ovariectomized female rats. Twenty antidromically evoked spikes were obtained in the medial basal hypothalamus, including the arcuate and ventromedial nuclei, by electrical stimulation of the median eminence. Sixteen of them had a notch in the rising phase and fractionation of the initial segment (IS)- and somatodendritic (SD)-spikes was elicited by repeated stimulation at frequencies higher than 10 Hz. The application of 0.5-1.5 mM GABA to the incubation medium inhibited SD spikes in 7 of these 16 neurons. The latency, amplitude and threshold of IS spikes were not affected by GABA except for one spike whose latency fluctuated. On the remaining 9 neurons having the notch, no effect of 5-10 mM GABA was discernible. Four of 20 antidromically evoked spikes, which had a smooth rising phase and a shorter duration, were not inhibited by 5-10 mM GABA, but a fluctuation of the latency was observed in one neuron. Fifteen neurons having spontaneous unit activity were also obtained in the arcuate nucleus and its adjacent area and tested with GABA. In 10 of the 15 neurons, spontaneous unit activity disappeared following 0.1-1.5 mM GABA perfusion, while the firing rate in the remaining 5 neurons was not affected by 5-10 mM GABA. These results provide evidence for a direct inhibitory effect of GABA on TI neurons and support the involvement of GABAergic neurons in regulating neuroendocrine functions.

The effect of implantation of cholecystokinin(CCK)-8 into the medial preoptic area (MPO) through the chronically implanted guide cannula on the release of LH was examined in the ovariectomized estradiol-primed rat. An inner cannula with CCK-8 at the tip, as well as an empty inner cannula as the control, was implanted into the MPO at 12.00 h. Blood samples were obtained at 11.00, 12.00, 16.00, 18.00 and 20.00 h, and serum concentration of LH was measured by RIA. There was no significant fluctuation over time in the mean serum LH values for each sampling time in the group of animals implanted with empty cannulae in the MPO. In the group of animals implanted with CCK-8 into the MPO, the fluctuation of the mean LH values over time was significant, with a marked and significant elevation of LH values peaking at 16.00 h. It was strongly suggested that CCK-8 was capable of stimulating LH-RH release, acting at the MPO.

Effects of electrical stimulation of the lateral amygdaloid areas on ovulatory gonadotropin release were examined in adult Wistar female rats. Electrical stimulation was applied in rats in proestrus under ether anesthesia with square wave pulses of 0.5 ms duration and 100 Hz frequency for 30 min (30 sec on and off). Stimulation of the lateral amygdala blocked ovulation in 50% of animals when it was applied between 13:30 and 14:30 with a current of 300 muA. Stimulation of the medial amygdala under the same experimental condition was absolutely ineffective to block ovulation. Sham stimulation was also ineffective. In determining the gonadotropin concentration in serum, the stimulation into the lateral amygdala was observed to inhibit the ovulatory release of LH, FSH and prolactin. It may be said that the lateral amygdaloid area participates in the control of gonadotropin release in an inhibitory manner.

In order to investigate the physiological role of endogenous opioid substances in the regulation of gonadotropin secretion, we studied the effect of Naltrexone (Nalt), a morphine antagonist, on serum luteinizing hormone(LH) concentrations in ovariectomized estrogen-treated rats. The site of action of Nalt and its interaction with other putative neurotransmitters on LH secretion were investigated in hypothalamic deafferentated rats and in animals treated with pharmacological inhibitors of the actions of neurotransmitter substances. Nalt injection increased serum LH levels at dosed 0.08 to 2 mg/kg BW. The response was dose-dependent but higher doses of Nalt had less effect. In inducing this response pattern, mediobasal connection between the anterior hypothalamus and the mediobasal hypothalamus was essential, but posterior input to the mediobasal hypothalamus was not necessary. Excepting alpha-adrenergic blocker, all the blockers used, i.e beta-adrenergic, serotonin, dopamine and acetylcholine blockers were effective in eliminating the LH release evoked by Nalt injection. These results suggest that endogenous opioid substances might inhibit LH secretion tonically through aminergic and/or cholinergic neurons and that the mediobasal neural connections to the mediobasal hypothalamus are indispensable for this inhibition.

Effects of electrical stimulation of the hippocampus (HPC), lateral amygdala (1-AMYG) and midbrain central gray matter (CG) on the release of ovulatory gonadotropin were examined using proestrous Wistar rats with or without pretreatment with reserpine, atropine or p-chlorophenylalanine (PCPA) at such dosage that had been confirmed not to block ovulation. Electrical stimulation of the HPC, 1-AMYG or CG under light ether anesthesia just before the critical period prevented a rise in serum LH, FSH and prolactin levels at 18:00. Pretreatment with atropine (200 mg/kg body wt, sc) was effective to abolish this inhibitory effect of the HPC stimulation on the release of LH and FSH, whereas reserpine treatment (1mg/kg body wt, ip) did not affect the effect. The inhibitory effect of the 1-AMYG or CG stimulation on LH and FSH release was abolished by treatment with PCPA (150 mg/kg body wt, ip), while neither atropine nor reserpine had any effect. The inhibitory effect of the HPC stimulation on the release of these hormones was also blocked by PCPA treatment. In regard to the prolactin release, it was inhibited by the stimulation of the HPC, 1-AMYG or CG in both the non-treated rat and in the atropine or PCPA-treated one, while in the reserpine-treated rat it was not inhibited but rather was facilitated by these stimulations. It was assumed that the normal maintenance of both cholinergic and serotonergic neural links for the expression of the HPC inhibition on ovulatory LH, FSH and prolactin secretion and that of serotonergic link for the expression of the 1-AMYG or CG inhibition are needed. The inhibitory action on prolactin release changed into facilitation under the depletion of monoamines, but the mechanism is unknown.

The detailed profile of prolactin (PRL) secretion in 22-24 and 29-31 days old female rats was investigated by serial blood sampling through an intracardiac cannula at 15-min intervals for each of the 9 or 10-h periods beginning at 09.00 or 10.00 and 22.00 h. By analysis of the power spectrum and the least squares method the time series of PRL concentrations which were measured by RIA were found to have approximately a 3-h period ultradian rhythm in either sampling period of both the 22-24 and 29-31 days old rats. The peak times calculated based on the acrophase estimated through the calculation of periodicity were concentrated around 12.00, 15.00 and 18.00 h for the sampling period 10.00-19.00, and 24.00, 03.00 and 06.00 h for the sampling period 22.00-07.00 h. However, in more than half of the animals at 22-24 days of age, one secretory episode around 12.00 h, and two secretory episodes around 24.00 and 03.00 h had markedly small amplitudes, making the remaining secretory episodes distinct diurnal and nocturnal surges, respectively. In the animals at 29-31 days of age, the amplitudes of the PRL episodes occurring around 12.00 h were markedly small, making the remaining two episodes as diurnal surges, whereas the amplitudes of PRL secretory episodes during the period 22.00-07.00 h were analogous to each other. These findings indicate that the semicircadian rhythm of PRL secretion is established on the basis of PRL secretion with the 3.0-h period ultradian rhythm.

To get an electrophysiological basis for understanding the sexual and age-related changes in the brain mechanisms controlling gonadotropin secretion, we studied single unit responses to electrical stimulation in the preoptico-arcuate, amygdalo-preoptic and hippocampo-preoptic neural connections in the rat. Orthodromic unit responses to single pulse electrical stimulation were recorded in young mature and aged, male and female rats by post-stimulus histogram analysis. Electrical stimulation of the medial preoptic area elicited responses in one-half of the hypothalamic arcuate neurons tested in female rats while the stimulation induced responses in less than one-third of the arcuate neurons in the male. The percentage of arcuate neurons responding to preoptic stimulation did not differ between young and aged animals. Stimulation of the medial amygdala evoked responses in a larger number of preoptic neurons in young mature rats than in aged animals in both sexes. There was no sexual difference in the number of responding neurons to amygdaloid stimulation. Percentages of preoptic neurons responding to the hippocampal stimulation were greater in young females showing regular estrous cycles than in males or aged females which showed constant estrous vaginal smears. In aged females whose vaginal cycles were induced by daily treatment with progesterone, more preoptic neurons responded to stimulation of the amygdala and the hippocampus than in aged constant estrous rats. The young cycling and aged progesterone-recycling rats had similar preoptic neuronal responses to the limbic stimulation. Stimulus thresholds for inducing these arcuate and preoptic unit responses were almost equivalent among different groups of animals. This suggests that the alteration in unit responses observed was not due to a change in neuronal excitability in the sites of stimulation in different animal groups. These results indicate that there are sex differences in the synaptic functions which transmit preoptic signals to the arcuate neurons and hippocampal impulses to the preoptic neurons. It is also suggested that the amygdalo-preoptic and hippocampo-preoptic neural pathways may be functionally modified in aged rats because of the absence of estrous cycles.

In the present study, the role of the medial preoptic area (MPO) in the neural control of the nocturnal prolactin (PRL) surge was investigated in ovariectomized rats. Cervical stimulation (CS) or bilateral MPO lesions caused a marked nocturnal PRL surge at 0400 h on the fourth day after CS or the lesions in ovariectomized rats in which blood samples were obtained by decapitation. However, operation for indwelling a catheter and serial blood collection completely eliminated the MPO lesion induced nocturnal surge while they did not affect the CS-induced surge. On the other hand, MPO lesions could not induce the nocturnal PRL surge in neonatally androgenized female rats. These results suggest that the MPO not only tonically inhibits the initiation of the nocturnal PRL surge but also has a buffer action on the PRL surge-suppressing action of stress. Furthermore, it may be possible that the failure of CS to initiate and maintain the nocturnal PRL surge in neonatally androgenized rats is not due to the inability of CS to disinhibit the inhibitory action of the MPO, but rather due to the extinction of the circadian rhythm itself of the nocturnal PRL surge in these rats.

The effect of electrochemical stimulation of the medial preoptic area (MPO) and the hypothalamic arcuate nucleus (ARC) on LH release was compared among long-term ovariectomized (OVX), ovariectomized rats treated with estradiol benzoate (OVX-E) and proestrous (PE) rats under pentobarital anesthesia. The MPO stimulation failed to facilitate LH release on OVX rats whereas the stimulation increased serum LH levels in both OVX-E and PE rats. The ARC stimulation resulted in an elevation of serum LH in OVX and PE rats. The time course of serum LH elevation after the electrochemical stimulation of the MPO or the ARC was different between OVX-E and PE rats. In PE rats serum LH concentrations began to rise 20-30 min after the stimulation of the MPO or the ARC with peak values obtained around 70-90 min. Serum LH started to elevate immediately with peak values in 10-20 min following the ARC stimulation in OVX rats and the MPO stimulation in OVX-E rats. The time courses for changes in serum LH concentrations following LH-RH administration were similar in OVX and PE rats. From these results, the lack of circadian rhythm in serum LH levels in long-term OVX rats may be due to the inability of the MPO to re- spond to the stimulus generated from circadian pacemaker(s). The cause of the delay observed between the stimulation of the MPO and LH rise in PE rats may not exist in the levels of neural transmission between the MPO and the ARC or the pituitary, but in the level of the mediobasal hypothalamus.

The sites and mechanism of the ovulation-inducing action of estradiol benzoate (EB) were studied by brain implantation of the crystalline steroid through chronically implanted outer cannula at 12:00 on diestrus day 2 in the 5-day cyclic rat. EB implantation in the medial amygdala or the bed nucleus of stria terminalis advanced cyclic changes in vaginal smears, timing of ovulatory LH release, and ovulation by 1 day, resulting in 4-day cycle. When implants in the bed nucleus of stria terminalis were placed for a shorter period of time on diestrus day 2, from 12:00 to 20:00, advancement of these parameters were similarly observed. Serum concentration of FSH and that of prolactin were significantly elevated at 20:00 on the same day in the rats implanted with EB in the medial amygdala or the bed nucleus of stria terminalis, compared with those in the non-treated controls. LH was not affected. The implantation in the arcuate nucleus was also effective to advance ovulation, but the anterior deafferentation prevented the effect. In contrast, EB implantation in the medial septal nucleus, the medial preoptic area, or the medial basal prechiasmatic area was consistently ineffective to advance vaginal cycle and ovulation. Multiunit activity in the arcuate nucleus showed an afternoon elevation on the day of implantation in these areas and as well on the day following, while it did not show such elevation on the day of implantation in the medial preoptic area. It is concluded that EB acts on the medial amygdala and the bed nucleus of stria terminalis in the mid-diestrus in 5-day cycle to stimulate FSH and prolactin release without affecting LH, which changes trigger a chain of reproductive events inducing early release of ovarian steroid responsible for early ovulatory gonadotropin release. The arcuate nucleus in one of the sites of stimulatory action of estrogen, but it requires the neural influence presumably from the medial amygdala and the bed nucleus of stria terminalis via the preoptic area for stimulating the ovulatory hormone release. EB exposure is considered to be endowed with the increase of its responsibility to this neural influence.

Effect of implantation of estrogens or catecholamines into the medial preoptic area through chronically implanted double cannula on the release of LH was examined in the ovariectomized estradiol-primed rat. Implantation of estrone at 12:00 on the second or third day after subcutaneous injection of estradiol benzoate (20 mug) increased the serum concentration of LH at 18:00 compared with that in the non-implanted controls, whereas implantation of estradiol benzoate on the third day did not affect it. Dopamine implantation at 12:00 on the third day also induced a significant increase of LH concentration at 18:00, and, in contrast, norepinephrine implantation decreased the concentration at 18:00. It may be said that the medial preoptic area is responsive to estrone and can induce LH release, whereas it is not to estradiol. Furthermore, dopamine was effective for the activation of the medial preoptic area in relation to the inducement of LH release.

The effect of a potent inhibitor of dopamine-beta-hydroxylase, diethyldithiocarbamate (DDC) or fusaric acid, on the antidromically evoked potentials by electrical stimulation of the preoptic suprachiasmatic area (POSC) was examined. Thirty seven neurons in the ventrolateral part of medulla oblongata (VLMO) were successfully tested after DDC injection. Nineteen of them decreased in amplitude to approximately 50% of control levels at 120 min after DDC injection, 20% at 180 min. These 19 neurons were characterized by having a notch in the rising phase and taking a longer time to complete the initial deflection. After fusaric acid injection, ten neurons in the VLMO were successfully tested and four of them decreased in amplitude similar to DDC injection. Effects of receptor blockers of noradrenaline, dopamine and serotonin on the single unit responses of the POSC neurons to the VLMO stimulation in proestrous rats were examined in order to know which of noradrenaline, dopamine or serotonin are involved in the neural transmission from the VLMO to the POSC. After the injection of phenoxybenzamine, an alpha-adrenergic receptor blocker, the rates of facilitatory and inhibitory responses in the POSC neurons to the VLMO stimulation were significantly decreased. Percentages of facilitatory and inhibitory responses were not changed significantly after injection of pimozide, a dopaminergic receptor blocker, or methysergide, a serotonergic receptor blocker. These findings suggest that noradrenaline might be involved in the transmission of the VLMO stimulation effect on the POSC neurons and that VLMO noradrenaline-containing neurons send their axons directly to the POSC.

Recently we reported that the immature female rat spontaneously expressed two daily prolactin (PRL) surges; one surge at 1800 h (diurnal) and the other at 0600 h (nocturnal), but the nocturnal surge ceased occurring as puberty approached. In the present experiment, the effects of ovariectomy and administration of ovarian hormones on the two surges were investigated. Ovariectomy at the age of 23 days did not abolish the two surges occurring on day 29 to 30 and their timing and magnitude were not different from those in the sham-ovariectomized rat. In the intact female rat at the ages of 29-30 days, an injection of 2.5 microgram estradiol benzoate at 1200 h progressively elevated PRL levels, whereas 0.5 mg progesterone injected at 1200 and 2400 h increased the magnitude of diurnal and nocturnal surges. The results indicate that the mechanism for two daily PRL surges in the immature female rat is capable of functioning independently of the ovarian activity, although the latter may exert certain modulatory influence.

The role of the lower brain stem in controlling preovulatory gonadotropin surges was investigated in female rats under acute experimental conditions. Electrolytic lesions or diethyldithiocarbamate implantations in the ventrolateral part of the medulla oblongata (VLMO), which were carried out at 1100-1330 h on the day of proestrus, resulted in a blockade of the preovulatory surges of LH, FSH and PRL as well as subsequent ovulation. Such treatments in the dorsomedial part of the medulla oblongata did not affect gonadotropin surges or ovulation. By means of electrolytic lesions in the VLMO, norepinephrine concentrations were significantly reduced in the preoptic-anterior hypothalamic area at 1700-1800 h on proestrus, though they did not change in the mid-posterior hypothalamus. Electrochemical stimulations of the suprachiasmatic part of the preoptic area or norepinephrine injections into the third ventricle at 1400-1500 h on proestrus in animals with VLMO lesions succeeded in induce gonadotropin surges and ovulation. These results suggest that the lower brain stem is involved in the induction of preovulatory gonadotropin surges and that the process may be mediated by the ascending noradrenergic system which originates in the VLMO.

Intracranial actions of estradiol benzoate (EB) in modulating the characteristics of pulsatile LH secretion were examined in ovariectomized rats. A minute amount of crystalline EB was implanted into the medial basal hypothalamus (MBH) or the preoptic suprachiasmatic area (POSC) and blood samples were collected at 6-min intervals for radioimmunoassay of serum LH concentrations. In rats with EB implanted in the MBH, the LH pulse amplitude decreased while the frequency did not change for 3 h after implantation. In rats bearing EB implants in the POSC, in contrast, the LH pulse frequency decreased without a significant variation in the pulse amplitude. Control animals with empty cannulae inserted into the MBH or POSC exhibited unchanged amplitude and frequency of LH secretory pulses. These results indicate that estrogen can regulate the amplitude and frequency of pulsatile LH secretion via mechanisms independent of each other. It appears that MBH and/or the pituitary is a site of the action of estrogen in decreasing LH pulse amplitude, while the POSC is a site of the steroid action in decreasing the pulse frequency.

Injections of 2 mg of progesterone into ovariectomized estrogen-primed rats significantly increased serum LH and FSH concentrations 3, 5 and 8 hr later. Receptor blockers of noradrenaline (NA), dopamine (DA) or acetylcholine (ACH), phenoxybenzamine (20 mg/kg body weight), pimozide (1mg/kg body weight) or atropine (700 mg/kg body weight), respectively, prevented the progesterone-induced gonadotropin release. On the other hand, none of them blocked the gonadotropin release following unilateral electrochemical stimulation (100 microA for 60 sec) of the medial preoptic area which occurred 0.5 and 1.5 hr later, although pimozide or atropine reduced serum LH concentrations at 4.0 hr after stimulation. Furthermore, the sites of action of NA, DA and ACH with respect to LH release were examined by intracerebral implantation in ovariectomized estrogen-primed rats DA or ACH, when implanted unilaterally into the medial preoptic urea, induced a significant increase in serum LH 5 hr later, whereas NA decreased LH levels. Implantations of NA or ACH into the bed nucleus of the stria terminalis or the medial amygdala increased serum LH although the effect of NA into the latter was not statistically significant. Only implantations of NA among the three substances into the lateral septum induced LH release. These results suggest that all of NA, DA and ACH play stimulatory roles in the regulation of gonadotropin secretion, and that there are regional differences of their effectivenesses in releasing LH within the limbic-preoptic area.

Serum LH and FSH levels were determined before and after LH-RH injection (100 micrograms, i.m.) in patients with prostatic cancer who were chronically treated with either chlormadinone acetate (CMA, 100 mg/day) or ethynylestradiol (EE, 1 mg/day). In patients treated with EE, the levels of serum LH and FSH before and after injection of LH-RH were significantly lower than those in controls. On the other hand in patients treated with CMA, the basal levels of serum gonadotropins did not differ from those in controls, and the increase in gonadotropin after LH-RH injection was comparable to that in controls. To examine the effects of these steroids on the hypothalamo-hypophysial axis in the regulation of gonadotropin secretion, CMA or EE was implanted in castrated male rats. CMA, EE or cholesterol (control) was implanted in the hypothalamic median eminence-arcuate nucleus region through a stainless doublecannula. EE implantation resulted in a 75% decrease in serum LH (p < 0.001) and a 38% decrease in serum FSH (p < 0.05) from the control levels on day 5 of implantation. On the other hand, CMA implantation induced a 33% decrease in serum LH (p < 0.05) from the control level on day 3 of implantation, but no significant change in serum FSH levels. The injection of 2 micrograms/kg of LH-RH on day 7 of implantation induced significant lowering of LH and FSH levels. There was no significant difference between serum levels of the hormones 20 min after LH-RH injection for these two groups and those for the control group. These studies suggest that EE has a potent negative feedback effect on both LH and FSH secretion, and that CMA has a mild negative feedback effect on LH secretion.

Fifty-one neurons in the ventrolateral part of the medulla oblongata were antidromically activated by electrical stimulation of the suprachiasmatic part of the preoptic area in urethane-anestetized, ovariectomized and estrogen-primed female rats. Two types of antidromic responses were distinguished on the basis of their spike configurations and antidromic spike latencies. One type ("fast spikes") was characterized by a fast and smooth rising phase and a shorter duration of the initial positive deflection. The other type ("slow spikes") had a notch in the rising phase and took a longer time to complete the initial deflection. Mean antidromic spike latency for the fast spikes was 9.8 msec while the value for the slow spikes was 30.2 msec. Ionophoretic injection of estradiol was accomplished on 37 of the 51 antidromically identified cells, of which 21 showed slow responses and 16 responded with fast spikes. In cells with slow spikes, estradiol facilitated (n = 9) or suppressed (n = 3) their generation of action potentials. None of cells with fast responses changed their activity in response to estradiol. It is evident from the present experiment that neurons in the ventrolateral part of the medulla oblongata send their axons directly to the suprachiasmatic part of the preoptic area which plays an important role in the control of the ovulatory surge of LH and that some of these neurons themselves are the sensitive sites of estradiol.

The fornix was sectioned in the frontal plane by means of a razor blade knife, and acute and chronic effects of this section on gonadotropin secretion were estimated. The 5-day cyclic rat which received the section of fornix under either anesthesia at 12:00 on the day of diestrus II showed advancement of the proestrous and estrous vaginal smears and as well as ovulatory gonadotropin release by one day. It was revealed that the primary effect was the inducement of FSH release on the day of section. The 4-day cyclic rat bearing the fornix section chronically resumed vaginal cyclicity after elapsing the diestrous period for 18 to 25 days. The rat ovulated normally and mean number of ova inoviducts was not different from that in the intact rat. However, the sectioned rat hadan higher concentrations of pituitary and serum FSH on the day of diestrus II than thatin the intact rat, and had an higher concentration of serum LH on the day of estrus. These results indicate that the hippocampus exerts the inhibitory influence on LH and FSH release and if this is eliminated the facilitatory influence dominates the brain mechanism controlling gonadotropin release, resulting in the advancement of estrous cycle (the acute effect) or the increase of gonadotropin release (the chronic effect).

The effect of spinal transections on the preovulatory release of gonadotropins and PRL was investigated in female rats. A preovulatory rise in serum LH, FSH and PRL and subsequent ovulation were prevented by complete spinal transections (CST) at high thoracic levels (T3-T7), but not at low thoracic and lumbar levels (T8-L5), performed at 1000-1230 h on proestrus. Norepinephrine (NE) concentrations in the preoptic-anterior hypothalamic area at 1700-1800 h on proestrus were also significantly reduced by CST at high thoracic levels, but not at lumbar levels. Either electrochemical stimulation of the suprachiasmatic part of the preoptic area or NE injection into the third ventricle at 1400-1500 h on proestrus restored ovulation in animals with CST at high thoracic levels. Animals with CST at lumbar levels exhibited relatively regular 4-day cycles, but those with CST at high thoracic levels showed prolonged periods of diestrous (8-20 days) before they resumed cyclicity. In the case of partial transections, bilateral transections of the lateral columns, but not transections of the dorsal or medial columns, of the spinal cord at T4-T5 significantly blocked the preovulatory gonadotropin release and the occurrence of ovulation. Unilateral transections of the lateral columns of the spinal cord or unilateral electrolytic lesions of the ventrolateral part of the medulla oblongata (VLMO) failed to block ovulation. When combinations of them were performed ipsilaterally, ovulation occurred, but when they were performed contralaterally, the incidence of ovulation was significantly decreased.(ABSTRACT TRUNCATED AT 250 WORDS)