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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.

In order to investigate the mechanism of PRL action on dopamine synthesis in tuberoinfundibular dopaminergic (TIDA) neurons, in vitro dopamine synthesis in the median eminence of hypothalamic slices was compared between hyperprolactinemic and hypoprolactinemic rats, Hyper- and hypoprolactinemia were induced in ovariectomized rats by repetitive injections of the dopamine antagonist haloperidol (Halo) and the dopamine agonist bromocriptine (Bromo), respectively. In vitro dopamine synthesis in TIDA neurons was estimated by measuring 3,4-dihydroxyphenylalanine (DOPA) accumulated in the median eminence after incubation of hypothalamic slices with a DOPA decarboxylase inhibitor. Treatment with Halo or Bromo produced increases or decreases, respectively, in the concentration of PRL in serum and in in vivo DOPA accumulation in the median eminence, as compared with vehicle treatment. The basal rate of in vitro DOPA accumulation in the median eminence was increased in Halo-treated rats and was decreased in Bromo-treated rats. The increase in basal DOPA accumulation after Halo treatment was inhibited by Ca2+ removal from medium or tetrodotoxin addition. A CA2+ -dependent increase in DOPA accumulation in the median eminence by depolarization was greater in Halo-treated rats than in Bromo-treated rats. This difference in DOPA accumulation was due to the changes in PRL secretion after Halo and Bromo treatments, since hypophysectomy abolished it. Incubation of hypothalamic slices in Na+-free media to increase the intracellular concentration of Ca2+ through inhibition of Na+-Ca2+ exchange caused an increase in DOPA accumulation. The rate of DOPA accumulation in Na+-free media was increased in Halo-treated rats and was decreased in Bromo-treated rats. On the other hand, neither Halo nor Bromo treatment altered the increase in DOPA accumulation induced by (Bu)2cAMP or forskolin. These results support the view that PRL stimulates dopamine synthesis in TIDA neurons by mechanisms which include an increase in the firing rate of TIDA neurons and increased depolarization-induced synthesis due to an enhanced response of the component that regulates dopamine synthesis to intracellular Ca2+.

The mechanism of the inhibitory effect of long term treatment with estradiol on dopamine synthesis in tuberoinfundibular dopaminergic (TIDA) neurons was studied by using hypothalamic slices from ovariectomized rats. Treatment with 2 mg estradiol valerate (EV) at a 3-week interval increased the weight of the anterior pituitary gland and the concentration of serum PRL. In vivo and in vitro dopamine synthesis in TIDA neurons were estimated in EV-treated animals by 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence after injections of 3-hydroxybenzylhydrazine (NSD 1015), a DOPA decarboxylase inhibitor, and after incubation of hypothalamic slices with NSD 1015, respectively. In vivo DOPA accumulation in the median eminence was less in EV-treated rats than in control rats. The basal rate of in vitro DOPA accumulation in the median eminence of hypothalamic slices from EV-treated rats was lower than that in control rats. Ca2+-dependent DOPA accumulation in the median eminence, determined by incubation in medium containing depolarization agents such as 50 mM K+ and veratridine, was decreased in EV-treated rats. Furthermore, cAMP-dependent DOPA accumulation, determined by incubation with Bu2cAMP or forskolin, was also suppressed in EV-treated rats. The decreased depolarization-induced DOPA accumulation in the median eminence recovered after cessation of EV treatment. Hyperprolactinemia lasting for 6 weeks, achieved by transplantation of anterior pituitaries under the kidney capsule, increased the rate of depolarization-induced DOPA accumulation in the median eminence. On the other hand, EV treatment was effective in inhibiting depolarization-induced DOPA accumulation in hypophysectomized rats regardless of the presence of anterior pituitary transplants. These results suggest that chronically administered estradiol inhibits dopamine synthesis in TIDA neurons via a direct action on the hypothalamus and overcomes the facilitatory action of PRL on dopamine synthesis; and estradiol inhibits all three distinct systems that regulate basal, Ca2+-dependent, and cAMP-dependent dopamine synthesis in TIDA neurons.

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.

The regulation of dopamine biosynthesis in tuberoinfundibular dopaminergic (TIDA) neurons by adenosine 3′,5′-cyclic monophosphate (cAMP) was investigated in the present study. Dopamine biosynthesis in TIDA neurons was estimated by the rate of in vitro dihydroxyphenylalanine (DOPA) accumulation in the median eminence of rat hypothalamic slices after incubation with a DOPA decarboxylase inhibitor. Addition of dibutyryl cAMP (db-cAMP) into medium caused an increase in the rate DOPA accumulation in the median eminence in a dose- and time-dependent manner. 8-Bromo-cAMP also increased the rate of DOPA accumulation in the median eminence ind cAMP was less effective than db-cAMP whereas neither adenosine nor sodium butyrate altered the rate of DOPA accumulation. An increase in the concentration of endogenous cAMP achieved by addition into medium of isobutylmethylxanthine, a phosphodiesterase inhibitor, or forskolin, an adenylate cyclase activator, was associated with an increase in the rat of DOPA accumulation in the median eminence, db-cAMP, however, had an almost negligible effect on the secretion of dopamine from the median eminence. The stimulatory effect of db-cAMP on DOPA accumulation in the median eminence was not dependent upon the presence of extracellular Ca 2+ and was not blocked by tetrodotoxin. Furthermore, the stimulation of DOPA accumulation in the median eminence induced by db-cAMP was additive with that induced by high potassium depolarization, which was Ca 2+ -dependent. These results suggest that dopamine biosynthesis in TIDA neurons is regulated by two distinct mechanisms, one of which involves cAMP, and another of which involves Ca 2+ .

(1) No significant alterations in the phase and amplitude of LH release were seen in proestrous rats whose peak corticosterone levels were at 09:00 hr after having been exposed to a restricted feeding schedule (fed from 10:00 to 13:00 hr) under the light-dark cycle. (2) Periodic feeding in rats kept under constant illumination induced peak corticosterone levels at 09:00 hr but did not affect the constant vaginal estrous state, and an injection of progesterone in such rats induced a marked elevation of serum LH levels at the same time as that observed in rats kept under the light-dark cycle. (3) In rats kept in the light-dark cycle, serum FSH levels decreased prior to the feeding period and increased during and after feeding, showing a different pattern from that of LH. Prolactin release in the light-dark cycle was parallel with LH release under the restricted feeding schedule, but under constant illumination, the peak levels were shifted to prior to the feeding period. (4) The results indicate that the circadian rhythm of LH release does not entrain the feeding schedule, but the rhythms of FSH and prolactin release appear to be affected by the feeding schedule.

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.

The responses of neurons in the arcuate nucleus (ARC) to gamma-aminobutyric acid (GABA), glycine, serotonin (5-HT), norepinephrine (NE), and dopamine (DA) were examined in hypothalamic slices from ovariectomized rats either treated or untreated with estrogen. In estrogen-untreated preparations, spontaneous unit activity of the majority of neurons identified antidromically by the median eminence (ME) stimulation was depressed by GABA and glycine. When effective, 5-HT was predominantly excitatory and DA was predominantly inhibitory, while NE evoked both excitatory and inhibitory responses. The IS-SD block in evoked spikes was also occasionally observed following GABA and glycine perfusion. The responsiveness of unidentified neurons, which were not activated by the ME stimulation, to agents other than 5-HT did not differ from that of antidromically identified neurons. Responses of unidentified neurons to 5-HT were predominantly inhibitory. Subcutaneous injection of 20 micrograms of 17 beta-estradiol-benzoate 2 days prior to the experiments did not significantly affect the responsiveness of both identified and unidentified neurons to GABA, 5-HT, DA, and NE. Contrarily, preincubation of slices in 17 beta-estradiol-containing medium (10 nM) for 1 h significantly increased the responsiveness of unidentified, but not identified, neurons to the inhibitory action of NE. These results suggest that GABA, glycine, 5-HT, NE, and DA play some functional roles in controlling the excitability of tuberoinfundibular (TI) neurons in the ARC. It is also suggested that the negative feedback effect of estrogen is mediated, at least in part, by increased responsiveness of ARC neurons other than TI neurons to the inhibitory action of NE.

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.These results suggest that the ascending neural pathway in the lateral columns of the spinal cord projecting to the ipsilateral VLMO participates in the neural mechanisms controlling the preovulatory release of gonadotropins and PRL by maintaining NE levels in the preoptic-anterior hypothalamic area.

The microinjection of vasoactive intestinal peptide (VIP) into the medial preoptic area rapidly inhibited pulsatile luteinizing hormone (LH) secretion within 1 h in ovariectomized rats. A transient but significant increase in prolactin (PRL) secretion occurred after VIP injection coincidentally with the initiation of the inhibition of LH pulses. Preoptic microinjections of secretin or peptide histidine isoleucine amide (PHI) did not have such effects. These results suggest specific actions of VIP, despite its structural similarity to secretin and PHI, on the preoptic mechanisms governing LH and PRL secretion in ovariectomized rats.

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.

Effects of microelectrophoretic application of cholecystokinin-8, gastrin-17, vasoactive intestinal peptide and secretin on activities of septal-preoptic neurons were examined in ovariectomized rats. All of the 4 peptides produced either excitatory or inhibitory responses in some neurons tested. No consistent relationship was observed between effects of different peptides, even between the peptides of the same family. These results provide electrophysiologic evidence for the action in the septal-preoptic region of these peptides, and suggest that there may be specific interneurons sensitive to a corresponding peptide with some overlapping.

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.

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.

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.

Studies were carried out on the role of vasoactive intestinal polypeptide (VIP) in the regulation of secretion and blood flow in the rat salivary gland. The first experiments to investigate the spontaneous secretory pattern revealed a clear diurnal fluctuation with a significant increase at night, so that the subsequent experiments were performed during the daytime where the secretion was consistently low. Intravenous administration of VIP at a dose smaller than 40 pmole caused a dose-dependent vasodilatory response, but at a high dose such a local effect was hampered by a decrease in systemic blood pressure. VIP potentiated the acetylcholine chloride (AcCho)--evoked salivary secretion, but VIP (0-100 pmole/kg) alone did not cause salivary secretion. Atropine reduced the salivary secretion evoked by AcCho and VIP, and the blood flow change evoked by AcCho. However, the blood flow change evoked by VIP was not affected by atropine. Hexamethonium exerted no significant effect on the response to administration of AcCho or VIP. The results indicate that VIP has a significant vasodilatory action and cooperates with AcCho in the regulation of salivary secretion in the rat, and VIP effects are atropine resistant, as in other species of animals.

The effects of implantation of gamma-aminobutyric acid (GABA) and serotonin (5-HT) into the arcuate nucleus (ARC) on serum luteinizing hormone (LH) and prolactin (PRL) were examined in the conscious ovariectomized rat. Following implantation of 5-HT, but not GABA, serum PRL was significantly increased. While 5-HT did not affect pulsatile LH secretion, GABA significantly decreased the mean LH concentration and pulse frequency, but not amplitude.

The anterior pituitary glands of sterilized rats secured by subcutaneous injection of 50μg estradiol benzoate contained a higher content of FSH and a little lower content of LH than those of normal proestrous glands. Serum contents of these hormones were not so different from those in normal proestrus and estrus. Electrical stimulations (0.1ms, 100Hz, 100μA) through chronically implanted electrodes into the medial preoptic area and medial part of amygdala induced the increases of serum LH and FSH. Stimulation into the dorsal hippocampus increased serum and pituitary concentrations of FSH and serum concentration of LH. Serum and pituitary concentrations of prolactin in the estrogenized rat were almost equivalent to those in normal proestrus and estrus, and stimulation of the hippocampus induced an increase of serum one. These results show that the brain of estrogenized rat differs in the function of hippocampus from that of normal cycling rat and in the function of amygdala from that of androgenized rat. However, the dramatic increase of serum and pituitary concentrations of both LH and FSH in response to bilateral ovariectomy indicates the right existence of steroid sensitive component in the brain.

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

Equimolar replacement of Na + in medium with choline chloride or sucrose and experimental manipulations known to increase [Na + ] i , such as ouabain addition and K + deprivation from medium, caused a marked increase in in vitro DOPA synthesis in the median eminence of rat hypothalamic slices in a Ca 2+ -dependent manner. These results suggest that a Na + −Ca 2+ exchange mechanism is closely involved in the regulation of dopamine biosynthesis in tuberoinfundibular neurons.

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.

This experiment examined whether various catecholaminergic synthesis inhibitors and receptor blockers affect the inhibitory effect on the frequency of pulsatile luteinizing hormone (LH) secretion induced by local application of estradiol benzoate (EB) into the preoptic suprachiasmatic area (POSC) of ovariectomized rats. Ovariectomized rats were pretreated with either alpha-methyl-p-tyrosine (AMPT; tyrosine hydroxylase inhibitor), AMPT plus threo-dihydroxyphenylserine (DOPS; norepinephrine precursor), diethyldithiocarbamate (DDC; dopamine-beta-hydroxylase inhibitor), pimozide (dopaminergic receptor blocker), phenoxybenzamine (alpha-adrenergic receptor blocker), propranolol (beta-adrenergic receptor blocker), or their vehicles. EB implantation into the POSC reduced the frequency of existing pulsatile LH secretion in vehicle-treated rats. Pretreatment of the rat with AMPT, AMPT plus DOPS, or pimozide did not affect the basal pulsatile LH secretion but eliminated the suppressive effect of EB implantation on the LH pulse frequency. In rats pretreated with DDC or phenoxybenzamine, basal pulsatile LH secretion was significantly inhibited, and EB implantation did not lower serum LH further in these rats. Propranolol had no obvious effect on either basal pulsatile LH secretion or EB-induced suppression of LH pulse frequency. These findings suggest that, in addition to the alpha-adrenergic involvement in maintaining the basal pulsatile LH secretion, the intact dopaminergic system is required for the suppression of the frequency of LH pulses induced by estrogen.

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.

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 enkephalin on dopamine synthesis in vitro in tuberoinfundibular dopaminergic (TIDA) neurons was investigated in rat hypothalamic slices. Dopamine synthesis in vitro in TIDA neurons was estimated by 3,4-dihydroxyphenylalanine (DOPA) accumulation in the median eminence after incubation of slices with a DOPA decarboxylase inhibitor. The enkephalin agonist [D-Ala2]Met-enkephalinamide (ENKamide) decreased the rate of basal DOPA accumulation in the median eminence portion of hypothalamic slices from ovariectomized rats at concentrations over 2 microM. The inhibitory action of ENKamide was more pronounced in hypothalamic slices from haloperidol-treated rats in which basal DOPA accumulation in the median eminence was stimulated by increased PRL secretion. In contrast, ENKamide decreased neither the rate of depolarization- induced CA2+-dependent DOPA accumulation nor the rate of (Bu)2cAMP- or forskolin-induced DOPA accumulation in the median eminence of normal or haloperidol-treated rats. The rank order of the potencies of enkephalins and their analogs for inhibition of DOPA accumulation in the median eminence was similar to that of their binding capacities for opioid receptors. ENKamide inhibited basal DOPA accumulation even when hypothalamic slices were incubated in Ca2+-free medium to which tetrodotoxin was added or when the median eminence was incubated alone without the remainder of the hypothalamic slice. These results suggest that enkephalin, by acting directly on axon terminals of TIDA neurons in the median eminence, inhibits basal dopamine synthesis.

Neural tissues including A 6 group noradrenaline neurons in the locus ceruleus or A 10 group dopamine neurons in the substantia nigra were transplanted into the third ventricle at the preoptic-anterior hypothalamic level of rats made hypertensive by salt loading. Either transplant exerted a long-lasting depressor effect.

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.

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.

It was investigated whether Ca 2+ is involved in the regulation of basal and depolarization-induced dopamine biosynthesis in tuberoinfundibular dopaminergic neurons. The rate of dopamine biosynthesis was estimated by in vitro dihydroxyphenylalanine (DOPA) synthesis in the median eminence following incubation of rat hypothalamic slices with a DOPA decar☐ylase inhibitor. Depolarizing agents such as K + and veratridine increased the synthesis rate of DOPA in the median eminence in a dose-dependent manner with a maximal synthesis rate obtained at concentrations of 50 mM and 50 μM, respectively. Removal of Ca 2+ and addition of EGTA (1 mM) into the medium did not influence basal DOPA synthesis in the median eminence but blocked the K + - and veratridine-induced DOPA synthesis. The Ca 2+ channel blockers verapamil (100 μM) and Co 2+ (4 mM) were effective in reducing the depolarization-induced DOPA synthesis. A23187 (10 μM), a Ca 2+ ionophore, stimulated basal DOPA synthesis in the median eminence. On the other hand, tetrodotoxin (2 μM), a Na + channel blocker, did not change the basal and K + -induced DOPA synthesis in the median eminence whereas it completely inhibited the veratridine-induced DOPA synthesis. These results suggest that depolarization-induced synthesis of dopamine in tuberoinfundibular neurons requires Ca 2+ influx through voltage-dependent Ca 2+ channels.

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.

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.

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 secretory pattern of growth hormone (GH) in 29 to 31 day old immature rats during long-term thiopental anesthesia was studied. Individual GH secretory profiles were determined by sequential blood sampling at 10 min intervals for 6.0 hr starting from about 1.0–1.5 hr after the onset of thiopental administration. EEG was concurrently monitored throughout the experimental period, and the animals were maintained in stable patterns. GH levels in whole blood samples were determined by radioimmunoassay and were subjected to analysis by power spectrum and least-squares methods. The results showed that all 25 immature rats exhibited episodic secretion of GH. Twenty-four of them (96%) showed GH surges occurring with periodicities within the mean ± 2SD range for the results from unanesthetized, sham-operated animals. The mean periodicity for the 25 animals was 3.25 ± 0.12 (SE) hr. The results suggest that the rhythmicity of GH secretion observed in the freely-moving condition is maintained during anesthesia.

Serum luteinizing hormone (LH) and prolactin (PRL) concentrations were measured in young (3-4 month old) and middle-aged (10-12 month old) intact female rats on proestrus, in ovariectomized rats after two estrogen injections (estradiol benzoate; EB, 10 micrograms/100 g body weight, s.c.) or after preoptic stimulation in EB-primed ovariectomized rats. Only animals showing regular 4-day estrous cycles were selected for the experiment. The magnitude of proestrous LH surge was significantly smaller in middle-aged than in young rats. Two BE injections, at noon on Days 0 and 3, in ovariectomized middle-aged rats failed to induce surges in LH secretion on Day 4 whereas the same treatment produced LH surges in ovariectomized young rats. The preoptic electrochemical stimulation (50 microA for 60 sec) produced a prompt rise in serum LH levels in ovariectomized EB-primed young but not in middle aged rats. The preoptic stimulation with a larger current (200 microA) induced LH secretin in middle-aged rats. In none of these situations serum PRL concentrations were different between young and middle-age rats. These results suggest differential aging rates in the preoptic mechanisms governing LH and PRL secretion in the rat. The function of the preoptic ovulatory center in responding to the estrogen positive feedback action and inducing LH secretion may become impaired and independent of the PRL control mechanism, even before the regular estrous cycle terminates.

To investigate the possibility that nerve growth factor (NGF) may play some role in the development of kindling, rabbit anti-NGF serum was injected into the third ventricle on the first 3 days of daily electrical stimulation of the basolateral amygdala. The number of stimulations required to reach full amygdaloid kindling increased significantly in the rat injected with anti-NGF serum compared to that in the rat injected with normal rabbit serum. It was confirmed that anti-NGF serum did not act as an anticonvulsant. The results demonstrate that NGF may be important for the long-lasting neuronal changes induced by daily electrical stimulation of the amygdala.

The rates of basal and cyclic AMP-dependent DOPA accumulation in the median eminence of hypothalamic slices were not different between ovariectomized young and aged rats. However, the rate of Ca2+-dependent, depolarization-induced DOPA accumulation was smaller in aged rats, suggesting that the Ca2+ system in the regulation of dopamine biosynthesis in tuberoinfundibular dopaminergic neurons is altered by aging.

The age at vaginal opening, estrous cyclicity, serum levels of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) on the day of proestrus, and number of ova and ovarian weight as measured on the day of estrus in spontaneously hypertensive (SH) and genetically matched normotensive Wistar Kyoto (WKY) female rats were compared. In SH rats, there was a significant delay in the vaginal opening, but the regular 4-day estrous cycle followed afterwards. No significant changes were observed in the afternoon increase in serum LH, FSH and PRL on the day of proestrus in SH and WKY rats, although the basal levels of LH and PRL in the morning (11:00 h) were lower in SH rats than in WKY rats. The mean number of ova in SH rats was also less than in WKY rats, whereas the ovarian relative weight was similar in both species of rats. It can be said that SH rats undergo certain, but not critical, endocrine and/or neuroendocrine changes related to reproduction.

The role of secretin in systemic blood pressure was examined in conscious rats. Either i.c.v. (0.5 or 1.0 micrograms/2 microliter 0.9% saline) or i.v. (1.0 or 2.0 micrograms/10 or 25 microliter saline) administration of secretin in conscious rats resulted in a significant fall in blood pressure. In consideration of previous findings that many other peptides such as substance P, enkephalin, bradykinin, neurotensin and vasoactive intestinal polypeptide cause pressor effects when administered centrally, but produce depressor responses after peripheral administration, the hypotension induced as both central and peripheral effects of secretin is unique and significant.