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We previously described sex differences in the number of corticotropin-releasing hormone-immunoreactive (CRH-ir) neurons in the dorsolateral division of the bed nucleus of the stria terminalis (BSTLD). Female rats were found to have more CRH neurons than male rats. We hypothesized that testosterone exposure during the critical period of sexual differentiation of the brain decreased the number of CRH-ir neurons in the hypothalamus, including the BSTLD and preoptic area. In the present study we confirm that testosterone exposure during the neonatal period results in changes to a variety of typical aspects of the female reproductive system, including estrous cyclicity as shown by virginal smear, the positive feedback effects of estrogen alone or combined with progesterone, luteinizing hormone secretions, and estrogen and progesterone-induced Fos expression in gonadotropin-releasing hormone neurons. The number of CRH-ir neurons in the preoptic area did not change, whereas CRH-ir neurons in the BSTLD significantly decreased in estrogen-primed ovariectomized rats exposed to testosterone during the neonatal period. These results suggest that the sexual differentiation of CRH neurons in the BSTLD is a result of testosterone exposure during the critical period and the BSTLD is more fragile than the preoptic area during sexual differentiation. Furthermore, sex differences in CRH in the preoptic area may not be caused by testosterone during this period.

The suprachiasmatic nucleus of the hypothalamus (SCN) is the site of the endogenous biological clock that regulates circadian rhythms in mammals. It has been shown that the gap junction couples neurons in the rat SCN. To determine the effects of estrogen on gap junction communication in the SCN, we examined the effects of estrogen on the expression of connexin32 and connexin43 mRNAs in the SCN and cerebral cortex (CX) of female rats. Ovarectomized adult rats were injected with 20 microg of 17beta-estradiol and killed 24 h after the treatment. Northern blot revealed that the expression of connexin32 mRNA was significantly increased in the SCN but not in the CX. On the other hand, estrogen increased the connexin43 mRNA level in the CX but not in the SCN. These results suggest that the gap junction with connexin32 is specifically regulated by estrogen in the SCN of female rats.

We determined whether estrogen regulates the expression of arginine-vasopressin (AVP) receptor mRNA in the preoptic area (POA) of female rats. By reverse transcription-polymerase chain reaction (PCR), we found that all three types of the AVP receptor mRNA, V1a, V1b and V2, were expressed in the POA, though the amount of PCR products was apparently different among them. In situ hybridization indicated that AVP V1a receptor mRNA was densely expressed in the POA, especially in the anteroventral periventricular nucleus of the POA; in contrast, AVP V1b and V2 receptor mRNAs were not abundant in this area. Finally, we demonstrated by Northern blot that estrogen significantly increased the expression of AVP V1a receptor mRNA in the POA of young ovariectomized rats. However, this regulation by estrogen was lost in middle-aged rats, indicating an age-related impairment in the regulation of AVP V1a receptor mRNA by estrogen in the POA.

We studied a sighted woman with premenstrual syndrome who showed menstrual changes in circadian rhythms. She showed alternative phase shifts in the sleep rhythm in the menstrual cycle: progressive phase advances in the follicular phase and phase delays in the luteal phase. Rectal temperature rhythm also showed similar menstrual changes, but the phase advance and delay started a few days earlier than changes in sleep-wake rhythm so that the two rhythms were dissociated around ovulation and menstruation. These results suggest that her circadian rhythms in sleep and temperature are under the control of ovarian steroid hormones and that these two rhythms have different sensitivity to the hormones.

Organotypic hypothalamic cultures of neonatal rats comprising the suprachiasmatic nuclei (SCN) produce stable 20 h release patterns of vasoactive intestinal polypeptide (VIP) and arginine-vasopressin (AVP). Compared with rats, voles show variably expressed circadian activity patterns. In this study we measured neuropeptidergic release patterns in organotypic SCN cultures of neonatal common voles (Microtus arvalis, n = 6). Slices were prepared at postnatal day 6. After 14 days of incubation, 2 h samples of medium were collected during 50 h. None of the vole SCN slices showed a circadian modulation in VIP release, Peaks in AVP occurred, 20 h apart from each other, in four of six vole SCN slices. These findings contrast with the concurrent release patterns of VIP and AVP in rat SCN slices. The results suggest an independent role of both neuropeptides in the oscillatory output pathways of the circadian pacemaker in the common vole. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.

To know whether insulin-induced hypoglycemia (IIH), which has been shown to inhibit pulsatile luteinizing hormone (LH) secretion, also affects the surge of LH secretion, adult female rats were injected with insulin (5 units/rat) or saline intravenously at 1300 or 1600 h on the day of proestrus and serum concentrations of LH and blood glucose were determined during the period from 1100 to 2100 h. The injection of insulin at neither 1300 nor 1600 h affected the surge of LH secretion, but it significantly decreased the blood glucose. Together with our recent hypothesis that the pulsatile and surge of LH secretion are controlled by separate gonadotropin-releasing hormone (GnRH) mechanisms, we conclude that the GnRH surge generator is less sensitive to IIH than the GnRH pulse generator.

To see changes in the number of luteinizing hormone-releasing hormone (LHRH) neurons during aging, young (3 months old)- and old (26 months old)-aged female rats, four of each, were used 4 weeks after ovariectomy for immunocytochemistry. The estimated total number (mean +/- SEM) of LHRH immunoreactive (ir) neurons in the forebrain of old-aged rats (353 +/- 34) was significantly smaller than that in young-aged rats (672 +/- 51). In the forebrain, the decrease in the number of LHRH-ir neurons with age in rostral parts was greater than that in caudal parts, suggesting heterogeneous impairment of LHRH neurons in the rat forebrain during aging.

Extracellular levels of acetylcholine (ACh) in the hippocampus were measured by the microdialysis method in freely moving young (3–4 months old) and old (18–24 months old) female rats over a period of 24 h to examine the effect of aging on hippocampal ACh release. Hippocampal ACh release during a 24-h period exhibited a diurnal variation with higher levels during the dark cycle than during the light cycle in old rats as well as young rats. The present study suggests that a diurnal variation in ACh release is maintained fairly well until the rats are at least 24 months old.

In order to evaluate the possible involvement of a GABAB-receptor mechanism in the control of prolactin release, the effect of baclofen, a GABAB-receptor agonist, on prolactin release was studied in male and female rats. Baclofen at a dose of 2–12 mg/kg was injected i.v. and blood samples were collected through an intraatrial cannula in freely moving rats. Baclofen caused an immediate increase in the prolactin concentration in serum, peaking at 6 min, in female rats. The injection also caused an increase in prolactin in male rats, the increase being smaller than in female rats. Together with the previous finding that the pituitary gland does not have GABAB receptors, the results support the concept that GABA plays a role in the central stimulation of prolactin release, mediated by GABAB receptors.

In the present study, we examined sex-specific changes in luteinizing hormone (LH) secretion and Fos expression in gonadotropin-releasing hormone (GnRH) neurons in response to naloxone in young (3 months old) and old (24 months old), gonadectomized male and female rats. We revealed by immunocytochemistry that, regardless of age and sex, naloxone significantly increased the number of GnRH neurons expressing Fos, which was associated with increased LH secretion. Additionally, although the magnitude of the increase in Fos-expressing GnRH neurons did not change in old males compared to young males, it was attenuated by almost half in old females compared to young females. LH levels decreased 60% in old males compared to young males and 15% in old females compared to young females. These results suggest LH secretion is impaired with age, but the ability of GnRH neurons to be stimulated by naloxone is preserved. However, the opioid-controlling mechanism is more fragile in females than males during aging.

Because there are sex differences in feeding-related behavior and orexin neurons are involved in feeding, we looked for a possible sex difference in the response of orexin neurons in the lateral hypothalamic area to fasting, using the phosphorylated cyclic AMP response element-binding protein (pCREB) as a marker of neural activity. Intact male and female rats at proestrus, estrus, or diestrus, were fed normally or fasted for 48h. After fasting, they were intravenously injected with saline or glucose and subjected to immunohistochemical processing for the detection of orexin and pCREB. In the rats fed normally and injected with saline, only a small population of orexin neurons expressed pCREB in both male and female rats. However, fasting increased the number of orexin neurons with pCREB (double-stained cells) in female rats regardless of the estrous day but not in male rats, revealing a significant sex difference in the response of orexin neurons to fasting. Glucose injection in fasted rats decreased the number of double-stained cells in female rats, and the magnitude of glucose-dependent decrease was greater at proestrus and estrus than at diestrus 2. We also found that female rats, but not male rats, showed an increase in total food intake after fasting (rebound feeding). We speculate that the demonstrated sex differences in the response of orexin neurons to fasting reflect the vulnerability of feeding mechanisms in females.

There are sex differences in nociceptive behavior induced by formalin in rats. To determine whether these sex differences are the result of the sexual differentiation of the brain, that is masculinization and defeminization [A.P. Arnold, R.A. Gorski, Gonadal steroid induction of structural sex differences in the central nervous system, Annu. Rev. Neurosci. 7 (1984) 413-442; M.M. McCarthy, A.T.M. Konkle, When is a sex difference not a sex difference? Front Neuroendocrinol. 26 (2005) 85-102], some female rats were injected with testosterone propionate (TP, 100 microg/25 microl/rat) on the day of birth and on the following day. As controls, other female rats and all male rats were injected with the same volume of sesame oil. They were castrated at the age of 8 weeks, and implanted with a silicon tube containing 20% of 17beta-estradiol or cholesterol. Two weeks after the implantation, rats were injected with 50 microl of 2% formalin in the right hind paw and their behavioral changes were observed for 1h. In cholesterol-implanted rats, all rats exhibited three typical phases of pain response and there were no significant differences in the scores of nociceptive behavior. In 17beta-estradiol implanted rats, female and TP-treated female rats had a significantly higher score of nociceptive behavior than male rats. These results indicate that estrogen produces sex differences in nociceptive behavior induced by formalin, and suggest that these differences are not due to the sexual differentiation of the brain, since the dose and the timing of the TP treatment effectively defeminize and masculinize female rats. Alternatively, sexual differentiation of the brain response to formalin-induced nociceptive behavior may be different from ordinary sexual differentiation.

To gain better understanding of the effects of bisphenol A (BPA) in the adult brain, a cDNA expression array was used to screen possible candidates for BPA-inducible genes in the medial preoptic area (MPOA). Adult ovariectomized rats were given a subcutaneous injection of 10 mg BPA or sesame oil alone as a control. Twenty-four hours after the injection, the MPOAs were dissected and total RNAs were extracted. When expression levels of cDNAs derived from pooled samples were compared to controls, the expression levels of some genes in BPA-injected rats appeared to be different from those in sesame oil-injected rats. Among the candidate genes, we focused on an increase in the expression of transforming growth factor (TGF)-beta3 mRNA. To quantify the change in TGF-beta3 mRNA by BPA, we examined the effects of 10 mg BPA (n=6), 1 microg 17beta-estradiol (n=6), or oil injection (n=6) on the expression of TGF-beta3 mRNA in the MPOA of ovariectomized rats by Northern blot. The TGF-beta3 mRNA level in the MPOA of BPA-injected rats was significantly increased compared to the level in oil-injected rats (p0.05). Injection of 1 microg 17beta-estradiol did not have any significant effect. The results suggest that, in the adult female rat, BPA acts on the MPOA by altering the expression of the TGF-beta3 gene in a manner distinct from that of estrogen.

We recently reported that neuromedin U (NMU) and fasting synergistically suppressed the pulsatile LH secretion, even though NMU has been shown to act as a satiety factor. In the present study, we examined whether this synergistic effect on the pulsatile LH secretion was mediated via corticotropin-releasing hormone (CRH) neurons. Adult ovariectomized (OVX) rats were stereotaxically implanted with a guide cannula into the third ventricle. After 2 weeks of recovery, blood samples were taken under freely-moving conditions at 6-min intervals for 180 min from 09:00 to 12:00 h in OVX rats that had been fasted for 48 h. After first 60 min of blood sampling, astressin (2 nmol/3 microl), a CRH receptor antagonist, dissolved in artificial cerebrospinal fluid (aCSF) or aCSF (3 microl) was injected as a control into the third ventricle. Thirty minutes after the first injection, the rats were injected with NMU (1 nmol/3 microl) into the third ventricle. We found that pre-treatment with astressin completely blocked the prolongation of the interpulse interval, which should be induced by NMU. We confirmed that a single intracerebroventricular injection of astressin per se did not affect the pulsatile LH secretion. The present study suggests that synergistic inhibitory effect of NMU and fasting on the pulsatile LH secretion is at least in part mediated via CRH neurons.

Since we found that the stress response of acetylcholine release in the hippocampus was attenuated in male rats living in a small cage, we individually housed female rats in a large (diameter=35 cm) or a small (19 cm) cylindrical cage for 4 days and examined the response to restraint stress which was applied from 12:00 h to 13:00 h. Both groups of rats showed a significant increase in the acetylcholine release in the hippocampus, as well as an increase in corticosterone release. But neither housing condition affected the expression of the 4-day estrous cycle. We surmise that there may be a sex difference in the effect of housing condition in the stress response of acetylcholine release in the hippocampus.

The effect of pentobarbital sodium (PB) on noradrenaline (NA) release in the medial preoptic area was studied in ovariectomized rats by a microdialysis method coupled to HPLC. Dialyzates were collected at 6-min intervals before and after the injection in freely moving rats. PB injected i.p. at a dose of 35 mg/kg caused a marked, approximately 40-50% decrease in NA release, which became evident 50 min after the injection and lasted for another 60 min during which the observation was performed. The results suggest that the anesthetic effect of barbiturates is related to the reduction in NA release in the brain.

We previously reported that the restriction of environmental space attenuates spontaneous locomotor activity and hippocampal acetylcholine release. To examine the effect of the restriction of environmental space on spatial learning function, male rats were individually housed in a cylindrical large cage (diameter=35 cm) or small cage (diameter=19 cm) for 5 days. Eight-arm radial maze performance was examined to evaluate spatial learning and memory functions. The task was performed once a day between 21:00 and 22:00 h in the dark phase. Although all rats learned and performed the task, those in the small cage had lower scores and took more trial time than those in the large cage. These results suggest that the restriction of environmental space impairs spatial learning in the dark phase in rats.

We measured levels of basal and norepinephrine (NE) -induced melatonin release in cultured pineal bodies of rats just after and 2 days after irradiation (20 or 200 Gy) or sham-irradiation. On the day of irradiation, basal and NE-induced melatonin release were not affected by irradiation. Two days after irradiation, basal melatonin levels in both irradiated groups were significantly lower than those in control group, whereas NE-induced melatonin levels in the irradiated groups were higher than those in the control group. These results suggested that irradiation decreases basal melatonin synthesis in pineal glands but that melatonin response to NE is increased after irradiation.

To elucidate hippocampal dysfunctions following chronic stress exposure, we evaluated the effect of chronic stress on maze learning performance, as assessed by a radial eight-arm maze task. In the 12-week stress sessions, male rats in the stress group were exposed to the stress of a 15-min immersion in cold water once a day and, rats in the control group were slightly handled. Rats in the stress group performed significantly poorly during the acquisition period (P < 0.01) and required more trials to attain at least seven correct choices in the first eight choices for five consecutive trials (P < 0.05). Together with our previous findings that chronic stress exposure damages the hippocampus histologically, we concluded that chronic stress exposure resulted in an impairment of maze learning performance, probably due to hippocampal damages.

In the present study, we examined the effect of arginine-vasopressin (AVP) V1 receptor antagonist ([Deamino-Pen 1 , Tyr(Me) 2 , Arg 8 ]-vasopressin) injection (1 μ g/2 μ l saline) into the third ventricle in the proestrous rat on serum concentrations of luteinizing hormone (LH) and prolactin (PRL) which were determined between 1100 and 2100 h at 1-h intervals. Control rats were injected with 2 μ l saline. The injection of AVP receptor antagonist at 1300 h resulted in significant decreases in both LH and PRL secretion compared to saline-injected rats. The present study provides evidence supporting our hypothesis that endogenous AVP plays a role in the induction of the surge of LH and PRL secretion as a stimulatory factor.

The effects of aging on the stress response of the septo-hippocampal cholinergic neurons was assessed by monitoring hippocampal acetylcholine (ACh) release. Young (3–4 month old) and aged (23–24 month old) male rats were subjected to restraint stress for 1 h. ACh was elevated within 15 min of the onset of restraint stress (177.5% of basal level) in young rats but not in aged rats. Corticosterone concentration was significantly elevated by restraint stress in young rats but not in aged rats. The present results suggest that stress response of the septo-hippocampal cholinergic neurons and the hypothalamic-pituitary-adrenocortical axis is attenuated during aging.

The present study examined the influence of long-term glucocorticoid treatment on a maze learning task on a radial 8-arm maze in rats. Either 100 mg cholesterol (as a control), or corticosterone, bead was implanted in rats for a period of 3 months, beginning at 12 weeks of age. The effect of this treatment on the maze learning task was evaluated during or 4 weeks after the treatments. In both experiments, corticosterone-implanted rats showed an increase in number of trials to attain at least seven correct choices in the first eight choices in five consecutive trials (P < 0.05). We concluded that long-term glucocorticoid exposure resulted in an impairment of the hippocampal functions, i.e. learning and memory, similar to that found in aged hippocampus.

The present study examined the role of corticotropin-releasing factor (CRF) in the acute stress-induced release of prolactin (PRL) in ovariectomized estrogen-primed female rats. Acute immobilization stress induced a marked increase in serum PRL levels in animals treated with saline intraventricularly (i.c.v.). However, a prior icv injection of alpha-helical CRF(9-41), a CRF antagonist, completely eliminated the immobilization-induced PRL release in the majority of animals, providing evidence for involvement of CRF in the acute stress-induced PRL release. On the other hand, an i.c.v. injection of CRF did not affect basal PRL release at any dose in non-stressed animals, suggesting that the peptide plays a permissive role which enables other undefined stress mediator(s) to stimulate PRL release.