Phosphorylation of cAMP response element-binding protein in the extended amygdala of male rats is induced by novel environment and attenuated by estrous female-bedding.

OBJECTIVE: We examined whether female pheromone, which would be contained in female-soiled bedding, affected the expression of phosphorylated cAMP response element-binding protein-like (pCREB) immunoreactive cells in the extended amygdala. METHODS: Male rats were exposed to following conditions: maintained in their home cage (home cage group), or relocated to a cage containing clean bedding (clean-bedding exposed group), ovariectomized (OVX) rat-soiled bedding (OVX-bedding exposed group) or estrogen-treated OVX rat-soiled bedding (OVX+E2-bedding exposed group). Rats were sacrificed 10-20 min after exposure and brain sections were subject to immunocytochemical processing. RESULTS: In the medial subdivision of the bed nucleus of the stria terminalis (BST) and the central amygdala (CeA), the number of pCREB immunoreactive (pCREB-ir) cells in the clean-bedding exposed group was significantly larger than in the home cage group, while the number of pCREB-ir cells in the OVX+E2-bedding exposed group did not differ from that in the home cage group. The bedding soiled by OVX rats was less effective. No significant difference in the number of pCREB-ir cells was detected in the other regions of the extended amygdala among all groups. CONCLUSIONS: The present study suggests that the exposure of clean bedding to male rats induces the expression of pCREB-ir in the medial BST and the CeA; exposure to female pheromone attenuates this expression.

Gonadal steroids maintain 24 h acetylcholine release in the hippocampus: organizational and activational effects in behaving rats.

Extracellular acetylcholine (ACh) levels in the dorsal hippocampus increases during learning or exploration, exhibiting a sex-specific 24 h release profile. To examine the activational effect of gonadal steroid hormones on the sex-specific ACh levels and its correlation with spontaneous locomotor activity, we observed these parameters simultaneously for 24 h. Gonadectomy severely attenuated the ACh levels, whereas the testosterone replacement in gonadectomized males or 17beta-estradiol replacement in gonadectomized females successfully restored the levels. 17beta-Estradiol-priming in gonadectomized males could not restore the ACh levels, and testosterone replacement in gonadectomized females failed to raise ACh levels to those seen in testosterone-primed gonadectomized males, revealing a sex-specific activational effect. Spontaneous locomotor activity was not changed in males by gonadectomy or the replacement of gonadal steroids, but 17beta-estradiol enhanced the activity in gonadectomized females. Gonadectomy severely reduced the correlation between ACh release and activity levels, but the testosterone replacement in gonadectomized males or 17beta-estradiol replacement in gonadectomized females successfully restored it. To further analyze the sex-specific effect of gonadal steroids, we examined the organizational effect of gonadal steroids on the ACh release in female rats. Neonatal testosterone or 17beta-estradiol treatment not only increased the ACh levels but also altered them to resemble male-specific ACh release properties without affecting levels of spontaneous locomotor activity. We conclude that the activational effects of gonadal steroids maintaining the ACh levels and the high correlation with spontaneous locomotor activity are sex-specific, and that the organizational effects of gonadal steroids suggest estrogen receptor-mediated masculinization of the septo-hippocampal cholinergic system.

Food availability affects orexin a/ hypocretin-1-induced inhibition of pulsatile luteinizing hormone secretion in female rats.

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

The cAMP response element-binding protein in the bed nucleus of the stria terminalis modulates the formalin-induced pain behavior in the female rat.

Abstract Differences in male and female responses to pain are widely recognized in many species, including humans, but the cerebral mechanisms that generate these responses are unknown. Using the formalin test, we confirmed that proestrus female rats showed nociceptive behavior, modulated by estrogen that was distinct from male rats, particularly during the interphase period. We then explored the brain areas, which were involved in the female pattern of nociceptive behavior. We found that, after a formalin injection and at the time corresponding to the behavioral interphase, the number of phosphorylated cAMP response element-binding protein (pCREB)-immunoreactive neurons observed by immunocytochemistry increased in the dorsolateral division of the bed nucleus of the stria terminalis (BSTLD) in female but not male rats. There were no significant sex differences in pCREB expression following formalin in any region other than the BSTLD. The increased pCREB in female rats was eliminated after an ovariectomy and restored with 17beta-estradiol treatment. Neither an orchidectomy nor 17beta-estradiol treatment affected the pCREB response in male rats. The increase in pCREB expression in the BSTLD in female rats after formalin injection was confirmed with immunoblotting. To determine the role of CREB in the BSTLD, adenovirus-mediated expression of a dominant-negative form of CREB (mCREB) was carried out. The nociceptive behavior during interphase was significantly attenuated by injection of virus carrying mCREB into the BSTLD in female rats but not in male rats. These results suggest a novel role for CREB in the BSTLD as a modulator of the pain response in a female-specific, estrogen-dependent manner.

Gonadal steroid hormones maintain the stress-induced acetylcholine release in the hippocampus: simultaneous measurements of the extracellular acetylcholine and serum corticosterone levels in the same subjects.

To examine the role of gonadal steroid hormones in the stress responses of acetylcholine (ACh) levels in the hippocampus and serum corticosterone levels, we observed these parameters simultaneously in intact, gonadectomized, or gonadectomized steroid-primed rats. In both sexes of rats, neither gonadectomy nor the replacement of gonadal steroid hormone affected the baseline levels of ACh. However, gonadectomy severely attenuated the stress response of ACh, whereas the replacement of corresponding gonadal hormone successfully restored the response to intact levels. The gonadal hormones affected the serum corticosterone levels in a different manner; the testosterone replacement in orchidectomized rats suppressed the baseline and the stress response of corticosterone levels, whereas the 17beta-estradiol replacement in ovariectomized rats increased the levels. We further found that letrozole or flutamide administration in intact male rats attenuated the stress response of ACh. In addition, flutamide treatment increased the baseline levels of corticosterone, whereas letrozole treatment attenuated the stress response of corticosterone. Moreover, we found a low positive correlation between the ACh levels and corticosterone levels, depending on the presence of gonadal steroid hormone. We conclude that: 1) gonadal steroid hormones maintain the stress response of ACh levels in the hippocampus, 2) the gonadal steroid hormone independently regulates the stress response of ACh in the hippocampus and serum corticosterone, and 3) the sex-specific action of gonadal hormone on the cholinergic stress response may suggest a neonatal sexual differentiation of the septohippocampal cholinergic system in rats.

Postpubertal feeding experience affects sex-specific spatial ability in rats.

We previously reported that feeding with powdered diet after weaning (3 weeks of age) enhanced spatial ability, and increased the amount of acetylcholine (ACh) released in the dorsal hippocampus in female rats. In the present study, to specify the time when feeding conditions caused these effects, a radial 8-arm maze task and an in vivo microdialysis study were performed in both sexes of rats. In rats fed standard laboratory diet (i.e., pelleted diet), males learned the radial 8-arm maze faster than females. Moreover, the ACh release showed a distinct diurnal rhythm which was high during the dark phase and low during the light phase, but males showed a greater amount of ACh released in the dorsal hippocampus than females. However, in rats fed powdered diet after 6 weeks of age, no significant sex differences were observed in the radial 8-arm maze task or in the amount of ACh released, since feeding with powdered diet enhanced spatial ability, and increased the amount of ACh released only in females. These results suggest that feeding conditions after 6 weeks of age may contribute to the sex difference in the spatial ability associated with the changes in the amount of ACh released in the dorsal hippocampus in rats.

Reduced Luteinizing Hormone Induction Following Estrogen and Progesterone Priming in Female-to-Male Transsexuals.

Anatomical studies have suggested that one of the brain structures involved in gender identity is the bed nucleus of the stria terminalis, though this brain structure is probably not the only one to control gender identity. We hypothesized that, if this brain area also affected gonadotropin secretion in humans, transsexual individuals might produce different gonadotropin levels in response to exogenous stimulation. In the present study, we examined whether estrogen combined with progesterone might lead to a change in luteinizing hormone (LH) secretion in female-to-male (FTM) transsexual individuals. We studied female control subjects (n = 9), FTM transsexual subjects (n = 12), and male-to-female (MTF) transsexual subjects (n = 8). Ethinyl estradiol (50 µg/tablet) was administered orally, twice a day, for five consecutive days. After the first blood sampling, progesterone (12.5 mg) was injected intramuscularly. Plasma LH was measured with an immunoradiometric assay. The combination of estrogen and progesterone resulted in increased LH secretion in female control subjects and in MTF subjects, but this increase appeared to be attenuated in FTM transsexual subjects. In fact, the %LH response was significantly reduced in FTM subjects (P < 0.05), but not in MTF subjects (P > 0.5), compared to female control subjects. In addition, the peak time after progesterone injection was significantly delayed in FTM subjects (P < 0.05), but not in MTF subjects (P > 0.5), compared to female control subjects. We then compared subjects according to whether the combination of estrogen and progesterone had a positive (more than 200% increase) or negative (less than 200% increase) effect on LH secretion. A χ2 analysis revealed significantly different (P < 0.05) effects on LH secretion between female controls (positive n = 7, negative n = 2) and FTM transsexual subjects (positive n = 4, negative n = 8), but not between female controls and MTF transsexual subjects (positive n = 7, negative n = 1). Thus, LH secretion in response to estrogen- and progesterone priming was attenuated in FTM subjects, but not in MTF subjects, compared to control females. This finding suggested that the brain area related to gender identity in morphological studies might also be involved in the LH secretory response in humans. Thus, altered brain morphology might be correlated to altered function in FTM transsexuals.

Sex difference in the 24-h acetylcholine release profile in the premotor/supplementary motor area of behaving rats.

The sex differences in various motor functions suggest a sex-specific neural basis in the nonprimary or primary motor area. To examine the sex difference in the 24-h profile of acetylcholine (ACh) release in the rostral frontal cortex area 2 (rFr2), which is equivalent to the premotor/supplementary motor area in primates, we performed an in vivo microdialysis study in both sexes of rats fed pelleted or powdered diet. The dialysate was automatically collected from the rFr2 for 24 h under freely moving conditions. Moreover, the number of cholinergic neurons in the nucleus basalis magnocellularis (NBM) was examined. Further, to confirm the relation between ACh release in the rFr2 and motor function, the spontaneous locomotor activity was monitored for 24 h. Both sexes showed a distinct 24-h rhythm of ACh release, which was high during the dark phase and low during the light phase. Female rats, however, showed a greater ACh release and more cholinergic neurons in the NBM than male rats. Similarly, spontaneous locomotor activity also showed a 24-h rhythm, which paralleled the changes in ACh release in both sexes, and these changes were again greater in female rats than in male rats. In addition, feeding with powdered diet significantly increased the ACh release and spontaneous locomotor activity. The present study is the first to report the sex difference in the 24-h profile of ACh release in the rFr2 in rats. The sex specific ACh release in the rFr2 may partly contribute to the sex difference in motor function in rats.

Effects of neonatal testosterone treatment on sex differences in formalin-induced nociceptive behavior in rats.

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.

Bisphenol A induces transforming growth factor-beta3 mRNA in the preoptic area: a cDNA expression array and Northern blot study.

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 (p<0.05). 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.

Sex difference in the response of melanin-concentrating hormone neurons in the lateral hypothalamic area to glucose, as revealed by the expression of phosphorylated cyclic adenosine 3',5'-monophosphate response element-binding protein.

Because there are sex differences in feeding behavior in rats, we looked for a possible sex difference in the response to glucose of melanin-concentrating hormone (MCH) neurons in the lateral hypothalamic area using phosphorylated cAMP response element-binding protein (pCREB) as a marker of neural activity. Intact male rats and female rats at diestrus 2, proestrus, or estrus were fed normally or fasted for 48 h and injected with saline or glucose (400 mg/kg). Thereafter, preparations were subjected to immunohistochemical processing for the double staining of MCH and pCREB. Fasting increased the ratio of MCH neurons with pCREB (double-stained cells) in both male and female rats. In fasted rats, glucose injection decreased the ratio of double-stained cells more promptly in females than in males. The magnitude of decrease caused by glucose was greater at proestrus and estrus than at diestrus 2. Gonadectomy in males enhanced and in females attenuated the response of MCH neurons to glucose. Testosterone and estrogen replacement in males and females, respectively, restored the response of MCH neurons to glucose. The demonstrated sex differences in the response of MCH neurons to glucose correlated well with the gonadal steroid milieu; thus, MCH neurons may play an important role in sex differences in feeding behavior.

Nicotine inhibits pulsatile luteinizing hormone secretion in human males but not in human females, and tolerance to this nicotine effect is lost within one week of quitting smoking.

CONTEXT AND OBJECTIVE: Despite having increased knowledge of the adverse reproductive effects of smoking, it is unclear whether nicotine affects the pulsatile LH secretion in humans. We addressed this issue in male and female smokers and nonsmokers. SUBJECTS AND METHODS: Twenty-nine male and 16 female nonsmokers and smokers were recruited as volunteers. In male smokers, nicotine effect was also studied before and after quitting smoking. In females, cyclic ovulatory function was assessed by measuring basal body temperature, and sampling studies were performed during the follicular phase. In the morning of the sampling day, an iv catheter was inserted into an anterobrachial vein, and blood samples (1.0-1.5 ml each) were taken at 10-min intervals for 480 min, during which, at 240 min, nicotine was administered via a transdermal patch (Nicotinell transdermal therapeutic system) containing 17.5 mg nicotine. Plasma LH was measured by immunoradiometric assay kits. RESULTS: Nicotine significantly lengthened the interpulse interval of pulsatile LH secretion in male nonsmokers but not in female nonsmokers. In male smokers, nicotine did not lengthen the interpulse interval, and in female smokers it was also ineffective. After quitting cigarette smoking in male smokers, the refractory to nicotine effect disappeared within 1 wk. CONCLUSIONS: We conclude that nicotine inhibits pulsatile LH secretion only in males, and the tolerance developed to the nicotine effect disappears within 1 wk of quitting cigarette smoking. However, we cannot deny the possibility that nicotine effect would have been detected in females if more subjects had been studied.

Feeding with powdered diet after weaning increases visuospatial ability in association with increases in the expression of N-methyl-D-aspartate receptors in the hippocampus of female rats.

We determined whether feeding with powdered diet improved the visuospatial ability in female rats by checking the expression of N-methyl-D-aspartate receptor (NMDAR) subunit 1 (NR1) mRNA in the hippocampus. In rats fed standard pelleted diet, males performed better than females in a radial 8-arm maze task as we reported previously. We found that the expression of NR1 mRNA, which may be the key mediator in visuospatial ability in the hippocampus, was also higher in males than in females. However, in rats fed powdered diet, no sex difference was seen in the radial 8-arm maze task and the expression of NR1 mRNA in the hippocampus, since feeding with powdered diet improved the visuospatial ability with increases in the expression of NR1 mRNA in the hippocampus in females. We suggest that the sex difference in visuospatial ability is at least in part due to feeding conditions.

Sex differences in feeding behavior in rats: the relationship with neuronal activation in the hypothalamus.

There is general agreement that the central nervous system in rodents differs between sexes due to the presence of gonadal steroid hormone during differentiation. Sex differences in feeding seem to occur among species, and responses to fasting (i.e., starvation), gonadal steroids (i.e., testosterone and estradiol), and diet (i.e., western-style diet) vary significantly between sexes. The hypothalamus is the center for controlling feeding behavior. We examined the activation of feeding-related peptides in neurons in the hypothalamus. Phosphorylation of cyclic AMP response element-binding protein (CREB) is a good marker for neural activation, as is the Fos antigen. Therefore, we predicted that sex differences in the activity of melanin-concentrating hormone (MCH) neurons would be associated with feeding behavior. We determined the response of MCH neurons to glucose in the lateral hypothalamic area (LHA) and our results suggested MCH neurons play an important role in sex differences in feeding behavior. In addition, fasting increased the number of orexin neurons harboring phosphorylated CREB in female rats (regardless of the estrous day), but not male rats. Glucose injection decreased the number of these neurons with phosphorylated CREB in fasted female rats. Finally, under normal spontaneous food intake, MCH neurons, but not orexin neurons, expressed phosphorylated CREB. These sex differences in response to fasting and glucose, as well as under normal conditions, suggest a vulnerability to metabolic challenges in females.

Rats living in small cages respond to restraint stress with adrenocortical corticosterone release but not with hippocampal acetylcholine release.

We previously reported that the restriction of environmental space attenuated the hippocampal acetylcholine release and impaired spatial learning function. To examine the effect of the restriction of environmental space on the stress response of the hippocampal acetylcholine release, an in vivo microdialysis study was performed in male rats after 4 days of housing in a large cylindrical cage (diameter=35 cm) or a small cylindrical cage (diameter=19 cm). Significant stress response of the hippocampal acetylcholine release was observed in rats in the large cages (N=5), but it was not observed in rats in the small cages (N=5). The corticosterone concentration in serum was significantly increased by the restraint stress in both groups of rats. Although cage size does not influence stress-induced secretion of corticosterone, rats housed in a small cage exhibit lower levels of stress-induced ACh release than rats living in a large cage.

Exposure to bisphenol A during gestation and lactation causes loss of sex difference in corticotropin-releasing hormone-immunoreactive neurons in the bed nucleus of the stria terminalis of rats.

It has been suspected that endocrine disrupters induce abnormal differentiation and development of reproductive organs. In the present study, we examined whether exposure to bisphenol A (BPA), a known endocrine disrupter, during gestation and lactation affects sex difference in the number of corticotropin-releasing hormone-immunoreactive neurons (CRH neurons) in the preoptic area (POA) and the bed nucleus of the stria terminalis (BST). For that purpose, pregnant female Wistar rats (n=8-11 per treatment group) were treated with either 0.1% ethanol (control group) or 10 mg/l BPA (BPA group) dissolved in their drinking water until their offspring were weaned. In the control group, we confirmed a previous report that the POA of female rats contained significantly more CRH neurons than that of male rats (p<0.05). This significant sex difference was also evident in the BPA group, indicating that BPA exposure used in the present study had no effect on the sex difference in CRH neurons in the POA. We also found in the control group that the BST of female rats contained significantly more CRH neurons (p<0.05) than that of male rats. However, this significant sex difference was not observed in the BPA group (p>0.05), suggesting that BPA exposure affected the sex difference in CRH neurons in the BST. Since there was no statistically significant difference in the number of CRH neurons between the control and the BPA group, irrespective of the sex, the results suggested that a loss of sex difference in CRH neurons was due to both an increase in CRH neurons in male rats and a decrease in CRH neurons in female rats. The present study indicates that there is a significant sex difference in the number of CRH neurons in the BST as well as in the POA and that exposure to BPA during gestation and lactation causes a loss of this sex difference in the rat BST, but not in the POA. We suggest that CRH neurons in the BST are more susceptible to endocrine disrupters than those in the POA, irrespective of the sex.

Nicotine inhibition of pulsatile GnRH secretion is mediated by GABAA receptor system in the cultured rat embryonic olfactory placode.

In past work, we suggested that nicotine inhibition of in vivo pulsatile LH release is not mediated by opiate receptors known to be involved in the inhibition of LH release. In the present study, we examined whether nicotine inhibits the pulsatile gonadotropin-releasing hormone (GnRH) release, and whether this inhibition of GnRH release by nicotine is mediated by the GABA receptor system, by checking in vitro pulsatile GnRH release from cultured GnRH neurons obtained from olfactory placodes of rat embryos at E13.5. The mean interpulse interval of pulsatile GnRH release into the medium was 34.2+/-2.0 min in the control period and increased to 95.3+/-19.0 min (n=6) in the period of nicotine treatment at a concentration of 500 nM, showing an inhibitory effect of nicotine on pulsatile GnRH release. The GABA(A) receptor antagonist bicuculline used alone at a concentration of 20 microM caused no significant changes in the pulsatile GnRH release, but when used in combination with 500 nM of nicotine, bicuculline blocked the nicotine inhibition of GnRH release. In a separate experiment, nicotine treatment at a concentration of 500 nM significantly increased GABA release. These results suggest that, in the cultured embryonic olfactory placode, nicotine stimulates GABA release, which then inhibits GnRH release through GABA(A) receptor system.

Sexual dimorphism in the GABAergic control of gonadotropin release in intact rats.

GABA is a potent regulator of gonadotropin release both in male and female rats. We reported 24 h profiles of GABA release in the medial preoptic area (MPO) where gonadotropin-releasing hormone (GnRH) surge generator resides in female rats. In this article, we review the sex difference in 24 h profiles of GABA release. GABA release is high and episodic in male rats without any time dependency, but female rats showed a surge-like secretion of GABA in the early morning of the proestrous day. GABA release rapidly decreased until the afternoon of the day of proestrus followed by the preovulatory luteinizing hormone (LH) surge. The peak time of GABA episodes changes with estrous cycle in female rats. Fitting with the double cosinor method demonstrated that the acrophase of the GABA release in proestrous female rats occurs in the early morning, whereas the acrophases in diestrous females, estrous females and males occur at various time of day. Proestrous female rats showed significant difference in the peak time and acrophase of the GABA release compared with other estrous stages of female and male rats. These results demonstrated further sexual dimorphism of GABA release in the MPO, suggesting that coupling between the GABA release and the circadian clock may be a determining factor in the sex difference of the hypothalamo-pituitary-gonadal (HPG) axis in rats.

Intracerebroventricular injection of corticotropin-releasing hormone receptor antagonist blocks the suppression of pulsatile luteinizing hormone secretion induced by neuromedin U in ovariectomized rats after 48 hours of fasting.

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.

Female rats living in small cages respond to restraint stress with both adrenocortical corticosterone release and acetylcholine release in the hippocampus.

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.