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Using phosphorylated cyclic AMP response element-binding protein (pCREB) as a marker of neural activity, we previously suggested that orexin neurons and melanin-concentrating hormone (MCH) neurons play distinct roles in feeding behavior. In the present study, we examined the expression of pCREB during ad-libitum feeding; previously, only fasted animals were examined. MCH neurons, but not orexin neurons, expressed pCREB during spontaneous food intake. The induction of pCREB expression did not differ by sex, but attenuation seemed to occur faster in females than in males. On the basis of the results of the present study, we speculate that MCH neurons respond to nutrition-related feeding, but the feeding-related activity of orexin was not evident unless hunger was accompanied by stress, such as the stress caused by the absence of food in the case of fasting. Therefore, the desire to eat under normal conditions does not drive orexin neurons, but it does drive MCH neurons. We tested this hypothesis by examining the effects of consuming glucose or saccharin, a nonmetabolized sweetener, in fasted male and female rats. Glucose and saccharin were equally effective in reducing pCREB expression in the orexin neurons of female rats. In MCH neurons, glucose attenuated the expression of pCREB, but saccharin had no effect, irrespective of sex. Taken together, the results indicate that MCH and orexin peptides play physiologically distinct roles in feeding behavior.

Reproductive success depends on a robust and appropriately timed preovulatory luteinizing hormone (LH) surge, which is induced by the activation of gonadotropin-releasing hormone (GnRH) neurons in response to positive feedback from increasing estrogen levels. Here we document an increase in postsynaptic GluR2-lacking Ca2+-permeable AMPA-type glutamate receptors (CP-AMPARs) at synapses on GnRH neurons on the day of proestrus in rats, coincident with the increase in estrogen levels. Functional blockade of CP-AMPARs depressed the synaptic responses only on the day of proestrus and concomitantly attenuated the LH surge. Thus, the phasic synaptic incorporation of postsynaptic CP-AMPARs on GnRH neurons is involved in the generation of the LH surge.

The formalin test for nociception shows characteristic sex differences in the pain response during the interphase period of the test. However, the mechanism underlying these differences remains unclear. We have recently reported the sex-specific involvement of the lateral subdivision of the bed nucleus of the stria terminalis (BSTL) in the formalin test in rats. Here, we evaluated whether sex-specific differences in the pain response were modulated by the dopamine system in the BSTL. We first examined the effects of injecting a dopamine D1 receptor agonist, dihydrexidine, or antagonist, SCH23390, into the BSTL on the formalin test. During the interphase of the formalin test, injection of the D1 receptor agonist exerted no effect in male or female rats. The antagonist significantly enhanced the nociceptive response in female rats but not in males, indicating a sex difference in the involvement of the dopamine system in the formalin test. Next, we examined the expression of dopamine D1 receptors in the BSTL. Immunohistochemical analysis showed that the dopamine D1 receptor was expressed in the BSTL in both sexes but showed stronger immunoreactivity in male rats than in females. These results suggest sex-specific differences in the formalin test in which the response of dopamine neurons projecting to the BSTL plays a role in attenuating pain in female rats.

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.

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.

Nicotinic cholinergic receptors play a role in cardiovascular regulation in the lower brain stem. Herein, we present evidence that l-3,4-dihydroxyphenylalanine (DOPA), a putative neurotransmitter in the central nervous system, is involved in the depressor response to microinjection of nicotine into the nucleus tractus solitarii (NTS). Microinjection of nicotine into the medial area of the NTS led to decreases in arterial blood pressure and heart rate in anesthetized rats. Mecamylamine, a nicotinic receptor antagonist, microinjected into NTS, blocked the depressor and bradycardic responses to nicotine. Nicotine-induced depressor and bradycardic responses were blocked by DOPA cyclohexyl ester (DOPA CHE), an antagonist for DOPA. DOPA CHE did not modify the action of carbachol on excitatory postsynaptic potential in rat cortical slices. These results suggest that endogenous DOPA is involved in nicotine-induced depressor responses in the NTS of anesthetized 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.

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.

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.

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.

We have reported in the past that female rats fed a powdered diet showed better spatial learning and memory functions than female rats a fed pelleted diet. In the present study, we examined the effects of feeding with powdered diet on acetylcholine release in the hippocampus in both sexes of rats. After weaning (3 weeks of age), rats were fed either standard pelleted diet or powdered diet, and after maturation (9-12 weeks of age), they were used in an in vivo microdialysis study, in which no eserine (a cholinesterase inhibitor) was added to the perfusate. The dialysate was collected from the dorsal hippocampus at 20-min intervals under freely moving conditions for more than 24 h. Acetylcholine in the dialysate was measured by high performance liquid chromatography. As we reported previously, the acetylcholine release showed a clear daily rhythm in both sexes, and males showed significantly greater acetylcholine release in the hippocampus than females in rats fed pelleted diet. Conversely, in rats fed powdered diet, no sex difference in the acetylcholine release was observed, since feeding with powdered diet significantly increased the acetylcholine release only in females. To further examine the number of cholinergic neurons in the medial septum and horizontal limb of the diagonal band of Broca, immunocytochemistry for choline acetyltransferase was performed in both sexes of rats fed either standard pelleted diet or powdered diet. However, neither sex nor feeding conditions affect the number of choline acetyltransferase immunoreactive cells in the areas. These results suggest that powdered diet after weaning enhances spontaneous acetylcholine release in the hippocampus in female rats without changes in the number of cholinergic neurons in the areas. It is possible that this effect of feeding contributes to improve the performance in spatial learning and memory functions in female rats fed powdered diet.

Alkylphenols, such as p-nonylphenol (NP) and 4-tert-octylphenol (OP) and bisphenol A (BPA) are thought to mimic oestrogens in their action, and are called endocrine disrupters. We examined whether these endocrine disrupters affected progesterone receptor (PR) mRNA expression in the adult female rat neocortex. In one experiment, at 12.00 h, ovariectomized rats were given a subcutaneous injection of 10 mg of NP, 10 mg of OP or 10 mg of BPA, or sesame oil alone as control. Twenty-four hours after injection, the left side of the frontal cortex, parietal cortex and temporal cortex was collected. In a second experiment to study the time-course of the effects of BPA on PR mRNA, the ovariectomized rats were given a subcutaneous injection of 10 mg of BPA and killed 0, 6, 12 and 24 h after injection. In addition to the frontal cortex and temporal cortex, the occipital cortex was also collected. Northern blotting revealed that, in the first experiment, injection of NP, OP or BPA significantly increased PR mRNA expression in the frontal cortex but not in the parietal cortex. In the temporal cortex, BPA significantly decreased PR mRNA, but NP and OP produced no significant changes. The second experiment revealed that, in the frontal cortex, BPA induced a significant increase in PR mRNA expression at 6 h after injection, which lasted until 24 h after injection. In the temporal cortex, PR mRNA expression was significantly decreased 6 h after injection of BPA and was still significantly low 24 h after injection. No significant change was observed in the occipital cortex. These results suggest that, even in adult rats, endocrine disrupters alter the neocortical function by affecting the PR system, although the physiological significance of PR in the affected area is unknown.

The present study was performed to record the electrical activity of olfactory placode neurons and to check the effect of GABA and bicuculline on it. Olfactory placodes obtained at day 13.5 of gestation were cultured for 1 week on multi-electrode dishes. Olfactory placode neurons showed spontaneous firing, with firing rates of 0.77 +/- 0.05 Hz (0.03-3.82 Hz, n = 12), but there was no bursting activity. Perfusion with 10 microM GABA almost immediately inhibited 8 of 11 firing activities (we could not test it in 1 activity). In contrast, perfusion with 10 microM bicuculline induced facilitation in 5 of 12 activities and did not induce any change in 7 other activities. Statistical analysis by chi(2)-test showed a significant difference in the response of neurons to the two drugs. Fisher's exact probability test showed that the inhibitory effect of GABA was significant (p0.05) whereas neither the facilitatory effect nor the lack of effect of bicuculline was significant (p0.1). These results suggest that cultured olfactory placode neurons, even in a probably immature stage, respond to GABA with inhibition, as generally observed at mature stages.

We examined the effects of intracerebroventricular injection of neuromedin U (NMU), at a dose that is reported to induce satiety in rats, on the pulsatile luteinizing hormone (LH) secretion in adult ovariectomized (OVX) rats under a normal feeding or a 48-h fasted condition. In OVX rats under the normal feeding condition, injection of NMU (1 nmol/3 microl) significantly decreased the mean LH concentration without affecting the frequency or amplitude of LH pulses, but under the 48-h fasted condition, it significantly decreased the mean LH concentration and the frequency of LH pulses without affecting the amplitude. The interpulse interval was significantly lengthened by NMU injection under the normal and the 48-h fasted condition, but the effect under the 48-h fasted condition was greater than under the normal feeding condition. We also confirmed that the 48-h fasted condition per se did not affect the pulsatile LH secretion in OVX rats. We suggest that NMU and fasting synergistically inhibit the pulsatile LH secretion, even though NMU has been said to act as a satiety factor.

A ketogenic diet was recently shown to reduce glutamate accumulation in synaptic vesicles, decreasing glutamate transmission. We questioned whether a ketogenic diet affects hippocampal function, as glutamate transmission is critically involved in visuospatial ability. In the present study, male Wistar rats were maintained on a ketogenic diet containing 10% protein and 90% fat with complements for 3 weeks to change their energy expenditure from glucose-dependent to fat-dependent. Control rats were fed a diet containing 10% protein, 10% fat, and 80% carbohydrates. The fat-dependent energy expenditure induced by the ketogenic diet led to decreased body weight and increased blood ketone production, though the rats in the two groups consumed the same number of calories. The ketogenic diet did not alter food preferences for the control or high-fat diet containing 10% protein, 45% fat, and 45% carbohydrates. Anxiety in the open field was not altered by ingestion the ketogenic diet. However, rats fed the ketogenic diet performed better in the Y-maze test than rats fed the control diet. No difference was observed between the two groups in the Morris water maze test. Finally, Western blot revealed that the hippocampal expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-type glutamate receptor subunit 1 (GluR1) was significantly increased in mice fed a ketogenic diet. These results suggest that hippocampal function is not impaired by a ketogenic diet and we speculate that the fat-dependent energy expenditure does not impair visuospatial ability.

Pituitary adenylate cyclase-activating peptide is densely distributed in the suprachiasmatic nucleus, which functions as the circadian pacemaker. A receptor for pituitary adenylate cyclase-activating peptide, denoted as PAC 1 , exists in six variant forms. We used reverse transcriptase–polymerase chain reaction to identify the PAC 1 variants that are expressed in the suprachiasmatic nucleus. Dominant variant forms of PAC 1 in the suprachiasmatic nucleus were PAC 1 short , PAC 1 hip , and PAC 1 hop1 . By in situ hybridization, we examined 24-h profiles of mRNAs for the identified receptor variants in the suprachiasmatic nucleus in constant darkness and during the light–dark cycle. In constant darkness there were clear circadian rhythms in PAC 1 short mRNA with a peak at circadian time 4 but no rhythmicity was observed in PAC 1 hip mRNA or PAC 1 hop1 mRNA. In light–dark cycles, on the other hand, PAC 1 hip mRNA displayed a bimodal rhythm with troughs at zeitgeber time 4 and 16 but PAC 1 hop1 mRNA stayed constant during the day. These results suggest that PAC 1 splice variants are differentially regulated in the rat suprachiasmatic nucleus.

Intrauterine growth restriction (IUGR) is a risk factor for memory impairment and emotional disturbance during growth and adulthood. However, this risk might be modulated by environmental factors during development. Here we examined whether exposing adolescent male and female rats with thromboxane A2-induced IUGR to social defeat stress (SDS) affected their working memory and anxiety-like behavior in adulthood. We also used BrdU staining to investigate hippocampal cellular proliferation and BrdU and NeuN double staining to investigate neural differentiation in female IUGR rats. In the absence of adolescent stress, IUGR female rats, but not male rats, scored significantly lower in the T-maze test of working memory and exhibited higher anxiety-like behavior in the elevated-plus maze test compared with controls. Adolescent exposure to SDS abolished these behavioral impairments in IUGR females. In the absence of adolescent stress, hippocampal cellular proliferation was significantly higher in IUGR females than in non-IUGR female controls and was not influenced by adolescent exposure to SDS. Hippocampal neural differentiation was equivalent in non-stressed control and IUGR females. Neural differentiation was significantly increased by adolescent exposure to SDS in controls but not in IUGR females. There was no significant difference in the serum corticosterone concentrations between non-stressed control and IUGR females; however, adolescent exposure to SDS significantly increased serum corticosterone concentration in control females but not in IUGR females. These results demonstrate that adolescent exposure to SDS improves behavioral impairment independent of hippocampal neurogenesis in adult rats with IUGR.

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

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.

In order to investigate the possibility that i.p. injection of pentobarbital sodium (PB, 32 mg/kg bw) potentiates the GnRH pulse generator activity, effects of i.v. infusions of an opiate receptor antagonist naloxone (NAL, 2 mg/h) on the pulsatile LH secretion were compared in saline (SAL)- and PB-injected rats in proestrus and diestrus 1. In SAL-injected rats in proestrus, NAL infusions significantly increased both the frequency and amplitude of LH pulses, and also the overall mean LH concentration. In PB-injected rats in proestrus, all the parameters of the pulsatile LH secretion were similar to those in SAL-injected rats in proestrus. The NAL infusion in PB-injected rats caused an increase in the frequency, but it was similar to that in SAL-injected rats. But, increases in the amplitude and the overall mean LH observed during NAL infusions in PB-injected rats were greater than in SAL-injected rats. In SAL-injected rats in diestrus 1, NAL infusions increased all the parameters, as in rats in proestrus. In PB-injected rats in diestrus 1, LH secretion was severely suppressed. NAL infusions recovered the pulsatile LH secretion, but the frequency and the overall mean LH of the secretion were smaller than those obtained during NAL infusions in SAL-injected rats. In addition, characteristic increases in the MUA (volleys), which occur in association with the initiation of an LH pulse and thus are considered to represent an increased activity of the GnRH pulse generator, appeared more frequently during NAL infusions in PB-injected rats in proestrus than in SAL-injected rats. These results suggest that the GnRH pulse generator in rats in proestrus, but not in rats in diestrus 1, is refractory to PB and further is potentiated by PB in the response to NAL. Together with the fact that this dosage of PB blocks the surge of LH secretion in rats in proestrus, the concept of the existence of separate neuronal mechanisms responsible for the surge and pulsatile secretion of LH are supported.

To examine developmental changes in the number of gonadotropin-releasing hormone (GnRH) neurons activated by an opioid receptor antagonist in female rats, blood sampling and double-labeled immunocytochemistry for Fos and GnRH were performed after the injection of naloxone (NAL) in immature (postnatal d16 and d30) and mature female rats. Three age groups of rats were perfused with 4% paraformaldehyde-PB 90 min after the subcutaneous injection of NAL (2.5 mg/kg) or saline. All tissue incubation and staining for double-labeled immunocytochemistry were simultaneously performed. Although no significant developmental change was observed in the total number of GnRH neurons (p>0.05), NAL-induced increases in serum luteinizing hormone (LH) concentrations were much greater in the d16 group than those in the d30 and mature groups (p

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

Double immunocytochemistry for Fos and GnRH was performed after the intravenous infusion of naloxone or bicuculline. Either naloxone or bicuculline significantly increased serum LH concentrations. After the naloxone infusion, some Fos-positive GnRH neurons were found from the preoptic area to the basal hypothalamic area, whereas those were found from the diagonal band of Broca to the preoptic area after the bicuculline infusion. These results suggest the distribution of GnRH neurons activated by naloxone and bicuculline in intact male rats.

Whether nicotine inhibits the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator to suppress pulsatile LH secretion, and whether this suppression of LH secretion by nicotine is mediated by opioid neurons, were studied in ovariectomized rats by examining changes in LH secretion and the multiunit activity (MUA) of the medial basal hypothalamus. Intravenous (i.v.) injection of nicotine (nicotine bitartrate, 100 micrograms) significantly increased the interval between characteristic increases (volleys) in MUA and LH pulses. This inhibitory effect of nicotine on the GnRH pulse generator activity was not blocked by the prior injection of an opiate receptor antagonist naloxone (naloxone hydrochlolide, 2 mg/kg bw), which was effective in significantly decreasing the interval between MUA volleys. The results suggest that nicotine alters the activity of the GnRH pulse generator, and that cholinergic neurons appear to be directly involved in suppressing pulsatile secretion of LH.

We investigated the effect of glucose and its related substrates on the recovery of pulsatile luteinizing hormone (LH) secretion which was suppressed by insulin in estrogen-primed ovariectomized rats. We also examined the effect of glucose on the electrical activity of the gonadotropin-releasing hormone (GnRH) pulse generator which was suppressed by insulin. The intravenous (i.v.) injection of insulin (5 units/rat) suppressed the pulsatile LH secretion for 3 h in estrogen-primed ovariectomized rats. This suppressive effect of insulin on the LH secretion was rapidly reversed by the i.v. injection of glucose and mannose but not by the injection of lactate and saline. Fructose could recover the LH secretion suppressed by insulin, but took a longer time than glucose did. By monitoring the electrical activity of the GnRH pulse generator, we found that i.v. injection of insulin suppressed the pulsatile LH secretion by decreasing the activity of the GnRH pulse generator. Again, the i.v. injection of glucose, but not saline, immediately recovered the decrease in the electrical activity of the GnRH pulse generator. Fructose could recover the activity of the GnRH pulse generator, but it took a longer time than glucose did. We suggest that glucose availability, but not simply a metabolic state such as the ATP level, is an essential factor for maintaining the electrical activity of the GnRH pulse generator which is responsible for pulsatile LH secretion.

Many vasoactive intestinal polypeptide (VIP) neurons are distributed in the suprachiasmatic nucleus (SCN) and most of them arborize throughout the nuclei. We determined temporal patterns of VIP receptor (VPAC2 receptor) mRNA in the rat SCN in constant darkness and light-dark cycles, by means of in situ hybridization histochemistry. Amounts of VIP precursor mRNA in the SCN were also quantified in adjacent sections to compare its temporal profile with that of VPAC2 receptor mRNA. In constant darkness no circadian rhythms in VIP precursor mRNA or VPAC2 receptor mRNA in the SCN were detected. In light-dark cycles VIP precursor mRNA and VPAC2 receptor mRNA in the SCN showed robust but different daily rhythms. VPAC2 receptor mRNA decreased over the course of the light period and then gradually recovered during the dark period. VIP precursor mRNA was also reduced in response to light but the reduction was transient. This difference in the nature of light responsiveness between VIP mRNA and VPAC2 receptor mRNA suggests that pre- and postsynaptic events of the VIP transduction system represent different stages of light information processing in the SCN.

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.

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.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.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.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.

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.

Prostaglandins (PGs), whose synthesis is catalyzed by the rate-limiting enzyme cyclooxygenase (COX) including COX-1 and COX-2, are among the important mediators involved in the regulation of gonadotropin-releasing hormone (GnRH) secretion. However, the cellular origin of PGs remains obscure in terms of its relationship to GnRH neurons. The present study was therefore aimed to clarify the anatomical relationship between COX-1-producing microglia and GnRH neurons in the preoptic area (POA), and to examine possible influence of ovarian steroids. We performed a triple labeled immunofluorescent histochemistry of COX-1, CD11b (a specific marker for microglia) and GnRH in the POA of ovarian steroid-primed and non-primed ovariectomized rats. The result confirmed our previous study suggesting COX-1 immunoreactivity in the vicinity of, but not within, GnRH neurons in the POA. COX-1 around GnRH cells was entirely (100%) localized in cells containing CD11b regardless of steroid replacement in ovariectomized rats. These CD11b-immunoreactive cells had small cell bodies and highly branched fibers characteristic of ramified microglia. Three-dimensional reconstruction of confocal images revealed close proximity of some COX-1-containing microglia and GnRH neurons. These results showed selective and constitutive expression of COX-1 in ramified microglia in the vicinity of GnRH neurons, providing evidence for intercellular communication, mediated by PGs, from microglia to GnRH cells.

We investigated whether a decrease in masticatory work affected not only jaw bone growth but also radial eight-arm maze learning, and whether there was a sexual difference in this effect, if any. Male and female rats, weaned at 3 weeks of age, were fed either pelleted or powdered chow until 16 weeks of age and learning experiments were conducted at 10–13 weeks of age. Almost all of the five dimensions of the jaw bones were greater in rats fed pelleted chow than in rats fed powdered chow in both sexes. The number of correct choices in the last five trials was significantly greater in female, but not in male, rats fed powdered chow, and the number of trials to attain at least seven correct choices in the first eight choices in five consecutive trials was greater in female rats fed pelleted chow than in female rats fed powdered chow and in male rats fed either powdered or pelleted chow. These results suggest that 1) a decrease in masticatory work due to soft-diet feeding during development enhances later learning ability preferentially in female rats, and 2) the reported sexual inferiority of female rats in learning and memory functions is due to hard-diet feeding as the standard laboratory condition.

To study mechanisms underlying the modulation of luteinizing hormone-releasing hormone receptor (LHRH-R) during lactation and the estrous cycle, we used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to generate a probe for rat LHRH-R messenger RNA (mRNA). Using primers based on the mouse sequence, we amplified an approximately 300 bp fragment from rat pituitary complementary DNA. This PCR product was shown to be part of LHRH-R cDNA by direct sequencing and by comparing to the rat LHRH-R cDNA reported recently. Then, this PCR fragment was used as a probe for northern blotting analysis. The level of LHRH-R mRNA in the pituitary was significantly decreased during lactation, by approximately 80%, compared to that of ovariectomized and intact (diestrous and metestrous cycling) rats while no statistical difference in glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mRNA level was observed between groups. During the estrous cycle, the level of LHRH-R mRNA in the pituitary was about two-fold higher on diestrous day 2 and the morning of proestrus than that on diestrous day 1 and quickly returned toward control level by noon of proestrus. In addition, we found that GAPDH mRNA levels from a so-called housekeeping gene often thought to be unchanged under different conditions, were significantly higher on proestrus while levels of 18S rRNA were not significantly changed. The large decrease in LHRH-R mRNA during lactation could account for the changes in LHRH binding previously reported.

To gain a better understanding of the existence of an ultra-short feedback mechanism controlling LHRH secretion in the medial preoptic area (MPO), we examined the effects of microinjections of LHRH or the LHRH antagonist [D-pGlu1, D-Phe2, D-Try3,6] into the MPO on pulsatile secretion of LH in ovariectomized rats and on the surge of LH secretion in proestrous rats. Neither the injection of 10 ng LHRH nor 100 ng its antagonist into the MPO had any effect on pulse frequency or the mean LH concentration in ovariectomized rats. The injection of 100 ng LHRH antagonist or 10 ng LHRH delayed or advanced, respectively, the LH surge in proestrous rats. Taking these results together with our previous report, the present study indicates that 1) endogenous LHRH in the MPO is involved in the ultra-short feedback regulation of LHRH release and 2) the ultra-short positive feedback mechanism in the MPO acts at the time of the proestrous LH surge but not under a hormonal milieu as in ovariectomized rats.

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.

We examined the induction of progesterone receptor-immunoreactive (PR-ir) cells by estrogen in the rat preoptic area and ventromedial hypothalamic nucleus. Ovariectomized young (3-month-old) and old (24-month-old) female rats were treated with estrogen or cholesterol for 4 days. Estrogen significantly increased PR-ir cells in the preoptic area and ventromedial hypothalamic nucleus in young rats. Cholesterol-treated old rats had very few PR-ir cells; estrogen treatment significantly increased the number of PR-ir cells in both the preoptic area and the ventromedial hypothalamic nucleus in old rats, although less than in young rats. Therefore, the ability of estrogen to induce PR immunoreactivity in the hypothalamus in ovariectomized rats is attenuated in old rats compared with young rats.

To identify cellular sites of prolactin receptor messenger RNA synthesis in the rat brain, we used a combined reverse transcriptase-polymerase chain reaction protocol to generate single stranded DNA probes for in situ hybridization. The results of these experiments identify the epithelial cells of the choroid plexus as a major site of prolactin receptor gene expression in the rat central nervous system.

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.

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.

The impact of the estrous cycle on the nociceptive response in middle-aged female rats was assessed using the formalin test and c-Fos immunoreactivity as a marker of neural activation. Young (2-month-old) and middle-aged (11-month-old) rats were examined, dividing the middle-aged rats into two groups based on their estrous cycle: regular 4-day estrous cycle and irregular estrous cycle. The right hind paw was subcutaneously injected with 50microl of 2% formalin or saline as a control. Behavioral changes were observed for 1h. Cycling rats were used during proestrus. Middle-aged female rats had a significantly higher score for nociceptive behavior compared to young rats, irrespective of estrous cyclicity, which suggests that aging, not the ability to maintain estrous cyclicity, causes hypersensitivity to the formalin injection. Immunohistochemical analysis found that the brain response to formalin injection was also more sensitive in middle-aged rats than young rats; a significant increase in the number of c-Fos immunoreactive cells was found in the ventral portion of the lateral septum of middle-aged rats injected with formalin compared to young and middle-aged rats injected with saline, irrespective of estrous cyclicity. Based on these results, we conclude that the sensitivity to painful stimuli in middle-aged female rats, which are in a neuroendocrine state similar to pre- and peri-menopausal women, is associated with age and not affected by reproductive ability.

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 17β-estradiol replacement in gonadectomized females successfully restored the levels. 17β-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 17β-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 17β-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 17β-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.

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 17β-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.

Strategies and methods for experiments on sex and gender-differences in the rat and human brain are discussed taking our experiments as examples. As sex differences that should be biologically present in "the old brain", control mechanism for the GnRH secretion in the rat, nicotine effects on pulsatile LH secretion in the human and feeding behavior in the rat are mentioned. Then, the absence of sex-difference in the spatial learning memory in the rat that has been fed soft diet, instead of ordinary hard diet, and also the absence of sex-difference in the overall cognitive function in the aged human, in accordance with our concept that gender-differences are produced in response to environmental stimuli in "the new brain", are mentioned.

The sex difference in the emotional response to stress suggests a sex-specific stress response in the amygdala. To examine the sex difference in extracellular levels of serotonin (5HT) and dopamine (DA) in the basolateral amygdala (BLA) and their responses to restraint stress, in vivo microdialysis studies were performed in male and female rats. In experiment I, dialysates were collected from the BLA at 15-min intervals under the freely moving condition. Mean extracellular levels of 5HT or DA in the BLA were higher in male rats than in female rats. In experiment II, rats were subjected to restraint stress for 60 min to examine the stress response of 5HT or DA levels. Although restraint stress significantly increased extracellular 5HT levels in both sexes of rats, female rats showed a greater response than male rats. Moreover, restraint stress significantly increased extracellular DA levels in female rats, but not in male rats. In experiment III, rats were subjected to restraint stress for 30 min to examine behavioral responses to restraint stress. Although no sex difference was observed in the number of audible vocalizations, male rats defecated a larger number of fecal pellets than female rats. In experiment IV, rats were tested for freezing behavior to examine contextual fear responses. Conditioned male rats showed a longer freezing time than conditioned female rats. We found sex differences in the extracellular levels of 5HT and DA in the BLA and their responses to restraint stress, which may be involved in the sex-specific emotional response to stress 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.

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.

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

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.

l -3,4-Dihydroxyphenylalanine (DOPA) is a neurotransmitter candidate. To map the DOPAergic system functionally, DOPA-induced c-Fos expression was detected under inhibition of central aromatic l -amino acid decarboxylase (AADC). In rats treated with a central AADC inhibitor, DOPA significantly increased the number of c-Fos-positive nuclei in the paraventricular nuclei (PVN) and the nucleus tractus solitarii (NTS), and showed a tendency to increase in the supraoptic nuclei (SON), but not in the striatum. On the other hand, DOPA with a peripheral AADC inhibitor elevated the level of c-Fos-positive nuclei in the four regions, suggesting that DOPA itself induces c-Fos expression in the SON, PVN and NTS. In rats treated with 6-hydroxydopamine (6-OHDA) to lesion the nigrostriatal dopamine (DA) pathway, DOPA significantly induced c-Fos expression in the four regions under the inhibition of peripheral AADC. However, under the inhibition of central AADC, DOPA did not significantly increase the number of c-Fos-positive nuclei in the four regions, suggesting that DOPA at least in part induces c-Fos expression through its conversion to DA. It was likely that the 6-OHDA lesion enhanced the response to DA, but attenuated that to DOPA itself. In conclusion, we proposed that the SON, PVN and NTS include target sites for DOPA itself.