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We earlier reported female-biased, sex-specific involvement of the dorsolateral bed nucleus of the stria terminalis (dl BST) in the formalin-induced pain response in rats. The present study investigated pain effects on mice behaviors. Because the dl BST is densely populated with corticotropin-releasing hormone (CRH) neurons, we examined sex differences in these parameters for the dl BST CRH neurons in male and female mice of a mouse line for which the CRH gene promoter (corticotropin-releasing factor [CRF]-Venus ΔNeo) controls the expression of the modified yellow fluorescent protein (Venus). Approximately 92% of Venus-positive cells in the dl BST were also CRH mRNA-positive, irrespective of sex. Therefore, the cells identified using Venus fluorescence were regarded as CRH neurons. A female-biased sex difference was observed in pain-induced behaviors during the interphase (5–15 min after formalin injection) but not during the later phase (phase 2, 15–60 min) in wild-type mice. In CRF-Venus ΔNeo mice, a female-biased difference was observed in either the earlier phase (phase 1, 0–5 min) or the interphase, but not in phase 2. Patch-clamp recordings taken using an acute BST slice obtained from a CRF-Venus ΔNeo mouse after formalin injection showed miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs). Remarkably, the mEPSCs frequency was higher in the Venus-expressing cells of formalin-injected female mice than in vehicle-treated female mice. Male mice showed no increase in mEPSC frequency by formalin injection. Formalin injection had no effect on mEPSC or mIPSC amplitudes in either sex. Pain-induced changes in mEPSC frequency in putative CRH neurons were phase-dependent. Results show that excitatory synaptic inputs to BST CRH neurons are temporally enhanced along with behavioral sex differences in pain response, suggesting that pain signals alter the BST CRH neurons excitability in a sex-dependent manner.

Background The signal transducer and activator of transcription 6 (STAT6) transcription factor plays a vitally important role in immune cells, where it is activated mainly by interleukin-4 (IL-4). Because IL-4 is an essential cytokine for myotube formation, STAT6 might also be involved in myogenesis as part of IL-4 signaling. This study was conducted to elucidate the role of STAT6 in adult myogenesis in vitro and in vivo. Methods Myoblasts were isolated from male mice and were differentiated on a culture dish to evaluate the change in STAT6 during myotube formation. Then, the effects of STAT6 overexpression and inhibition on proliferation, differentiation, and fusion in those cells were studied. Additionally, to elucidate the myogenic role of STAT6 in vivo, muscle regeneration after injury was evaluated in STAT6 knockout mice. Results IL-4 can increase STAT6 phosphorylation, but STAT6 phosphorylation decreased during myotube formation in culture. STAT6 overexpression decreased, but STAT6 knockdown increased the differentiation index and the fusion index. Results indicate that STAT6 inhibited myogenin protein expression. Results of in vivo experiments show that STAT6 knockout mice exhibited better regeneration than wild-type mice 5 days after cardiotoxin-induced injury. It is particularly interesting that results obtained using cells from STAT6 knockout mice suggest that this STAT6 inhibitory action for myogenesis was not mediated by IL-4 but might instead be associated with p38 mitogen-activated protein kinase phosphorylation. However, STAT6 was not involved in the proliferation of myogenic cells in vitro and in vivo. Conclusion Results suggest that STAT6 functions as an inhibitor of adult myogenesis. Moreover, results suggest that the IL-4-STAT6 signaling axis is unlikely to be responsible for myotube formation.

Recent studies of the ketogenic diet, an extremely high-fat diet with extremely low carbohydrates, suggest that it changes the energy metabolism properties of skeletal muscle. However, ketogenic diet effects on muscle metabolic characteristics are diverse and sometimes countervailing. Furthermore, ketogenic diet effects on skeletal muscle performance are unknown. After male Wistar rats (8 weeks of age) were assigned randomly to a control group (CON) and a ketogenic diet group (KD), they were fed for 4 weeks respectively with a control diet (10% fat, 10% protein, 80% carbohydrate) and a ketogenic diet (90% fat, 10% protein, 0% carbohydrate). After the 4-week feeding period, the extensor digitorum longus (EDL) muscle was evaluated ex vivo for twitch force, tetanic force, and fatigue. We also analyzed the myosin heavy chain composition, protein expression of metabolic enzymes and regulatory factors, and citrate synthase activity. No significant difference was found between CON and KD in twitch or tetanic forces or muscle fatigue. However, the KD citrate synthase activity and the protein expression of Sema3A, citrate synthase, succinate dehydrogenase, cytochrome c oxidase subunit 4, and 3-hydroxyacyl-CoA dehydrogenase were significantly higher than those of CON. Moreover, a myosin heavy chain shift occurred from type IIb to IIx in KD. These results demonstrated that the 4-week ketogenic diet improves skeletal muscle aerobic capacity without obstructing muscle contractile function in sedentary male rats and suggest involvement of Sema3A in the myosin heavy chain shift of EDL muscle.

Tuberoinfundibular dopaminergic (TIDA) neurons in the arcuate nucleus (ARC) of the hypothalamus play a role in inhibiting prolactin (PRL) secretion from the anterior pituitary. PRL is involved in a variety of behaviors, including feeding. Consequently, we hypothesized that fasting might reduce the activity of TIDA neurons, which might alter PRL secretion. However, direct examinations of TIDA neuron activity are difficult. Recently, transgenic mice were generated that expressed green fluorescent protein (GFP) under the control of the rat tyrosine hydroxylase gene. We first determined that GFP in the dorsomedial ARC was a reliable marker of TIDA neurons. Then, we performed electrophysiology and immunocytochemistry in GFP-labeled TIDA neurons to examine whether different feeding conditions could change their activity. Eight-week-old male mice were fed or fasted for 24 h. After sacrifice, we prepared acutely isolated brain slices for conducting whole-cell voltage-clamp recordings. TIDA neurons were identified with fluorescence microscopy. The mean amplitude of miniature excitatory postsynaptic currents (mEPSCs) was significantly reduced in fasting mice compared to fed mice, but different feeding conditions did not affect the mean mEPSC intervals. This result suggested that fasting reduced the number of excitatory synaptic inputs to TIDA neurons. To determine whether a reduction in excitatory synaptic inputs would cause a reduction in TIDA neuron activity, we examined the effect of 24-h fasting on c-Fos expression in the ARC. We found that fasting significantly reduced the number of Fos-positive TIDA neurons. In addition, serum PRL levels were significantly increased. Taken together, the present findings suggested that short-term fasting attenuated TIDA neuron activity.

Prostaglandin E2 (PGE2) promotes gonadotropin secretion by regulating the activity of neurons that release gonadotropin-releasing hormone (GnRH) in the hypothalamus. However, the mechanisms of action of PGE2 at these neurons have yet to be fully explored. We examined the effects of PGE2 on the generation of miniature excitatory postsynaptic currents (mEPSCs) at GnRH neurons as measured by whole-cell, patch-clamp recordings. GnRH neurons were identified in slices prepared from the preoptic areas of female GnRH-EGFP rats. Exposure to PGE2 significantly increased the frequency, but not the amplitude, of the mEPSCs generated on the day of proestrus, but neither frequency nor amplitude was altered on day 1 of diestrus. These data suggest that the action of PGE2 on mEPSC frequency varies depending on the stage of estrous. An estrogen-dependence of PGE2's action was further supported by the increased frequency, but not amplitude, of mEPSCs generated at GnRH neurons prepared from estrogen-primed ovariectomized rats. Conversely, PGE2 had no effect on mEPSC frequency or amplitude at GnRH neurons in cholesterol-treated rats. Subsequent experiments to identify candidate receptors for PG2E's action revealed that exposure to a PGE2 receptor 4 (EP4) agonist, but not EP1 or EP2 agonists, mimicked the effects achieved by PGE2 exposure. These effects of mEPSCs could be reversed using an EP4 antagonist, illustrating the specificity of the effect. Collectively, these data demonstrate that PGE2 can alter excitatory synaptic neurotransmission at GnRH neurons via EP4 signaling at presynaptic site(s) in an estrogen-dependent fashion during proestrus.

Skeletal muscle regeneration is mostly dependent on muscle satellite cells. Proper muscle regeneration requires enough number of satellite cells. Recent studies have suggested that the number of satellite cells in skeletal muscle declines as we age, leading to the impairment of muscle regeneration in older population. Our earlier study demonstrated that zinc finger transcription factor early growth response 3 (Egr3) plays an important role for maintaining the number of myoblasts, suggesting that age-related decrease in muscle satellite cell should be associated with the expression levels of Egr3. The aim of this study was to investigate whether aging would alter the Egr3 expression in satellite cells. A couple groups of male C57BL/6J mice were examined in this study: young (3 Mo) and old (17 Mo). Immunohistochemical staining showed that the satellite cell number decreased in normal and injured muscles of old mice. In fluorescence-activated cell sorting-isolated muscle satellite cells from normal and injured muscles, the mRNA expression of Egr3 was significantly decreased with age regardless of injury. In harmony with these results, Pax7 mRNA levels also decreased in the satellite cells from old mice. Alternatively, inhibition of Egr3 expression by shRNA decreased Pax7 protein expression in cultured myoblasts. These results suggest that Egr3 is associated with the age-related decline of muscle satellite cells in older population. Also, Egr3 might be implicated in the regulation of Pax7. Therefore, the loss of Egr3 expression may elucidate attenuated MSCs function and muscle regeneration in older age.

Satellite cell proliferation is a crucially important process for adult myogenesis. However, its regulatory mechanisms remain unknown. Early growth response 3 (Egr3) is a zinc-finger transcription factor that regulates different cellular functions. Reportedly, Egr3 interacts with multiple signaling molecules that are also known to regulate satellite cell proliferation. Therefore, it is possible that Egr3 is involved in satellite cell proliferation. Results of this study have demonstrated that Egr3 transcript levels are upregulated in regenerating mouse skeletal muscle after cardiotoxin injury. Using C2C12 myoblast culture (a model of activated satellite cells), results show that inhibition of Egr3 by shRNA impairs the myoblast proliferation rate. Results also show reduction of NF-кB transcriptional activity in Egr3-inhibited cells. Inhibition of Egr3 is associated with an increase in annexin V+ cell fraction and apoptotic protein activity including caspase-3 and caspase-7, and Poly-ADP ribose polymerase. By contrast, the reduction of cellular proliferation by inhibition of Egr3 was partially recovered by treatment of pan-caspase inhibitor Z-VAD-FMK. Collectively, these results suggest that Egr3 is involved in myoblast proliferation by interaction with survival signaling. J. Cell. Physiol. 232: 1114-1122, 2017. © 2016 Wiley Periodicals, Inc.

The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc.

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.

Satellite cell proliferation is a crucially important process for adult myogenesis. However, its regulatory mechanisms remain unknown. Early growth response 3 (Egr3) is a zinc-finger transcription factor that regulates different cellular functions. Reportedly, Egr3 interacts with multiple signaling molecules that are also known to regulate satellite cell proliferation. Therefore, it is possible that Egr3 is involved in satellite cell proliferation. Results of this study have demonstrated that Egr3 transcript levels are upregulated in regenerating mouse skeletal muscle after cardiotoxin injury. Using C2C12 myoblast culture (a model of activated satellite cells), results show that inhibition of Egr3 by shRNA impairs the myoblast proliferation rate. Results also show reduction of NF-кB transcriptional activity in Egr3-inhibited cells. Inhibition of Egr3 is associated with an increase in annexin V

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 17β-estradiol treatment. Neither an orchidectomy nor 17β-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.

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.

Recently, there have been increasing reports that nano-sized component of particulate matter can reach the brain and may be associated with neurodegenerative diseases. Previously, our laboratory has studied the effect of intranasal instillation of nano-sized carbon black (CB) (14 nm and 95 nm) on brain cytokine and chemokine mRNA expressions and found that 14-nm CB increased IL-1 beta, TNF-alpha, CCL2 and CCL3 mRNA expressions in the olfactory bulb, not in the hippocampus of mice. To investigate the effect of a single administration of nanoparticles on neurotransmitters and proinflammatory cytokines in a mouse olfactory bulb, we performed in vivo microdialysis and real-time PCR methods. Ten-week-old male BALB/c mice were implanted with guide cannula in the right olfactory bulb and, 1 week later, were instilled vehicle or CB (14 nm, 250 microg) intranasally. Six hours after the nanoparticle instillation, the mice were intraperitoneally injected with normal saline or 50 mug of bacteria cell wall component lipoteichoic acid (LTA), which may potentiate CB-induced neurologic effect. Extracellular glutamate and glycine levels were significantly increased in the olfactory bulb of CB-instilled mice when compared with vehicle-instilled control mice. Moreover, we found that LTA further increased glutamate and glycine levels. However, no alteration of taurine and GABA levels was observed in the olfactory bulb of the same mice. We also detected immunological changes in the olfactory bulb 11 h after vehicle or CB instillation and found that IL-1 beta mRNA expression was significantly increased in CB- and LTA-treated mice when compared with control group. However, TNF-alpha mRNA expression was increased significantly in CB- and saline-treated mice when compared with control group. These findings suggest that nanoparticle CB may modulate the extracellular amino acid neurotransmitter levels and proinflammatory cytokine IL-1 beta mRNA expressions synergistically with LTA in the mice olfactory bulb.

Summary Background Tributyltin (TBT) compounds have been widely used as antifouling biocides on ships and are known to be endocrine disrupters. However, little is known about the influence of TBT on emotion and atopic dermatitis. Objective We evaluated the effects of TBT on the emotional behavior of C57BL/6 mice and on development of AD-like skin lesions in DS-Nh mice, which develop dermatitis spontaneously under conventional conditions. Methods Five-week-old C57BL/6 mice were fed 0, 50, 100, 200, 300, 400 or 500 ppm TBT diet for 2 weeks. At the end of the exposure period, an open-field test was performed. Six-week-old DS-Nh mice were fed 200 ppm TBT for 14 weeks. Skin eruption scores were checked every week. Skin biopsy was performed from ears. Results No significant difference was found in mortality or body weight among the groups receiving 0–200 ppm TBT diet during the course of the study. In the open-field test, mice fed 200 ppm TBT showed lower activity and higher frequency of defecation than did controls. These figures represent a high level of anxiety and fear and were significant in male mice compared with control mice. The skin eruption score was significantly higher in the DS-Nh mice fed TBT than in control mice. In the DS-Nh mice fed TBT, acanthosis of epidermis and infiltration of inflammatory cells in the dermis were more severe than those in controls. Conclusion TBT diet induced alterations in emotional behavior in C57BL/6 mice, and also induced early onset and deterioration of AD-like lesions in DS-Nh mice.

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.

The mechanisms that underlie the complex process of muscle regeneration after injury remain unknown. Transient receptor potential cation channel vanilloid 1 (TRPV1) is expressed in several cell types, including skeletal muscle, and is activated by high temperature and by certain molecules secreted during tissue inflammation. Severe inflammation and local temperature perturbations are induced during muscle regeneration, which suggests that TRPV1 might be activated and involved in the process. The aim of this study, was to clarify the role of TRPV1 in the myogenic potential of satellite cells responsible for muscle regeneration. We found that mRNA and protein levels of TRPV1 increased during regeneration after cardiotoxin (CTX)-induced muscle injury in mice. Using isolated mouse satellite cells (i.e., myoblasts), we observed that activation of TRPV1 by its agonist capsaicin (CAP) augmented myogenin protein levels. Whereas CAP did not alter myoblast proliferation, it facilitated myoblast fusion (evaluated using myonucleii number per myotube and fusion index). In contrast, suppression of TRPV1 by siRNA impaired myoblast fusion. Using mice, we also demonstrated that intramuscular injection of CAP facilitated muscle repair after CTX-induced muscle injury. Moreover, we showed that these roles of TRPV1 might be mediated by interleukin-4 and calcium signaling during myoblast fusion. Collectively, these results suggest that TRPV1 underlies normal myogenesis through promotion of myoblast fusion. J. Cell. Physiol. 231: 2275-2285, 2016. © 2016 Wiley Periodicals, Inc.

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.

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 µg 17β-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.

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.

Obesity is characterized by whole-body insulin resistance, yet the expression of many insulin-stimulated genes, including leptin, is elevated in obesity. These observations suggest that insulin resistance may depend on tissue type and gene. To address this hypothesis, we examined the regulation of immediate-early gene expression in liver and adipose tissue after injection of insulin and glucose, in lean insulin-sensitive, and in A(y)/a obese insulin-sensitive and obese insulin-resistant mice. Expression of hepatic jun-B mRNA was robustly increased after insulin injection in lean insulin-sensitive a/a mice and insulin-sensitive A(y)/a mice. In contrast, induction of hepatic jun-B and c-fos gene expression by insulin was markedly attenuated in obese insulin-resistant mice. Surprisingly, induction of adipose jun-B and c-fos gene expression by insulin was markedly enhanced in obese insulin-resistant mice. Furthermore, the expressions of jun-B and leptin were also enhanced in insulin-resistant mice after injection of glucose. Leptin mRNA was positively correlated with blood glucose levels and jun-B mRNA in lean but not insulin-resistant mice. Multiple regression analysis indicated that the correlation between leptin mRNA and jun-B mRNA was significant even after removing the effect of blood glucose, but the correlation between leptin mRNA and glucose was no longer significant after removing the effect of jun-B mRNA. These data suggest that some impairments in biosynthetic responses to insulin are manifest primarily in the liver, leading to hyperinsulinemia and stimulating the expression of some adipose insulin-stimulated genes, including leptin. These studies demonstrate the utility of immediate-early gene expression in the analysis of biosynthetic mechanisms of insulin resistance.

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.

Recently, we reported that bisphenol A (BPA), an endocrine disrupter, increased progesterone receptor (PR) mRNA in the preoptic area (POA) in adult ovariectomized rats. In the present study, we examined whether BPA also induced expression of PR proteins in both the POA and the ventromedial hypothalamic nucleus (VMH), and whether those proteins were involved in the induction of sexual behaviour. Two weeks after ovariectomy, rats received a subcutaneous (s.c.) injection of BPA, 17 beta-oestradiol or vehicle. Twenty-four hours after the injection, the rats were killed and their tissues were examined by immunocytochemistry. Some rats that received a s.c. injection of BPA, E2 or vehicle alone on the day before were injected with progesterone at 15.00 h and examined for sexual behaviour 5-7 h later. As expected, injection of 10 microg E2 significantly increased the number of PR immunoreactive cells in both the POA and the VMH compared to the number after injection of vehicle alone. In both the POA and the VMH, injection of BPA at a dose of 10 mg also significantly increased the number of PR immunoreactive cells compared to the number after injection of sesame oil alone. Furthermore, BPA induced a dose-dependent increase in the number of PR immunoreactive cells in both the POA and the VMH, demonstrating that the number of PR cells was significantly increased by as little as 100 microg of BPA. Ovariectomized (OVX) rats that were primed with 10 mg of BPA, followed by 1 mg of progesterone, displayed mainly rejection behaviour, but not lordosis as typically observed in OVX rats primed with E2 followed by progesterone. The present study suggests that BPA influences reproductive functions, including sexual behaviour even in adulthood, by altering the PR system in the hypothalamus.

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.

Cognitive function can be affected by the estrous cycle. However, the effect of the estrous cycle on synaptic functions is poorly understood. Here we show that in female rats, inhibitory-avoidance (IA) task (hippocampus-dependent contextual fear-learning task) drives GluA2-lacking Ca2+-permeable AMPA receptors (CP-AMPARs) into the hippocampal CA3-CA1 synapses during all periods of the estrous cycle except the proestrous period, when estrogen levels are high. In addition, IA task failed to drive CP-AMPARs into the CA3-CA1 synapses of ovariectomized rats only when estrogen was present. Thus, changes in the stoichiometry of AMPA receptors during learning depend on estrogen levels. Furthermore, the induction of long-term potentiation (LTP) after IA task was prevented during the proestrous period, while intact LTP is still expressed after IA task during other period of the estrous cycle. Consistent with this finding, rats conditioned by IA training failed to acquire hippocampus-dependent Y-maze task during the proestrous period. On the other hand, during other estrous period, rats were able to learn Y-maze task after IA conditioning. These results suggest that high estrogen levels prevent the IA learning-induced delivery of CP-AMPARs into hippocampal CA3-CA1 synapses and limit synaptic plasticity after IA task, thus preventing the acquisition of additional learning.

Butyl benzyl phthalate (BBP) and bisphenol A (BPA), termed endocrine disrupters, are known to mimic oestrogen in their actions, and therefore there is concern about their effect on reproductive functions. Since it is reported that the inhibitory action of oestrogen on the pulsatile secretion of luteinizing hormone (LH) is enhanced under insulin-induced hypoglycaemia, whether this also applies to BBP and BPA was examined in the present study. In adult ovariectomized (OVX) rats, the pulsatile LH secretion 24 h after subcutaneous injection of 10 mg BBP (BBP-treated), 10 mg BPA (BPA-treated) or 100 ng 17β-oestradiol (E 2 -treated), all of which were dissolved in sesame oil, was not changed significantly compared to that after the injection of sesame oil only. Furthermore, in oil-treated OVX rats, the pulsatile LH secretion immediately after intravenous injection of insulin (1.0 U) was not changed compared to that after saline injection. In BBP-treated OVX rats, the injection of insulin (1.0 U) significantly decreased the number of LH pulses as in E 2 -treated OVX rats. The injection of insulin did not significantly affect the amplitude of LH pulses in BBP-, BPA- and E 2 -treated OVX rats. The results indicate that the oestrogenic action of BBP is significantly enhanced by insulin-induced hypoglycaemia and thus the pulsatile LH secretion is inhibited. We suggest that weak oestrogenic endocrine disrupters may become harmful to reproductive functions even in adult female rats, if acting under a low energy state.

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.

To determine whether the suprachiasmatic nucleus can drive a circadian release of gonadotropin-releasing hormone (GnRH) in the preoptic area, we measured the release of GnRH, arginine-vasopressin and vasoactive intestinal polypeptide (VIP) in cocultures of the preoptic area and the suprachiasmatic nucleus at 2-h intervals over a period of 120 h. The release of GnRH in cocultures exhibited a significant circadian rhythm in the presence of oestrogen but not in the absence of oestrogen. The period of the GnRH circadian rhythm was the same as that of the arginine-vasopressin circadian rhythm, and different from the VIP circadian rhythm in each coculture. Furthermore, the peak phase of the GnRH rhythm occurred at the time same as that of the arginine-vasopressin rhythm in each coculture. However, the peak phase of the GnRH rhythm was not always the same as that of the VIP rhythm. Administration of arginine-vasopressin significantly increased GnRH release in single preoptic area cultures in the presence of oestrogen, but VIP did not. The result suggests that, in cocultures of the suprachiasmatic nucleus and the preoptic area, arginine-vasopressin neurones drive the circadian release of GnRH in the presence of oestrogen. We suggest that arginine-vasopressin neurones in the suprachiasmatic nucleus mediate the clock information to GnRH neurones in vivo as well.

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.

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

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.