Analysis of the Anticipatory Behavior Formation Mechanism Induced by Methamphetamine Using a Single Hair.

While the suprachiasmatic nucleus (SCN) coordinates many daily rhythms, some circadian patterns of expression are controlled by SCN-independent systems. These include responses to daily methamphetamine (MAP) injections. Scheduled daily injections of MAP resulted in anticipatory activity, with an increase in locomotor activity immediately prior to the time of injection. The MAP-induced anticipatory behavior is associated with the induction and a phase advance in the expression rhythm of the clock gene Period1 (Per1). However, this unique formation mechanism of MAP-induced anticipatory behavior is not well understood. We recently developed a micro-photomultiplier tube (micro-PMT) system to detect a small amount of Per1 expression. In the present study, we used this system to measure the formation kinetics of MAP-induced anticipatory activity in a single whisker hair to reveal the underlying mechanism. Our results suggest that whisker hairs respond to daily MAP administration, and that Per1 expression is affected. We also found that elevated Per1 expression in a single whisker hair is associated with the occurrence of anticipatory behavior rhythm. The present results suggest that elevated Per1 expression in hairs might be a marker of anticipatory behavior formation.

Differences in recovery processes of circadian oscillators in various tissues after sevoflurane treatment in vivo.

The inhalation anesthetic sevoflurane reversibly suppresses Period2 (Per2) mRNA expression in the suprachiasmatic nucleus (SCN). However, a discrepancy exists in phase shifting of the Per2 expression rhythm between sevoflurane application in rats (in vivo application) and explants (ex vivo application). This investigation aimed to resolve this issue. First, tissues from the SCN, choroid plexus in the lateral ventricle (CP-LV), and choroid plexus in the fourth ventricle (CP-4V), which are robust circadian oscillators, and pineal gland (PG) tissue, which is a circadian influencer, were prepared from Per2::dLuc transgenic rats. Significant phase responses of bioluminescence rhythms for different preparation times were monitored in the four tissue explant types. Second, tissue explants were prepared from anesthetized rats immediately after sevoflurane treatment, and bioluminescence rhythms were compared with those from non-anesthetized rats at various preparation times. Regarding bioluminescence rhythm phases, in vivo application of sevoflurane induced phase shifts in CP-LV, CP-4V, and PG explants according to the times that rats were administered anesthesia and the explants were prepared. Phase shifts in these peripheral explants were withdrawn due to the recovery period after the anesthetic treatment, which suggests that peripheral tissues require the assistance of related tissues or organs to correct phase shifts. In contrast, no phase shifts were observed in SCN explants. These results indicated that SCN explants can independently correct bioluminescence rhythm phase. The bioluminescence intensity of explants was also decreased after in vivo sevoflurane application. The suppressive effects on SCN explants were withdrawn due to a recovery day after the anesthetic treatment. In contrast, the suppressive effects on the bioluminescence intensities of CP-LV, CP-4V, and PG explants remained at 30 days after anesthesia administration. These results suggest that anesthetic suppression is imprinted within the peripheral tissues.

The analysis of Period1 gene expression in vivo and in vitro using a micro PMT system.

To detect a small amount of Period1 (Per1) expression, we developed a micro-photomultiplier tube (µPMT) system which can be used both in vivo and in vitro. Using this system, we succeeded in detecting Per1 gene expression in the skin of freely moving mice over 240 times higher compared with that of the tissue contact optical sensor (TCS) as previously reported. For in vitro studies, we succeeded in detecting elevated Per1 expression by streptozotocin (STZ) treatment in the scalp hairs at an early stage of diabetes, when glucose content in the blood was still normal. In addition, we could detect elevated Per1 expression in a single whisker hair at the time of diabetes onset. These results show that our µPMT system responds to minute changes in gene expression in freely moving mice in vivo and in mice hair follicles in vitro. Furthermore, Per1 in the hair can be used for a marker of diabetic aggravation.

Characterization of the circadian oscillator in the choroid plexus of rats.

Circadian rhythms are a fundamental biological phenomena that control various physiological functions. The suprachiasmatic nucleus (SCN) is a master clock that integrates various peripheral clocks. Recently, the choroid plexus (CP) was reported to be one such peripheral clock, a circadian oscillator that might conversely affect the SCN. Hence, the principle aim of our study was to unravel the circadian oscillator within the CP. Quantitative PCR against rPer1, rPer2, and rBmal1 showed that CP in the lateral ventricle (CP-LV) and fourth ventricle (CP-4V) has a robust circadian oscillator. The phases of the CP oscillator are between those of the pineal gland (PG) and SCN. Bioluminescence monitoring of explants showed that the intrinsic circadian period of CP-LV and CP-4V was approximately 21 h, which is shorter than SCN and PG. It is possible that interaction between oscillators of the CP-LV, CP-4V, PG, and SCN ensures the SCN adopts a stable 24 h rhythm, with each of the regions having an intrinsic oscillator with different phases and periods. In situ hybridization analysis revealed that dusk-to-dawn variation of rPer2 expression was found in epithelial cells of the CP only. Furthermore, the CP circadian oscillator might control cerebrospinal fluid secretion. However, no dusk-to-dawn variation in expression of the water channel, aquaporin 1, was observed. Further investigations are needed to clarify the involvement of circadian rhythm on CP.

Morphological Analysis of Trafficking and Processing of Anionic and Cationic Liposomes in Cultured Cells.

Liposomes, artificial phospholipid vesicles, have been developed as a non-viral drug delivery system to allow contained agents to be efficiently delivered to target sites via systemic circulation. Liposomes have been used as a gene transfer tool with cultured cells; however, their precise trafficking and processing remain uncertain. Furthermore, liposomes with different surface charges are known to exhibit distinct properties. The purpose of the current study was to elucidate the intracellular trafficking and processing of liposomes with anionic and cationic surface charges from a morphological view point. We found that cationic liposomes (CLs) were more effectively taken by the cells than anionic liposomes (ALs). Confocal laser scanning microscopy and transmission electron microscopy demonstrated distinct intracellular localization and processing patterns of ALs and CLs. ALs and their contents were localized in lysosomes but not in cytosol, indicating that ALs are subjected to the endosome-lysosome system. In contrast, contents of CLs were distributed mainly in the cytosol. CLs appear to disturb the cell membrane and then collapse to release their contents into the cytosol. It is feasible that the contents of CLs enter the cytosol directly rather than via the endosome-lysosome system.

Effectiveness of Radiofrequency Hyperthermia for Treating Cartilage in Guinea Pigs with Primary Osteoarthritis.

Objective Autophagy was reported to be essential for maintaining chondrocyte function, and reduced autophagy leads to osteoarthritis (OA). Previous studies showed involvement of heat shock stress in the control of autophagy in cells. This study sought to investigate the effect of hyperthermia on the expression of autophagy-related proteins in articular cartilage and the progression of naturally occurring OA in Hartley guinea pigs. Design Radiofrequency pulses of 13.56 MHz were applied to the animals' knees for 20 minutes to induce hyperthermia. The knee joints were resected at 8 hours, 24 hours, 72 hours, 7 days, and 6 months after hyperthermia. Serial sections of knees were examined for histopathological changes. The expression levels of Unc-51-like kinase 1 (ULK1) and Beclin1 were analyzed by immunohistochemistry. Results Analysis of the distribution of positive cells showed that, in cases of moderate OA, ULK1 and Beclin1 expression levels were significantly decreased in the superficial zone (SZ) and middle zone (MZ) ( P < 0.01) compared with normal cartilage. Seven days after exposure to radiofrequency waves, expression levels of ULK1 and Beclin1 were augmented in the SZ in animals with mild OA. The severity of cartilage degradation was significantly reduced ( P < 0.01) in the radiofrequency-treated knees versus the untreated knees. Conclusions This study showed that heat stimulation enhanced autophagy in healthy knee chondrocytes and chondrocytes in knees with mild OA. The study also showed that long-term periodic application of hyperthermia suppresses aging-related progression of OA. The activation of autophagy by radiofrequency hyperthermia may be an effective therapeutic approach for osteoarthritis.

Development of an imaging system for in vivo real-time monitoring of neuronal activity in deep brain of free-moving rats.

We have newly developed a system that allows monitoring of the intensity of fluorescent signals from deep brains of rats transgenically modified to express enhanced green fluorescent protein (eGFP) via an optical fiber. One terminal of the optical fiber was connected to a blue semiconductor laser oscillator/green fluorescence detector. The other terminal was inserted into the vicinity of the eGFP-expressing neurons. Since the optical fiber was vulnerable to twisting stresses caused by animal movement, we also developed a cage in which the floor automatically turns, in response to the turning of the rat's head. This relieved the twisting stress on the optical fiber. The system then enabled real-time monitoring of fluorescence in awake and unrestrained rats over many hours. Using this system, we could continuously monitor eGFP-expression in arginine vasopressin-eGFP transgenic rats. Moreover, we observed an increase of eGFP-expression in the paraventricular nucleus under salt-loading conditions. We then performed in vivo imaging of eGFP-expressing GnRH neurons in the hypothalamus, via a bundle consisting of 3000 thin optical fibers. With the combination of the optical fiber bundle connection to the fluorescence microscope, and the special cage system, we were able to capture and retain images of eGFP-expressing neurons from free-moving rats. We believe that our newly developed method for monitoring and imaging eGFP-expression in deep brain neurons will be useful for analysis of neuronal functions in awake and unrestrained animals for long durations.

Kisspeptin expression is decreased in the arcuate nucleus of hypothyroid female rats with irregular estrus cycles.

Insufficiency of thyroid hormones inhibits gonadotropin release and results in dysregulation of reproductive function, although the precise mechanism of this disrupted gonadotropin secretion remains unclear. Kisspeptin is a neuropeptide that strongly stimulates gonadotropin secretion and plays an important role in reproductive function. To examine the involvement of kisspeptin in the dysregulation of gonadotropin secretion in hypothyroidism, we investigated Kiss1 mRNA expression and kisspeptin immunoreactivity in the hypothalamus of female rats treated with propylthiouracil (PTU). In the PTU-treated rats, serum thyroxine (T4) was significantly decreased, whereas thyroid stimulating hormone (TSH) levels were significantly increased. In addition, irregular estrus cycles were observed in PTU-treated rats. In situ hybridization and immunohistochemistry revealed significant reductions in the number of Kiss1 mRNA-expressing neurons and kisspeptin-immunoreactive neurons in the arcuate nucleus (ARC) but not in the anteroventral periventricular nucleus (AVPV) of the PTU-treated rats. Although the serum levels of luteinizing hormone (LH) and estradiol (E2) were unaffected, serum prolactin levels were significantly increased after PTU treatment. These data indicate that kisspeptin expression in the ARC is suppressed under thyroid hormone insufficiency, suggesting that the dysregulation of reproductive function in hypothyroidism is caused by inhibition of kisspeptin neurons in the ARC.

Establishment of an in vitro cell line experimental system for the study of inhalational anesthetic mechanisms.

General anesthesia affects the expression of clock genes in various organs. Expression of Per2, a core component of the circadian clock, is markedly and reversibly suppressed by sevoflurane in the suprachiasmatic nucleus (SCN), and is considered to be a biochemical marker of anesthetic effect in the brain. The SCN contains various types of neurons, and this complexity makes it difficult to investigate the molecular mechanisms of anesthesia. Here, we established an in vitro experimental system using a cell line to investigate the mechanisms underlying anesthetic action. Development of the system comprised two steps: first, we developed a system for application of inhalational anesthetics and incubation; next, we established cultures of anesthetic-responsive cells expressing mPer2 promoter-dLuc. GT1-7 cells, derived from the mouse hypothalamus, responded to sevoflurane by reversibly decreasing mPer2-promoter-driven bioluminescence. Interestingly, the suppression of bioluminescence was found only in the serum-starved GT1-7 cells, which showed neuron-like morphology, but not in growing cells, suggesting that neuron-like characteristics are required for anesthetic effects in GT1-7 cells.

Characterization of sevoflurane effects on Per2 expression using ex vivo bioluminescence imaging of the suprachiasmatic nucleus in transgenic rats.

The inhalation anesthetic sevoflurane suppresses Per2 expression in the suprachiasmatic nucleus (SCN) in rodents. Here, we investigated the intra-SCN regional specificity, time-dependency, and pharmacological basis of sevoflurane-effects. Bioluminescence image was taken from the SCN explants of mPer2 promoter-destabilized luciferase transgenic rats, and each small regions of interest (ROI) of the image was analyzed. Sevoflurane suppressed bioluminescence in all ROIs, suggesting that all regions in the SCN are sensitive to sevoflurane. Clear time-dependency in sevoflurane effects were also observed; application during the trough phase of the bioluminescence cycle suppressed the subsequent increase in bioluminescence and resulted in a phase delay of the cycle; sevoflurane applied during the middle of the ascending phase induced a phase advance; sevoflurane on the descending phase showed no effect. These results indicate that the sevoflurane effect may depend on the intrinsic state of circadian machinery. Finally, we examined the involvement of GABAergic signal transduction in the sevoflurane effect. Co-application of both GABAA and GABAB receptor antagonists completely blocked the effect of sevoflurane on the bioluminescence rhythm, suggesting that sevoflurane inhibits Per2 expression via GABAergic signal transduction. Current study elucidated the anesthetic effects on the molecular mechanisms of circadian rhythm.

The increase in the number of spines on the gonadotropin-releasing hormone neuron across pubertal development in rats.

The onset of puberty is initiated by an increase in the release of the gonadotropin-releasing hormone (GnRH) from GnRH neurons in the hypothalamus. However, the precise mechanism that leads to the activation of GnRH neurons at puberty remains controversial. Spines are small protrusions on the surface of dendrites that normally receive excitatory inputs. In this study, we analyzed the number and morphology of spines on GnRH neurons to investigate changes in synaptic inputs across puberty in rats. For morphological estimation, we measured the diameter of the head (DH) of each spine and classified them into small-type (DH < 0.65 µm), large-type (DH > 0.65 µm) and giant-type (DH > 0.9 µm). The greatest number of spines was observed at the proximal dendrite within 50 µm of the soma. At the soma and proximal dendrite, the number of spines was greater in adults than in juveniles in both male and female individuals. Classification of spines revealed that the increase in spine number was due to increases in large- and giant-type spines. To further explore the relationship between spines on GnRH neurons and pubertal development, we next analyzed adult rats neonatally exposed to estradiol benzoate, in which puberty onset and reproductive functions are disrupted. We found a decrease in the number of all types of spines. These results suggest that GnRH neurons become to receive more and greater excitatory inputs on the soma and proximal dendrites as a result of the changes that occur at puberty and that alteration to spines plays a pivotal role in normal pubertal development.

Rapid modulation of hypothalamic Kiss1 levels by the suckling stimulus in the lactating rat.

In mammals, lactation suppresses GnRH/LH secretion resulting in transient infertility. In rats, GnRH/LH secretion is rescued within 18-48 h after pup separation (PS) and rapidly re-suppressed by subsequent re-exposure of pups. To elucidate the mechanisms underlying these rapid modulations, changes in the expression of kisspeptin, a stimulator of GnRH secretion, in several lactating conditions (normal-lactating; 4-h PS; 18-h PS; 4-h PS +1-h re-exposure of pups; non-lactating) were examined using in situ hybridization. PS for 4 h or 18 h increased Kiss1 expressing neurons in both the anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC), and subsequent exposure of pups re-suppressed Kiss1 in the AVPV. A change in Kiss1 expression was observed prior to the reported time of the change in GnRH/LH, indicating that the change in GnRH/LH results from changes in kisspeptin. We further examined the mechanisms underlying the rapid modulation of Kiss1. We first investigated the possible involvement of ascending sensory input during the suckling stimulus. Injection of the anterograde tracer to the subparafascicular parvocellular nucleus (SPFpc) in the midbrain, which relays the suckling stimulus, revealed direct neuronal connections between the SPFpc and kisspeptin neurons in both the AVPV and ARC. We also examined the possible involvement of prolactin (PRL). Administration of PRL for 1 h suppressed Kiss1 expression in the AVPV but not in the ARC. These results indicate that suckling stimulus rapidly modulates Kiss1 expression directly via neuronal connections and indirectly through serum PRL, resulting in modulation in GnRH/LH secretion.

Maternal high-fat diet during lactation increases Kiss1 mRNA expression in the arcuate nucleus at weaning and advances puberty onset in female rats.

Nutrition has significant influences on the development of reproductive functions. Post-weaning manipulation of nutritional status has been shown to alter puberty onset accompanied by changes in the expression of kisspeptin, a neuropeptide encoded by the Kiss1 gene which plays important roles in pubertal development. However, information about the influence of overnutrition during early development is sparse. In this study, we examined pubertal development and Kiss1 mRNA expression in female pups reared by dams fed a high-fat diet (HFD) during lactation. Maternal HFD significantly increased body weight and accelerated puberty onset of female offspring. The number of Kiss1-expressing neurons in the arcuate nucleus (ARC) at weaning was significantly greater in pups of HFD-fed dams than in pups of dams fed a normal diet (ND), whereas no significant difference was observed in the anteroventral periventricular nucleus, the other Kiss1-expressing nucleus. Because adipocyte size and serum leptin level were increased in HFD offspring, we examined the effects of exogenous leptin during the pre-weaning period on Kiss1 expression. Unexpectedly, exogenous leptin had no effect on Kiss1 expression. In summary, we demonstrate that a maternal HFD during the early postnatal period induces increased Kiss1 expression in the ARC and early puberty onset in female offspring.

Effect of sex steroid hormones on the number of serotonergic neurons in rat dorsal raphe nucleus.

Disorders caused by the malfunction of the serotonergic system in the central nervous system show sex-specific prevalence. Many studies have reported a relationship between sex steroid hormones and the brain serotonergic system; however, the interaction between sex steroid hormones and the number of brain neurons expressing serotonin has not yet been elucidated. In the present study, we determined whether sex steroid hormones altered the number of serotonergic neurons in the dorsal raphe nucleus (DR) of adult rat brains. Animals were divided into five groups: ovariectomized (OVX), OVX+low estradiol (E2), OVX+high E2, castrated males, and intact males. Antibodies against 5-hydroxytryptamine (5-HT, serotonin) and tryptophan hydroxylase (Tph), an enzyme for 5-HT synthesis, were used as markers of 5-HT neurons, and the number of 5-HT-immunoreactive (ir) or Tph-ir cells was counted. We detected no significant differences in the number of 5-HT-ir or Tph-ir cells in the DR among the five groups. By contrast, the intensity of 5-HT-ir showed significant sex differences in specific subregions of the DR independent of sex steroid levels, suggesting that the manipulation of sex steroid hormones after maturation does not affect the number and intensive immunostaining of serotonergic neurons in rat brain. Our results suggest that, the sexual dimorphism observed in the serotonergic system is due to factors such as 5-HT synthesis, transportation, and degradation but not to the number of serotonergic neurons.

Visualization of Kisspeptin Binding to Rat Hypothalamic Neurons.

The neuropeptide kisspeptin plays an important role in fertility and the onset of puberty, stimulating gonadotropin-releasing hormone (GnRH) neurons to activate the hypothalamic-pituitary-gonadal axis. Several studies have demonstrated a morphological interaction between kisspeptin- and GnRH-expressing neurons; however, few have addressed the interaction of kisspeptin with other neuronal subtypes. We recently showed that fibers immunoreactive for kisspeptin were densely distributed in the dorsal part of the arcuate nucleus. These fibers were found in close proximity to GnRH and tuberoinfundibular dopamine (TIDA) neurons. In the present study, using biotinylated kisspeptin, we established a visualization method for identifying kisspeptin binding sites on TIDA neurons. Biotinylated kisspeptin bound to the cell bodies of TIDA neurons and surrounding fibers, suggesting that TIDA neurons express sites of action for kisspeptin. Our assay also detected biotinylation signals from kisspeptin binding to GnRH fibers in the median eminence, but not to cell bodies of GnRH neurons in the medial preoptic area. Positive signals were completely eliminated by addition of excess non-labeled kisspeptin. This method enabled us to detect kisspeptin binding sites on specific neural structures and neuronal fibers.

Neurokinin B- and kisspeptin-positive fibers as well as tuberoinfundibular dopaminergic neurons directly innervate periventricular hypophyseal dopaminergic neurons in rats and mice.

Neurons co-expressing kisspeptin, neurokinin B (NKB), and dynorphin in the hypothalamic arcuate nucleus, named KNDy neurons, are directly affected by sex hormones, and are well known for regulating the secretion of gonadotropin-releasing hormone. However, recent studies have shown that KNDy neurons also project and terminate to tuberoinfundibular dopaminergic (TIDA) neurons, suggesting a role in prolactin secretion. Moreover, there is a possibility that other neurosecretory dopaminergic neurons regulating prolactin secretion, such as periventricular hypophyseal dopaminergic (PHDA) neurons, may also be innervated by KNDy neurons. In the present study, by means of double immunohistochemistry and retrograde neural tracer, we examined whether KNDy neurons project directly to PHDA neurons that project to blood vessels, as well as to TIDA neurons. The results revealed that KNDy neurons are widely projecting to neurosecretory dopaminergic neurons of the PHDA and TIDA neurons in rats and mice. Secondary, presence of a major receptor for NKB, neurokinin-3 receptor (NK3R), in PHDA and TIDA neurons was examined and it appeared that most TIDA and PHDA neurons possess NK3R. These findings indicate that, in rodents, KNDy neurons widely project to neurosecretory dopaminergic neurons distributed in the hypothalamus, and may affect them via the NKB-NK3R signaling pathway.

Epigenetic suppression of mouse Per2 expression in the suprachiasmatic nucleus by the inhalational anesthetic, sevoflurane.

BACKGROUND: We previously reported that sevoflurane anesthesia reversibly suppresses the expression of the clock gene, Period2 (Per2), in the mouse suprachiasmatic nucleus (SCN). However, the molecular mechanisms underlying this suppression remain unclear. In this study, we examined the possibility that sevoflurane suppresses Per2 expression via epigenetic modification of the Per2 promoter. METHODS: Mice were anesthetized with a gas mixture of 2.5% sevoflurane/40% oxygen at a 6 L/min flow for 1 or 4 h. After termination, brains were removed and samples of SCN tissue were derived from frozen brain sections. Chromatin immunoprecipitation (ChIP) assays using anti-acetylated-histone antibodies were performed to investigate the effects of sevoflurane on histone acetylation of the Per2 promoter. Interaction between the E'-box (a cis-element in the Per2 promoter) and CLOCK (the Clock gene product) was also assessed by a ChIP assay using an anti-CLOCK antibody. The SCN concentration of nicotinamide adenine dinucleotide (NAD(+)), a CLOCK regulator, was assessed by liquid chromatography-mass spectrometry. RESULTS: Acetylation of histone H4 in the proximal region of the Per2 promoter was significantly reduced by sevoflurane. This change in the epigenetic profile of the Per2 gene was observed prior to suppression of Per2 expression. Simultaneously, a reduction in the CLOCK-E'-box interaction in the Per2 promoter was observed. Sevoflurane treatment did not affect the concentration of NAD(+) in the SCN. CONCLUSIONS: Independent of NAD(+) concentration in the SCN, sevoflurane decreases CLOCK binding to the Per2 promoter E'-box motif, reducing histone acetylation and leading to suppression of Per2 expression.

Direct and specific effect of sevoflurane anesthesia on rat Per2 expression in the suprachiasmatic nucleus.

BACKGROUND: Our previous studies revealed that application of the inhalation anesthetic, sevoflurane, reversibly repressed the expression of Per2 in the mouse suprachiasmatic nucleus (SCN). We aimed to examine whether sevoflurane directly affects the SCN. METHODS: We performed in vivo and in vitro experiments to investigate rat Per2 expression under sevoflurane-treatment. The in vivo effects of sevoflurane on rPer2 expression were examined by quantitative in situ hybridization with a radioactively-labeled cRNA probe. Additionally, we examined the effect of sevoflurane anesthesia on rest/activity rhythms in the rat. In the in vitro experiments, we applied sevoflurane to SCN explant cultures from Per2-dLuc transgenic rats, and monitored luciferase bioluminescence, representing Per2 promoter activity. Bioluminescence from two peripheral organs, the kidney cortex and the anterior pituitary gland, were also analyzed. RESULTS: Application of sevoflurane in rats significantly suppressed Per2 expression in the SCN compared with untreated animals. We observed no sevoflurane-induced phase-shift in the rest/activity rhythms. In the in vitro experiments, the intermittent application of sevoflurane repressed the increase of Per2-dLuc luminescence and led to a phase delay in the Per2-dLuc luminescence rhythm. Sevoflurane treatment did not suppress bioluminescence in the kidney cortex or the anterior pituitary gland. CONCLUSION: The suppression of Per2-dLuc luminescence by sevoflurane in in vitro SCN cultures isolated from peripheral inputs and other nuclei suggest a direct action of sevoflurane on the SCN itself. That sevoflurane has no such effect on peripheral organs suggests that this action might be mediated through a neuron-specific cellular mechanism or a regulation of the signal transduction between neurons.

Morphofunctional changes of the astrocyte in rat hippocampus under different corticosteroid conditions.

In the present study, we examined the changes in the morphofunction of astrocytes in rat hippocampus under different circulating corticosteroid conditions by immunohistochemistry analysis of glial fibrillary acidic protein (GFAP) and ultra-high-voltage electron microscopy. Each GFAP-immunoreactive cell showed a hypertrophic appearance with well-developed thicker fibrous processes, and the number and the density of GFAP-immunoreactive cells were increased 4 weeks after adrenalectomy, whereas the changes were restored to the sham-control level with corticosterone replacement. The morphometric changes were observed in particular around the pyramidal neurons of CA1 and in the subgranular layer of dentate gyrus. The quantitative analysis clearly showed a significant increase in the number and the density of GFAP-immunoreactive cells in the adrenalectomy group; following corticosterone replacement, these increases were returned to the sham-control level. These changes were also specifically revealed by stereo-observation with ultra-high-voltage electron microscopy. The astrocyte showed more complicated fine three-dimensional branching after adrenalectomy. These results suggested that both the structure and function of astrocytes were modulated by corticosteroids via glucocorticoid receptor.

Immunohistochemical analysis of the colocalization of corticotropin-releasing hormone receptor and glucocorticoid receptor in kisspeptin neurons in the hypothalamus of female rats.

Kisspeptin, a neuropeptide encoded by Kiss1 gene, plays pivotal roles in the regulation of reproductive function. Recently various stressors and stress-induced molecules such as corticotropin-releasing hormone (CRH) and corticosterone have been shown to inhibit Kiss1 expression in rat hypothalamus. To determine whether CRH and glucocorticoids directly act on kisspeptin neurons, we examined the colocalization of CRH receptor (CRH-R) and glucocorticoid receptor (GR) in kisspeptin neurons in the female rat hypothalamus. Double-labeling immunohistochemistry revealed that most kisspeptin neurons in the anteroventral periventricular nucleus and periventricular nucleus continuum (AVPV/PeN), and arcuate nucleus (ARC) expressed CRH-R. We also observed a few close appositions of CRH immunoreactive fibers on some of kisspeptin neurons in AVPV/PeN and ARC. On the other hand, most kisspeptin neurons in AVPV/PeN expressed GR, whereas only a few of kisspeptin neurons in ARC expressed GR. Altogether, our study provides neuroanatomical evidence of the direct modulation of kisspeptin neurons by CRH and glucocorticoids and suggests that stress-induced CRH and glucocorticoids inhibit gonadotropin secretion via the kisspeptin system.