HPC-1/syntaxin 1A is believed to regulate the exocytosis of synaptic vesicles. In order to examine the physiological function in vivo, we have produced HPC-1/syntaxin1A knock-out mice. Surprisingly, the null mutant mice revealed normal development and basal synaptic transmission in cultured hippocampal neurons appeared to be normal. In this study, we have examined the learning and behavioral phenotypes of mutant mice using behavioral approach. It was appeared that consolidation of conditioned fear memory was impaired in homozygous mutant mice but not in heterozygote. The mice exhibited impairment of cued fear memory extinction. We have also examined latent inhibition of cued fear memory (LI) to access behavioral property. Interestingly, LI was impaired both in heterozygous and homozygous mutant mice unlike the case of conditioned fear memory test. Similar results were observed in pre-pulse inhibition with acoustic startle reflex. Implication of these behavioral abnormalities in HPC-1/syntaxin1A knock-out mouse will be discussed. [J Physiol Sci. 2006;56 Suppl:S206]

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Osteoarthritis (OA) is an age-related involutional degenerative joint disease. OA attacks the knee joint particularly, and is associated with chronic pain. Intra-articular injection of mono-iodoacetate (MIA) has been used for the development of experimental pain-related OA model of osteoarthritis. Usually young rats (less than 7 weeks) were used in this model as MIA acts as metabolic inhibitor of the chondrocytes, and no report of the MIA model in the adult and aged rats was found expect no insert age in the papers until now.The objective of this study was to investigate the difference of pain related behaviors (lifting hindpaw) between the young (7w) and adult (over 24 month) rats in the MIA (60mg/ml, 50 μl) model of OA. The pain-related behaviors were evoked using von Frey hairs (2-17g) and electrically rotating brush and counted the number of lifting among 10 trials. The surface skin temperature at the knee joint were measured, as an index of joint inflammationIn pain-related behaviors, the young rats showed more frequent lifting of the hind-paw to intense von Frey filament compared with those of the adults 14 days after injection. The baseline temperature of skin surface in the young rat was lower than that of adult, and it clearly increased over 10 days after the MIA injection in the young rats. On the other hand, no clear influence was observed in the adults.These results suggest that the young rat is more suitable for the MIA injection model for studying the OA and its associated pain-related behaviors. [J Physiol Sci. 2006;56 Suppl:S207]

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Orexin-B (ORX-B) acts upon orexin 1 (OX₁R) and orexin 2 (OX₂R) receptors, and is involved in the regulation of feeding, sleep-wakefulness and locomotor activity. Orexin neurons project to the shell of the nucleus accumbens (AccSh), which plays an important role in the regulation of reinforced behaviors including feeding, and may participate in the control of feeding behavior through the modulation of AccSh neuron activity. Therefore, the effects of ORX-B on AccSh neurons were investigated using cytosolic Ca²⁺ concentration ([Ca²⁺]_i) imaging study in rat brain slice preparations. When ORX-B was applied to AccSh neurons in artificial cerebrospinal fluid (ACSF), it increased [Ca²⁺]_i dose-dependently. The ORX-B-induced increase of [Ca²⁺]_i was also seen in the presence of tetrodotoxin, suggesting that ORX-B directly affects AccSh neurons, but not via synaptic transmissions. Increase of [Ca²⁺]_i induced by ORX-B in normal ACSF disappeared completely when tested in N-methyl-D-glucamine (NMDG)-containing ACSF. [Ca²⁺]_i increase induced by ORX-B in normal ACSF was also attenuated in about 60% of neurons tested in Ni²⁺-containing ACSF. Results suggest that in many AccSh neurons, orexins increase [Ca²⁺]_i via non-selective cation channels and Ni²⁺-sensitive voltage-dependent Ca²⁺ channels, and that orexins may participate in the regulation of feeding behavior through the activation of AccSh neurons. [J Physiol Sci. 2006;56 Suppl:S207]

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The mesolimbic system and its related structures are considered to be important for ingestive behavior. We have revealed that these structures are involved in overconsumption of normally preferred taste stimuli. However, the processing of taste information in palatability-induced intake of food and fluid is still unclear. To obtain further information concerning the role of these structures in ingestion of taste stimuli, we recorded single neuron activities in the nucleus accumbens shell, ventral tegmental area, ventral pallidum, and central and basolateral nuclei of the amygdala during ingestion of taste solutions in freely behaving rats. In the experimental chamber, the rats were previously trained to lick distilled water from each spout of the bottles through a small hole on the chamber's wall. In the recording session, the rats were presented with distilled water and various taste stimuli at each trial in pseudorandom order. After a 2.5 kHz cue tone presentation for 2.5 s, rats were allowed 5 s to access the spout. Thirty to fifty percent of units in each region showed facilitation or inhibition in firing rates during licking behavior. Although in most cases these changes in the firing pattern during licking did not depend on the taste presented at each trial, a small number of units in the ventral tegmental area and amygdala responded to a specific taste. These results suggest that the mesolimbic system is implicated in taste-guided licking behavior. [J Physiol Sci. 2006;56 Suppl:S207]

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Our previous study has demonstrated that excitation of neurons in the rat ventromedial hypothalamus (VMH) induced running behavior. In this study we examine the effect of neuropeptide oxytocin injection into the VMH on the running behavior. Microinjection of oxytocin into the VMH induced a dose-dependent increase in the running behavior in male rats. On the other hand, simultaneous injection of oxytocin receptor antagonist, d(CH2)5-Tyr(Me)2-Orn8-Vasotocin, with oxytocin inhibited the running behavior. Oxytocin administration into the VMH of ovariectomized (OVX) female rats also induced running behavior. In the estrogen-treated OVX rats oxytocin injection into the VMH further increased the running behavior than OVX rats. In the histological study, effective cites of oxytocin injection on inducing running behavior is located in the ventro-lateral part of the VMH. These findings suggest that oxytocin receptor in the VMH is involved in the induction of the running behavior, and in female rats estrogen facilitate the effect of oxytocin on running behavior. [J Physiol Sci. 2006;56 Suppl:S207]

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Sleep-related masticatory muscle activity has been reported to occur in association with physiological changes of sleep process (e.g., arousal) in humans. This study aimed to assess masticatory muscle activities during wakefulness and sleep in the guinea pig. Animals were prepared for chronic experiments to monitor electroencephalogram, electro-occulogram and electromyograms from dorsal neck, bilateral masseter and digastric muscles in freely moving states. After the animals recovered from surgery, these signals were recorded for 6 to 8 hours during light-phase. Sleep stages were scored for every 10-second epoch. Muscle activities were integrated for each epoch and compared between ingestion and three quiet states (quiet wakefulness, non-REM and REM sleep). All muscles showed the highest activity during ingestion. Neck muscle activity decreased clearly from quiet wakefulness to non-REM sleep and further to REM sleep. Masticatory muscle activity was 5 to 8 times lower in quiet states than during ingestion. However, the difference between the quiet states was less evident in the masticatory muscles. These results suggest that, when epoch-based analysis was done, masticatory muscle activity in the quiet states is usually very low and the state-dependent muscle activity may be different between masticatory and neck muscles. [J Physiol Sci. 2006;56 Suppl:S208]

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It is widely recognized that fluctuations of steroid hormones during women's menstrual cycle induce various physiological and psychological changes and sometimes cause undesirable condition such as premenstrual syndrome (PMS). In the present study we take advantage of menstrual distress questionnaire (MDQ) to screen subjects having normal menstrual cycle but being aware of distresses especially on luteal phase. Healthy women subjects (N=16, aged22±1) are divided into two groups of those who marked high scores on this questionnaire (MDQH) and others who scored relatively low (MDQL). Their steroidal hormones (cortisol, estradiol, progesterone and testosterone) concentrations in saliva are analyzed with ELISA and restoring time of skin temperature after cold stimulation (RT) is measured which indicates the autonomic nervous activity of cutaneous peripheral artery. Then the salivary hormonal levels of MDQH subjects are rather higher than of MDQL while they showed less fluctuation and RT is significantly longer throughout the cycle. The acute stress-loading test is also demonstrated on these two groups (N=22, aged24±3) by means of Stroop's color and word confliction task. And subjects of MDQH are found to be less responsive for acute stress and show no changes in salivary cortisol level and RT. Based on these results, we consider that the hyporesponsiveness for stress or stimulation could correlate with women's menstrual distresses out of the therapeutic range and thus it should be taken into account to cope with their complaints. [J Physiol Sci. 2006;56 Suppl:S208]

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In mammals,the circadian peacemaker is located in the suprachiasmatic nucleus(SCN).Many of the SCN neurons exhibit spontaneous firing rhythms with periods specific to each neuron,suggesting that neuronal interaction is critical for the SCN to function as a master peacemaker.Hundreds of gene transcriptions are circadianly regulated,yet,a little is known about the intracellular transduction of the circadian signal.The purpose of our study is to identify the molecules which transmit the circadian signal to the membrane potential.Tetrodotoxin(TTX) inhibits synaptic communication,but a line of evidence supports it does not affect the intracellular circadian oscillation.To exclude the effect of the circadian rhythm of the membrane potential on the intracellular signal transduction,we applied TTX to pull out Na⁺ channel independent genes.We cultured the SCN slice of 300μm thick from the adult mouse using culture membranes.We separately monitored Per1-luciferase expression rhythms in the SCN to determine the timing of TTX administration.The SCN slices were sampled for 24h in 4 h intervals.Using serial frozen sections,we systematically analyzed circadian expression rhythms of clock and clock controlled genes by in situ hybridization.Our results indicated a number of genes showed a robust circadian expression rhythm under the TTX treatment,and some gene expressions were severely dumped by the treatment while others were not.These findings suggest that the rhythmic input is necessary for the former,while the latter involves in the core oscillation and/or rhythmic signal transduction. [J Physiol Sci. 2006;56 Suppl:S208]

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The aim of the present study was to examine whether a 3-h spontaneous wheel-running could entrain the mouse circadian rhythm under constant darkness (DD). Four-month-old, male, wild type mouse (n=27, C57BL6J, CLEA Japan, Inc) were used. They were raised in own animal quieter where environmental conditions were kept constant (light from 6:00 to 18:00, light intensity ca 100 lx, room temperature 22±2°). At 4 month old age, they were transferred to individual cage without running-wheel. The animals were exposed to DD for 4 weeks to establish stable free-running rhythm. Then the cage were exchanged with a cage out-fitted with a running-wheel (10cm diameter) for 3-h, 3-h from 6:00 to 9:00 of local time. Wheel running treatment was implemented for 80-100 days with a fixed period of 24-h. Afterwards, the spontaneous locomotor activity was measured in DD for 4 weeks. As a result, 15 of 23 mice entrained the scheduled 3-h wheel-running session. The timing of wheel setting during entrainment was related (-0.644, p<0.008) to free-running period before treatment under DD, when 3-h wheel running commenced the late subjective night (CT 17-22, CT12 was activity onset of animal). 6 animals showed longer free-running periods than 24-h ranged 24.04-24.44, and their free-running periods were significantly shortened after 3-h wheel treatment (M±SD, 23.93±0.07h). A possible role of 3-h wheel running was demonstrated as the non-photic zeitgeber. [J Physiol Sci. 2006;56 Suppl:S208]

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A master circadian clock of mammals is located in the suprachiasmatic nucleus (SCN) which is entrained by the light-dark (LD) cycle. Transcription-translation feedback loops involving several clock genes are the molecular machinery for rhythm generation, where Per gene plays a key role. Adaptation to the photoperiod is an essential strategy for organisms living in the high latitudes, and the mechanism is currently explained by two mutually coupled circadian oscillators. However, the localization of these oscillators remains elusidated. Adult mice carrying luciferase reporter genes at downstream of the mPer1 promoter were used. They were housed in three LD conditions, LD12:12, LD6:18 or LD18:6 for three weeks. The anterior and posterior SCN slices were cultured separately to monitor Per1 expression for 5days. In addition, spontaneous locomotor activity was monitored by thermal sensors. When transferred from LD12:12 to LD18:6, the activity time was compressed from 12.4h to 7h. On the other hand, in LD6:18 the activity time was lengthened by 1.6h with the activity-onset phase-advanced. In the SCN from LD12:12, the circadian peak in the posterior SCN phase-leaded the peak in the anterior SCN by 2.5h. In LD6:18, the peaks of both rhythms were located at nearly the same time of day, whereas in LD18:6, two peaks were observed in the anterior SCN which were evident in the first several days of culture, but eventually merged into one single peak. The results suggest that the two distinct oscillators reside in the SCN which respond differently to different photoperiods. [J Physiol Sci. 2006;56 Suppl:S209]

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Circadian rhythms are intrinsic oscillations which control various physiological and behavioral phenomena. In mammals, the master circadian pacemaker (or circadian clock) is located in the suprachiasmatic nuclei (SCN) of the ventral hypothalamus. Although the environmental light/dark cycle (LD) is the most potent synchronizer of the circadian pacemaker in the SCN, it is well known that exercise has effects on various circadian oscillations. Actually, novel wheel-running, which are called non-photic stimuli, induce phase advances in the circadian pacemaker. Recently, it is reported that circadian oscillators can be found in most tissues. Therefore, to explore the effect of voluntary running activity on gene expression under the LD cycle, we examined the daily expression of clock gene such as mPer1, mPer2, and mBmal1 in the central and peripheral tissues of mice. We found that daily exercise induced changes in the expression of clock genes in both central and peripheral tissues. These results suggest that daily exercise regulates the circadian oscillators in both central and peripheral tissues . [J Physiol Sci. 2006;56 Suppl:S209]

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The effects of taurine on the behaviour of male mice were investigated using operant conditioning task. This amino acid is found in high concentrations in the central nervous system of mammals and human maternal milk, has been shown to be essential for the development. Male mice (C57BL/6) were used as subjects and divided into four groups, 1) Long-loading: Taurine (400 mg/kg in a day) dissolved in distilled water was provided as drinking water. In the prenatal period, the taurine was given to through subjects' mothers, from the birth to weaning period, through maternal milk and after the weaning, from the drinking water by the end of the experiment. 2) Loading in pre-weaning: subjects were exposed to the taurine by the weaning. 3) Loading in adult: from after the weaning. 4) Control: no loading (drinking distilled water). Subjects were trained for reacting to illuminated key as soon as possible. As indexes of learning, correct reaction ratio and reaction time was subjected for analysis.All subjects acquired this discrimination task in one month period of the training. The learning speed of each experimental group was different depending on the timing but not length of the loading. [J Physiol Sci. 2006;56 Suppl:S209]

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In mammal, circadian rhythms are driven by a pacemaker located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The pacemaker is entrained to environmental light-dark cycle via the retino-hypothalamic tract, which terminates predominantly in the ventral SCN. The main neurotransmitter of the tract is glutamate. Previous studies show that the ventral part of the SCN responds to light more quickly than the dorsal SCN. However, it is unknown that how the dorsal SCN is entrained to the ventral. The dorsal part of the SCN exhibits an endogenous rhythm of arginine vasopressin (AVP) in vivo and in vitro. Measuring AVP releasing rhythm in the SCN slice culture, we examined phase-shifting effect of N-methyl-D-aspartate (NMDA) on the rhythm of the dorsal SCN. The pulse application of NMDA induced phase delays at early subjective night and phase advances at late subjective night. Even in the presence of bicucullin, a GABA-A antagonist, NMDA also induced phase shift of the rhythm. On the other hand, VIP antagonist inhibited NMDA-induced phase shift. These results suggest that VIP but not GABA would concern the photic entrainment of the dorsal SCN. [J Physiol Sci. 2006;56 Suppl:S209]

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Orexins are synthesized by lateral hypothalamic neurons which appear to have extensive projections throughout the neuraxis. Orexins have been shown to influence a variety of homeostatic mechanisms such as feeding and drinking behavior. Orexins act on two receptor subtypes, orexin-1 (OX1R) and orexin-2 (OX2R) receptors. Orexin-A (OXA) binds selectively to OX1R, whereas OX2R binds both OXA and orexin-B (OXB). The presence of OX1R in the bed nucleus of stria terminalis (BST) is verified. OXA microinjections into the BST evoked an increase in liquid food intake (FI) and water intake (WI) in a dose dependent manner. Here, the effect of selective OX1R antagonist SB334867 was examined in the BST in male Wistar rats. 0.26 nmol SB334867 was bilaterally microinjected into the BST alone or 15 min before 0.14 nmol OXA microinjections, whether the increases in liquid FI and WI following OXA applications can be antagonized. OXA enhanced liquid food consumption and increased WI as well. SB334867 pretreated OXA groups did not show any significant differences compared to the control groups neither in liquid food, nor in water ingestion. These findings show that the effects of OXA can be antagonized by SB334867 whereas SB334867 alone did not alter intakes indicating that the effects of OXA are mediated by OX1Rs in the BST.This work was supported by National Research Fund of Hungary (C012, M036687), and by the HAS. [J Physiol Sci. 2006;56 Suppl:S210]

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In the present experiments homeostatic consequences of orbitofrontal cortical (OBF) interleukin-1β (IL-1β) microinjection were investigated. Food intake (FI), water intake and body temperature (BT) were measured after bilateral OBF microinjection and i.p. injection of IL-1β. Metabolic alterations, namely, blood glucose levels (BGLs), plasma concentrations of insulin, leptin, cholesterol, triglycerides and urate were also determined. Similar to consequences of i.p. administration, short term FI was suppressed, whereas BT was raised remarkably after OBF microinjection of IL-1β. Central application of the cytokine led to a diabetes-like prolonged elevation of BGL, furthermore, plasma levels of insulin and triglycerides were found decreased, whereas that of uric acid increased. In our single unit recording study high proportion of OBF neurons were responsive to the microelectrophoretically applied IL-1β. An overwhelming majority of these neurons were also responsive to microelectrophoretically applied D-glucose, i.e., proved to be the elements of the central glucose-monitoring neural network (GMNN). Our findings, therefore, confirm that neocortically organized IL-1β mediated adaptive reactions through the orbitofrontal cortical GMNN play important roles in the central homeostatic control.Supported by: National Research Fund of Hungary (T 042721, M 036687), the Hungarian Academy of Sciences and Richter Gedeon Pharmaceutical Co., Ltd., Hungary. [J Physiol Sci. 2006;56 Suppl:S210]

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The effects of D-allose, one of the rare sugars, against ischemia injury in rat retina were determined. Retinal ischemia was induced by intraocular pressure to 130mmHg and maintaining that level for 45min. Morphological analysis was performed to study the effects of D-allose on histological changes in the rat retina induced by ischemia. Glutamate release from retina was monitored by using a microdialysis biosensor. Vitreal PO2 was measured oxygen sensitive microelectrode during and after ischemia. Seven days after ischemia, significant reductions in both number of ganglion cells and thickness of the inner plexiform layer were detected. Pretreatment of D-allose significantly inhibited the ischemic injury of the inner retina. A large amount of glutamate was released during ischemia. After recirculation, glutamate levels were increased again and reached a maximum in 20min. This increase of released glutamate was clearly suppressed by the pretreatment of 200mg/kg D- allose. D-allose also attenuated the increase in vitreal PO2 values during repurfusion. These results suggest that D-allose may protect neurons by decreasing extra-cellular glutamate and attenuating oxidative stress in ischemic injury. injury in rat brain. [J Physiol Sci. 2006;56 Suppl:S210]

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As superoxide and hydroxyl radical have been implicated in pathogenesis of Parkinson disease, free radical scavenging and antioxidant have attracted attention as way to prevent progression. We examined effects of eugenol, an essential oil extracted from clove, on 6-hydroxydopamine (6-OHDA)-induced dopamine reduction in mouse striatum. Eugenol administration 3 days before and 7 more days following one intracerebroventricular 6-OHDA injection prevented reductions of striatal DA and its metabolites and increases of thiobarbituric acid-reactive substances, an indicator of lipid peroxidation. Eugenol did not change catalase, glutathione peroxidase, and superoxide dismutase like activities. Eugenol has known to have superoxide and hydroxyl radical scavenging activities in vitro. These results suggest that eugenol prevents 6-OHDA induced DA depression by preventing lipid peroxidation directly. Effects of eugenol treatment in this model suggest possible value in treatment of Parkinson disease. [J Physiol Sci. 2006;56 Suppl:S211]

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It is well established that the phosphorylation of tyrosine hydroxylase (TH) at Ser⁴⁰ is critical in regulating the catalytic activity of the enzyme. However, the influence of the phosphorylation of Ser⁴⁰ on the intracellular stability of TH protein has not been investigated. This study was performed to estimate such a possibility. Although the treatment of rat pheochromocytoma cell line PC-12 cells with forskolin increased the amount of TH phosphorylated at Ser⁴⁰ in the cells, it did not affect the total amount of TH in the cells. Next, human TH type 1 (hTH1) of wild-type and a mutant missing the first 52 amino acid residues were expressed as histidine-tagged forms in PC-12 cells, and then the cells were treated with forskolin. However, the phosphorylation of hTH1 at Ser⁴⁰ did not affect the amount of the wild-type hTH1 protein present in PC-12 cells. Finally, wild-type and a mutant Ser⁴⁰ of which was replaced by Asp (S40D, a mimic of TH phosphorylated at Ser⁴⁰) were expressed in PC-12 cells as histidine-tagged forms or untagged forms. Neither histidine-tagged nor untagged forms showed any difference in their amounts of wild-type hTH1 and S40D hTH1 present in the cells. Collectively, these results indicate the fact that the phosphorylation of Ser⁴⁰ does not affect the stability of TH protein in PC-12 cells. However, we still do not deny the possibility that the successive phosphorylation of Ser¹⁹ and Ser⁴⁰ may affect the intracellular stability of TH protein. [J Physiol Sci. 2006;56 Suppl:S211]

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It has been reported that corticotropin-releasing factor (CRF) is involved in the regulation of norepinephrine neuron responses to stress such as an immobilized stress. Furthermore, systemic lipopolysaccharide (LPS) injection upregulates the transcription of the genes encoding CRF and CRF type 1 receptor in the paraventricular nucleus of the hypothalamus. We have already reported that an increase in norepinephrine turnover within the murine locus coeruleus is accompanied by septic shock triggered by LPS intraperitoneal injection. We also elucidated that the expression levels of the enzymes involved in the catecholamine biosynthesis were altered by peripheral LPS injection. Collectively, the effects of CRF on the expression levels of the enzymes at murine locus coeruleus were investigated by peripherally injecting CP-154,526 (CP-154), a CRF receptor type 1 antagonist. Pretreatment with CP-154 attenuated the increase in expression levels of GTP cyclohydrolase I mRNA due to intraperitoneal LPS injection at 4 h after the injection. However, no effects on the expression level of tyrosine hydroxylase mRNA at the site were observed. Taken together with the fact that LPS injection enhances tetrahydrobiopterin biosynthesis at locus coeruleus, CP-154 may attenuate the increase of NE turnover by way of suppressing the enhanced GCH expression level at the site caused by peripheral LPS injection. [J Physiol Sci. 2006;56 Suppl:S211]

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Asparagine-linked oligosaccharide (N-glycans) on the cell surface undergo dynamic changes during embryogenesis; i.e., in the peripheral tissues, mannose type N-glycan is changed to complex type N-glycan. On the contrary, in the brain, mannose type N-glycan appears to be retained in adulthood. In the last meeting, we have investigated the carbohydrate diversity in embryonic mouse brain and showed the expression of complex type N-glycan on the cell surface of the subventricular zone cells by immunohistochemical analysis. In this study, we performed quantitative analysis of embryonic brain cells by using Fluorescence-activated cell sorting (FACS) system, and revealed that a half of the nestin positive cells in the embryonic mouse brain boar complex type N-glycan. We, then, tried to purificate the neural stem cells (NSC) from mouse brain through complex type N-glycan on surface. As a result, complex type N-glycan positive cells enable to form neurosphere at greater extent (36.7 fold) compared to N-glycan negative cells, and enable to differentiate to neurons and glial cells. These results indicate that unique expression of complex N-glycans are valuable substances for the prospective purification of living NSC. [J Physiol Sci. 2006;56 Suppl:S211]

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Selective vulnerability of late oligodendrocyte progenitors (preOLs: NG2-positive and O4-positive cells) to hypoxic-ischemia (H-I) was reported, explaining the etiology of periventricular leukomalacia (PVL). In rats, preOLs is detected in white matter at postnatal day 2 (P2)-P4. To investigate whether H-I targeting to preOLs caused pathological changes more similar to PVL and whether erythropoietin (EPO) has effect to reduce brain damage by H-I, right common carotid artery occlusion (RCAO) was carried out in P2-P4 rats followed by hypoxic condition (6% 0₂) for various time (0-90 min). RCAO with 6% O₂ for 60 min resulted in high proportion of death: 64% in P2, 50% in P3, and 89% in P4. Histological examination 2 days after H-I revealed that no obvious change was shown in P2 rats. However, typical histological changes of PVL were found in most of surviving P3 rats, suggesting that RCAO followed by 6% 0₂ for 60 min in P3 pups induced histological changes more similar to human PVL in P3 pups. Various dose of EPO (1-30,000 U/kg, i.p.) was treated to animals just before H-I, and the mortality and histological alterations were assessed. With lower concentration of EPO (50-100 U/kg), death rate became to 27-38%, and the damage in cortical and callosal white matter was decreased, indicating that low dose of EPO is protective to cerebral white matter damage in P3 rats. [J Physiol Sci. 2006;56 Suppl:S212]

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Dopamine(DA)ergic differentiation from ES cells was induced by 5 steps in McKay^,s method: stage 1, maintenance of ES cells; stage 2, formation of EBs; stage 3, selection of nestin-positive cells; stage 4, expansion of nestin (+) cells; stage 5, induction to DAergic neurons. To investigate whether physiological low oxygen found in development and cytokines expressed in the DA-depleted striatum increase the production of DA neurons from ES-derived neural progenitor cells (NPCs), NPCs were treated with cytokine mixtures (100 pg/ml IL-1β, 1 ng/ml IL-11, 1 ng/ml LIF, 1 ng/ml GDNF) or lowered O₂ (3.5%) on stage 4 and stage 5, followed by tyrosine hydroxylase (TH) immunostaining. Low oxygen from stage 4 increased total number of TH(+) cells (1.78-fold) and number of TH(+) cells per sphere (1.58-fold of control ) as compared to normal O₂. Cytokine mixture significantly increased TH(+) cells (2.13-fold) compared to non-treated control. IL-1β during stage 4 exhibited major contribution in the effect of cytokine mixtures. Data suggest that physiologically relevant low oxygen in development and cytokines and trophic factors that were enhanced in injured brain cause in increase of DAergic neurons from ES-derived NSCs. [J Physiol Sci. 2006;56 Suppl:S212]

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Persistent inflammation and successive innate immune reaction have been considered to cause Parkinson's disease (PD). Recently, we have found that peripherally injected lipopolysaccharide (LPS) induced tumor necrosis factor (TNF)-α production in astrocytes in the olfactory bulb (OB) and enhanced the number of apoptotic cells in the OB. This finding could be one of the explanations for the olfactory dysfunction observed frequently and early in the patients suffering from PD. Herein, we attempted to clarify the relative roles of TNF-α playing in the induction of programmed cell death after peripheral LPS injection, in which TNF receptor-deficient mice (TNFR^−/−) were used. The intraperitoneal injection of 50 μg LPS into wild-type mice induced the successive increases in mRNA expression levels in the OB of the genes encoding TNF-α and caspase-8. The number of TUNEL-positive cells in the OB increased to a significant level in LPS-injected wild-type mice at 24 h after the LPS injection. On the contrary, LPS (50 μg)-injected TNFR^−/− mice did not reveal any increase in caspase-8 mRNA expression level and the number of apoptotic cells in the OB, despite the significant increase in the TNF-α mRNA expression level at the site. These findings suggest the fact that a crucial role should be assigned to TNF-α in the induction of programmed cell death triggered by peripheral LPS injection. [J Physiol Sci. 2006;56 Suppl:S212]

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Caffeine causes the periodical Ca²⁺ release from the Ca²⁺ store in cultured bullfrog sympathetic ganglion cells. We have already reported that the time course of cytoplasmic Ca²⁺ increment does not reflect faithfully that of Ca²⁺ decrease in the Ca²⁺ store. To understand how this difference arise, the dependency of the amount of Ca²⁺ release from the Ca²⁺ store on caffeine concentration applied was investigated. Dependency of Ca²⁺ increase in cytoplasm on caffeine concentration was different from that of Ca²⁺ decrease in the Ca²⁺ store. At the application of 5mM caffeine, the amount of Ca²⁺ released from the store was half of that at the application of 10mM caffeine, and the amount of cytoplasmic Ca²⁺ increase was about 20%, suggesting that all amount of Ca²⁺ released from the Ca²⁺ store did not cause the cytoplasmic Ca²⁺ increment. [J Physiol Sci. 2006;56 Suppl:S212]

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Regeneration of spinal cord injury is important thema in rehabilitation science as well as basic on. This experience of aim is to recognize the process after spinal cord injury by asphyxia. To establish an animal model of spinal cord injury produced by asphyxia, we used adult rats with aorta occulusion . Fifteen minutes after occulusion electrical reflex activity of apinal cord disappeared. After thirty minutes occulusion , irreversible function changes appeared, long term depression of reflex activities and disorder of motor functions. We traced time course of electrical and functions change after thity minutes occulusion for twelve hours. [J Physiol Sci. 2006;56 Suppl:S213]

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Changes in the chloride homeostasis-related genes in trigeminal nuclei of the adult rats with nerve injuryBing Wei, Atsuo FukudaDepartment of Physiology, Hamamatsu University School of Medicine, Hamamtsu, Shizuoka 431-3192, JapanAbstractAccording the modern pain control theory predicts that a loss of inhibition in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. It involves a trans-synaptic reduction in the expression of the potassium-chloride exporter KCC2. we have previously shown KCC2 mRNA downregulation in injured facial motoneurons, that impairs Cl¯ homeostasis and makes GABA act depolarizing. We report here changes in the expression level of the KCC2 and NKCC1 mRNAs in the trigeminal nuclei of an experimental rat model of trigeminal neuropathic pain produced by a chronic constriction injury to the infraorbital nerve. By means of in situ hybridization histochemistry, our data indicates that 1 to 3 weeks after the operation the KCC2 mRNA was downregulated in the spinal nucleus. The result suggests that the KCC2 downregulation may play pivotal roles in the trigeminal neuralgia by rendering GABA action depolarizing. [J Physiol Sci. 2006;56 Suppl:S213]

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In anesthetized rats, responses of extracellular dopamine release in the nucleus accumbens to innocuous and noxious mechanical cutaneous stimulation of the back were investigated. Coaxial microdialyis probe was stereotaxically implanted in the nucleus accumbens and dialysed with artificial cerebrospinal fluid at a speed of 2 μl/min. Dialysate output from the probe was directly injected into the high liquid chromatograph and the amount of dopamine released from the nucleus accumbens for 5 min was consecutively measured with electro-chemical detector. Innocuous stroking stimulation for 5 min applied to the bilateral back significantly increased dopamine release for 10 min after the onset of stimulation. The increase in dopamine release was observed by stroking of the contralateral, but not iplilateral back. Bilateral noxious pinching stimulation had no effect on dopamine release in the nucleus accumbens. These results indicate that excitation of the innocuous mechanical afferents can stimulate dopamine release without emotional influence. [J Physiol Sci. 2006;56 Suppl:S213]

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Our new project is to detect minimal brain injury in the shaken-baby syndrome. We will take a rat model that was established by Ueda et al (Neuroscience Letters, 385 82-86 2005). A high-spatial resolution image and a diffusion analysis can be useful to detect and follow pathological changes in the brain. It is reasonable to take a higher field to get a better image of small brains of neonatal, infantile, and juvenile rats that are similar size to mouse. In order to establish a basic system in vertical high-field MR micro-imaging system, we had made a probe with the stereotaxic coordinates for mice inside a gradient system. A set of the conventional fixation devices (a bite bar and a pair of ear bars) was installed in an acrylic tube. The position of the bite bar can be adjusted to the level position. 1% enflurane in a gas mixture of 36% O₂-2%CO2-62% NO delivered through the tracheal canula by artificial ventilator. The body temperature was kept in adequate range by a hot water circuit, and the temperature was monitored by fluorescence thermometer. ECG was also monitored by PowerLab. Using this probe, we can get reasonable quality of gradient-echo image, spin-echo diffusion image, and multi-slice multi-spin-echo image in 100 μm pixel resolution with a slice thickness of 0.5 mm. Vital conditions of mice kept constant for 4 hours and longer. We are now trying to detect a minimal hemorrhage in the infant rat brain that was shaken by a shaking machine. [J Physiol Sci. 2006;56 Suppl:S213]

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Microglia are resident macrophages in the central nervous system. In response to injuries or inflammation, microglia are stimulated to exhibit drastic changes in morphology and functions, and called as "activated microglia". Activated microglia proliferate vigorously, migrate and accumulate to the site of the inflammation, and phagocytose pathogens and cellular debris. Further, activated microglia produce various bioactive molecules to assist repair and regeneration of the nervous system. In this report, to elucidate molecular mechanisms of microglia activation, we focused on signaling molecules including small G protein Rac and microglia-specific calcium binding protein Iba1. Activated microglia exhibit extremely active motility and phagocytosis, suggesting the existence of microglia-specific mechanisms regulating the actin cytoskeleton. We previously reported that expression of Iba1 is upregulated during microglia activation, and that Iba1 is involved in motility and phagocytosis of microglia. Furthermore, Iba1 was shown to be cooperating with Rac in regulation of the actin cytoskeleton. Herein, we demonstrated intracellular behavior of these signaling molecules in microglia activation. [J Physiol Sci. 2006;56 Suppl:S214]

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We have previously showed that endogenous vasopressin increases the vasopressin (AVP) and oxytocin (OT) mRNA production in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus after central salt loading in conscious rats. We reached this conclusion by exposing hybridization signals to x-ray films. However, we are also asked to confirm this results by showing the specific signals on the brain region using double labeling procedure (Fos-like immunoreactivity: FLI and mRNA). For this purpose, all hybridized sections were dipped in emulsion (Art emulsion, Fuji film) and then stored at 4°C for 14 days and 28 days for AVP and OT assays after completion of immunostaining of FLI. The distribution of the hybridization signal on the sections were similar to that of FLI in the SON. From these results, we confirmed that central vasopressin affects its own synthesis and OT synthesis in the SON. [J Physiol Sci. 2006;56 Suppl:S214]

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We investigated the structural changes in asparagine-linked sugar chains of rat sciatic nerve glycoproteins during aging and reported previously that high-mannose-type sugar chains were abundant in younger animals, whereas two sialylated/sulfated oligosaccharides, OIBA1 and OIBA2, increased during maturation. We investigated further on the sugar chains increased by aging and found that there were several oligo-sialylated sugar chains (referred to as OIBA-3. OIBA-4, and OIBA-5). The common structure of these sugar chains was determined to be a bi-antennary complex-type oligosaccharide; Galβ1-4GlcNAcβ1-2-Manα1-6(Galβ1-4GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc. A sulfate residue linked to the 6-O-GlcNAc on the C3 antenna. The difference among them was revealed to be sialic acid contents. We analysed the structures of these sugar chains by exo-glycosidase digestion, HPLC analysis, and MALDI-TOF-MS. The oligosaccharide structures of the OIBA familiy were determined as follows; (Sia)_0-3-Galβ1-4GlcNAcβ1-2-Manα1-6((Sia)_0-1-Galβ1-4(SO₄-6)GlcNAcβ1-2Manα1-3)Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc. These data indicated that highly sialylated oligosaccharides increased in peripheral nerve glycoproteins of aged rats. [J Physiol Sci. 2006;56 Suppl:S214]

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During neural development, guidance molecules such as semaphorins and netrin play crucial roles in neuronal network formation by controlling axonal pathfinding. Semaphorin4A (Sema4A), a member of class 4 semaphorin induced growth cone collapse of hippocampal neurons. The binding of Sema4A to the growth cone indicated the presence of a receptor transmitting signals to intracellular effectors to induce growth cone collapse of hippocampal neurons (Yukawa K et al. Int J Mol Med 16: 115-118, 2005). Transfection experiments of receptor candidate genes into Cos7 cells demonstrated that Sema4A could bind to guidance receptors including Plexin-B1, Plexin-B2 and Plexin-B3. To identify functional Sema4A receptor and the signal transduction machinery, we performed Cos7 cell contraction assay where the completion of intracellular signal transmission induced contraction of Cos7 cells. Expression vectors for receptor candidate genes and Rho family GTPase (Rnd1, Rnd2 or Rnd3) were transfected into Cos7 cells. As a result, we confirmed that Plexin-B1 could transmit intracellular signal of Sema4A through Rnd1. Our data also suggested that Plexin-B2 and B3 could transmit intracellular signal of Sema4A through Rho family GTPase different from Rnd1. Concomitantly, our mouse behavioral analyses now screen knockout mice of semaphorins and guidance receptors to get a clue whether the guidance receptor system controls mouse behavior. The screening disclosed a hyperactive behavior of knockout mice of Sema4D, another class 4 semaphorin. [J Physiol Sci. 2006;56 Suppl:S214]

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Leptin promotes fatty acid oxidation in skeletal muscle by activating α2 AMP-kinase (AMPK), directly at the muscle level and via the hypothalamic-sympathetic nerve. We examined the role of AMPK upstream kinase such as LKB1, Atm, and Ca²⁺/calmodulin-dependent protein kinase kinase β (CaMKKβ) in leptin-induced AMPK activation in C2C12 muscle cell line that expresses leptin receptor Ob-Rb. Leptin produced a biphasic activation of α2 but not α1 AMPK in C2C12 cells with an early peak by 1 hr and a late peak by 12-24 hr. Antisense RNA of Atm abolished the 1st peak, while RNAi of CaMKKβ suppressed the late peak. RNAi of LKB1 decreased neither the 1st nor 2nd peak. Consistently, leptin induced expression of CaMKKβ and transcription factor Nur77 from 6 to 24 hr. Nur77 increased CaMKKβ expression via unique NGF1-B (Nur77)-response element. Leptin stimulated Nur77 expression by PI3-kinase (PI3K)/PKCζ pathway, promoting JAK2/IRS1/PI3K complex. Furthermore, leptin increased intracellular Ca²⁺ from 6 to 24 hr through PI3K/PLCγ- and Ca²⁺-channel-activation. Nur77 and CaMKKβ expression decreased in skeletal muscle of leptin-deficient mice (ob/ob). In sum, leptin activates α2 AMPK in C2C12 muscle cells through two distinct mechanisms: the early activation is mediated by Atm and the late activation is due to PI3K/Nur77/CaMKKβ. Significant decrease of Nur77 and CaMKKβ expression in ob/ob mice suggests that the signaling pathway as well as Atm is important for leptin-induced AMPK activation in skeletal muscle. [J Physiol Sci. 2006;56 Suppl:S215]

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Two primary systems are particularly involved in setting on the physiological stress response, HPA and sympathetic-adrenomedullary (SAM) systems. The present study compared the responses of both the HPA and SAM activities to the acute psychological stress between genders. Subjects were selected according to their score in Spielbergers Trait Anxiety Inventory (STAI) to assess the predisposition to personal anxiety, and high (score >55) and low (score <45) anxiety groups were included. The video of corneal surgery was served as the stressor for 15 min. Salivary cortisol and amylase were assayed as indexes of the HPA and SAM activities, respectively. Salivary β-endorphin was also assayed as a possible index of HPA activity. There were no differences of all the resting salivary parameters levels among the groups. As expected, during the stressful video viewing, all the salivary parameters were significantly increased in all groups. There were no differences in amylase levels between high and low anxiety of both genders. However, cortisol and β-endorphin levels of high anxious females were significantly lower than those of high anxious males. Thus, in contrast to the traditional view, high anxious females exhibited lower levels of HPA hormones than high anxious males during stressful video viewing. Our findings suggest that high trait anxiety in females may be associated with an inability to respond to sufficient activation of HPA to acute psychological stress. [J Physiol Sci. 2006;56 Suppl:S215]

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Abnormal cerebellar development of staggerer (sg) mouse has been reported due to the mutation of retinoic acid receptor-related orphan nuclear receptor (ROR)α, a member of the steroid, thyroid, and retinoid receptor superfamily. However, the role of RORα on cerebellar development has not yet been clarified. The appropriate mRNA levels of neurotrophins and their receptors are necessary for development of cerebellum. However, the changes in expressions of these genes in sg mouse cerebellum have not yet been studied. To understand whether neurotrophins and their receptors are involved in cerebellar impairments of sg mouse, we investigated these gene expression profiles during postnatal development by RT-PCR and in situ hybridization. The decreased mRNA levels of BDNF, NT-3 and NT-4 at P15 or P21 and increased mRNA levels of NGF, TrkA and TrkB were shown in homozygous sg mouse compared with those in wild type. Interestingly, the expression patterns of these mRNA in sg mouse are similar to those in hypothyroid animals, suggesting a possible cross-talk between RORα and thyroid hormone receptors. The hybridization signals for BDNF, NT-3 and TrkB mRNAs were mostly located in the granule cells, suggesting the important roles of neurotrophins in granule cells during sg postnatal development. These results indicate that the mutation of RORα alters the expression of neurotrophins and their receptors, which may be partly responsible for abnormal cerebellar development of sg mouse. [J Physiol Sci. 2006;56 Suppl:S215]

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Polychlolinated biphenyls(PCBs) are known as environmental contaminants that cause various effects in many organs including the central nerves system.We have previously reported that PCB5005 (hydroxylated PCB) suppressed Thyroid hormone(TH) receptor (TR)-mediated transcription on several artificial TH-response elements (TREs).In this study, we examined TR-mediated transcription on native TRE. We used malic enzyme (ME) response element contains three TRE half sites. Transcriptional activity of ME was measured by using transient transfection based repotor gene assay in fibroblast-derived clonal cells (CV-1) and neuroblastoma-derived clonal cells (TE671).TR-mediated transcription was suppressed by PCB5005.However PCB6002 did not affect on the transcription.On the other hand, PCB187 induced the TR-mediated transcription congeners.Next, to study the effects of these congeners on endogenous ME gene expression, we applied the reverse-transcriptional polymerase chain reaction (RT-PCR). PCB5005 exposure suppressed the expression of ME mRNA. These results indicate that exposure of PCBs affects TR-mediated transcription on native promoter. [J Physiol Sci. 2006;56 Suppl:S216]

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It has been known that oxytocin receptors are implicated in autism. Autistic subjects have been reported to have lower serum concentrations of oxytocin. Genetic linkage studies and single nucleotide polymorphism analyses for autistic disorder also show positive association of the oxytocin receptor with autism. Furthermore, mice lacking the oxytocin receptor gene show autistic phenotypes, such as socio-behavioral deficits. On the other hand, secretin, a gastrointestinal hormone, has been considered as a possible treatment for autism. However, the relationship between oxytocin and secretin remains to be determined. In the present study, we investigated whether secretin activates oxytocin neurons. We measured plasma oxytocin concentrations after an intracerebroventricular (icv) injection of secretin. The icv injection of secretin caused a dose-dependent increase in plasma oxytocin concentrations. The results indicated that secretin induces the activation of oxytocin neurons, consistent with a hypothesis that the effect of secretin on autism is mediated by oxytocin/oxytocin receptor system. [J Physiol Sci. 2006;56 Suppl:S216]

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The regulation of transcription of target genes by nuclear hormone receptors, such as estrogen receptor (ER) and progesterone receptor (PR), is related to the growth and development of many organs and tumors such as breast cancer. Recently, it has been reported that steroid and xenobiotic receptor (SXR) is expressed in a series of breast cancer cells. It is known that more than 60% of prescribed drugs bind to SXR to induce the transcriptional activities of target genes such as cytochrome P450 (CYP) 3A4. In the present study, to investigate the action of SXR on PR-mediated transcription, we performed transient cotransfection-based reporter assays using African green monkey kidney fibroblast-derived CV-1 cells. SXR activated the PR-mediated transcription through progesterone response element (PRE) in the presence of PR agonist up to 3-fold in a receptor-concentration dependent manner . On the other hand, without PR, SXR did not activate the transcription through PRE. These results indicate that SXR may directly or indirectly interact with liganded PR, but not with PRE in the presence of agonist. These results indicate that SXR may alter the effect of progesterone in breast cancer cells. In conclusion, together with the effect of SXR on ER-mediated transcription, SXR may deteriorate the breast cancer, and may be a useful clinical marker to classify breast cancers. [J Physiol Sci. 2006;56 Suppl:S216]

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GABA is known to produce a depolarization or secretion in adrenal medullary (AM) cells of various species. However, whether the GABAergic system is intrinsic or extrinsic in the adrenal medulla is ambiguous. Thus, we addressed this issue using immunological techniques. The immunoblotting and immunohistochemistry revealed that GAD, a GABA synthesizing enzyme, was present in rat AM cells, but not adrenal cortical cells. VGAT, a vesicular GABA transporter, was also found in the rat adrenal medulla. The fractionation study with bovine adrenal medulla showed that GAD and VGAT were recovered in a crude membrane fraction, but not in a chromaffin granule fraction, suggesting GABA is stored in synaptic-like microvesicles, but not chromaffin granules. Perfusion of a GABA-containing solution in the rat AM cells loaded with Fura-2 resulted in an increase in Ca ²⁺ signal in some, but not all, of AM cells that responded to electrical stimulation. The maximum response of Ca ²⁺ signal evoked by both electrical stimulation and GABA did not differ from that elicited by electrical stimulation alone. This result suggests that GABA did not produce an inhibition of membrane excitability through a shunt effect. The immunoblot showed that GABA receptors in rat AM cells consisted of at least α 1, α 3, and γ 2 subunits. The results suggest that GABA functions as a paracrine or autocrine in rat AM cells [J Physiol Sci. 2006;56 Suppl:S216]

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In adult female rats, estrogen has crucial effects on the estrus cycle, reproduction and sexual behavior. The hypothalamus is the brain area most obviously related to estrogen actions on these physiological events. Electrophysiologically, it was demonstrated that estrogen exerts different actions between the anterior part containing the preoptic area and the posterior part containing ventromedial hypothalamus. Total RNA was prepared from the anterior and the posterior parts of the hypothalamus of 7 week-old ovariectomized rats after the treatment with estrogen or without the treatment. To evaluate the profile of estrogen responsiveness of genes, total RNA was subjected to cDNA microarray analysis. Based on the results of microarray and quantitative real-time PCR analyses, we selected four neuronal system-related genes, acetylcholine receptor α 4, GABA-A receptor δ , serotonin receptor 6 and GABA transporter 2. In the previous meeting, we presented the estrogen-mediated transcriptional regulation of the four selected genes. The examination of the regulation of these genes at the translational level is now in process by Western-blotting and immunohistochemistry. Recently, we obtained results that the expression of the acetylcholine receptor α 4 protein differed depending on the area of the hypothalamus and the time of exposure to estrogen. We present here the recent results and discuss the molecular bases for the site- and time-specific regulation of neuronal system-related genes in the hypothalamus by estrogen. [J Physiol Sci. 2006;56 Suppl:S217]

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In the hypothalamus, there are two sexually dimorphic nuclei, the anteroventral periventricular nucleus (AVPV) and the sexually dimorphic nucleus of the preoptic area (SDN-POA), which have been associated with sex-specific regulation of reproductive neuroendocrinology and behavior. The AVPV is sexually dimorphic with over three times as many dopaminergic neurons in the female rat compared with male. On the other hand, the volume of the SDN-POA is several times smaller in female than in male. Estrogen, derived from circulating testosterone from pup testis, masculinizes the developing AVPV and SDN. However, molecular mechanisms of estrogen signaling in the sexual differentiation of the brain have not been clarified hitherto. In this report, using a customized DNA microarray with selected estrogen-responsive genes (172 genes), we attempted to show the gene cascades to establish the sexual dimorphism in the AVPV and SDN of rat brain. Several genes of the postnatal day 5 (PD5; the day of birth: PD1) female brain were up- or down- regulated, significantly, by masculinization with estrogen treatment on PD1. Six of these genes showed the same expression pattern between the AVPV and SDN. We are currently investigating estrogen-responsive gene expressions in the PD1–PD4 female brains to profile estrogen function. The data would contribute to clarify the molecular mechanisms of estrogen signaling in the sexual differentiation of the brain. [J Physiol Sci. 2006;56 Suppl:S217]

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Gonadotropin-releasing hormone (GnRH) neurons in the hypothalamus constitute the final common path for the neuroendocrine control of reproduction. The activity of GnRH neurons is modulated by cyclic fluctuations in circulating concentration of estrogen. However, the nature of estrogen action upon these cells has not been clarified. We report here a direct action of estrogen on the regulation of potassium current, which is closely related to the neuronal excitability, in GnRH neuronal cell line, GT1-7. Delayed rectifier potassium current (I_K) and calcium-activated voltage-gated potassium (BK) current were recorded by perforated patch clamp configuration in GT1-7 cells cultured in DMEM with 10% fetal bovine serum for 3 days. BK current was increased by addition of 17β-estradiol (E2) in culture medium in a concentration-dependent manner. This action of E2 was completely blocked by ICI-182,780, a potent estrogen receptor antagonist. Acute application of E2 had no effect on the BK current. We further examined the calcium currents whether the effect by E2 on BK currents was mediated through augmentation of calcium currents. E2 had no effect on the calcium currents in GT1-7 cells. E2 exerted no effect on I_K. These results indicate that E2 increases the BK current by activating estrogen receptor without affecting calcium currents. [J Physiol Sci. 2006;56 Suppl:S217]

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Estrogen stimulates cell proliferation in anterior pituitary gland, which is one of the typical estrogen-responsive tissues. Recently, we have found that 17-β estradiol (E2) had estrogen receptor-mediated mitogenic action as well as anti-mitogenic (in presence of IGF-1 or insulin) action on rat lactotroph primary cell culture. However, at present, the cellular and molecular mechanisms underlying the IGF-1- or insulin-dependent anti-mitogenic action of estrogen upon lactotroph remain unknown. In this study, primary cell culture of the anterior pituitary cells was treated for 4 hours with IGF-1 (50 ng/ml) alone or with IGF-1 and E2 (1 nM) in combination. 1 nM E2 was chosen because anti-mitogenic action is typically demonstrated by such concentration of E2. After the treatment, total RNA was isolated from the sample and used for genome-wide gene expression analysis with CodeLink^TM Rat Whole Genome Bioarrays (33,849 gene probes, Amersham Biosciences). Our data analysis revealed that 0.49% (165/33,849) of the gene probes were down-regulated and 0.38% (129/33,849) were up-regulated by E2 in presence of IGF-1. These affected genes turned out to be cell cycle-related genes, growth factors, and signal transducers. Expression analysis by quantitative RT-PCR validated the results obtained by CodeLink Bioarray analysis, and thus our data strongly implied some involvement of the E2-affected genes in the IGF-1-dependent anti-mitogenic action of estrogen on the lactotroph primary cell culture. [J Physiol Sci. 2006;56 Suppl:S217]

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It is well known that the limbic system and the hypothalamo-pituitary-adrenocortical (HPA) axis is activated in response to stressful stimuli in rats. In addition, estrogen enhances the stress responses in the female rat, but it remains unknown how estrogen affects stress responses. In the present study, we examined the possible pathway of estrogen affecting stress reponses. Adult female rats of Wistar-imamichi strain were ovariectomized 2 weeks prior to the experiment. The stainless steel tube containing cholesterol (Chol) or 17β-estradiol (E₂) (Chol : E₂ = 100 : 1 by weight) was stereotaxically implanted in the paraventricular nucleus (PVN) or the amygdala. Three days after the implantation, blood samples (300 μl) were taken every 1 h through an indwelling cardiac catheter from 1100 h to 1600 h to monitor the serum corticosterone (CS) concentration as a marker of HPA axis activity, and restraint stress was applied from 1200 h to 1300 h. The serum CS concentration was assayed by a radioimmunoassay. The serum CS concentration increased at the onset of restraint, and gradually decreased after the release from the restraint in all experimental groups. E₂ implantation in the amygdala caused a significant enhancement of the CS release response during the restaint, comparing Chol. On the other hand, no significant difference was observed between the two groups implanted with E₂ or Chol in the PVN. The implantation did not affect the vaginal smear and the serum E₂ concentration. These results suggest that amygdala is the possible pathway of E₂ enhancing stress responses. [J Physiol Sci. 2006;56 Suppl:S218]

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To confirm a role of prolactin-releasing peptide (PrRP) on stress response, we examined the effects of restraint stress (RTS), nociceptive stimulus and acute inflammatory stress in rats on the expression of the PrRP gene in the hypothalamus and medulla oblongata using in situ hybridization histochemistry. Moreover, we examined the effects of pretreatment with indomethacin on acute inflammation-induced PrRP gene expression and pretreatment with an anti-PrRP antibody on nociceptive stimulus-induced c-fos gene expression in the hypothalamic paraventricular nucleus (PVN). RTS, nociceptive stimulus and acute inflammatory stress upregulated the PrRP gene expression in the medulla oblongata. Acute inflammation-induced increase in the PrRP gene expression was significantly attenuated almost completely by prereatment with indomethacin. Pretreatment with anti-PrRP antibody attenuated significantly nociceptive stimulus-induced c-fos gene expression in the PVN. RTS, nociceptive stimulus and acute inflammatory stress activate PrRP neurons. Especially, activation of PrRP neurons by acute inflammation was mediated mainly by prostaglandins. Pretreatment with anti-PrRP antibody attenuated stress response via neurons in the PVN. These results suggested that PrRP is a potent and important mediator of stress response in the hypothalamic PVN in rats. [J Physiol Sci. 2006;56 Suppl:S218]

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Activated estrogen receptor binds to specific genomic DNA termed estrogen response element (ERE) and enhances the transcription of target genes. There are numerous studies to examine ERE transcriptional activation in cell lines or tumor cells but little is known about primary culture cells especially in the pituitary. To determine the direct transcriptional activity of ERE in primary culture cells, we established the adenovirus-mediated reporter assay system for ERE transcriptional activation in lactotroph cells. 2×ERE, which was originally constructed and the most responsive for estrogenic induction among 1× to 4×ERE, was used for reporter DNA construct. Because the pituitary cell population consists of several cell types including estrogen-responsive gonadotroph cells, Cre-loxP system was used to localize the luciferase gene expression in lactotroph cells. Cre recombinase gene with nuclear localization signal was expressed under the control of prolactin promoter and the luciferase reporter gene was flanked by loxP sites. Double-fluorescence immunocytochemistry revealed that both Cre recombinase and luciferase proteins were expressed specifically in lactotroph cells when pituitary cells in primary culture were infected with two adenovirus vectors carrying these proteins. Treatment with 1 nM estradiol increased the ERE activity about 9-fold in the presence of dextran/charcoal-treated serum. This system will be useful to detect the ERE transcriptional activity in primary lactotroph cells. [J Physiol Sci. 2006;56 Suppl:S218]

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Estrogen receptor (ER) is a key regulator of proliferation and differentiation in breast cancer cells. On the other hand, steroid and xenobiotic receptor (SXR) regulates the expression of the cytochrome P-450 3A (CYP3A) gene family. It has been reported that SXR is expressed in breast cancer cells. To study whether SXR affects on estrogen action, we performed transient transfection-based reporter assays. SXR potentiated ER-mediated transcription in the presence of estradiol in MCF-7 breast cancer cells. To study the mechanism of SXR potentiation on ER-mediated transcription, we performed GST pull down, mammalian two hybrid, and electropholetic mobility shift assays. We showed that (i) SXR did not bind with ER, (ii) SXR did not bind to ERE. Thus, we focused on the effect of SXR on the binding between ER and corepressors. In reporter assays, silencing mediator for retinoid and thyroid receptors (SMRT) reversed the potentiation of ER activity by SXR. The SMRT was dissociated from ER by SXR in a receptor concentration-dependent manner. These results suggest that SXR induced ER-mediated transcription by squelching limiting amounts of SMRT. In conclusion, our results indicated that SXR induces ER signaling, which may play crucial role for cell growth, cell differentiation in breast cancer cells. [J Physiol Sci. 2006;56 Suppl:S218]

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Polychlorinated biphenyls (PCBs) are environmental chemicals that may cause a series of abnormal effects. Due to its stability and lipophilicity, they are accumulated in liver and/or adipose tissues. It has been speculated that PCB may associate with the generation and development of breast cancer. Thus, we focused on the effect of PCBs on estrogen rceptor (ER)- or progesterone receptor (PR)-mediated transcription using transient transfection-based reporter assays. In CV-1 cells, hydroxylated (OH)-PCB activated the estradiol (E2)-induced ER-mediated transcription in a dose dependent manner at concentration from 10⁻¹² to 10⁻⁹ M. However, the transcriptional activity is suppressed at higher concentrations. The magnitude of induction was about 2.5 fold compare to the level without PCB. On the other hand, no significant difference was observed when PR was cotransfected in the presence of OH-PCB. Since steroid and xenobiotic receptor (SXR) potentiated the ER-mediated transcription (Rokutanda et al, personal communication), we also investigated the effect of PCBs on SXR-induced ER-mediated transcription. OH-PCB further activated the transcription in both CV-1 and MCF-7 breast cancer derived cells. These results indicate that PCBs potentiated the ER-mediated transcription in breast cancer cells. In conclusion, PCB may associate with the production and development of breast cancer. [J Physiol Sci. 2006;56 Suppl:S219]

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The present work was carried out to examine the "Escape from High Na⁺ Hypothesis" presented by S. Yoshida for mitochondria, i.e., α-probacteria (the ancestor of mitochondrion with flagella) were so susceptible to high Na⁺ concentration that they escaped into eukaryotic cells and transformed into mitochondria (symbiosis). The movement of sperm, comparable to α-probacteria, was estimated using a phase-contrast microscope and a high-speed video camera designed by T. Etoh; the beat frequency was monitored at 250–500 frames/s. Bathing media always contained glucose, since it is essential for sperm motility. When [Na⁺]_o was raised from normal (140 mM) to 190 mM or to 240 mM, beat frequency of the sperm gradually decreased with time. This effect cannot be ascribed to elevated osmolarity, because adding mannitol to the medium did not introduce any significant changes to sperm motility. In addition, a Na⁺ ionophore (monensin) suppressed sperm movement when it was added to the normal 140 mM Na⁺ solution. In order to observe changes in [Na⁺]_i in response to [Na⁺]_o alteration, Na⁺ dynamics was monitored with a Na⁺ indicator, SBFI, using ARGUS-50 (Hamamatsu Photonics). The fluorescence image analysis showed that [Na⁺]_i increased in the midpiece but not in the head of the sperm when [Na⁺]_o was raised or when monensin was added to the normal solution. In summary, an increase in [Na⁺]_o generates [Na⁺]_i elevation in the mouse sperm, especially in the flagellar portion of the mouse sperm, and it causes a slowdown in sperm activity. [J Physiol Sci. 2006;56 Suppl:S219]

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In breeding season, the sexually mature male newt, Cynops pyrrhogaster, vibrates the tail in front of the female at an early stage of courtship. The effects of intracerebroventricular (ICV) and intraperitoneal (IP) injections of ovine prolactin (PRL), antiserum against newt PRL, and antibody against the newt PRL receptor on the expression of the tail-vibrating behavior of male newts were studied to see whether PRL acts centrally or peripherally to induce the behavior. Both of ICV and IP injections of PRL to gonadotoropin-primed males enhanced the expression of the behavior dose-dependently. The minimum effective dose of PRL was 0.1 μg for ICV injection, whereas it was 100 μg for IP injection. The minimum effective amount of the antiserum was 0.05 μl for ICV injection, whereas it was 20 μl for IP injection. Neither ICV nor IP injection of preimmune rabbit serum affected the expression of the behavior. Furthermore, ICV, but not IP, administration of 0.3 μg of anti-newt PRL receptor antibody blocked the spontaneously occurring courtship behavior in sexually developed males. Neither ICV nor IP injection of the same amount of normal rabbit IgG affected the expression of the behavior. These results strongly suggest that endogenous PRL enhances the behavior by acting centrally through the PRL receptors localized in the brain area. [J Physiol Sci. 2006;56 Suppl:S219]

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