Although it is said that daily exercise habit improves cognitive function, direct evidence on the effect remains unknown. We hypothesized whether 1) ergometer exercise with a moderate intensity could improve cognitive function, and 2) neuronal activation in the frontal cortex played a role in improving cognitive function. To test the hypotheses, we asked 18 subjects to perform a Stroop color-word test (SCWT) before and 5 min after exercise for 15 min at 0% (without exercise), 20%, 40%, and 60% of the maximum exercise. The total time and the number of errors in the SWCT were recorded as an index of cognitive function. To estimate cerebral blood flow in the frontal brain, oxygenated hemoglobin concentration (O₂Hb) was continuously monitored with near-infrared spectroscopy. The total time for SCWT was not influenced by 0% and 20% exercise but shortened by 40% and 60% exercise. The number of errors was not altered by any intensity of exercise. When measuring O₂Hb during 40% exercise, we found a gradual and sustained increase in O₂Hb, which lasted for more than 5 min following exercise. O₂Hb gradually increased in the SCWT and peaked 40-50 s after SCWT onset. Its relative increase of O₂Hb by SCWT was the same before and after exercise. These results indicate that 1) dynamic exercise with a moderate intensity improves cognitive function and 2) the changes of cerebral oxygen metabolism in the frontal cortex may be in association with improved cognitive function. [J Physiol Sci. 2007;57 Suppl:S166]

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Neurogenesis, which occurs not only in the developing brain but also in restricted regions in the adult brain including the forebrain subventricular zone (SVZ), is regulated by a variety of environmental factors, extracellular signals, and intracellular signal transduction pathways. We investigated whether the cAMP/PKA/cAMP response element(CRE)-binding protein (CREB) signaling pathway is involved in the regulation of cell proliferation of neural stem cells (NSCs) isolated from SVZ of adult mice. Treatment of NSCs with the PKA inhibitor H89 and KT5720 inhibited epidermal growth factor (EGF)-stimulated NSC proliferation. Similar inhibition was also observed when a dominant-negative mutant of CREB was expressed by adenovirus vector infection. EGF treatment increased CRE-mediated transcriptional activity, but this increase was much less than that by treatment with forskolin, an adenylate cyclase activator. Forskolin treatment changed neither basal nor EGF-stimulated proliferation. The PKA inhibitors blocked neither EGF-induced phosphorylation of extracellular signal-regulated kinase (ERK), a protein kinase mediating the EGF mitogenic action, nor nuclear translocation of phosphorylated ERK. These results suggest that basal activity of the cAMP/PKA/CREB pathway is required for the mitogenic signaling of EGF in NSCs at a step downstream of ERK activation. [J Physiol Sci. 2007;57 Suppl:S166]

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We developed a simple learning and memory test on the basis of repetitive counting locomotion activity and rearing for one hour in an open field. Here we tested whether the causal relationship between brain chemical elements and administrated Pb-induced behavioral changes can be detected. Correlation analysis was applied for 4320 pairs; 6 brain areas that are reportedly involved in learning, memory, and emotion (prefrontal cortex, nucleus accumbens, amygdala, dorsal hippocampus, ventral hippocampus, and cerebellum punched or cut out from brain slices) x (15 chemical elements [Mg, Al, P, Ca, Mn, Fe, Co, Ni, Cu, Zn, Rb, Sr, Ag, Ba, and Pb] measured by Inductively Coupled Plasma Mass Spectroscopy) x behavioral data (locomotor activity and rearing). We detected the significance of brain area-specific positive and negative correlations between chemical elements themselves then between chemical elements and the behavioral indexes of our developed method, which seems to be practical and useful for the initial behavioral test of learning and memory regarding developmental neurotoxicology. [J Physiol Sci. 2007;57 Suppl:S166]

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Karuta is a traditional Japanese card game, in which the players compete with their opponents to find the cards with the latter half of the 'Tanka,' which the reader reads out aloud. Players need excellent memory and quick movement for getting a card. We recorded the event-related potential P300 and near-infrared optical topography of Karuta players during an auditory oddball task which is similar to Karuta, in an attempt to investigate the information processing in the brain of Karuta players. The subjects were 27 university students who were Karuta players, and the control group consisted of 12 university students who were not Karuta players. We used Neuropack μ(Nihon Koden) to record the event-related potential and ETG-100 (Hitachi Medico) to measure the cerebral blood volume. The followings were observed in Karuta players. (1)The response time of the event-related potential was shorter than the latency of P300. (2)P300 appeared 300msec after the target stimuli. (3)A large positive wave was also observed 300msec after non-target stimuli. (4)Cerebral blood volume was markedly increased on both sides in the beginner of Karuta players. In middle-level players, the cerebral blood flow on the left side was slightly reduced, although that on the right side remained high. In the expert players, the cerebral blood flow on both sides was significantly. In conclusion, Karuta training may increase the active memory area and may cause cerebral blood volume changes. [J Physiol Sci. 2007;57 Suppl:S167]

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To investigate the involvements of the rat hippocampus (Hip) and prefrontal cortex (PFC) in inhibitory control of behavior, we recorded local field potentials (LFPs) from the Hip and PFC during an operant discrimination task and analyzed the LFPs during stimulus presentation for no-reward (S-). Rats received the discrimination training using light and sound as the discrimination stimuli (one for S+, and the other for S-) until a stable discriminative performance was achieved. After that, recording electrodes were implanted into Hip and PFC, and we recorded the LFPs during the discrimination task. Obtained data were filtered with band-pass filters of 8-16, 16-32, 32-64 and 64-128Hz, and those activities were analyzed with time resolution of 1 ms. The Hip 8-16Hz LFP activities increased gradually across time during S- presentation, and the activities of 64-128Hz LFPs increased during S- presentation in both Hip and PFC. These results suggest that Hip and PFC higher gamma-band activities relate to inhibitory control of behavior. Given the involvement of PFC in inhibitory control of behavior, PFC higher gamma-band activities may reflect some inhibitory function and may cause the increase of Hip higher gamma-band activities. [J Physiol Sci. 2007;57 Suppl:S167]

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To investigate whether local field potentials (LFPs) reflect learning and memory, we recorded LFPs from the rat hippocampus (Hip) and prefrontal cortex (PFC) during reversal discrimination task. Rats received a discrimination training using high and low frequency sound as the discrimination stimuli. One was for reward (S+), and the other was for no-reward (S-). The training was continued until a stable discriminative performance was achieved. Then, recording electrodes (tungsten, differentially recording) were implanted into Hip and PFC. After the recovery, each rat received the reversal discrimination training and the LFPs were recorded during the training. In this study, we analyze 2 types of event triggered averaging in which the triggered points were onsets of discrimination stimuli, and lever-press responses, respectively. We could not find a significant learning-related change in the discrimination stimulus-triggered averaging, one of commonly used analyses. On the other hand, the lever-press response-triggered averaging revealed negative components immediately before lever-press responses. The amplitudes changed with learning sessions in both Hip and PFC. These results suggest that some learning related effects are reflected in LFPs, at least in Hip and PFC in this learning. [J Physiol Sci. 2007;57 Suppl:S167]

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In rat primary somatosensory cortex, neurons which dominantly respond to a same whisker (principal whisker) cluster and form a cortical column. Here, we performed in vivo two-photon calcium imaging to investigate whether spontaneous activities were synchronized among them and how neurons with different direction selectivities are spatially arranged within a single column. We loaded a region of the cortex (250-300 μm in diameter) in layer II with a calcium indicator in anesthetized or sedated and immobilized rats. We recorded spontaneous and stimulus-evoked fluorescent changes from a population of single neurons simultaneously. Additionally, we recorded action potentials of single neurons juxta-cellularly. We deflected the principal whisker in the rostral-to-caudal and caudal-to-rostral directions. Fluorescent changes recorded from soma correlated occurrences of action potentials of the recorded cells in almost one-to-one manner. Spontaneous activities were usually not synchronized among neurons. Once a while, however, a large proportion of neurons synchronized their spontaneous activities. The results suggest that there were two states of spontaneous activities in layer II. About 60% of dye-loaded cells (includin astrocytes) responded to whisker deflection. The direction preference of the neurons changed gradually across a column in some cases, while it did not systematically change in the other cases. [J Physiol Sci. 2007;57 Suppl:S167]

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Characteristics of human stereoscopic-depth discrimination depend on temporal properties of visual stimuli such as presentation duration and temporal frequency. The human stereo system may consist of different sub-mechanisms, each processing visual inputs with different temporal properties. Here we test this possibility by considering recent physiological findings that neurons in the dorsal visual pathway compute the disparity energy whereas neurons in the ventral pathway compute the global-match solution to the stereo correspondence problem. Subjects were required to report depth embedded in dynamic random-dot stereograms (RDSs). We manipulated the refresh rate of dot patterns of RDSs across blocks of trials. In a single block, the sign of disparity and the ratio of binocularly correlated and anti-correlated dots were altered to examine the underlying computation. In trial blocks with fast refresh rate, subjects perceived reversed depth for anti-correlated RDSs despite the lack of global match. When correlated and anti-correlated dots nearly match in proportion, subjects performed at chance. In trial blocks with slow refresh rate, reversed depth perception for anti-correlated RDSs was weak. However, correct depth was perceived near the matching point at which no depth was perceived in the fast condition. These results suggest that the disparity energy computation accounts for stereoscopic depth perception when monocular images change rapidly in time, whereas the stereo correspondence computation underlies depth perception for slowly changing images. [J Physiol Sci. 2007;57 Suppl:S168]

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Mere exposure effect was first proposed by Zajonc (1968) as a phenomenon that "the repeated exposure of a stimulus object to an individual seems to enhance his attraction to the object". We tried to measure brain activity when MEE is occurring by comparing BOLD signal change when subjects made a choice according to preference between 2 stimuli; one was pre-exposed (subliminal exposure) to subjects and the other was novel. The preference ratio of this choice (preference judgment: PJ) was 57.8±12.7% for pre-exposed figures and higher than chance level (p=.03). As a control, subjects made a choice depending on whether they remembered stimuli (memory judgment: MJ).The different brain regions were activated in PJ and MJ. In PJ, bilateral insular cortices were activated in addition to a part of prefrontal and parietal cortices. In MJ, parts of occipital, parietal, prefrontal cortical areas and left insular cortex were activated. The right prefrontal region (inferior frontal gyrus) showed higher activity during PJ than MJ, and this region is supposed to be responsible for "the judgment to choose preferred stimuli". In contrast, a region that showed higher activity when subjects chose pre-exposed stimuli rather than novel stimuli was right insula. This region may be related to attraction to pre-exposed stimuli. [J Physiol Sci. 2007;57 Suppl:S168]

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The prefrontal cortex has been shown to be involved in the regulation of emotions. It has been also reported that the prefrontal cortex related to the positive mood in mothers viewing pictures of their newborn infants. This study sought to determine the influence of sex and parental status on the prefrontal activity elicited by discrimination tasks of infant facial expressions. We measured NIRS activity of the prefrontal cortex in adults (male group, non-parent female group and mother group) when they see pictures on screen to judge a) the kind of pictures, b) the sex of adults, c) the emotion of adult facial expressions, d) the sex of infants, e) the emotion of infant facial expressions. This study was approved by the ethics committee of Nagasaki University. When subjects judge the emotion of infant, the concentration of delta oxygenated hemoglobin (oxyHb) was significantly increased in the right prefrontal cortex only in mother group compared with male and non-parent female groups (One way ANOVA followed by bonferoni / Dunn post hoc test; p<0.05). However, the left prefrontal cortex activity was not increased in any groups. No significant differences were found among the groups in the other discrimination tasks. These results suggest that the right frontal cortex is involved in maternal attachment behaviors. [J Physiol Sci. 2007;57 Suppl:S168]

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One of the most prevailing views on the functional localization of human cognition is the functional hemispheric asymmetry, wherein the left hemisphere is implicated primarily in verbal processing whereas the right hemisphere is implicated primarily in nonverbal processing. Inhibitory control has been known to be involved in the lateral prefrontal cortex, particularly in the posterior inferior frontal gyrus (IFG). However, it remains unclear what factor determines the hemispheric dominance of the activation in the IFG during inhibitory control. In this fMRI study we tested whether the hemispheric asymmetry principle applies to the IFG activation during inhibitory control. A flanker task, one of the major tasks used to examine inhibitory control, was devised that allowed us to manipulate the modality of the presented flanker stimulus (color word or colored patch) from which interference effects originate, keeping the total stimulus modality effects balanced. Consistent with previous studies of the flanker task, the posterior part of the IFG was prominently activated. Although the dorsal frontal areas showed prominent hemisphere-by-modality interaction in simple visual effects, the IFG never showed the hemisphere-by-modality interaction in inhibitory control of flanker interference. These results suggest that the left and right IFG activation during inhibitory control is independent of the modality of presented stimulus from which the interference effects originate. [J Physiol Sci. 2007;57 Suppl:S168]

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To investigate neural mechanisms underlying behavioral rule-shift, we trained animals to perform a path-planning task, which required planning of actions in multiple steps of cursor movements to reach a goal by operating manipulanda with either arm. To dissociate the movements of the arms and cursor, we trained the monkeys to perform the task with three different arm-cursor assignments. The assignment was changed without instructions, and the monkeys were required to adapt to new assignments with trial and errors. We recorded neuronal activity from the anterior cingulated motor area, while animals performed the path-planning task. We found neurons that were selectively activated when the assignment was changed. The present findings suggest that the Anterior cingulated cortex is involved in monitoring outcomes of action and updating behavioral rule to be followed. [J Physiol Sci. 2007;57 Suppl:S169]

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Recent technical advance can allow us to measure cortical activations of children non-invasively in daily life situation. Reading to children, Yomikikase is now thought to be a powerful method for parents to communicate with their children even in infancy. In the field of developmental psychology, there are studies for effects of Yomikikase such as developing abilities to listen and understand, growing an ability to image from language, facilitating communication between book reader and its listener etc. In this study, we measured cortical blood flow (CBF) of both mother and child during Yomikikase by fNIRS, and investigated the effects of Yomikikase from neural aspects. Eight mothers and of their fourteen children were participated. Written informed consents were obtained. We measured the frontal CBF by the fNIRS (OMM-3000, Shimadu). When we compared the frontal CBF between Yomikikase and simple book reading, in 6 mothers, the frontal CBFs were greater during Yomikikase. However, in 6 children, the frontal CBFs were decreased during Yomikikase. We further tested several control tasks and will discuss the meaning of the decrease in frontal CBF during Yomikikae in children. [J Physiol Sci. 2007;57 Suppl:S169]

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The hippocampus is important for declarative memory in humans. Long-term potentiation (LTP) is a form of synaptic plasticity, which has been studied extensively in the rodent hippocampus. However, information about LTP in the primate hippocampus is limited, which is primarily due to difficulties in the precise implantation of electrodes within deep structures of the primate brain. We have been developed a technique to record evoked potentials in the monkey hippocampus, overcoming the difficulty by combination of MRI with stereotaxic procedure. Using this model, we investigated the induction and maintenance of hippocampal LTP in monkeys and compared its properties with those of rats. Stimulation and recording electrodes were implanted in the perforant pathway and dentate gyrus, respectively. The effects of high-frequency stimulation (HFS, 400 Hz, 20 pulses x 20) on evoked potentials were tested in an awake condition. As results, HFS reliably induced LTP in the hippocampus of both species. LTP was maintained stably for more than 4 weeks in monkeys, whereas it declined to the basal level within a week in rats. These data suggest that 1) the present experimental model of evoked potential recording is suitable for testing long-term synaptic plasticity, and 2) synaptic plasticity in the hippocampus is more stably maintained in monkeys than in rats, which could be involved in the specific difference of duration for which temporary graded retrograde amnesia is observed. [J Physiol Sci. 2007;57 Suppl:S169]

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Tool-use represents an origin of human intelligence. Besides only non-human primates and corvids have been known to posess its rudimentary capacity, we have recently proved, for the first time, tool-use in rodents -the degu (Octodon degu)-that they can be trained to use a rake-like tool to retrieve a reward. Hippocampal dentate gyrus is known to generate new neurons throughout adulthood, which exhibit morphology and physiological properties of established neurons. Such neurogenesis is particularly well studied in rodents to be enhanced during learning and memory processes. To elucidate cellular mechanisms of tool-use learning, we examined the effects of training to use a tool on the adult neurogenesis in the hippocampal dentate gyrus using this novel rodent model.We found significantly more numbers of new neurons in tool-use trained, compared to sham-trained, group indicating that tool-use learning enhances adult dentate neurogenesis. [J Physiol Sci. 2007;57 Suppl:S169]

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The sex difference in emotional response to stress suggests a sex-specific stress response in the amygdala. To examine the sex difference in serotonin and dopamine release response to stress in the basolateral amygdala (BLA), in vivo microdialysis studies were performed in male and female rats. In experiment I, dialysates were collected from the BLA at 15-min intervals under the freely moving condition. Mean serotonin or dopamine release in the BLA were higher in male rats than in female rats. In experiment II, rats were subjected to restraint stress for 60 min to examine the stress response of serotonin or dopamine release. Although restraint stress significantly increased serotonin release in both sexes of rats, female rats showed a greater response than male rats. Moreover, restraint stress significantly increased dopamine release in female rats, but not in male rats. In experiment III, rats were subjected to restraint stress for 30 min to examine behavioral responses. Although no sex difference was observed in the number of audible vocalizations, male rats defecated a larger number of fecal pellets than female rats. In experiment IV, rats were tested for freezing behavior to examine contextual fear responses. Conditioned male rats showed a longer freezing time than conditioned female rats. We found sex differences in the serotonin and dopamine release in the BLA and their responses to restraint stress, which may be involved in the sex-specific emotional response to stress in rats. [J Physiol Sci. 2007;57 Suppl:S170]

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Oxytocin is known as a peptide hormone that causes uterine contruction and facilitates the delivery of the newborn, and stimulates the milk ejection during motherhood. Abundant oxytocin receptors also appear in central nervous system (CNS), especially in limbic system. Oxytocin is involved in the regulation of social behaviors, including maternal care, affiliation and social behaviors in CNS. Moreover, recent studies have shown that oxytocin has anti-anxietic effect during motherhood. However, the exact molecular mechanism is unclear. In the present study, we identified a crucial molecule involved in the regulation of anti-anxiety by oxytocin and regulated the expression by oxytocin. Oxytocin induced the expression of regulator of G-protein signaling2 (RGS2), which is known as a modulator of anxiety, in the amygdala slices of female mice. RGS2 expression in the amygdala of lactating mice was significantly higher than that of virgin mice. Moreover, the expression of RGS2 in virgin mice undergone restraint stress was facilitated compared with that of control mice. These results suggest that oxytocin may downregulate anxiety through the induction of the expression of RGS2 gene. [J Physiol Sci. 2007;57 Suppl:S170]

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The subfornical organ (SFO) is a circumventricular structure and related to drinking behavior. The nucleus can be affected directly by blood-born peptides, because of lacking the blood-brain-barrier. Peripheral and/or central administrations of ghrelin induce food intake while those of amyline inhibit it. Recently, it has been reported that ghrelin and amylin influence separate subpopulations of SFO neurons using patch-clamp recording at dissociated cells. In the present study, we investigated effects of both peptides on spontaneous electrical activity of SFO neurons at slice preparations using multi-electrodes extracellular recording system (MEA system). Bath application of amylin at 100 nM excited 75% of SFO neurons tested. On the other hand, that of ghrelin at 100 or 300 nM did not only excite in 25% of SFO neurons, but also inhibited in 25% of SFO neurons. Similar to the results in a previous study, amylin-sensitive SFO neurons did not show ghrelin-induced excitability. Interestingly, some of them showed ghrelin-induced inhibition. These results suggest that although SFO neurons are excited by either ghrelin or amylin, some amylin-sensitive SFO neurons are inhibited by ghrelin, at least, at slice preparation level. [J Physiol Sci. 2007;57 Suppl:S170]

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We have recently reported that galanin inhibits activation of angiotensin II (ANG)-sensitive neurons in the subfornical organ. Since the nucleus is related to ANG-induced drinking behavior and cardiovascular response, galanin may inhibit the physiological responses. In the present syudy, we investigated the effect of galanin on ANG-induced physiological responses in conscious rats. The increment of water intake by intracerebroventricular injection of ANG at 100 pmol was significantly suppressed by co-injection of galanin over 25 pmol, but those by cholinergic stimulations were not. On the other hand, the intracerebroventricular injection of galanin alone from 10 pmol to 5 nmol did not affect drinking behavior. However, concerneing to cardiovascular response, the intracerebroventricular injection of galanin alone induced transient increments of mean blood pressure and heart rate. The intracerebroventricular injection of ANG at 100 pmol increased long-lastingly mean blood pressure with slightly decrease of heart rate. By co-injection of galanin at 50 pmol with ANG suppressed significantly long-lasting phase of the ANG-induced increase of blood pressure. These results indicate that galanin inhibits ANG-induced physiological responses in the brain in conscious rats. [J Physiol Sci. 2007;57 Suppl:S171]

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It is known that estrogen and estrogen receptor (ER) are related to progression and differentiation of breast cancer. Steroid and xenobiotic receptor (SXR) regulates target genes including Cytochrome P450 monooxygenase 3A4 (CYP3A4). Recently, it has been reported that SXR is expressed in breast cancer cells. In addition, we reported that SXR augments the ERα-mediated transcription. Polychlorinated biphenyls (PCBs) have been known as environmental chemicals that cause various effects on endocrine system or associate with the production of carcinomas. Using transient transfection-based reporter gene assays, we showed that PCB augmented the SXR-mediated transcription in MCF7 breast cancer cells but not in CV-1 cells. ERα did not affect the SXR-mediated transcription in CV-1cells. Combination of PCB and SXR additively induced the ERα-mediated transcription. Cofactors including steroid receptor coactivator-1 (SRC-1), SRC-3, silencing mediator for retinoid and thyroid hormone receptors (SMRT) and nuclear receptor corepressor (N-CoR) were excluded from the alternation of cofactor bindings to SXR using a series of mammalian two-hybrid assays. These results indicate that unknown factors in MCF7 cells may be required to the augmentation by PCB. Induction by PCBs may result in disruption of drug metabolism, in addition to native endocrine systems. [J Physiol Sci. 2007;57 Suppl:S171]

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Transcriptional regulation by estrogen receptor (ER) and progesterone receptor (PR) are related to proliferation and differentiation of breast cancer. On the other hand, it is speculated that polychlorinated biphenyl (PCB) is related to progression of breast cancer. In the present study, we analyzed the effect of PCB on the regulation of PR-mediated transcription. In MCF-7 cells, low dose of PCB activated PR/ progesterone response element (PRE) transcription. However, at higher concentrations, the transcriptional activities were gradually decreased to the level activated by agonist. In CV-1 cells, PCB suppressed PR/PRE transcription at higher concentrations. These results suggest that the nuclear factor(s) in MCF-7 cells may be related to the augmentation by PCB in MCF-7 cells. We cotransfected ER&alpha with PR/PRE into CV-1 cells. The transcription was slightly suppressed by PCB, suggesting that the effect of ER was excluded from the augmentation by PCB at lower concentrations. We confirmed that PCB that we used in the present study did not affect the cell death. To analyze the effect of PCB on bindings of cofactors to PR, we are currently performing a series of mammalian two-hybrid assays. In addition, we analyzed the effect of PCB on the proliferation of MCF-7 cells. The number of cells was induced by PCB. In conclusion, PCB may disrupt PR-mediated transcription in breast cancer cells. [J Physiol Sci. 2007;57 Suppl:S171]

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Polychrolinated biphenyls (PCBs) and dioxins (PCDD, PCDF, coplaner-PCB) are known as the environmental chemicals that may cause adverse effects of many organs including developing brain. WHO determined toxic index of dioxins as toxicity equivalent factor (TEF). We have previously reported that the magnitude of suppression of thyroid hormone (TH) receptor (TR)-mediated transcription by environmental chemicals does not always correlate to their TEFs. The magnitude of suppression by each congener was not correlated with the degree of dissociation. These findings have let us hypothesize that environmental chemicals may act on DNA binding domain of TR.In this study, we have investigated which domain of TR is responsible for such action of dioxins and PCBs. For this purpose, we performed transient cotransfection experiments using several chimeric proteins constructed by combination of glucocorticoid and TH receptors. EDCs suppressed the transactivation mediated by chimeric receptors that contain DNA binding domain (DBD) of TR. We also performed electrophoretic mobility shift assay (EMSA) with chimeric receptors and obtained that PCBs dissociated chimeric receptors containing DBD of TR from HRE. These results suggest that PCBs may affect TR-mediated transcription through DBD. [J Physiol Sci. 2007;57 Suppl:S171]

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Hypothalamic hormones including dopamine regulate critical functions of pituitary cells via the cyclic AMP-protein kinase A (PKA) pathway. The PKA-downstream transcription factor cAMP response element-binding protein (CREB) is an integrating molecule activated by many other protein kinase pathways. We investigated the involvement of CREB in the regulation of cell proliferation and the PRL promoter of rat lactotrophs in primary cell culture. Adenoviruses were used for efficient gene delivery into pituitary cells. Bromocriptine, a dopaminergic agonist known to decrease intracellular cAMP concentrations, caused inhibition of PRL promoter activity and lactotroph proliferation, which was accompanied by decreases in CRE-mediated transcription and CREB phosphorylation in lactotrophs. Expression of a dominant-negative CREB (MCREB), which was effective in suppressing CRE-mediated transcription induced by forskolin, inhibited basal and forskolin-induced PRL promoter activity. MCREB expression lowered basal proliferative levels and blocked forskolin-induced proliferation of lactotrophs. IGF-1 did not affect intracellular cAMP concentrations but transiently increased lactotroph CREB phosphorylation. MCREB expression also inhibited IGF-1-induced lactotroph proliferation. These results suggest that CREB is involved in the regulation of cell proliferation and the PRL promoter in normal lactotrophs. [J Physiol Sci. 2007;57 Suppl:S172]

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We have previously demonstrated that GABA system (GABA_A receptors, GABA synthesizing enzyme, and vesicular GABA transporter) existed in rat adrenal medullae (AM) at mRNA and protein levels but not in adrenal cortical (AC) cells, and have conclude GABA may function as a paracrine or autocrine in AM cells. In the present study, we examined vesicles of bovine AM cells immunologically and morphologically with the sucrose density gradient and electron microscopy to identify which organelle contains GABA. The fractionation study showed that vesicular GABA transporter (VGAT), which was a necessary enzyme to accumulate GABA in vesicles from cytosol, was found mainly in the low density fractions (10 and 15%), while DβH, a marker protein of chromaffin granules, was presented in both low (10–25%) and high density fractions (50–65%) but not in the middle density fractions (30–45%) where synaptophysin, a marker protein of synaptic-like vesicles, was enriched. Catecholamine distribution over the sucrose gradient was nearly consistent with DβH distribution. EM study demonstrated that there was a difference in the size distribution of vesicles in different fractions. Fifteen percent and 60% fractions mainly contained granules with a median of diameter of 210 nm and 237 nm respectively while a 25% fraction contained synaptic-like vesicles with 189 nm. These results suggest that chromaffin granules are heterogeneous and GABA may be stored in chromaffin granules with a low density, but not in synaptic-like vesicles. [J Physiol Sci. 2007;57 Suppl:S172]

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Galanin-like peptide (GALP) is a 60 amino-acid peptide, and the GALP mRNA is restricted to neurons in the hypothalamic arcuate nucleus (Arc) and pituicytes in the posterior pituitary gland (PP). Chronic osmotic stimulus caused by dehydration and salt loading induced the GALP gene expression in the PP of rats, but it remains unknown whether acute osmotic stimulus such as intraperitoneal (i.p.) injection of hypertonic saline induces the GALP gene expression, and the time-course pattern of the GALP gene expression after acute osmotic stimulus. We examined the dose-response (2% BW, 2.8, 4.5, 6.0 and 9.0% NaCl) and time-course (6.0% NaCl, 1, 3, 6, 12 and 24 h) effects of acute osmotic stimulus on GALP mRNA levels in the Arc and PP of rats, using in situ hybridization histochemistry. After i.p. injection of hypertonic saline (6.0%), plasma osmolality increased significantly at 1 h, and plasma sodium concentration also increased significantly at 1 and 3 h. The GALP mRNA level in the PP increased significantly 6 h after i.p. injection of hypertonic saline (6.0 and 9.0%), but that in the Arc did not change. The GALP mRNA level in the PP also increased significantly 3 and 6 h after i.p. injection of hypertonic saline (6.0%). These results showed that the GALP gene expression in the PP was inducted 3 h after acute osmotic stimulus, and the gene expression might be regulated by plasma osmolality and/or plasma sodium concentration. [J Physiol Sci. 2007;57 Suppl:S172]

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Salusin-α, and -β were newly discovered as bioactive endogenous peptides (Shichiri et al., 2003). Salusin-β stimulates the secretion of arginine vasopressin (AVP) from perifused rat hypophysis, and coexists with AVP in the rat hypothalamic magnocellular supraoptic (SON) and paraventricular nuclei. In the present study, we investigated the effects of osmotic stimuli on salusin-β-like immunoreactivity (LI) in the rat hypothalamo-neurohypophyseal system, and also examined the effects of salusin-β on the synaptic inputs to the rat magnocellular neurosecretory cells (MNCs) of the SON and the AVP / oxytocin (OXT) release from the rat freshly dissociated SON and neurohypophysis. We demonstrated that salusin-β-LI exhibited markedly increase in the hypothalamo-neurohypophyseal system after osmotic stimuli, and evoked AVP / OXT releases from the neurohypophysis, but not cause significant changes in the excitatory and inhibitory postsynaptic currents of the MNCs. These results suggest that salusin-β is increased by osmotic stimuli, secreted from nerve endings, acting as autocrine / paracrine modulator to stimulate the AVP / OXT release from the neurohypophysis and involved in the regulation of water balance. [J Physiol Sci. 2007;57 Suppl:S172]

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We developed enhanced green fluorescent protein (eGFP) monitoring system which is able to measure eGFP that appeared to the living cell by the optical fiber, continuously. This system irradiates the excitation light to the neuron and the nerve ending where eGFP exists and the optical fluorescent signal is converted into the electric signal. In this study, using this system, we continuously measured change of fluorescence in arginine vasopressin (AVP)-eGFP transgenic rat posterior pituitary (PP) with several physiological stimulations. High potassium solution, salusinβ, and galanin-like peptide (GALP) showed a marked decrease of fluorescent signal that suggested AVP secretion from the nerve endings in the PP. These results suggest that this monitoring system has the possibility that AVP secretion can be indirectly monitored, as index the change of fluorescence. [J Physiol Sci. 2007;57 Suppl:S173]

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Adrenomedullin 2 (AM2)/intermedin belongs to the super family of AM and calcitonin gene-related peptide (CGRP). Centrally administered AM and AM2 activated hypothalamic oxytocin (OXT)-secreting neurons and increased plasma OXT level in rats. We examined the effects of centrally administered AM2 on neuroendocrine and autonomic nervous functions in comparison with those of AM in conscious rats. Plasma concentrations of OXT were significantly increased 10 and 30 min after central administration of AM and AM2. The effects of centrally administered AM2 were more potent stronger than those of AM. After pretreatmen of the CGRP receptor antagonist and AM receptor antagonist, the effects of centrally administered AM were completely inhibited on plasma OXT levels and on the c-fos gene expression in the supraoptic and paraventricular nuclei of the hypothalamus, whereas those of AM2 were incompletely inhibited. The mean arterial pressure (MAP) and heart rate were increased potently for an extended period after icv injection of AM and AM2 in urethane-anesthetized rats. The effects of AM2 were more potently stronger and long-lasting than those of AM on MAP. These data suggested that AM2 would have unknown receptor except CGRP and AM receptor in the central nervous system. [J Physiol Sci. 2007;57 Suppl:S173]

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We have previously shown that voltage-dependent L-type Ca²⁺ channels provide a pathway for extracellular Ca²⁺ entry in cultured parathyroid cells isolated from patients with secondary hyperparathyroidism. We then asked whether normal parathyroid cells exhibit L-type Ca²⁺ channel activity. The normal parathyroid cells, loaded with either fluo-3AM or fluo-4AM, were bathed in Hepes-buffered solution containing 1.5 or 2.0 mM Ca²⁺. Fluorescence signal was detected with a Nipkow-type confocal microscopy system. Pressure-application (10 s) of the solution containing either 150 mM K⁺ or 2.5-3.0 mM Ca²⁺ evoked a transient increase in fluo-3 or fluo-4 fluorescence (Ca²⁺ transient). In nominally Ca²⁺-free solution, the high-K⁺ solution failed to evoke the Ca²⁺ transient. In the presence of 10 μM nitrendipine or nicardipine, both high-K⁺-induced Ca²⁺ transients and high-Ca²⁺-induced Ca²⁺ transients were strongly inhibited. Similar inhibitory effects were observed in the presence of 0.2-0.5 mM Cd²⁺. In contrast, FPL64176, an L-type Ca²⁺ channel agonist, significantly enhanced high-K⁺-induced Ca²⁺ transients in 1.2-1.5 mM extracellular Ca²⁺. These results suggest that L-type Ca²⁺ channels provide a pathway for extracellular Ca²⁺ entry in hyperplasic and normal parathyroid cells. [J Physiol Sci. 2007;57 Suppl:S173]

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There is circumstantial evidence in relationship between exercise strength and endorphin contents. However, little is known about the relationship between exercise frequency and changes in beta-endorphin levels. Therefore, the present study was carried out to examine the relationship between exercise frequency and beta-endorphin levels using rat experimental model.F344 male rats, 6 weeks of age, were exercised in treadmill at 10 and 20 m/min twice or three times per day. One hour after the exercise, hypothalamus was taken from the whole brain and the beta-endorphin level in water extracts of the hypothalamus was measured by ELISA.Exercise at 10 m/min did not affect the beta-endorphin levels: the beta-endorphin levels in those rats were nearly identical to that observed in controls. On the other hand, exercise at 20 m/min for three times per day, but not twice per day, affected the endocrine system and caused significant increase in beta-endorphin levels as compared with those in control. These results suggest that low strength levels of exercise, such as 10 m/min did not affect endocrine system, but much higher strength levels of exercise caused changes in endocrine system. [J Physiol Sci. 2007;57 Suppl:S173]

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LPS, a bacterial endotoxin, is known to affect various functions of the central nervous system, including stimulation of the hypothalamo-pituitary-adorenal axis. We have previously reported that LPS increased immunoreactivity of corticotropin-releasing hormone (CRH) in the PVN in association with inhibition of gonadotropin secretion. LPS also produces deficits in learning and memory. In the present study, we examined whether LPS treatment may differentially influence c-Fos expression in the PVN and the hippocampus which is believed to be crucial for learning and memory. We also examined whether the LPS-induced change in c-Fos expression in the hippocampus may be mediated by the LPS-induced release of glucocorticoid. Ovariectomized rats were treated with an intravenous (iv) injection of LPS (40 μg/kg) or saline. Two hours after the injection, the brains were fixed and immunohistochemistry against c-Fos was performed. In the PVN, LPS treatment induced a dramatic increase in the number of c-Fos immunoreactive cells as reported previously. By contrast, LPS treatment significantly decreased c-Fos expression in the DG. In the CA3, there was no difference in c-Fos immunoreactive cell number between LPS treated rats and their controls. Pretreatment with metyrapone (75 mg/kg), an inhibitor of glucocorticoid synthesis, had no effect on LPS induced decline in c-Fos expression in the DG. These results show that LPS treatment selectively decreases c-Fos expression in the DG in a glucocorticoid independent manner. [J Physiol Sci. 2007;57 Suppl:S174]

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Sodefrin, a peptide pheromone, has been considered to be a major female-attr acting substance released from the abdominal gland of the male newt, Cynops pyrrhogaster. Recently, we found that a considerable amount of steroidal substances such as pregnenolone, progesterone, androstenedione and estradiol exsists in the abdominal gland of the male newt. Moreover it was confirmed that pregnenolone and androstenedione are released into the duct of the abdominal gland, As a step to elucidate the role of these substances, they were tested for their female-attracting activity employing the method developed by Toyoda et al.(1994). As a result, these two steroidal substances to be contained in 0.01 part of the abdominal gland was revealed to have a female-attracting activity. It was also revealed that a mixture of these steroids and sodefrin exerts more potent attracting activity than the steroids or sodefrin alone, These results strongly suggest that both steroids and the peptide pheromone are released from the gland and act together to attract sexually developed females during courtship.Toyoda et al. (1994). Physiology and Behavior, 55, 569-576. [J Physiol Sci. 2007;57 Suppl:S174]

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Estrogen influences on many basic molecular and cellular events in the brain of rats. Exposure of the developing brain to estrogen during the critical periods results in profound changes in morphology. In order to examine key factors, which are regulated by estrogen and induce sexual dimorphism in the brain, we focused on the expression of genes in the sexually dimorphic nucleus (SDN) and the anteroventral periventricular nucleus (AVPvN) of the preoptic area. Female rats injected with 100 μg 17β-estradiol on the day of birth or without injection were sacrificed 2 or 5 days later. Total RNA from SDN or AVPvN was subjected to analysis using a focused microarray (EstrArray), which consists of 173 estrogen-responsive rat genes. Microarray analysis was carried out three times to evaluate its accuracy and reproducibility, and revealed that 28 genes showed significant differences in the gene expression profiles between the tissues and the postnatal days. The major functional categories analyzed here included apoptosis, migration, nervous system development, cell growth and metabolism. The set of apoptosis-related genes showed significant correlations between the tissues and the postnatal days. These genes were also examined by real-time RT-PCR analysis. Among the apoptosis-related genes, Yars, c-Jun, Nckap1, and Prkcd were up-regulated in AVPvN more than SDN. These results suggest that the regulation of apoptosis-related genes by estrogen is a cue to induce the sexual differentiation of the rat brain during the critical periods. [J Physiol Sci. 2007;57 Suppl:S174]

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Somatostatin, originally identified as a hypothalamic peptide that inhibits the secretion of pituitary growth hormone, is widely distributed in the central nervous system and the periphery, and is implicated in neuronal survival or neurogenesis. By using non-isotopic in situ hybridization histochemistry, we report here that somatostatin gene is expressed transiently in the central part of the rat medial preoptic nucleus, which coincides with the sexually dimorphic nucleus of the preoptic area (SDN-POA), during, but not after, the establishment of sex difference. On postnatal day 1 (day of birth), somatostatin mRNA was detected in the SDN-POA of both sexes. On days 8 through 35, the area of somatostatin mRNA-positive cells was significantly larger in males than in females. In males, the area attained its maximum size on day 15 and diminished gradually thereafter. In females, the area did not change in size during this period. On day 60, expression of somatostatin mRNA was low and not different between sexes. Throughout the observed period, Nissl staining and calbindin immunohistochemistry enabled visualization of the typical SDN-POA in the same region. Orchidectomy of males on day 1 decreased, and administration of estradiol benzoate to females (10 µg in 0.02 ml sesame oil) on days 1 through 10 increased the volume of somatostatin mRNA-positive areas which corresponded to the SDN-POA, when observations were made on day 15. Sex-specific, transient transcription of the somatostatin gene may causally relate to the estrogen-dependent organization of the SDN-POA. [J Physiol Sci. 2007;57 Suppl:S174]

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The volume of the sexually dimorphic nucleus in the preoptic area (SDN-POA) is 2-4 times larger in male rat than in female, however organization of the SDN-POA and function of this nucleus have not well known. In contrast, estrogen causes the sexually dimorphism by acting in perinatal periods. Recently, transgenic rats expressing enhanced green fluorescent protein (EGFP) under the control of an estrogen receptor (ER) α gene promoter 0/B were generated to tag ERα-positive neurons in the brain (Mol. Brain Res., 139(2005)42-51, Hamada et al.). In the present study, we examined GFP expression could be used a marker for the SDN-POA. GFP labeled cells were distributed in the core of SDN-POA (GFP-SDN) of male and female transgenic rats and in the majority of these cells included ERα, immunohistochemically. Both distribution area and number of GFP expressed cells in the SDN-POA were larger in male than in female, however, female GFP cells in the SDN-POA showed concentrated distribution than male. In addition, these GFP cells expressed calbindin that was a marker for the core of SDN-POA, immunohistochemically. Administration on the day of birth of estrogen to females increases the volume of the GFP-SDN by 8 weeks. By demonstrating the sexual dimorphism and gonadal steroid modulation of the GFP-SDN, we hereby establish that GFP expression in SDN-POA could be useful in vivo marker to make clear the function of the SDN-POA. Moreover, ERα gene promoter 0/B plays a key role for organization of sex differentiation of the SDN-POA. [J Physiol Sci. 2007;57 Suppl:S175]

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In mammals, the sperm triggers a series of cytosolic Ca²⁺ oscillations that continue for -4 h, stopping at about the time of pronucleus (PN) formation. PLCζ, a strong candidate of the sperm factor, induces fertilization-like Ca²⁺ oscillations when expressed in mouse eggs by injecting its cRNA, and is subsequently accumulated into PN. To reveal the role of pronuclear translocation of PLCζ in the cessation of Ca²⁺ oscillations, the timing of these events was precisely analyzed. Ca²⁺ oscillations ceased around the time of PN formation by injecting with 0.1-0.5 μg/ml PLCζ cRNA. For K377E which lacks nuclear translocation ability, Ca²⁺ oscillations continued over 10 h with 0.1-0.5 μg/ml. Thus, nuclear sequestration of PLCζ can be a factor for cessation of Ca²⁺ oscillations. To record Ca²⁺ oscillations under the condition close to the physiological condition as far as possible, Eggs were fertilized and kept in culture until the start of [Ca²⁺]_i measurement. The timing of PN formation (T_PN) was determined by the first recognition of a large nucleolus. When [Ca²⁺]_i was recorded from 180 min, no Ca²⁺ spike was seen in half of eggs; that is, the last Ca²⁺ spike occurred at least 100 min prior to T_PN. ER staining showed the PN membrane was formed 45-60 min before T_PN. In these eggs, PLCζ was found to translocate into PN as soon as the PN membrane was formed. Taking this finding into account, Ca²⁺ oscillations could cease prior to PN formation after physiological fertilization. [J Physiol Sci. 2007;57 Suppl:S175]

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In Xenopus laevis, sperm bind to vitelline envelope (VE) that surrounds an egg, and undergo acrosome-reaction that enables sperm to penetrate the VE. Sperm-egg fusion activates an egg and resumes the cell cycle. Although there are considerable lines of evidence, molecular aspects of fertilization remain to be elucidated because of the conflicting results. In the present study, to understand molecular mechanism of sperm-egg interaction, we isolated and characterized a 26 kDa Ca²⁺-binding protein, Xenopus dicalcin, in Xenopus eggs. Xenopus dicalcin is localized prominently in VE and cytoplasm of the cortex of both the animal and the vegetal hemisphere of Xenopus eggs. Two VE glycoproteins bind to Xenopus dicalcin in a Ca²⁺-dependent manner. Since these two VE glycoproteins are considered to function as sperm-receptors in the VE, we tested the effect of dicalcin on fertilization in vitro. Preincubation of eggs with recombinant dicalcin reduced the success of fertilization to as little as 10% of control. In contrast, inhibition of intrinsic dicalcin by preincubation of anti-dicalcin antibody surprisingly increased the success of fertilization. These results indicated that dicalcin inhibits sperm-egg interaction and subsequent fertilization. Mechanism of dicalcin's action and future aims will also be discussed. [J Physiol Sci. 2007;57 Suppl:S175]

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Preference for social odors in the rat is sexually dimorphic, i.e. they prefer odor derived from sexually active, opposite-sex conspecifics to that from sexually inactive or same-sex individuals. In addition, male rats show clear preference for odor of castrated males to that of sexually active males. This preference toward same-sex odor is not appeared in females, presumably due to the property as territorial animals. Since neuroendocrine basis of those preferences is still unclear, we examined effects of ibotenic-acid lesion in the medial amygdala or the preoptic area, both belonging to the vomeronasal systems, on male rat preference toward conspecific odors. Preference was measured by a 3-compartment apparatus, in which experimental males was presented with one of stimulus-odor pairs, receptive female and sexually active male, receptive female and ovariectomized female, or sexually active male and castrated male. Nose-poking time to explore each stimulus was compared among medial amygdala lesioned, preoptic lesioned and sham-operated male rats. Medial amygdala lesions disturbed preference for receptive females to ovariectomized females, while preoptic lesions eliminated preference for castrated males to sexually active males. Although ejaculation latency was increased in the medial amygdala and preoptic lesioned males, no male showed a complete suppression of sexual behavior. The results suggest that the medial amygdala and the preoptic area play different roles in regulation of social behavior. [J Physiol Sci. 2007;57 Suppl:S175]

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The genetic background and key gene for congenital scoliosis has not yet been clarified. Ishibashi rats (IS) have congenital malformations of lumbar vertebrae leading to kyphoscoliosis similar to that seen in human. Analysis of IS may thus provide insights into the genetic causes of human congenital scoliosis. We characterized skeletal malformations of lumbar vertebrae in IS by roentgenographic study and double staining. Then, we screened for the difference of gene expression of Hox10 and 11 paralogues, which were well known playing critical roles in the determination of characteristics of lumbar and sacral vertebrae, between IS and Wistar strain rats by Real Time-PCR. Significant differences on skeletal structures between IS and Wistar were found: transitional vertebrae; anterior wedged vertebra; union of anterior vertebrae; an additional vertebra. Especially, transitional vertebra was frequently observed (77%). Double staining of IS fetuses appeared the union of primary ossification centers in the lumbar vertebral column, and these changes was not observed in other vertebral column.Analyses of Hox10 and 11 paralogues by quantitative RT-PCR, the expression level in lumbar/ sacral vertebral column was, relative to those of Wistar, dramatically decreased except for Hox a10 and c11.Hox 10 and 11 paralogues may play significant role in the elaboration of the transitional vertebrae. To elucidate the difference of expression pattern of Hox between IS and Wister, we started whole mount in-situ hybridization. [J Physiol Sci. 2007;57 Suppl:S176]

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This experiment aimed at exploring the effects of all trans-retinoic acid (ATRA) or combined with neurotrophic factors on the differentiation of epidermic growth factor (EGF)-responsive neural stem cells (NSCs). The forebrain SVZ region from neonatal SD rats (P1-P2) was dispersed and cultivated in the serum-free media (DMEM/F12 1:1, 2% B27 supplement) with EGF (20 ng/ml). The neurospheres of second passage were used to observe the effects of ATRA 1 μM alone, ATRA 1 μM + BDNF(brain-derived neurotrophic factor) 10 ng/ml or ATRA 1 μM + GDNF(glial cell line-derived neurotrophic factor) 10 ng/ml on differentiation in experimental groups. Blank control and vehicle control (DMSO 1:1000 v/v) were set in parallel. Immunocytochemistry was used to identify the expression of cell markers after 6-7 days of differentiation. The majority of cells in each group were still Nestin-positive. MAP-2-positive cells were significantly increased in all experimental groups, while no difference in the number of MAP-2-positive cells was observed either among 3 experimental groups or between 2 control groups. GFAP expressions were similar in all the groups. MBP-positive cells were seldom observed in each group. It indicated that ATRA could enhance the differentiation of EGF-responsive NSCs into cell phenotype of neurons. At the concentration of 10 ng/ml, neither BDNF nor GDNF had significant synergistic effect with ATRA in promoting the differentiation of NSCs. [J Physiol Sci. 2007;57 Suppl:S176]

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Alzheimers disease (AD), the most common form of dementia in the elderly population, is characterized by an insidious onset with memory impairment and an inexorable progression of cognitive decline. Amyloid β peptide (Aβ) is a product of sequential cleaving of amyloid precursor protein (APP) by two proteolytic enzymes, β- and γ-secretases, which cleave the APP at the N and C terminus of Aβ, respectively. The age-related activities of these secretase may induce changes in turnover of Aβ and elevate the risk of AD. Epidemiologic studies show a relation between dietary fish oil and AD. DHA supplementation was shown to improve cognitive function in the elderly. Therefore aging and depletion of DHA are risk factors for AD. We hypothesized that imbalance of Aβ production may occur by aging and depletion of DHA. We examined what mechanisms involved in impairment the balance of Aβ generation by aging and depletion of DHA. We examined age-related changes of mRNA expressions in these proteins. One group of rats fed a fish oil deficient diet (F1 diet) through three generations. Other group of rats fed MF (normal rodent diet). The hippocampus and cerebral cortex were dissected from young (7 weeks old) and adult (50 weeks old) F1 or MF fed rats. Expressions levels of APP, β- site APP-cleaving enzyme (BACE), presenilin 1 (γ-secretase) and neprilysin mRNA in these tissues were measured by Real-Time PCR. [J Physiol Sci. 2007;57 Suppl:S176]

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Our recent experiments demonstrated that the ovarian sympathetic nerves act as vasoconstrictor in anesthetized non-pregnant rats. It is important to study aging at an organ level in addition to at the cellular and an individual level. In the present study, we focused on the ovaries that lose their function earlier than individual death, and examined aging effects on neural regulation of ovarian blood flow. We measured responses of ovarian blood flow to stimulation of ovarian sympathetic nerve and conduction velocities of ovarian sympathetic nerves in adult (6 months old, regularly cycling) and aged (28 months old, constant diestrus) Wistar rats. The electrical stimulation (0.5 ms, 10 V, 20 Hz, 20s) of distal part of the severed ovarian plexus nerve decreased the ovarian blood flow to about 30% of the control in both adult and aged rats. Recording of compound action potentials from ovarian sympathetic nerves (ovarian plexus nerves) showed that the ovarian sympathetic nerves contain mostly unmyelinated C fibers. There were no differences between adult and aged rats in the conduction velocity. It was found that both conduction velocities and vasoconstrictor function were well-maintained in the ovarian nerve of aged rats without reproductive activity. [J Physiol Sci. 2007;57 Suppl:S176]

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Cholesterol cholelithiasis is one of the most rapidly increasing digestive diseases during the past 50 years in Japan . Although the etiology of gallstones is multifactorial, gallbladder dysmotility accelerated nucleation of cholesterol crystals in the gallbladder. Gallbladder contraction mainly depends on cholecystokinin (CCK)(via CCK-ARs). We have generated CCK-AR knockout (-/-) mice and found that one third of CCK-AR(-/-) male mice, not all, showed sludge and gallstone formation. The present study was designed to determine how much the gallbladder dysmotility contributed to gallstone formation as a primary cause and whether sex difference existed in gallstone formation, comparing CCK-AR(-/-) male and female mice, because it has been believed that gallstones were more popular in women than men. No sludge or gallstone formation was observed in any mouse at 6 months of age. The frequency of sludge and gallstone formation at 12 and 24 months of age tended to be higher in female CCK-AR(-/-) mice than the males, but the difference was not significant. In summary, the gallbladder dysmotility alone could accelerate sludge and gallstone formation, but its contribution to sludge and gallstone formation was limited below 50%. The 12-month-duration was required to produce gallstones, and after 12 months of age, ageing did not further increase the frequency of sludge and gallstone formation. No significant sex difference in the frequency of sludge and gallstone formation was observed. [J Physiol Sci. 2007;57 Suppl:S177]

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We have previously demonstrated that rhythmic exercises such as Zen abdominal breathing, gum-chewing, hula dancing and so on caused an increased 5-HT level in adult human. In this study, we evaluated urinary 5-HT level of pre-school children before and after the rhythmic exercise program (REP) including zigzag run, jumping rope, vaulting box, climbing bar, doing horizontal bar and overhead ladder etc. The subjects were 11 junior (4-5 years old) and 8 senior (5-6 years old) pre-school children. We found that the mean 5-HT level in senior pre-school children was higher than that in junior pre-school children before REP. This high 5-HT level in senior children may be due to a neuronal developmental of serotonergic system. REP induced an increase in 5-HT level in 6 of 11 junior children and 6 of 8 senior children. Although the rest children showed the decreased 5-HT level after REP, three of 5 junior children and one of 2 senior children had markedly high 5-HT level before REP. The decreased 5-HT level after REP may be derived from an exercise-induced fatigue. [J Physiol Sci. 2007;57 Suppl:S177]

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The QT interval indicates the repolarization process of myocardium, and is considerably affected by the preceding sinus cycle (RR interval). Since excessively prolonged QT intervals may contribute to the initiation of lethal cardiac arrhythmia, the lability of repolarization in adults can be evaluated using the variability index (QTvi). On the other hand, no reports have been available regarding QTvi in newborn babies and preschool children. Thus, QTvi has been anticipated to indicate the changes accompanied with postnatal growth, based on the characteristics of the immature autonomic nervous system and myocardium of such young children.Electrocardiograms were recorded at rest in a total of 60 healthy children aged 0 days to 6 years. RR and QT intervals of 120 consecutive heart beats were measured to calculate QTvi.A tendency towards a decrease in QTvi was observed in children aged from 0 days to 7 years (linear regression line: y = -1.076x + 0.007, r = -0.464). Significant difference (p=0.0021) was observed between the average QTvi (-1.57 ± 0.11) of preschool children (average age: 6.3 ± 0.5 years) and that (-1.19 ± 0.29) of neonates (average month: 7.0 ± 4.1 months).It was suggested that repolarization time would change along with the sinus cycle during postnatal growth, resulting in a reduction of lability of repolarization. [J Physiol Sci. 2007;57 Suppl:S177]

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It is well known that role of GABA_A-R mediated actions is important for early CNS development. The GABA_A receptor-mediate actions can regulate radial migration of cortical plate cells. GAD67-GFP knock-in mouse make it possible to investigate the effects of ambient GABA because of the decreased GABA content. It is known that GABA_A receptor is activated taurine as well as GABA. In this study, we investigate the influence of the ambient GABA and taurine to cortical plate cells by using GAD67-GFP knock-in mice and taurine metabolism blocker, D-cysteine sulfinate (D-CSA).The cortical plate cells which exist in ventricular zone at E14 were labeled by in utero electroporation. Three days after the electroporation, the distribution of labeled cells was analyzed. There was no significant difference in the distribution among genotypes of GAD67-GFP knock-in mice. GABA_A-R mediated currents were recorded by whole-cell recording from labeled cells. Evoked GABA current had dose-dependent manner and had no differences among genotypes. Ambient taurine was decreased by the i.p. injections of D-CSA. The radial migration of cortical plate cells was facilitated in D-CSA injected homozygous GAD67-GFP knock-in mice. Thus, taurine may have supplemental effect for GABA, that could play critical protective role in pathological condition of GABAergic system. [J Physiol Sci. 2007;57 Suppl:S177]

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In the mouse, opening of the vaginal cavity to the skin is a postnatal tissue remodeling, occurring around the fifth week of life. The tissue remodeling required to complete maturation of the female genital tract at the time of puberty depends on a hormonally triggered apoptosis-dependent process occurring in the lower part of the vaginal mucosa. However, the detailed mechanism of apoptosis induced by sex hormone is unknown. Our analysis disclosed that mice lacking the class IV semaphorin, Sema4D develop vaginal atresia resulting from the failure in vaginal opening. In order to analyze the mechanism of which Sema4D-deficient mice develop vaginal atresia, TUNEL assay was performed on vaginal mucosa from 5-week-old mice. As a result, apoptotic cells in vaginal mucosa were significantly less in Sema4D-deficient mice compared to wild-type mice. To demonstrate the apoptosis-inducing activity of Sema4D, we performed a rescue experiment by placing back recombinant Sema4D to cultured vaginal epithelial cells from Sema4D-deficient mice. Sema4D induced a significant apoptosis of vaginal epithelial cells. Thus Sema4D may play a non-redundant role in the tissue remodeling by inducing apoptosis in vaginal epithelial cells of 5-week-old mice. [J Physiol Sci. 2007;57 Suppl:S178]

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Propolis has been known to include couples of bioactive substances that inhibit growth of tumor cells. Among them, a plant flavonoid quercetin (QU) specifically antagonized proliferating rat C6 Glioma cells at a micoromolar concentration range, while QU left C6 cells intact when arrested at the G0/G1 phase of the cell cycle. Thus, QU seems to be a promising candidate for cancer chemotherapy. However, QU has been known to bind with serum albumin after being absorbed from digestive tract. This interaction with albumin makes effects of QU less predictable and may modulate biological actions of other drugs. To clarify binding property of QU with albumin, we have set off kinetic analysis of quercetin-albumin interaction with a BIACORE instrument, that is based on a surface plasmon resonance. It was found that QU binds to bovine serum albumin (BSA) with two different kinetics; fast and slow association mechanisms were recognized. The maximum binding of the slow component was highly dependent on temperature, being largest at a body temperature. The dissociation constant for a quercetin-albumin interaction is in submicoromolar range. Thus, almost all of QU in blood stream is likely bound to serum albumin, For an efficient and predictable chemotherapy, QU must be modified to avoid any interaction with serum albumin. [J Physiol Sci. 2007;57 Suppl:S178]

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We sought to clarify the role of brain polyamines on stress-induced hyperthermia (SIH), a model of anticipatory anxiety, after single injection of diazepam, polyamine or 14-days injections of α-difluoromethylornithine (DFMO), a specific inhibitor of polyamine synthesis. In addition, polyamine levels in the hippocampus and hypothalamus were measured after putrescine or DFMO treatments. Male C57BL/6J mice are subjected to two sequential rectal temperature measurements with a 10-min interval. The first measurements is the basal temperature (T₁) and the second is the stress-enhanced temperature (T₂) and the difference (delta-T=T₂-T₁) is regarded as SIH. In control mice, delta-T was nearly 1°C. Pretreatment with diazepam dose-dependently inhibited the SIH. Similarly, putrescine blocked delta-T although T₁ was decreased, dose-dependently. Furthermore, spermidine and spermine also lowered delta-T and T₁ at the doses lower than putrescine. In contrast, DFMO had no effect on T₁ but it increased delta-T. Pretreatment with putrescine or DFMO increased or decreased brain putrescine levels, respectively. These results suggest that endogenous brain putrescine and other polyamines might have an anxiolytic-like effect under stressful conditions. [J Physiol Sci. 2007;57 Suppl:S178]

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Many studies on long-term head-down bed rest (HDBR) used as a simulation of microgravity indicate that sweating response is reduced accompanying an elevation of core temperature, suggesting that the alteration of sweating response during HDBR may be caused by the dehydration induced. This study was aimed to investigate whether the reduced sweating response is prevented by HDBR in the absence of dehydration. In 12 healthy male volunteers, daily water intake was encouraged during 20 days HDBR to match the urine volume in the previous day. Six of twelve subjects exercised every day during HDBR. Before and after the session of HDBR, each subject was immersed his legs in hot water at 42 ^o C for 45 min in a sitting position. Core (tympanic) temperature, skin temperature, chest sweat rates and chest blood flow were recorded continuously. Body weight was not significantly changed after HDBR for each of the exercise and the control groups. Higher core temperatures and blood flow, greater sweat rate, and earlier onset of sweating were observed during the hot water immersion after HDBR. The regression line relating sweat rate to body temperature was not significantly shifted after HDBR in both of the exercise and the control groups, indicating that sweating function was not altered by the HDBR. It was concluded that encouraged water intake (i.e., euhydration) can prevent the reduced sweating response under microgravity condition such as during spaceflight. [J Physiol Sci. 2007;57 Suppl:S178]

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