The midline raphe nuclei may be involved in the control of respiration. Stimulation of the caudal raphe regions, including raphe obscurus (RO), pallidus (RP) and magnus (RM), causes a change in respiration. These regions contain serotonergic cells. However, the raphe nuclei also contain non-serotonergic neurons. This study was undertaken to investigate firing patterns and spinal projections of respiratory neurons in medullary raphe nuclei of cat, and examined whether these respiratory neurons were serotonergic or not. Extracellular spikes of 28 single respiratory neurons were recorded in midline medullary tegmentum in Nembutal anesthetized cats. A total of 28 respiratory neurons was recorded in the raphe obscurus, pallidus and magnus. The cells were classified as Late-inspiratory (I) (Late-I, n=5), inspiratory-expiratory (E) phase-spanning (I-E PS, n=5), E-I phase-spanning (E-I PS, n=8), expiratory-decrementing (E-DEC, n=4), E-frequency modulated (n=3), inspiratory-constant (I-CON, n=3) based on their firing pattern in relation to the phase of respiration. Out of the 18 respiratory neurons were tested for antidromic activation from the ventrolateral quadrant of the C4-5 spinal cord, two E-frequency modulated, one E-DEC and one E-I PS neuron were antidromically activated with the latencies that corresponded to axonal conduction velocities of 12.8–27.3 m/s. These results suggest that some non-serotoninergic respiratory neurons of the caudal raphe nuclei transmit respiratory activity to the spinal cord. [J Physiol Sci. 2008;58 Suppl:S116]

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We measured the oxygen cost of breathing during voluntary hyperventilation in 13 subjects to test the hypothesis that the oxygen cost of breathing at a given level of ventilation would vary, dependent on the combination of tidal volume and respiratory frequency. We also measured thoracoabdominal asynchrony indices with respiratory inductive plethysmography. Prior to experiments, resting tidal volume (100%VT) was determined in each subject. Subjects voluntarily increased ventilation with tidal volume fixed (100%VT, 150%VT, or 300%VT). Increased ventilation increased VO2 in proportional to the level of ventilation. Changes in VO2 during hyperventilation (delta VO2) were compared between VTs. Delta VO2 was significantly higher during 100%VT breathing than 300%VT breathing. These results suggest that the oxygen cost of breathing during hyperventilation is high when VT is fixed at the resting level. Respiratory inductive plethysmography showed that thoracoabdominal asynchrony indices (PhAng, PhRIB, PhREB, and PhRTB) significantly increased during 100%VT breathing with the level of ventilation increased. Such an increase was not found during 150%VT breathing or 300%VT breathing. However, correlation analyses revealed no significant correlation between delta VO2 and delta PhAng, delta PhRIB, delta PhREB, or delta PhRTB, suggesting that thoracoabdominal asynchrony does not explain differences in the oxygen cost of breathing between VTs. [J Physiol Sci. 2008;58 Suppl:S116]

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Previous research on respiration and psychology indicated that anxiety level has a close relationship with respiratory pattern. The objective of our study was to compare respiratory pattern and anxiety of rhythmic gymnasts to regular students to find characteristics of group sports athletes. Respiration of 15 female college level rhythmic gymnasts (AG) and 28 medical students (RG) were tested by the CO₂ rebreathing method using gas containing 5% CO₂ and 95% O₂. VE (minute ventilation), ETCO₂ (end-tidal CO₂), VT (tidal volume), and RR (respiratory rate) were measured throughout three stages: rest, rebreathing and recovery. Anxiety was measured by Spielberger's State-Trait Anxiety Inventory. CO₂ response was defined as the slope of the response curve and the respiratory pattern was sought by calculating the VT% and RR% contributions to the rise of VE during rebreathing. Data from 13 gymnasts and 26 students were used for analysis. As a result, AG was significantly higher on both the trait and state anxiety. The AG average VE was lower during rest stage and ETCO₂ had less variance. There was no significant difference in the CO₂ response and no correlation between anxiety levels and respiratory parameters. Although there were no significant differences in the average RR% and VT% contributions, there was significantly less variance in AG suggesting similar breathing rhythms. These results indicate that in comparison to regular students, rhythmic gymnasts are high in anxiety and similar in respiratory patterns. [J Physiol Sci. 2008;58 Suppl:S116]

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Analyses of respiratory neuron activity by voltage-sensitive dye imaging in the brainstem-spinal cord preparation of postnatal day 0 or 1(P0-P1) rat or mouse have revealed a characteristic activity pattern in the rostral ventral medulla; preinspiratory activity in the rostral medulla (corresponding to para-facial respiratory group) preceding inspiratory activity in the more caudal medulla (corresponding to pre-Botzinger complex) (e.g. Onimaru & Homma, 2003). On the other hand, postnatal changes of organization of the respiratory neuron network remain to be elucidated, whereas P4-P5 rat or mouse is limitation age for experimental usage of the en bloc reparation. In the present study, we analyzed respiratory neuron activity by voltage-sensitive dye imaging (network level) and whole-cell patch clamp recordings (individual cell level) in the ventral medulla of P0-P5 rats and mice. We found that basically same activity pattern was detected in P0-P4 rats and P0-P5 mice, while the fluorescence intensity (S/N ratio) tended to decrease with age especially in the rat. In the mouse, application of low concentration (0.1-0.2 μM) of adrenaline was effective to induce regular respiratory activity in P0-P1 preparations, whereas this treatment was not necessary to obtain regular C4 activity and conspicuous spatio-temporal pattern of respiratory neuron activity in P4-P5 preparations. Using whole-cell recordings, we also confirmed presence of subtypes of respiratory neurons that show burst pattern corresponding to imaging data. [J Physiol Sci. 2008;58 Suppl:S116]

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Last year we described the flow-volume curve (F-V curve) in rats. In that study rats were orally intubated using 14G (OD 1.75mm) cannulas. The F-V curve revealed a long ascending limb similar to that seen in human with extrathoracic upper airway obstruction. In the current study we examined the effect of tracheal cannula caliber on the F-V curve. The maximum cannula size used for Wistar or Norway Brown strain rats of 8-9 week of age was 13G (OD 1.95mm) for oral intubation and 14G (OD 1.95mm) for tracheostomy. The use of 12G or 13G cannulas respectively shortened the ascending limb of the F-V curve. The curve consisted of 3 phases: a steep ascending, a mild descending and a steep descending phase. Expiratory flow in the ascending limb increased along with increase in thoracic pressure while the mild descending limb was unaffected. When airways were constricted by inhaled methacholine the mild descending phase was shortened and vital capacity decreased, although the other phases were not affected. These findings suggest that large airway caliber is the crucial determinant of F-V curve in rats. To analyze F-V curves in rats maximal cannula size should be used. [J Physiol Sci. 2008;58 Suppl:S117]

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Aquatic frogs, Pipidae have some interesting characteristics from the viewpoint of comparative respiration physiology. They lack buccal ventilatory cycle, exhale air from the lung before aspirating air into the buccal cavity, and have inherent muscles suspected homologous to the mammalian 'diaphragm'. To study the reason why the aquatic frogs lack the buccal cycle, we recorded the respiratory motor outputs from the in vitro brainstem preparation of Xenopus laevis excluding the influence by the peripheral feedback mechanisms. The cranial nerve activities of the Xenopus exhibited the intermittent bursts with doublet, triplet or multiplet patterns, those were similar to the lung-breathing pattern in vivo, and lacked the rapid bursting activity corresponding to the buccal oscillation. The activation sequence of motor outputs within a burst complex was consistent with that of the normal ventilation in vivo. These results indicate that the intermittent pattern and the lack of buccal oscillation in the breathing of Xenopus are not caused by reflex mechanism but generated within the brainstem. This in vitro preparation may be useful for the comparative study as a model close to the ancestor of amiotes. [J Physiol Sci. 2008;58 Suppl:S117]

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Exhaled air contains numerous kinds of volatile chemicals derived from metabolic conditions in health and diseases. Breath chemicals such as nitric oxide and carbon monoxide are generally recognized as biomarkers for several diseases. However, there have been very few studies to refer whether mental status could be associated with volatile chemicals in breath. In the present study, we examined whether breath compositions or their concentration changes were associated with mental conditions. Seventy nine adults volunteered to this study. Current smokers and drinkers were excluded. Their heart rates and physical accelerations were monitored to determine their qualities of night sleep. Mental stress, depression and neurosis levels were evaluated by the General Health Questionnaires 28, Self-Depression Scale and Cornel Medical Index, respectively. Exhaled airs were collected into bags before and after night sleep and the concentrations of hydrogen, methane, carbon monoxide, acetone, acetaldehyde and ethanol were analyzed. There were significantly positive correlations among the concentration differences of expired carbon oxide before and after the sleep, total scores of GHQ and neurosis levels determined by the CMI scores. Breath hydrogen concentrations before night sleep were significantly correlated to depression levels. Another chemicals did not indicate significant correlations to neither mental status, nor sleep conditions determined by the heart rate variability and physical accelerations. These results suggested that exhaled air were closely associated with mental conditions. [J Physiol Sci. 2008;58 Suppl:S117]

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DSCAM1 on human chromosome 21q22 is a neural adhesion molecule that plays diverse roles in the neural development. We disrupted the Dscam1 locus in mouse and found that the Dscam1^−/− mice died within 24 hours after birth. Whole body plethysmography of the mice showed irregular respiration. Further, inspiratory activity of the C4 ventral root that drives diaphragm, assessed in a medulla-spinal cord preparation of Dscam1^−/− mice, also showed irregular rhythm with frequent apnea. In Dscam1^−/− mice, optical imaging of this preparation by using voltage-sensitive dye revealed that the synchroneity of pre-inspiratory (Pre-I) neuron activity in para-facial respiratory group (pFRG) as a respiratory rhythm generator was lost, while that of inspiratory neuron activity in pre-Botzinger complex as a pattern generator was preserved in rostral ventrolateral medulla (RVLM). In the immunohistochemical analysis with SMI311, that the marker for neuronal soma and dendrites, the number of SMI311-positive neurons was increased at dorsolateral facial nucleus. The VII nerve root activities that included Pre-I burst activity were decreased in Dscam1^−/− mice. According to the test of respiratory network induced by RVLM stimulation, bilateral neural connections were disrupted. These results suggest that the deficiency of Dscam1 selectively depressed Pre-I neuron network and its deficiency causes congenital central hypoventilation. [J Physiol Sci. 2008;58 Suppl:S117]

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Although respiratory sensation such as dyspnea, or breathlessness are known to be elicited by carbon dioxide, and hypoxia, it is not well known if there is a positive interaction between both chemical stimuli, in the same manner as in ventilation. We examined both ventilation and respiratory sensation during progressive hypoxia with three different CO2 levels using 14 young subjects. Ventilatory variables such as end-tidal CO2 (PetCO2) and O2 (PetO2), tidal volume (VT), respiratory frequency (f) and minute ventilation (VE) were monitored using an aeromonitor (Minato) and the arterial oxygen saturation (SpO2) was by a pulse oxymeter (Ohmeda, Biox). All of these data were stored into a personal computer with breath by breath fashion. We built the model to extract both sensitivities and thresholds of hypercapnia and hypoxic ventilatory responses and the sensitivity of their interaction. [J Physiol Sci. 2008;58 Suppl:S118]

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Respiratory long-term facilitation (LTF) is a long-lasting (> hr) augmentation of respiratory motor output that occurs even after cessation of hypoxic stimuli. Although ventilatory LTF has been reported in mice, phrenic LTF has not been reported in this species. We examined whether phrenic LTF can be observed in mice, a promising animal species for the future study with genetic engineering. Phrenic nerve activity was recorded with a pair of silver hook electrodes in urethane (1.3 g/kg, ip)-anesthetized, vagotomized, paralyzed (0.2 mg/kg panchronium bromide for every hour, ip), and artificially ventilated C57BL/6 male mice. Volume and frequency of the artificial ventilation was set so that the animal's PaCO₂ would be just above the apneic threshold. Isocapnic condition was maintained throughout the experiment by adding appropriate CO₂ to the inhaled gas mixture so that changes in expired PCO₂ would not exceed 0.2% from the baseline. We examined possible effect of episodic hypoxia (EH, inhalation of the gas mixture of FIO₂ = 0.10-0.15 and FICO₂ = 0.005-0.01 for 45 sec) and intermittent hypoxia (IH, 5 times of EH separated by 5 min of 100% O₂) on phrenic nerve activity for 1 hour. Amplitude of the integrated phrenic nerve activity gradually increased after cessation of IH. An increase in the burst frequency of the phrenic nerve activity was not so apparent. EH, on the other hand, did not affect amplitude or frequency of the phrenic nerve activity. We conclude that phrenic LTF occurs in mice as in a similar manner to that in cats and rats. [J Physiol Sci. 2008;58 Suppl:S118]

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Thirteen healthy adults were exposed to isocapnic sustained hypoxia to compare the effect of hypoxia on regional cerebral oxygenation states and to investigate the relation hypoxic ventilatory response (HVR) and deoxygenation level of brain tissue. Regional cerebral oxygenation profiles were monitored with near-infrared spectroscopy applying the probes to scalp at front and back of head. Arterial oxygen saturation declined from 97.6±1.2% (mean±SD) to 80.7±3.1% and HVR was -0.29±0.16L/min/%. Oxyhemoglobin (O2Hb) decreased 4.77±2.53uM at forehead region and 1.45±4.83 uM at suboccipital region. On the other hand, deoxyhemoglobin (HHb) increased 6.45±2.29 uM at front of head and 3.87±3.18 uM at back of head. The changes of O2Hb and HHb at back of head were smaller than front of head, though there was not significant difference. Tissue oxygenation index (TOI=O2Hb/(O2Hb+HHb)×100 (%) ) also decreased 8.8±3.3% at front of head and 2.2±4.7% at back of head. The brain tissue oxygenation levels at suboccipital region was significantly higher than forehead region. HVR correlated significantly to decrease in TOI at suboccipital region, but not at forehead region. These results suggest that suboccipital region is protected from hypoxic risk and we may speculate that local oxygenation level of respiratory center act a potential influence on hypoxic chemosensitivity. [J Physiol Sci. 2008;58 Suppl:S118]

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Ventilatory responses to acute hypoxia (HVR) are modulated by histamine H1 receptors via changes in the metabolic rate. Hypothalamic histamine contributes to an energy supply balance to the brain during starvation. We tested the hypothesis that HVR varies with feeding states and that histamine H1 receptors are also involved in the responses after fasting. Wild-type (WT) and histamine H1 receptor knockout (H1RKO) mice were exposed to hypoxic gas (7% O₂ + 3% CO₂ in N₂) for 20 min in a 12 h fasted state and in a fed state. In WT mice, HVR in the fed state showed an initial increase and then a sequential decline (hypoxic ventilatory decline, HVD); whereas, HVR in the fasted state was initially increased and thereafter maintained. A smaller reduction in O₂ consumption (VO₂) during hypoxia and a lower respiratory exchange ratio (R) were observed in the fasted state than in the fed one, suggesting that fasting diminishes HVD along with undamped VO₂ because of a predominant lipid metabolism. In H1RKO mice, HVR in the fed state showed an initial increase and its maintenance throughout hypoxia; conversely, HVR in the fasted state showed an initial increase and HVD. VO₂ during hypoxia was not significantly different between fed states, although CO₂ excretion was lower in the fasted state than in the fed one. These results suggested that HVR is affected by fed states because of a change in the balance between lipid and glucose metabolisms, and that histamine H1 receptors may be involved in the HVR depending on the fed states. [J Physiol Sci. 2008;58 Suppl:S118]

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5-HT release in the dorsomedial medulla oblongata (DMM), especially in the hypoglossal nucleus, affects genioglossus muscle activity. We previously showed that 5-HT release in the DMM is increased by CO₂ inhalation and that 5-HT2 activity in the DMM increases airway dilation and ventilatory volume in adult mice. However, whether there was dose dependency for inspired-CO₂-induced increase in 5-HT release in the DMM was not clear. Prematurity of medullary 5-HT neurons has been associated with sudden infant death syndrome. In the present study, 5-HT release in the DMM, airway and respiratory responses to CO₂ inhalation were investigated in infant rats. Infant Wister rats were anesthetized with pentobarbital sodium (0.5 mg/0.1 ml saline/10 g body wt i.p.) and locally anesthetized with 0.5% Marcain for insertion of a microdialysis probe in the DMM and placed in a double-chamber plethysmograph to record respiratory variables. After recovery from anesthesia, extracellular fluid was collected at 1.2 μl/min every 10 min. 5-HT was analyzed with an ECD-HPLC. Infant rats were exposed to 0, 5, 7 and 9% CO₂ in O₂ at intervals of 10 min. The 5-HT increase induced by CO₂ inhalation in the DMM of infant rats was dose-dependent. Specific airway resistance was increased by CO₂ inhalation in infant rats, as were tidal volume, respiratory rate, minute ventilation and mean inspiratory flow. Our results suggest that inspired-CO₂-induced increases in 5-HT release in the DMM were dose-dependent and occurred with airway narrowing and ventilatory augmentation in infant rats. The airway response differed from that seen in adult mice. [J Physiol Sci. 2008;58 Suppl:S119]

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To evaluate a mechanism underlying DT breathing, we previously examined the effect of DT breathing on EEG, and found the increase in high-frequency alpha activity (HF-alpha; 10-13Hz). Since the urinary serotonin (5-HT) level was increased after DT breathing, we hypothesized that the activity of the 5-HT neuron located in the brain stem may be involved in modulating the EEG power during DT breathing (Fumoto et al,Neuroscience Research, 50, 2004, 307-317). In the present study, we investigated EEG (Cz,Pz,Oz), and CBF in the prefrontal cortex as assessed by changes of hemoglobin oxygenation using 24-channal near-infrared spectroscopy(NIRS) in the healthy subjects during and after DT breathing. Blood serotonin(5-HT) levels were measured using a HPLC system. As a result, we found the augmentation of HF-alpha power on EEG during and after DT breathing. DT breathing evoked a gradual increase of CBF in the prefrontal cortex. Blood 5-HT levels increased after DT breathing. So we suggest that prefrontal cortex are activated by DT breathing, which cause augmentation serotonergic system of brain stem, in turn induce increase alphapower on EEG. [J Physiol Sci. 2008;58 Suppl:S119]

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We studied effects of vagotomy on the ventilatory response to hyperoxic hypercapnia in the awake neonatal rat at P0-3. The vagus nerves were cut bilaterally at mid-cervical level. Each rat was exposed successively to four levels of inspired CO ₂ (0, 3, 6, 9% CO ₂, mixed with 50% O ₂, balance N ₂). Tidal volume and respiratory frequency were determined by direct plethysmography. Under control conditions (room air), the breathing pattern, respiratory frequency and tidal volume were not strictly different between the vagus-intact and vagotomized neonatal rat. In the vagus-intact neonatal rat, the progressive hyperoxic hypercapnia increased both respiratory frequency and tidal volume. In the vagotomized neonatal rat, though the progressive hypercapnia increased tidal volume, this stimulus greatly decreased the respiratory frequency less than half. As a result, the increment of the minute ventilation evoked by hypercapnia was lower in the vagotomized neonatal rat than the vagus-intact neonatal rat. We also observed the vagotomized neonatal rat showed mouth-opening gasp-like breathing under these hypercapnic conditions. Thus the vagotomy affected not only respiratory frequency and volume control, but also breathing motor pattern at this neonatal period. We concluded that afferent feedback from the lung stretch receptor would be essential for the respiratory networks to organize proper respiratory motor pattern and keep proper lung ventilation in response to hypercapnia. [J Physiol Sci. 2008;58 Suppl:S119]

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The molecular and cellular mechanisms underlying experience-dependent plasticity of brain function are poorly understood. Recent in vitro studies have identified the regulated trafficking of AMPA receptors (-Rs) into synapses as a major molecular component of neural plasticity. Moreover it was shown that experience-driven plasticity in the developing rat barrel cortex is accompanied by and/or requires AMPA-R delivery to synapses. By combining in vivo gene delivery with in vitro recordings, experience drives recombinant GluR1, an AMPA-R subunit, into synapses formed between layer 4 and layer 2/3 neurons. These studies show that synaptic delivery of AMPA-Rs contributes to plasticity driven by natural stimuli in the mammalian brain.Although social isolation early in life has been shown to alter behaviors including learning and emotion, how neonatal isolation affects synaptic connection is poorly understood. Since whisker-barrel system is involved in social interaction, we examined if social isolation changes experience-dependent synaptic delivery of AMPA receptors in the developing rat barrel cortex. We found that neonatal social isolation disrupted in vivo trafficking of GluR1 and GluR4 into synapses formed from layer 4 to layer 2/3 of developing rat barrel cortex. These effects of social isolation are blocked by administration of the glucocorticoid receptor antagonist. Furthermore, injection of corticosterone mimics these effects of social isolation. [J Physiol Sci. 2008;58 Suppl:S119]

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Sensory deprivation in one modality can have striking effect on enhancement of the remaining modalities. However, little is known about the cellular and molecular mechanisms underlying this type of plasticity. Recent studies in brain slices have identified the regulated trafficking of AMPA receptors (-Rs) into synapses as a major molecular component of neural plasticity. We previously reported that whisker experience can drive AMPA-Rs into synapse in developing (PND 12 14) barrel cortex (Takahashi et al, 2003). Here we examined the trafficking of AMPA-Rs into synapses in the visually-deprived rat barrel cortex. Layer 2/3 pyramidal neurons were infected with Herpes viruses driving expression of GluR1 in PND21 rats in vivo. Animals were returned to their home cages for 2 days, either with their eyes intact or sutured. Subsequently, brain slices were prepared from these animals and transmission between layer 4 and layer 2/3 was examined. We found that recombinant GluR1 was delivered to synapses in rats with visual deprivation but not in intact rats, and these effects were mediated by an activation of serotonergic system. [J Physiol Sci. 2008;58 Suppl:S120]

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Corticospinal axons show regressions during development but the manner of regressions of those axons after innervation into the spinal gray matter is largely unknown. We examined the regressive events observed in the corticospinal slice coculture by way of confocal time-lapse imaging of single axons labeled with EYFP. We made daily or short interval (30 to 60 min) image acquisitions from 5 to 13 DIV. From daily images, we obtained (1) gradual shortening, (2) amputation of the branches, and (3) disappearance of large part within a day. Acquisitions at shorter intervals allowed us to analyze retractions and degenerations in detail. Retractions occurred intermittently: it retracted at a certain constant rate for a period and stopped, and then resumed retraction at the same rate and stopped again. The rate of retraction was calculated to be 224 ± 42 μm/day (mean ± s.e.m.), which was consistent with the gradual shortening observed in daily images. Degeneration occurred on isolated axons and the part undergoing degeneration disappeared in 3-4 hours. Degenerating axons showed bright beaded spots arranged periodically. Those bright spots changed fluorescence intensity during degeneration but individual spot did not alter its position. The present observation and its detailed analysis on retraction and degeneration provide important clues to understand mechanisms of regressive events in CNS axons. [J Physiol Sci. 2008;58 Suppl:S120]

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We showed the rat corticospinal (CS) synapses were formed all the gray matter at P6-7; but those in the ventrolateral area were eliminated from P8 to P10. However, after this synapse elimination, the "second wave" of the CS innervation arrived at P12: CS terminals again increase on the ventral and also dorsal side. In this study, the CS synaptic responses were directly shown in the acute cervical cord slices by optical imaging with the voltage sensitive dye. The ventralmost part of posterior funiculus was stimulated to activate CS axons and the response signal was recorded. The responses were disappeared after an electrolytic lesion made by DC current. The optical response in the VL area once peaked at P6-7, but reduced at P10, and increased again from P15. To study developmental change in corticoceptive spinal neurons, spinal cord cells were whole-cell patch clamped and electrophysiological properties were studied. CS-EPSC-positive cells were labeled with Neurobiotin^TM, which were later reconstructed on the two-dimensional plain by camera lucida drawing. Responsive neurons are widely distributed in the P6-7 neonates from the laminae III to IX, but neurons in the laminae IX became unresponsive at P10. The majority of the ventral neurons have multipolar, long and smooth dendrites with large (20-30 μm in the diameter) somata showing "repetitive" (trains of multiple spikes) or "phasic"(decreasing spike trains) firin. On the other hand, most of dorsal neurons have smaller (10-20 μm) somata and multipolar branches, and they have "repetitive", "phasic", or "delayed onset" firing. [J Physiol Sci. 2008;58 Suppl:S120]

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Type-1 metabotropic glutamate receptor (mGluR1) in cerebellar Purkinje cells (PCs) plays a central role in induction of cerebellar long-term depression (LTD), a form of synaptic plasticity crucial for cerebellar motor learning. We previously revealed complex formation between mGluR1 and B-type γ-aminobutyric acid receptor (GABA_BR) in PCs. Here we examined in cultured mouse PCs whether and how GABA_BR influences LTD of the postsynaptic glutamate-responsiveness (LTD_glu) that is induced by conjunctive dendritic glutamate application and somatic depolarization. GABA and baclofen, GABA_BR agonists increased the magnitude of LTD_glu. This effect was mimicked by mastoparan, a G_i/o protein activator and abolished by pertussis toxin, a G_i/o protein inhibitor. Baclofen augmented Ca²⁺ release from the intracellular stores, a response coupled to mGluR1 via phospholipase C. These findings suggest that GABA_BR enhances LTD_glu by potentiating the downstream signaling of mGluR1 via G_i/o protein. Moreover, in mouse cerebellar slices, CGP55845, a GABA_BR inhibitor decreased the magnitude of LTD at parallel fiber-PC synapses. GABA_BR-mGluR1 crosstalk may regulate the extent of cerebellar LTD in situ. [J Physiol Sci. 2008;58 Suppl:S120]

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The neurosteroid dehydroepiandrosterone sulfate (DHEAS) is known to improve memory and learning, however, little is known about the underlying synaptic mechanism. We examined the effects of chronic administration of DHEAS (20mg/kg for 7 days) on the plasticity of Schaffer collateral-CA1 synapse using an optical recording technique on rat hippocampal slices stained with a voltage-sensitive dye. DHEAS significantly facilitated the induction of frequency-dependent LTP. While tetanus of at least 50 pulses (at 100 Hz) was required to induce LTP in control rats, only 20 pulses were needed in DHEAS-treated animals. DHEAS did not alter the presynaptic glutamate release. Co-administration of the sigma 1 receptor antagonist NE100 with DHEAS perfectly inhibited the DHEAS-facilitated LTP. In slices from the DHEAS-treated rats, the NMDA-induced intracellular Ca²⁺ ([Ca²⁺]i) increase in CA1 pyramidal neurons was significantly potentiated. The sub-threshold tetanus markedly increased the tyrosine phosphorylation of Src in the DHEAS-treated rats, which critically depended on the potentiated [Ca²⁺]i increase. Conversely, the Src family kinase inhibitor PP2 attenuated the NMDA-induced [Ca²⁺]i increase and abolished the DHEAS-facilitated LTP, suggesting that the potentiated NMDAR function and Src activation are interdependent. This novel postsynaptic NMDAR-mediated signal amplification through [NMDAR/Ca²⁺-Src-NMDAR/Ca²⁺] cycle may play a pivotal role in the DHEAS-facilitated LTP induction. [J Physiol Sci. 2008;58 Suppl:S121]

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Neurons in avian nucleus magnocellularis (NM) receive synaptic inputs from auditory nerve fibers, and extract the timing information of sounds. NM is tonotopically organized such that characteristic frequency (CF) of neurons decreases from rostro-medial (high CF) to caudo-lateral (low CF) direction within the nucleus. NM neurons show several CF-dependent specializations, including properties of postsynaptic membrane and synaptic transmission; high-CF cells express K channels abundantly and receive a few large end-bulb synapses, while K channels are scarce and multiple small bouton synapses converge in low-CF cells. These specializations are crucial for encoding the timing information precisely at each CF. On the other hand, NM neurons show a systematic difference in the amplitude of spikes depending on the CF and it becomes smaller toward the low CF. This may indicate that the process of generating spikes is also specialized along the tonotopic axis. In this study, we examined electrophysiologically and immunohistochemically the distribution and the properties of Na channels in NM neurons along the tonotopic axis using the chicken brainstem slices. We found that NM neurons accumulated Na channels at the proximal axon and varied their distribution in a CF-dependent manner; they were more abundant at the low CF. Computer simulation showed that the abundance of Na channels in the low-CF neurons was crucial to increase the reliability and precision of spike generation during the convergence of multiple small inputs. [J Physiol Sci. 2008;58 Suppl:S121]

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Interaural time difference (ITD) is a major cue for sound source localization. However, the ITDs experienced by animals with a small head are extremely small, making ITD detection difficult particularly for the low-frequency sound. A precisely-timed inhibition is proposed to overcome this disadvantage in some mammals. Here, we found a sound intensity dependent mechanism of compensation for the small ITD cue in the coincidence detector neurons in nucleus laminaris (NL) of the chicken by in vivo unit recordings and the mechanism was confirmed by simulation. Loud sound made the contrast of ITD tuning robust in the low-frequency NL neurons by suppressing the firing activity at the worst-ITD. In contrast, the suppression was weaker in the middle- to high-frequency NL neurons. The contrast of ITD tuning was maximal at the intermediate sound intensity and was lost at the loud sound because of the increase of firing activity at the worst-ITD. The suppression of firing activity was through the activation of superior olivary nucleus (SON) and was eliminated by electrolytic lesion of SON. The frequency dependency of suppression reflected the robust projection of SON to low-frequency NL. Consequently, the small ITD cue in the low-frequency sound was compensated by the sound intensity dependent inhibition from SON and the ITD sensitivity was made nearly constant across frequencies. [J Physiol Sci. 2008;58 Suppl:S121]

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Transient rise of Ca²⁺ at presynaptic nerve terminals triggers transmitter release. Amplitude, kinetics and distribution of Ca²⁺ transient within the nerve terminal regulate release modality. At the calyx of Held, the giant presynaptic terminal in auditory brainstem, injection of EGTA into the terminal attenuated EPSC amplitude at postnatal day (P)7. This effect of EGTA was diminished at P14, suggesting that Ca domain size shrinks during development. However characteristics of Ca²⁺ transient within the nerve terminal are poorly understood and its developmental profile remains elusive. To investigate these issues, we tried imaging for a single action potential-evoked Ca²⁺ transient using a low affinity Ca²⁺ indicator Oregon Green BAPTA/5N. Confocal spot measurement enabled detection of Ca²⁺ gradients within the terminal. Ca²⁺ transients were mainly observed at the synaptic site of the terminal, but the spatial distribution was sparser in older animals (P13-14 vs. P7-P8). Amplitude of the transient was smaller in older animals. In contrast, the rise time was determined by action potential duration, and decay time was regulated by Ca²⁺ buffer concentration at the both age groups in similar manner. At P7 reducing functional Ca²⁺ channel number by applying ω-AgTX diminished the effect of EGTA injection on EPSC. These results indicate that decrease in Ca²⁺ channel density contributes to developmental shrinkage of Ca²⁺ domain size at this nerve terminal. [J Physiol Sci. 2008;58 Suppl:S121]

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The quantal nature of synaptic transmission has been demonstrated at various types of synapses. Although quantal neurotransmitter release is assumed to underlie the quantal nature of synaptic transmission, it has not been directly examined to date. In this study, we imaged glutamate release at single synapses for quantal analysis by using a fluorescent glutamate probe called EOS. Glutamate release evoked by a single action potential was successfully detected at single synapses in cultured hippocampal neurons. Also, successes of release were distinguishable from failures. The rate of success and the mean amplitude of EOS signals were well fitted with a binomial model, indicating that release events consist of quantal components. Furthermore, the fitting parameters suggest that single synapses typically contain multiple release units. Histogram analysis of glutamate release indicates that release events consist of an integral number of quanta. We conclude that quantal release of glutamate underlies the quantal nature of synaptic transmission at glutamatergic synapses. [J Physiol Sci. 2008;58 Suppl:S122]

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We addressed the mechanism ofsynaptic modulations induced by changes in the presynaptic membrane potential, using simultaneous pre- and postsynaptic whole-cell recordings at the calyx of Held in rat brainstem slices. A weak sustained depolarization (from -70 mV to -60 mV) of calyceal presynaptic terminal increased the EPSC amplitude despite that it diminished presynaptic action potential amplitude. However, as we further depolarized the terminal (to -50 mV), the amplitude of EPSC become eventually depressed concomitantly with a marked reduction in the presynaptic action potential amplitude. When the presynaptic action potential amplitude was gradually decreased by tetrodotoxin (TTX, 20-40 nM), the EPSCs amplitude remained the same as far as the action potential overshoot exceeded +10 mV, but upon further reduction of the overshoot EPSCs steeply diminished. When presynaptic Ca²⁺ currents (I_pCa), induced by an a.p.- waveform command pulse that is recorded from calyceal terminal, were used to evoke EPSCs, a weak sustained depolarization (15-20 mV, 1 sec) enhanced both I_pCa and EPSCs in parallel. Although this magnitude of depolarization did not evoke detectable Ca²⁺ currents, Ca²⁺ measurements using fluo-4 showed a clear increase in the Ca²⁺ concentration in the nerve terminal. We conclude that weak prolonged presynaptic depolarization increases Ca²⁺ into the nerve terminal, and the P/Q-type specific Ca²⁺ induced channel facilitation plays an essential role in the facilitation of transmitter release following presynaptic depolarization. [J Physiol Sci. 2008;58 Suppl:S122]

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Sensing interaural time difference (ITD) is essential for azimuthal sound source localization. In birds, bilateral sound signals first converge in nucleus laminaris (NL). NL neurons act as a coincidence detector of bilateral excitatory synaptic inputs, and encode ITD. Thus, the acuity of coincidence detection (CD) in NL neurons is critical for ITD calculation. NL neurons also receive GABAergic inputs, and these inhibitory innervations have been thought to improve the acuity of CD in NL. GABAergic inputs for NL arise mainly from the superior olivary nucleus (SON). SON neurons increase their activity with the increase of sound intensity, but poorly hold timing information. Therefore, SON neurons are thought to supply the tonic inhibition during sound presentation. Consistently, continuous application of GABA for slice preparation increases the shunting conductance of NL neurons, and improves the acuity of CD in NL. On the other hand, the precisely-timed inhibition is proposed to enhance ITD discrimination in mammals, but such inhibition has not been found in NL of birds. In this study, we show the possibility that NL neurons of birds also receive the precisely-timed inhibition, and this inhibition modulates the acuity of CD in NL. [J Physiol Sci. 2008;58 Suppl:S122]

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SNAP-25 is thought to play a key role in synaptic vesicle fusion for neurotransmitter release. PKC can phosphorylate SNAP-25 at Ser 187, but its outcome is yet unclear. We have addressed this question at the calyx of Held synapse, using knock-in mice with their Ser 187 of SNAP-25 being replaced by Ala. Between wild-type and knock-in mice, no significant difference was found in the amplitude, kinetics or frequency of spontaneous miniature (m) EPSCs. Although the mean amplitude of evoked (e) EPSCs in knock-in mice was slightly larger than that in wild-type mice, neither the transmitter release probability nor the size of readily releasable pool of synaptic vesicles was different.Interestingly, the PKC activator PDBu showed significantly weaker effect in increasing the mEPSC frequency in the knock-in mice compared with the wild-type mice. However, the magnitude of increase in the eEPSC amplitude by PDBu was comparable between knock-in and wild-type mice. At the calyx of Held presynaptic terminal, PDBu enhanced immuno-cytochemical colocalization of SNAP-25 and the vesicle marking protein in wild-type mice, but not in knock-in mice. We conclude that phosphorylation of SNAP-25 may enhance spontaneous interactions of SNAP-25 with vesicular proteins, thereby enhancing spontaneous fusions of synaptic vesicles in the nerve terminal. [J Physiol Sci. 2008;58 Suppl:S122]

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Neurotransmitters are released from presynaptic nerve terminals in two different ways, stimulus-evoked release and spontaneous release. It has been shown that a synaptic vesicle protein, synaptotagmin (syt) 1 serves as a Ca²⁺ sensor and synchronizes evoked transmitter release with a presynaptic action potential. However, a role of this protein for Ca²⁺-regulated spontaneous transmitter release remains uncertain. To address this question, we have studied spontaneous release from syt 1-deficient hippocampal neurons cultured on microislands. There were no significant differences in spontaneous release between wild-type and syt 1-deficient neurons, although the synchronous evoked release was abolished in syt 1 null neurons, consisting with previous results (Cell 79: 717). However, when null neurons were transfected with syt 1 harboring a mutation in either Asp309 or Asp363 in its Ca²⁺-binding domain, the frequency of spontaneous release was greatly reduced, but not its quantal size. Decreased frequency in spontaneous release is not due to less synapses in neurons transfected with syt 1 mutants, because the number of synapses were similar in both of wild-type neurons and those expressing mutants. These results indicate that spontaneous synaptic vesicle fusion at resting status are apparently regulated by Ca²⁺ binding to Asp309 and Asp363 of syt 1. Thus, syt 1 plays an important role in Ca²⁺-regulated spontaneous neurotransmitter release as well as evoked transmitter release. Supported by KAKENHI (18800027) [J Physiol Sci. 2008;58 Suppl:S123]

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In the literature, synaptic vesicle endocytosis has been attributed to three pathways: (1) kiss-and-run, (2) rapid endocytosis and (3) classical (clathrin-mediated) endocytosis. To examine endocytic pathways of superior cervical ganglion (SCG) neurons in long-term culture, we tested effects of endocytic inhibitors on synaptic transmission induced by various synaptic activities. QVPSRPNRAP (P4), a peptide of dynamin interacting with amphiphysin 1 inhibits clathrin-mediated endocytosis. Dynasore inhibits not only clathrin-mediated endocytosis but also other compensatory synaptic vesicle endocytosis. P4 introduced into the presynaptic neuron showed no change in the EPSP amplitudes evoked by paired action potentials (APs) with the interval of 20-2000 ms. However, P4 reduced EPSP amplitudes during repetitive firing at 5, 10, 20 and 30 Hz for 2 sec and slowed down the recovery after a train of APs at 5 Hz for 5 min. P4 gradually reduced the amplitude of EPSPs evoked by longer repetitive firing, even low frequency at 0.2 Hz for 60 min. Control scrambled peptide showed no reduction. Dynasore remarkably reduced EPSP amplitudes with various pattern of stimulation. These findings suggest that use-dependent endocytic pathways are induced in cultured presynaptic SCG neurons. [J Physiol Sci. 2008;58 Suppl:S123]

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We identified a novel ubiquitin ligase (E3), Kspot as a target molecule of a high titer autoantibody produced in the serum of a patient with stomach can cer, associated with psychiatric symptoms.Kspot is highly conserved througho ut species and expressed in the mammalian nervous system. In this study we d emonstrated that Kspot formed an SCF complex and mediated ubiquitination of cellular proteins. Furthermore, overexpression of Kspot in primary cultured rat hippocampal neurons resulted in a decrease in the miniature excitatory p ostsynaptic current frequency. We propose that Kspot plays an important role in the regulation of the neurotransmitter release at the presynaptic site. [J Physiol Sci. 2008;58 Suppl:S123]

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Syntabulin, a vesicle cargo-protein interacting with kinesin-1, transports syntaxin-1 and mitochondria in the axon to the presynaptic terminal. To examine functional significance of syntabulin as a vesicle cargo in presynaptic neuron, synaptic transmission was examined at a cholinergic model synapse formed between rat sympathetic ganglion neurons in culture. Expression of the kineshin-1 binding domain of syntabulin (KBD) in the presynaptic neuron should disrupt the interaction and significantly reduced the incidence of synaptic coupling, measured by recording excitatory postsynaptic potentials (EPSPs), on 10, 17 and 24 days in culture. This finding suggests that syntabulin plays an important role in the synapse development. Furthermore, in long-term cultured neurons, transfection of KBD in presynaptic neurons reduced the amplitude of EPSPs (48%) in comparison with that in control synapses transfected with EGFP-DNA. In general, the synaptic transmission reduces with repetitive stimulations. KBD transfection accelerated the reduction of synaptic transmission with high frequency stimulation at 1 Hz, but not with low frequency stimulation at 0.3 or 0.1 Hz. In addition, the recovery of EPSP amplitude after depletion of synaptic vesicles in the presynaptic terminal with train stimulation at 5 Hz for 4 min remarkably slowed down in the KBD expressed synapse. Injection of ATP reversed the slowed down recovery. These findings suggest that syntabulin interacting with kinesin-1 functions in a mature synapse to maintain synaptic transmission. [J Physiol Sci. 2008;58 Suppl:S123]

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GABA neurons make widespread connections within neuronal networks and thus are capable of controlling network oscillations and their patterns in the widely divergent species. In the terrestrial mollusk Limax valentianus, oscillatory activity of the procerebrum (PC) neurons is considered to encode the odor information. Previous studies of immunohistochemistry and electrophysiology showed that GABA is present in the CNS and may be involved in control of neuronal activity of Limax. In the present study, we have applied immunohistochemical methods to the sectioned preparations of Limax CNS to identify GABA-like immunoreactive neurons and to assess the spatial patterns of innervations by these cells in the CNS. The neuronal cell bodies and fibers showing GABA-like immunoreactivity were distributed widely throughout the CNS, and they were also extensively observed near the PC. Then, we examined the GABAergic effects on the generation of local field potential (LFP) in the PC and on the neuronal activity in the PC neurons. Application of GABA-receptor antagonists to the whole CNS decreased the frequency of the oscillatory neural activity as a periodic LFP. On the other hand, application of GABA-receptor agonists increased or decreased the LFP frequency. In cultured PC neurons, increased Ca²⁺ concentration was often observed by the application of GABA. These results suggested that the GABAergic synaptic transmission is involved in the oscillatory neural network of the PC in Limax and may play excitatory roles in the LFP generation. [J Physiol Sci. 2008;58 Suppl:S124]

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S100B is predominantly synthesized by astrocytes and secreted to the extracellular space. The secreted S100B exerts various effects through the activation of receptor for advanced glycation end-products (RAGE). We have previously shown that the amplitude of kainate-induced gamma oscillation in hippocampal CA1 stratum radiatum is significantly smaller in S100B knockout (KO) mice.To assess the contribution of extracellular S100B to the gamma oscillation, we performed local field potential recording with local infusion of S100B at CA1 str. radiatum in S100B KO mice in vivo. While local infusion of 10 μM S100B resulted in an increase of the gamma oscillation, rescuing S100B knockout effect. Post-hoc immunohistochemical observation indicated that the infused S100B was not taken up by astrocytes. In a separate set of experiments, we locally applied anti-S100B antibody for functional blockade in wild-type (WT) mice, resulting in a decreased amplitude of the gamma oscillation. Both results indicate that the presence of extracellular S100B contributes to the increased gamma oscillation. Next, we asked whether the effect of extracellular S100B was due to activation of S100B receptors. The amplitude of kainate-induced gamma oscillation at CA1 str. radiatum decreased significantly by blocking RAGE in WT mice. Furthermore, co-application of S100B and anti-RAGE antibody in S100B KO mice abolished the effect of S100B on the gamma oscillation. These data suggest that activation of RAGE significantly contributes to the increased gamma oscillation. [J Physiol Sci. 2008;58 Suppl:S124]

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In order to clarify the effect of age-related change in trigeminal nociception advancing age, phosphorylation of extracellular signal-regulated kinase (ERK) in trigeminal spinal subnucleus caudalis (Vc) neurons was studied in the aged rats following subcutaneous capsaicin injection into the whisker pad and the following results were obtained. Adult (9-12 months) and aged (30-34 months) Fischer male rats were used for the present study. Rats were injected with 10mM capsaicin into the right whisker pad subcutaneously and naloxone was injected intravenously 20 min before capsaicin injection. The mechanical escape threshold was significantly lower in aged rats compared with adult rats. A large number of pERK-LI cells were expressed in the superficial laminae of Vc in adult and aged rats following subcutaneous capsaisin injection into the whisker pad region. The number of pERK-LI cells was largest at about 2.0 mm caudal from the obex and gradually decreased in their numbers in more rostral and caudal sections. The rostro-caudal distribution profile of pERK-LI cells expressed after subcutaneous capsaicin injection into whisker pad was similar in adult and aged rats. The number of pERK-LI cells was slightly larger in aged rats compared with that of adults. The larger number of capsaicin-induced pERK-LI cells was expressed in adult rats than aged rats following intravenous administration of naloxone. The present findings suggest that the descending modulation system was impaired advancing age, resulting in the abnormal pain sensation in aged rats. [J Physiol Sci. 2008;58 Suppl:S124]

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Dysfunction of noradrenaline (NA) system in the brain is known to be closely related to the etiology of attention-deficit hyperactivity disorder (AD/HD). To determine whether the release of NA in the locus coeruleus (LC), the main source of NA in the brain, is also enhanced in the SHR compared to WKY, the NA content in the homogenate LC tissue and the extracellular levels of NA in the LC were quantified in the present study. The NA content in the homogenate LC tissue obtained from rats strictly at the period of juvenile (5 week-old), was measured by HPLC. Next, the extracellular levels of NA in the LC were measured at the period of juvenile (5 week-old) and adolescent (WKY: 12 week-old, SHR: 14 week-old), by using in vivo microdialysis, following the determination of the location of the LC. The results showed that the NA content in the homogenate tissue of the LC was significantly higher in SHR than that of WKY in juvenile animals, however, the significant difference was not detected in the extracellular levels of NA in the LC between SHR and WKY in both juvenile and adolescent animals. In conclusion, the present results indicate that the rate of NA synthesis in the LC is increased in SHR compared to that in WKY, whereas both release and reuptake of NA similarly function in these two strains or these functions are accelerated in SHR compared to WKY. [J Physiol Sci. 2008;58 Suppl:S124]

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It is known that exposure to stress during pregnancy causes behavioral disorders of offspring when they grow up. Exposure of glucocorticoids to fetus may be one of factors responsible for these impairments, since glucocorticoids affect neuronal proliferation, differentiation and maturation. Several studies suggest that amino acids important for CNS development fetal period. We have recently shown that GABA may play a pivotal role in radial migration of neocortical neurons. The fetal vulnerability to maternal stress is known to increase in this period. Thus, we hypothesized that maternal stress may affect GABA-related neocortical development. To investigate this GAD67-GFP knock in mice, in which GABA concentration is different by genotype, were used for maternal stress model. As the maternal stress, the restraint and light exposure were performed on pregnant mice three times per day for 3 days from embryonic day 15. Maternal and fetal body weights were significantly decreased after the stress. To confirm the effect of the maternal and fetal stress on a physiological parameter, serum corticosterone levels were measured by means of the radioimmunoassay method. The maternal serum corticosterone was significantly increased by the maternal stress. Fetuses from the stressed mice exhibited significantly higher levels of corticosterone than those of fetus obtained from non-stressed control mice. Thus the fetuses affected by the maternal stress could be useable for the "stress in utero" model. [J Physiol Sci. 2008;58 Suppl:S125]

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The δ2 glutamate receptor (δ2 receptor) is predominantly expressed at parallel fiber (PF)-Purkinje cell (PC) synapses. Knock-out of the δ2 receptor gene causes abnormal phenotype such as loss of LTD induction, impaired synaptogenesis between PF and PCs and persistent innervations of PCs by multiple climbing fibers, resulting in severe ataxia. The δ2 receptor is classified in ionotropic glutamate receptors; however, it has been elusive whether the δ2 receptor is activated by glutamate or related amino acids and working as a cation-permeable ion channel. We produced a δ2 receptor deletion construct that lacks conserved glutamate binding domains (S1 and S2) and ion-channel-pore-forming transmembrane domains (TM1–TM3). The construct (NTD-TM4-CTD) consists of the extracellular N-terminal domain (NTD), TM4 and intracellular C-terminal domain (CTD). Using lentiviral vectors, the NTD-TM4-CTD was expressed in P6 PCs of δ2 receptor-deficient mice, and the mice were analyzed at P25–P35. Surprisingly, the rotarod performance was significantly superior in infected mice to their non-infected littermates. Patch-clamp analysis showed that innervation of δ2-receptor-null PCs by multiple climbing fibers was significantly rescued by NTD-TM4-CTD expression. These results indicate that the δ2 receptor is neither a glutamate receptor nor an ion channel, but a membrane protein that is associated with cerebellar synapse formation presumably by interacting the extracellular NTD with an unidentified ligand. [J Physiol Sci. 2008;58 Suppl:S125]

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Most GABAergic interneurons of the rodent cortex originate from the ganglionic eminences (GEs). During perinatal days, they migrate tangentially through the marginal zone (MZ) and intermediate zone/subventricular zone (IZ/SVZ) towards the cortex. After reaching the cortex, they migrate into the cortical plate and incorporate into their appropriate laminar position. To investigate the dynamics of the migrating interneurons in living immature animal, we have developed in vivo time-lapse imaging with two-photon microscopy employed on immature transgenic mice expressing Venus in interneurons. At first, to establish the time-lapse imaging of immature mouse neocortex neurons, we designed for the specialized experimental preparations. For example, we developed four-direction restraint bars (front, back, left, and right side of the head) to alleviate heartbeat and breathing which are suitable for immature mouse. Before time-lapse imaging, to identify Venus labeling neurons, we tried double-labeling immunofluorescent staining against Venus fluorescence and GABA at P60 mouse. In result, GABA staining was mostly observed in Venus positive cells (more than 90%) in neocortex. In combination with these new methods with two-photon microscopy, we could observe the multidirectional tangential migration of GABAergic interneurons in the MZ in vivo. This approach will be a tool for understand neural activity in developing intact brain. [J Physiol Sci. 2008;58 Suppl:S125]

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In general, neural signals in the neocortex propagate by way of cortical neural circuits based on NMDA and non-NMDA receptor activities. Here we investigated contribution of these receptors to determination of the signal traveling routes between the primary (Oc1) and secondary visual cortices (Oc2) of rat brain slices. Electrical stimulation was delivered to the white matter in the Oc1 under bath-application of caffeine, and spatio-temporal aspects of traveling signals were observed by using optical recording methods. When 1/4 Hz-stimulation was applied, signal traveled horizontally along deep layers from Oc1 to Oc2, and climbed up into the Oc2, and then returned along layer II/III from Oc2 to Oc1. On the contrary, when stimulation frequency was changed to 1/64 Hz after 1/4 Hz-stimulation, signal climbed up into the Oc1, and traveled horizontally along layer II/III from Oc1 to Oc2 in parallel with signal traveling along deep layers, in which signal intensity in the layer II/III was much higher than that in the deep layers. The traveling signal in the former way was NMDA receptor activity-dependent, and that in the latter was non-NMDA recepor activity-dependent. These results suggest that the visual cortex has an ability to change routes of signal traveling in the visual cortical circuits based on the relative balance between NMDA and non-NMDA receptors. [J Physiol Sci. 2008;58 Suppl:S125]

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The senile plaque, extracellular deposit of amyloid-β (Aβ), is a pathological hallmark of Alzheimer's disease (AD). Recent studies have shown that Aβ, prior to accumulating extracellularly, is increased first intracellularly and may trigger neuronal dysfunction especially in the early stage of AD. We attempted to study the functional role of intracellular Aβ, by introducing Aβ protein into pyramidal neurons through patch pipettes in rat neocortical slices. With Aβ intracellularly injected, spike-induced Ca²⁺ increases, measured with fura-2-based fluorimetry, were greater than control. However, neither did Aβ injection alter Ca²⁺ current under voltage clamp nor cause Ca²⁺ release from intracellular Ca²⁺ stores. Instead, Aβ enlarged the spike width by suppressing large conductance Ca²⁺-activated potassium channels, thus increasing spike-induced Ca²⁺ influx. Given that the formation and secretion of Aβ have been shown to depend on neural activity, the present Aβ-promoted elevation of neural excitability can lead to a positive feedback increase in production of intracellular Aβ. Such self-reinforceing increase in intracellular Aβ may well rise extracellular Aβ, thus triggering further pathological changes such as synaptic dysfunction and neuronal loss. We propose that in the early stage of AD, intracellular increase in Aβ may render neurons more vulnerable to cell death and could be causative of the disease. [J Physiol Sci. 2008;58 Suppl:S126]

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Activity-dependent internalization of glutamate receptor (GluR) is an underlying mechanism for the long-term depression (LTD) of synaptic efficacy at synapse between the parallel fiber (PF)-Purkinje cell (PC) in the cerebellum. However, translocation of AMPA-receptor at the dendritic spine has not been examined by image-analysis in PC. Here, we visualize activity-dependent change in AMPA-R distribution by using a monoclonal antibody recognizing extracellular domain of GluR2 and GFP-labeled GluR2 in cultured PC. Surface-expression of GluR2, detected by the monoclonal antibody, was dense at the dendritic spines. Chemical stimulation (40μM Glu+40mM KCl) caused internalization of GluR2, which was enhanced by latrunculin A, a blocker of actin-polymerization, while it was suppressed by jasplakinolide, a blocker of actin-depolymerization. These observations suggest that actin-depolymerization underlies the induction of the activity-dependent GluR2-internalization. Then, we analyzed time-dependent change in the spine-shape and GluR2-distribution in dendrite. Chemical stimulation (Glu+KCl) caused persistent retraction of GFP-GluR2 from the surface of dendritic spines, but the length of spines, visualized by GFP, was shortened by the same stimulation only temporally. Thus, we conclude that LTD-inducing chemical stimulation (Glu+KCl) causes release of GluR2 from F-actin-based scaffolds and translocation of them from synaptic membrane to the intracellular storage site in the dendritic shaft of PC. [J Physiol Sci. 2008;58 Suppl:S126]

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In the cerebellum, four distinct types of glutamate transporters, GLAST, GLT-1, EAAC1 and EAAT4, are distributed especially near excitatory synapses in Purkinje cells (PCs). Glial transporters, GLAST and GLT-1, are co-localized in processes of Bergmann glia wrapping excitatory synapses on PCs, whereas neuronal transporters, EAAC1 and EAAT4, are expressed in extrasynaptic regions of PCs. To clarify differential roles of these transporters in the removal of synaptically released glutamate, we analyzed the kinetics of excitatory postsynaptic currents (EPSCs) in PCs in mice lacking either glial or neuronal transporter (the mice were kindly supplied by Dr. Kohichi Tanaka). GLAST contributed to uptake of glutamate that flooded out of the synaptic cleft at early times after transmitter release. It also played a critical role to maintain one-to-one relationship at the climbing fiber-PC synapses by preventing trans-cellular glutamate spillover. GLT-1 played a similar but minor role compared to GLAST. In contrast, the main role of EAAT4 was to remove low concentrations of glutamate that escaped from the uptake by glial transporters at late times and thus prevented the transmitter from spilling over to neighboring synapses in the same cell. Furthermore, it was intimately involved in the regulation of mGluR-mediated slow EPSCs induced by repetitive stimulation of parallel fibers. The contribution of EAAC1 to the removal of glutamate was almost negligible. [J Physiol Sci. 2008;58 Suppl:S126]

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Cerebellar outputs from the deep cerebellar nuclei (DCN) are critical to the generation and control of movement. Activity of DCN neurons is mainly controlled by GABAergic inhibitory transmission of Purkinje cells in the cerebellar cortex and is also modulated by nerve inputs originated from other brain regions in and out of the cerebellum. Serotonergic nerve fibers originated from the dorsal raphe nuclei has been known to send their input onto DCN. In this study, therefore, we examined modulatory effects of serotonin (5-HT) on GABAergic synapses in the DCN. We found that 5-HT decreased the amplitude of stimulation-evoked IPSCs (eIPSC) in DCN neurons, and their effect was abolished by a 5-HT_1B antagonist, SB224289. The decrease in IPSCs amplitude was associated with increase in paired-pulse ratio of eIPSC. 5-HT also decreased the frequency of miniature IPSCs without changing their amplitude. These data suggested that 5-HT presynaptically inhibited the eIPSC amplitude. Furthermore, slow inward currents were observed in DCN neurons during 5-HT application. Pharmacological studies revealed that 5-HT possibly activated 5-HT₅ receptor positively coupled to G-protein and elicited slow inward current through acceleration of I_h channel at the membrane of DCN neurons. In summary, 5-HT released onto DCN may pre- and post-synaptically play a regulatory role in the spike generation and the gain control of inhibitory GABAergic synapses, thereby promoting the voluntary activity in DCN neurons themselves. [J Physiol Sci. 2008;58 Suppl:S126]

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Ethanol (EtOH) modulates synaptic efficacy in various brain areas, including the cerebellum, which acts for the motor learning and memory. Previous studies have reported that EtOH exposure enhances tonic inhibition of cerebellar granule cells, which is thought to be a major reason for alcohol-induced impairment of motor behavior. However, the effects of EtOH on molecular layer interneurons (MLIs), such as basket and stellate cells in the mouse cerebellum have remained unknown. Here, we found that a sedative EtOH concentration (50 mM) efficiently enhanced spontaneous activity of MLIs and increased their firing rate. The EtOH-mediated excitation was blocked by inhibitors for adenylyl cyclase and the hyperpolarization-activation cation current, I_h, suggesting that the HCN channels were activated by EtOH. Furthermore, an anesthetic EtOH concentration (100 mM) induced facilitation of miniature IPSCs, which was mainly dependent on intracellular cyclic AMP, voltage-dependent Ca²⁺ channels, and intracellular Ca²⁺ stores, but not on I_h or PKA. Therefore, EtOH enhances GABAergic transmission onto Purkinje cells through I_h-dependent facilitation of MLI firing, and cyclic AMP-regulated and PKA-independent enhancement of Ca²⁺-triggered GABA release at presynaptic terminals. This dual modulation of GABAergic synaptic transmission could influence spontaneous activity of cerebellar Purkinje cells, leading to regulation of the cerebellar neuronal circuit processing. [J Physiol Sci. 2008;58 Suppl:S127]

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Cerebellum consists of ten lobules in mice and some functional differences between lobules have been reported. In this study, we focused on the developmental changes (postnatal days from P7-8, P14-18, P19-24 to P35-38) of GABA_B receptor (GABA_BR) mediated K⁺ current between lobules 3, 9, and 10. We recorded from Purkinje cells in sagittal section by whole-cell patch clamp at -70 mV in the presence of AMPA and GABA_A receptor antagonists. Upon application of GABA_BR agonist, baclofen, an outward current was evoked in all three lobules and at all developmental stages. The current amplitude showed a bell-shaped developmental change in all three lobules and the amplitude in lobule 3 was smaller than those in lobules 9 and 10 at all developmental stages. We previously showed that a similar outward current was observed only in lobule 10 by tetanic simulation of molecular layer (ML) in the presence of metabotropic glutamate receptor antagonist to block slow EPSC. The amplitude of ML-stimulation evoked outward current in lobule 10 also showed a bell-shaped developmental change. These results suggests that GABA_BR mediated K⁺ current is not qualitatively different between lobules 3, 9 and 10 but is different quantitatively, implying that there is a difference in the extend of GABA_BR mediated inhibitory effect on Purkinje cells between lobules 3, 9, and 10 during development. [J Physiol Sci. 2008;58 Suppl:S127]

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Viral-vector-mediated gene expression in a specific subset of developing cells is a useful approach for exploring nervous system development. Here we attempted to transduce developing Purkinje cells by injecting lentiviral vectors into neonatal rat cerebellum. We injected lentivectors expressing GFP into the cerebellar cortex of postnatal day 0 (P0) pups, and found robust GFP expression in Purkinje cells three days after the injection (P3). Electrophysiological analysis of P21-P23 Purkinje cells infected with lentiviral vectors at P0 showed no significant alteration compared with non-transduced Purkinje cells. Consistently, rotarod performance of P21-P23 rats treated neonatally with lentiviral vectors was very similar to that of non-treated littermates. These results suggest that infection of lentiviral vectors does not influence viability, or morphological or functional maturation of developing Purkinje cells. Interestingly, unlike mature granule cells, granule cell precursors in the external granular layer were efficiently transduced. Thus, the lentiviral vector is a versatile tool for the delivery of a foreign gene into developing neurons in the cerebellar cortex without significant influence on the process of their development, and can be used to unravel roles of genes involved in cerebellar development as well as gene therapy vector, for example, against congenital cerebellar diseases. [J Physiol Sci. 2008;58 Suppl:S127]

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In the hippocampus, it has been reported that perforant path-granule cell synapses show frequency-dependent and reversible synaptic depression; however the precise mechanism has not been elucidated. Here, we analyzed this frequency-dependent depression electrophysiologically in terms of the release probability and the pool size of synaptic vesicles. We recorded field excitatory postsynaptic potentials (fEPSPs) from medial and lateral perforant path (MPP and LPP) synapses as well as from Schaffer collateral synapses (CA1 synapses) of mouse hippocampal slices. When the stimulus frequency was increased from 0.033 to 1 Hz, fEPSPs decreased at MPP and LPP synapses, whereas it increased at CA1 synapses. Thus, both perforant path synapses exhibited clear frequency-dependent depression. During the depression, the paired-pulse ratio increased at MPP synapses, while it decreased at LPP and CA1 synapses, suggesting that the release probability decreased at MPP synapses, whereas it increased at LPP and CA1 synapses. Higher-frequency stimulation (20 Hz, 10 stimuli) further decreased fEPSPs at MPP and LPP synapses, and in this condition, CA1 synapses also exhibited depression. We also estimated the size of the readily releasable pool of these synapses and found that the vesicle pools of MPP and LPP synapses are much smaller than those of CA1 synapses. These results suggest that the frequency-dependent depression at perforant path synapses is caused mainly by the depletion of synaptic vesicles in the pool. [J Physiol Sci. 2008;58 Suppl:S127]

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Extra-synaptic NMDA-receptor (NMDA-R) -mediated plateau potential can be induced either by synaptic stimulation in the presence of glutamate transporter antagonist TBOA or by iontophoresis of glutamate in hippocampal CA1 pyramidal neurons (Suzuki et al., 2007, submitted). To investigate if the NMDA-R-mediated plateau potentials is accompanied by a rise in [Ca ²⁺]_i, we performed whole-cell recordings and Ca²⁺ imagings simultaneously from single CA1 pyramidal neurons in hippocampal slices. Neurons were loaded with Ca²⁺ indicator Fluo-4 (100μM). NMDA-R-mediated plateau potentials were induced by repetitively stimulating Schaffer collaterals or by applying glutamate iontophoretically in the presence of 10μM CNQX and 50μM DL-APV. Substantial increase in [Ca²⁺]_i accompanying the plateau potential was detected both at the dendrite and the soma. When TBOA was present, a slow rise in [Ca²⁺]_i was detected near the soma after the end of the plateau potentials. After adding Cd²⁺ and antagonists for metabotropic glutamate receptors, most of the Ca²⁺ rise was suppressed, leaving small but substantial Ca²⁺ rise in the dendrites. This Ca²⁺ elevation was abolished by applying 30μM 5,7-dCK, an antagonist for the glycine binding site of the NMDA-R. Our results show that the NMDA-R-mediated plateau potential is accompanied by substantial Ca²⁺ elevation mainly due to Ca²⁺ entry from voltage-gated Ca²⁺ channels and partly due to Ca²⁺ entry from NMDA-R channels. [J Physiol Sci. 2008;58 Suppl:S128]

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Among metabotropic glutamate receptors (mGluRs), type I mGluR modulates neuronal excitability through intracellular Ca²⁺ mobilization. Difference in Ca²⁺ mobilization between subclasses of the receptors has been reported, and this difference probably causes various neuronal modifications. In hippocampal interneurons, type I mGluRs (mGluR1 and mGluR5) have been immunohistochemically identified. The subclass-specific physiological effects of mGluRs on hippocampal synaptic transmission, however, have not been fully elucidated. In the present study, effect of type I mGluR agonist on intracellular Ca²⁺ concentration was investigated in slice preparation. DHPG increased intracellular Ca²⁺ concentration in hippocampal nonprincipal cells, and the Ca²⁺ elevation was partially inhibited by mGluR1-subtype-specific antagonist, CPCCOEt, or mGluR5-specific antagonist, MPEP. To examine the contribution of this DHPG-induced increment of cell excitability to inhibitory GABAergic signal transduction, spontaneous IPSCs (sIPSCs) were recorded from CA3 pyramidal neurons. Frequency and amplitude of sIPSCs were increased by DHPG application. This facilitation was prevented by CPCCOEt. In the absence of DHPG, perfusing CPCCOEt also inhibited sIPSCs. MPEP treatment does not markedly inhibited DHPG-induced facilitation of the sIPSCs. These results suggest that both subtypes of mGluRs are functionally expressed, and at least mGluR1 modulates IPSCs in this hippocampal area. [J Physiol Sci. 2008;58 Suppl:S128]

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