The abnormality of neonatal hippocampus may lead to the impairment of prefrontal cortex as observed in schizophrenia. Dopamine is related to such disorder. Recently the dysfunction of GABAergic interneurons has been suggested as the pathophysiology of schizophrenia. We have reported that dopamine profoundly suppresses GABAergic transmission in the neonatal rat hippocampus by a presynaptic manner (Noriyama et al., 2006). However, the effect of dopamine on spontaneous activities of GABAergic interneurons in the neonatal hippocampus is unknown. In this study, we studied the effect of dopamine on GABAergic interneurons in the neonatal mouse hippocampus by whole-cell patch clamp recordings from CA1 pyramidal cells. The amplitude of evoked GABA_A-PSCs was decreased by dopamine. In contrast to the inhibitory effect on evoked GABA_A-PSCs, the frequency and amplitude of spontaneous GABA_A-PSCs were increased by dopamine in the neonatal mice. The effect on the frequency was reduced by D1 antagonists. Dopamine had no effect on minuature GABA_A-PSCs. These results suggest that dopamine may increase the excitability of GABAergic interneurons. These complex effects of dopamine on GABAergic transmission may be related to the pathophysiology of schizophrenia. [J Physiol Sci. 2008;58 Suppl:S128]

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HPC-1/syntaxin1A is believed to play an important role in the exocytosis of synaptic vesicle. Previously, we have produced HPC-1/syntaxin1A knock-out mouse. In homozygous mutant (KO) mouse, latent inhibition of cued fear memory was attenuated. Surprisingly, the attenuation of latent inhibition in KO mouse was recovered by administration of 5-HT_2A-receptor agonist or SSRI, suggesting serotonergic disturbance. In order to examine the effect of 5-HT or cAMP, which is regulated via 5-HT_2A-receptor, on hippocampal neurons from KO mouse in vitro, hippocampal slices at 7days in vitro (DIV) and dissociated hippocampal neurons at 14DIV were treated with 5-HT or Sp-cAMPS. Then, they were fixed and immunostained for synaptophysin or synapsin. Among all groups, no difference was observed between wild type (WT) and KO neurons in the number of puncta of synaptophysin in both slice and dissociated cultures. On the other hand, the number of puncta of synapsin in dissociated KO neurons treated with Sp-cAMPS was lower than that in WT neurons. In the 5-HT-applied group, the number of puncta of synapsin was not different between genotypes. Implication of 5-HT signaling and behavioral abnormalities in HPC-1/synataxin1A knock-out mouse will be discussed. [J Physiol Sci. 2008;58 Suppl:S128]

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AAV vectors have gained a preeminent position in the field of gene delivery to the brain through their ability to achieve extensive and long-term transduction of neurons with no apparent toxicity. Although various AAV serotypes have recently become available, the transduction patterns of AAV serotypes within the hippocampal neurons have not yet been clarified. In this study, we set out to compare the expression patterns of enhanced green fluorescent protein (EGFP) in the adult mouse hippocampus by using titer-matched AAV vectors. Recombinant AAV serotypes 2, 8, and 9 incorporating chicken beta-actin (CAG) promoter-driven EGFP expression cassette were purified by an ion-exchange chromatography. By biophotonic imaging, the neuronal type affected viral tropism and transgene distribution. The EGFP expression levels with AAV8 were greater than that with the recombinant AAV2 or AAV9 in pyramidal neurons and granule cells, whereas interneurons were inefficiently transduced with the AAV8 vector. These results suggest that vectors based on distinct AAV serotypes can be adequately chosen for specific applications in the hippocampal neurons. [J Physiol Sci. 2008;58 Suppl:S129]

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Electrophysiological interaction between GABAergic transmission and encapsulate astrocytes is still unclear. Astrocytes could be affected by GABA spillover that could attribute to regulating extracellular Cl⁻ of synaptic clefts by causing passive and/or active Cl⁻ transport in themselves. To characterize GABA-induced currents and [Cl⁻]i alterations in CA1 astrocytes, whole cell patch-clamp recordings and simultaneous Cl⁻ imaging using fluorescent Cl⁻ indicator MEQ were obtained from mice (P17-30) hippocampal slices. The Cl⁻ concentration in intracellular solution was adjusted to physiological condition (40 mM). GABA-induced inward currents were observed from -100 to +40 mV. Picrotoxin (PTX) reduced, but failed to abolish these inward currents. Additional application of GABA transporter (GAT) inhibitors abolished the residual slow decay currents, suggesting that the GABA-induced inward currents were consisting of GABA_A receptor and GAT mediated currents. Cl⁻ imaging using MEQ revealed that GABA decreased [Cl⁻]i in a large part of cells, which was blocked by PTX. [Cl⁻]i increases were also observed in some cells accompanied by the large PTX-insensitive currents, suggesting Cl⁻ influx associated with GABA transport at GABAergic synapses. These data indicate that both GABA_A receptors and GATs activation depolarize CA1 astrocytes. Not only Cl⁻ efflux by GABA_A receptor-channels but also Cl⁻ influx by GATs exist and the Cl⁻ efflux/influx balance may differ cell by cell, hence it could modify synaptic environment. [J Physiol Sci. 2008;58 Suppl:S129]

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Our laboratory identified calcium-dependent activator protein for secretion 2 (CAPS2) as a gene differentially expressed in developing mouse cerebellum and recently showed that CAPS2 is highly related to autism, the most common neurodevelopmental disorder. CAPS2 is widely expressed in the mouse brain including cerebellum and hippocampus, and is involved in the secretion of brain derived neurotrophic factor(BDNF). BDNF secretion and the activation of its receptor TrkB are required for many of the cellular aspects during synaptic plasticity, including gene expression, protein synthesis, spine morphology and long-term preservation of synaptic transmission. Therefore, the function of CAPS2 is thought to be related to synaptic plasticity. To address this hypothesis, we examined the effect of CAPS2 deficiency on synaptic plasticity such as long-term potentiation (LTP) and long-term depression (LTD) in hippocampal CA3-CA1 synapse by comparing wild type and CAPS2 KO mice. [J Physiol Sci. 2008;58 Suppl:S129]

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The spontaneously epileptic rat (SER), a double mutant (zi/zi, tm/tm), exhibits both tonic convulsions and absence-like seizures from the age of 8 weeks. β₁ subunit plays critical roles in modulating the activation and inactivation kinetics of sodium current in epilepsy. Since the first point mutation in the voltage-gated sodium channel (VGSC) β₁ subunit in human generalized epilepsy with febrile seizures plus (GEFS+) was identified, more and more types of genetic epilepsy have been causally related to gene changes in VGSC. The present study was undertaken to detect sodium channel β₁ subunit expression and mutation analysis in hippocampus of SERs. In this study, the mRNA expression of β₁ subunit in SERs hippocampus was significantly higher than that in control Wistar rats hippocampus by RT-PCR; The protein expression of β₁ subunit as measured with immunofluorescence and western blot was significantly increased; However, no sequence mutation was detected in five exons of β₁ subunit by means of mutation analysis. Our study suggests for the first time that up-regulation of sodium channel β₁ subunit at the mRNA and protein levels of SER hippocampus that underlies the observed seizure phenotype in SER might be a secondary appearance of the epileptiform activity instead of the causal role. [J Physiol Sci. 2008;58 Suppl:S129]

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The effects of the toxin extracted from various tissues of wild and cultured puffer fish were investigated in voltage-dependent sodium current (INa) of single rat CA1 neurons, and compared the results to that of tetrodotoxin (TTX). Toxin extracts from wild puffer fish inhibited INa in a dilution-dependent manner, and the toxin extracts from liver or ovary produced 300-500 times greater inhibition than that from the muscle. The inhibition was stronger in the order of liver = ovary >>> eyeball = kidney = intestine > muscle > gills.The toxin extracted from puffer fish cultured in netcages or in tanks set up on land, in an attempt to isolate them from the food chain was also examined. The toxin extracts from these cultured puffer fish also suppressed INa, but the inhibition was much weaker, and the effects of toxin extracts were almost the same in all tissues examined including liver, ovary and muscle etc. We calculated the maximum edible amount for each tissue assuming that lethal dose of TTX is 1-10 μg / kg, and we found that liver or ovary were edible in the case of cultured puffer fish. [J Physiol Sci. 2008;58 Suppl:S130]

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The hypothalamic supraoptic nucleus (SON) is one of the major sites for synthesis of arginine vasopressin (AVP), and secretion of AVP is controlled by the electrical activities of magnocellular neurosecretory cells (MNCs), which in turn is regulated by neuronal excitatory and inhibitory synaptic inputs and humoral factors. Brain-derived neurotrophic factor (BDNF) mRNA was markedly increased by osmotic stress in the SON. In the present study, we examined the effect of hyper- and hypo-osmotic stimuli on spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from the MNCs in the rat SON, using whole-cell patch-clamp techniques in in vitro brain slice preparation. We also examined the effect of BDNF on sIPSCs recorded from the MNCs in the rat SON. (1) The frequency of sIPSCs, but not amplitude, was significantly increased by hypo-osmotic stimuli (250 mOsm/kg) after hyper-osmotic incubation (350 mOsm/kg) for more than 2 hours. This response was inhibited partially by the application of ruthenium red (10 μM), an inhibitor of the transient receptor potential vanilloid (TRPV) cation channels. (2) The frequency of sIPSCs, but not amplitude, was significantly decreased by the application of BDNF (2 nM). These results suggest that osmotic stimuli regulate the inhibitory synaptic transmission to the MNCs in the SON and BDNF may have a role on this regulation. [J Physiol Sci. 2008;58 Suppl:S130]

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Ghrelin is an orexigenic peptide discovered from stomach as a ligand of orphan G protein coupled receptor and participates in the regulation of growth hormone release. Previous studies have demonstrated that ghrelin suppressed water intake and stimulated the secretion of arginine vasopressin (AVP) in rats. The magnocellular neurosecretory cells (MNCs) in the supraoptic nucleus (SON) terminate their axons in the posterior pituitary and secrete AVP into the systemic circulation. In the present study, we examined the effect of ghrelin on the excitatory synaptic inputs to the MNCs in in vitro rat brain slice preparations, using whole-cell patch-clamp recordings. Application of ghrelin (0.1-1 μM) caused a significant increase in the frequency of the spontaneous excitatory postsynaptic currents (sEPSCs) without affecting the amplitude in a dose-related manner. The increased frequency of sEPSCs persisted in the presence of tetrodtoxin (1 μM). Ghrelin-induced potentiation of sEPSCs was significantly suppressed by previous exposure to transient receptor potential vanilloid (TRPV) blocker, ruthenium red (10 μM). These results suggest that ghrelin participates in the regulation of synaptic inputs to the MNCs in the SON and TRPV family may be involved in peptidergic modulation of sEPSCs to the MNCs in the SON. [J Physiol Sci. 2008;58 Suppl:S130]

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The amygdala and monoaminergic innervation thereonto are considered to cooperatively regulate emotional states and behaviors. In the previous study, we find that dopamine can induce a slow afterdepolarization (sADP) by activating both noradrenalin and serotonin receptors in lateral amygdala (LA) neurons. In the present experiments, we investigated how the monoaminergic inputs cooperatively induce the sADP on LA neurons by whole cell recordings in rat brain slices. Bath-application of high concentration noradrenalin (10μM) or serotonin (5μM) only induced sADP in LA neurons. This sADP lasted for more than 5 seconds and was similar to dopamine-induced sADP. Bath-application of low concentration noradrenalin (1μM) or serotonin (0.5μM) only failed to induce the full sADP. On the other hands, when low concentration noradrenalin and serotonin were combined together, they could induce the full sADP. These results suggest that monoaminergic inputs cooperatively modulate the excitability of amygdala neurons. [J Physiol Sci. 2008;58 Suppl:S130]

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An unique synaptic feature of ventrobasal (VB) thalamic relay neurons (which is a relay station of somatosensory information) is that, in addition to receiving primary sensory synapses, they receive massive feedback synapses originating from the neocortex. It is previously reported that cholinergic systems modulate neuronal activities in VB relay neurons. However, its mechanisms on synaptic transmission are not well examined. In the present study, we investigated the effects of exogenously applied acetylcholine (ACh) on synaptic transmissions of the mouse VB thalamic relay cell. Using whole-cell patch-clamp technique, bath-application of ACh (0.01-10 mM) elicited the direct postsynaptic inward current in a dose-dependent manner. Relatively high doses of ACh (1 and 10 mM) reduced amplitudes of both corticothalamic EPSCs and IPSCs mediated by thalamic reticular nucleus. On the other hand, ACh didn't affect EPSCs from the medial lemniscus tract which delivers somatosensory information. Both the direct excitation of the postsynaptic membrane and the regulation of synaptic transmissions of ACh were mimicked by a nicotinic ACh receptor agonist, but not by a muscarinic ACh receptor agonist. Thus, these results indicate that the activation of nicotinic ACh receptors resulted in reduction of non-lemniscal inputs and depolarization of relay cells in the VB thalamus. [J Physiol Sci. 2008;58 Suppl:S131]

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The nucleus tractus solitarius (NTS) is known to plays a major role in the regulation of cardiovascular, respiratory and gustatory functions. In addition, caudal NTS is believed to belong to the swallowing pattern generators, whereas the rostal NTS is part of the taste pathways. Neurotrophins, such as NGF and BDNF, promotes the development and neuronal survival mediated by activation of Trk receptor tyrosine kinases. Certain G-protein-coupled receptors (GPCRs) agonists have been shown to stimulate the tyrosine phosphorylation/trans-activation of growth factor receptors. It has been shown that activation of Trk receptor tyrosine kinases can interferes GPCRs mechanism. Voltage-dependent calcium channels (VDCCs) serve as crucial mediators of membrane excitability and calcium-dependent functions such as neurotransmitter release, enzyme activity and gene expression. We previously reported that Angiotensin 2 (Ang 2) and glutamate facilitate VDCCs mediated by GPCRs in NTS. Therefore, this study was designed to test the hypothesis that NGF and BDNF interferes with Ang 2- and glutamate-induced facilitation of VDCCs in NTS. Pretreatment with NGF and BDNF attenuated the Ang 2-induced facilitation of VDCCs but not glutamate-induced facilitation of VDCCs in NTS. NGF-induced attenuations were antagonized by pretreatment with TrkA receptor antagonist K-252a. These results indicated that NGF attenuated the Ang 2-induced facilitation of VDCCs mediated by TrkA receptor in NTS. [J Physiol Sci. 2008;58 Suppl:S131]

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The periaqueductal gray matter (PAG) has been identified as the main structure of the supraspinal pain modulation. Accumulating evidence shows that GABAergic and serotoninergic neurons in the PAG have an important role in the pain modulation. The aim of this report is to characterize glutamatergic synaptic transmission in neurons involving serotonin and/or GABA, and plastic changes of this neuronal network by neuropathic pain. We used the control ICR mouse and the mouse with partial ligation of the sciatic nerve. The synaptic current of the ventrolateral PAG (vlPAG) neurons were recorded to find NADA receptor mediated current and AMPA receptor mediated current using the whole cell patch-clamp recording technique, and then the mRNA for neurotransmitter expressed in the recorded neurons were detected using single cell RT-PCR technique. Within neuronal group not containing serotonin in the normal mouse, NMDA receptor mediated currents of GABAergic neurons were significantly larger than those of non-GABAergic neurons. There was a tendency that NMDA receptor mediated currents of neurons in the ligated mouse were larger than those of the normal mouse. These results suggest that NMDA component of the glutamatergic synaptic current is different between neurons with different transmitters and that during neuropathic pain NMDA component of the glutamatergic synaptic current increases in the vlPAG. [J Physiol Sci. 2008;58 Suppl:S131]

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Using voltage-sensitive dye recording, we surveyed neural responses related to the vagus nerve (N. X) in the embryonic chick brainstem. In our previous studies, we identified four N. X-related response areas in the brainstem. On the stimulated side, they included (1) the nucleus of the tractus solitarius (NTS) and (2) the dorsal motor nucleus of the vagus nerve (DMNV), whereas on the contralateral side, they corresponded to (3) the parabrachial nucleus (PBN: the second/higher-ordered nucleus) and (4) the medullary non-NTS region. In the present study, in addition to these areas, we identified another response area circumflex to the obex. The intensity of the optical signal in the response area was much smaller than that in the NTS/DMNV, and the spatio-temporal pattern could be discerned after signal averaging. The conduction rate to the response area was slower than that to the other four areas. Ontogenetically, the response area was distributed on the stimulated side at the 6-day embryonic stage, and it spread into the contralateral side in 7- and 8-day embryonic stages. These distribution patterns were consistent with projection patterns of vagal afferent fibers stained with a fluorescent tracer, suggesting that the response area included a primary sensory nucleus. In comparison with the functional development of the other four response areas, we traced the functional organization of N. X-related nuclei in the embryonic brainstem. [J Physiol Sci. 2008;58 Suppl:S131]

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Spontaneous correlated neural activity at early stages of development spreads like a wave and is considered to play a fundamental role in neural development. One important question is where the activity originates, especially at the earliest stage of wave expression. We assessed this issue by examining spatiotemporal patterns of the depolarization wave in the chick embryo using the optical imaging technique with voltage-sensitive dyes. We surveyed the region responsible for wave induction by applying electrical stimulation to various parts of the brain. At stage 24, in which the correlated activity first appears, propagating optical signals were evoked only when the stimulation was applied to the upper cervical cord near the obex. As development proceeded, regions responsible for wave induction extended rostrally and caudally, and reached the upper medulla and lumbosacral spinal cord by stages 27-28. When we monitored spontaneous depolarization waves, the origin of the wave was localized in the upper cervical region at stages 24-29, but was distributed through the whole spinal cord from stage 30. These results show that the correlated activity in the chick embryo initially originates in the upper cervical cord, but is generated by multiple regions at later stages. We suggest that regional and temporal differences in neuronal excitability might underlie the developmental profile of wave generation. [J Physiol Sci. 2008;58 Suppl:S132]

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The membrane properties and morphological characteristics of premotor neurons for the trigeminal motor nucleus (MoV) in the supratrigeminal region (SupV) were investigated in neonatal rat brainstem slice preparations using whole-cell recordings and immunohistochemical techniques. Premotor neurons in the SupV were identified on the basis of the intracellular calcium concentration rise in response to electrical stimulation of the MoV using a calcium imaging system. Whole-cell recordings were made from SupV neurons that responded to MoV stimulation. We divided these neurons into two groups according to their discharge pattern in response to a 1-sec depolarizing current pulse at the resting membrane potential. High-frequency firing neurons (HF neurons) showed regular high-frequency tonic firing. Low-frequency firing neurons (LF neurons) discharged at lower frequencies and displayed stronger firing frequency adaptation than HF neurons. In some HF neurons, membrane depolarization produced a rhythmical burst discharge. Biocytin was injected to the recorded neuron through patch pipettes, and the axons of biocytin-labeled SupV neurons projected in the ventral, medial-ventral or lateral-ventral directions to the MoV and terminated extensively within the MoV. These results suggest that premotor neurons for the MoV in the SupV show physiological and morphological diversity and therefore may be related to controlling the complicated and diverse motor patterns of mastication. [J Physiol Sci. 2008;58 Suppl:S132]

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It has been reported that peripheral glutamate is involved in the peripheral hyperalgia induced by an inflammation or a nerve injury. The interaction between capsaicin and glutamate receptors is thought to be somehow involved in the nerve injury- or inflammation-induced hyperalgesia. However, how the glutamates contributes to modulate the peripheral neuronal excitability is not known.The aim of the present study was to evaluate the interaction between glutamate and capsaicin receptors in the modulation of Vc neuronal excitability in rats. We divided rats into 5 groups depend on the subcutaneous injections of drugs, vehicle injection (Veh), capsaicin injection (Cap), glutamate injection (Glu), glutamate following capsaicin injection (Cap/Glu) and capsaicin following glutamate injection (Glu/Cap). The pERK immunohistochemistry was carried out in these five groups of rats. 1)The number of pERK-LI cells in all groups was significantly increased as compared to those of Veh group.2)The number of pERK-LI cells in Cap group was significantly decreased compared to Glu/Cap group, but not after Cap/Glu injection.3)The number of pERK-LI cells in Glu group was significantly increased compared to Cap/Glu group, but not after Glu/Cap injection.These findings indicate that glutamate is involved in an enhancement of the excitability of the capsaicin receptors as well as activation of glutamate receptors. [J Physiol Sci. 2008;58 Suppl:S132]

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Recent findings have indicated that a secondary neuronal damage following spinal cord injury is associated with prolonged purinergic receptor activation, which results in excitotoxicity of spinal motoneurons. In this study, we investigated the effects of extracellular ATP on lamina IX neurons of rat spinal cord slices by using the whole-cell patch-clamp technique. At a holding potential of -70 mV, ATPγS, a nonhydrolyzable ATP analog, generated an inward current, which was resistant to tetrodotoxin, in about a half of the lamina IX neurons tested. The ATPγS-induced inward current was not reproduced by α,β-methylene ATP, BzATP, UTP, or UDP, but mimicked by 2-methylthio ADP. The ATPγS-induced inward current was suppressed by the addition of GDPβS into patch-pipette solution, thus suggesting that metabotropic P2Y receptors were activated by ATPγS. The ATPγS-induced inward current was often accompanied by a significant increase in the frequency of glutamatergic spontaneous excitatory postsynaptic currents (sEPSCs). α,β-Methylene ATP did not change any holding currents, but instead increased sEPSC frequency in a subpopulation of lamina IX neurons. PPADS inhibited both the inward current and the increase in sEPSC frequency by ATPγS. On the other hand, MRS2179 blocked the ATPγS-induced inward current, but did not affect the ATPγS-induced increase in sEPSC frequency. These results indicate that extracellular ATP facilitates excitatory synaptic transmission by activating both P2X and P2Y receptors in a subpopulation of spinal motoneurons. [J Physiol Sci. 2008;58 Suppl:S132]

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Proteinase-activated receptors (PARs), PAR-1 to -4, are activated by selective proteinases which expose their new N-terminal regions as tethered ligands. PAR-1, -2, and -4 can be also activated by synthetic peptides corresponding to the individual tethered ligands without proteinases. Although PARs are reported to be involved in nociceptive transmission in peripheral terminals of primary afferents, it has not been clarified yet whether PARs are activated in the spinal dorsal horn. We previously found that PAR-1 agonist peptides, SFLLRN and TFLLR, reversibly increase the frequency of glutamatergic spontaneous excitatory postsynaptic current without a change in the amplitude in substantia gelatinosa (SG) neurons, which are thought to play a pivotal role in regulating nociceptive transmission. In the present study, we further examined the enhancement of excitatory transmission by PAR-1 agonists by applying the whole-cell patch-clamp technique to SG neurons in adult rat spinal cord slices. The presynaptic effect of SFLLRN was not seen in a nominally Ca²⁺-free solution. Similar facilitatory effects were produced by endogenous PAR-1 activating proteinases, thrombin and trypsin. All of the PAR-1 agonists tested did not alter holding currents at -70 mV. These results indicate that not only exogenous but also endogenous PAR-1 agonists enhance the spontaneous release of L-glutamate from nerve terminals in the SG, probably through Ca²⁺ entry from extracellular solution; this presynaptic action may play a role in producing nociception. [J Physiol Sci. 2008;58 Suppl:S133]

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A clinically-used analgesic tramadol reportedly not only activates μ-opioid receptors but also exhibits various actions including monoamine uptake inhibition in the CNS. Both opioids and monoamines such as norepinephrine (NE) and serotonin (5-HT) are known to modulate excitatory synaptic transmission in substantia gelatinosa (SG; lamina II) neurons of the spinal cord. In order to know which of μ-opioid, NE and 5-HT receptor activation is mainly involved in the antinociceptive action of tramadol, we examined the effects of a metabolite of tramadol, mono-O-demethyl-tramadol (M1; 1 mM), NE and 5-HT on glutamatergic excitatory transmission by applying the whole-cell patch-clamp technique to SG neurons of adult rat spinal cord slices. M1 produced an outward current mediated by μ-opioid receptors at -70 mV in about 40% of SG neurons examined; NE (20 μM) produced an outward current in a SG neuron which did not respond to M1. M1 persistently decreased the frequency of spontaneous EPSC by activating μ-opioid receptors whereas NE (20 μM) did not affect sEPSC frequency and 5-HT (40 μM) decreased sEPSC frequency, which was followed by its increase. It is concluded that M1 inhibits glutamatergic excitatory transmission in a manner different from those of NE and 5-HT. μ-Opioid receptor activation but not monoamine uptake inhibition could contribute to the antinociceptive action of tramadol at the spinal cord level. [J Physiol Sci. 2008;58 Suppl:S133]

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In line of development of inhibitory synapses, the transmission changes between the GABAergic and the glycinergic have been demonstrated in the brainstem and spinal cords. In this plasticity, it has been demonstrated that inhibitory transmitter released itself could developmentally change from GABA to glycine on the inhibitory afferent originated from the medial nucleus of the trapezoid body onto the lateral superior olivary nucleus (LSO). Because both GABA and glycine are carried into the synaptic vesicles by mean of inhibitory amino acid transporter, the transmitter released is supposed to be affected by the concentration of GABA and glycine in the terminals. Thus, we made whole-cell patch recordings in paired spinal cord culture neurons expressing both postsynaptic GABA_A- and glycine-receptors. The inhibitory postsynaptic currents (IPSCs) evoked by electrical stimulation of the presynaptic neuron, evoked IPSCs were either GABAergic, glycinergic, or GABA and glycine co-release. Perfusion with a high concentration glycine in presynaptic neuron changed GABAergic or co-release neuron to co-release or glycinergic neuron, respectively. Furthermore, the application of GABA at a 100mM into the presynaptic neurons changed the transmission to the GABAergic dominant. These results suggest that transmitter switching in development could be contributed by the change of the transmitter content in the developing presynaptic terminal. [J Physiol Sci. 2008;58 Suppl:S133]

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The spinal GABAergic system is reported to contribute to antinociceptive effects by activation of 5-HT₃ receptors. The purpose of the present experiments is to elucidate synaptic functions of 5-HT₃ receptors on GABAergic neurons in the spinal cord. Mice were anesthetized with pentobarbital. The spinal cord was removed and sectioned into 350 μm transverse slices. Under visual control using an infrared DIC microscope, whole-cell voltage clamp recordings at -70 mV were made from neurons located in the superficial dorsal horn. 2-Me-5-HT, a selective 5-HT₃ receptor agonist, was applied by local perfusion using another glass pipette placed by the recorded neuron. The neurons, which showed postsynaptic inward currents induced by 2-Me-5-HT, were sampled for single cell reverse transcription-polymerase chain reaction (RT-PCR) analysis using primers specific for GABA-synthesizing enzyme GAD67. The analysis showed that GAD67 mRNA was detected in a part of the neurons. Furthermore, in the presence of tetrodotoxin, miniature inhibitory postsynaptic currents (mIPSCs) mediated by GABA_A receptors were recorded. 5-Me-5-HT significantly increased the frequency of the GABAergic mIPSC without affecting their amplitude. These findings provide sound evidence that 5-HT₃ receptors located at the presynaptic terminals and somatic membrane control the release of GABA and might contribute to the modulation of nociception in the superficial dorsal horn. [J Physiol Sci. 2008;58 Suppl:S133]

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We have reported that melittin enhances excitatory and inhibitory transmission in substantia gelatinosa (SG) neurons of the spinal cord by activating phospholipase A₂ (PLA₂). Melittin-induced enhancement of GABAergic but not glycinergic inhibitory transmission was sensitive to a Na⁺-channel blocker tetrodotoxin, and glutamate receptor antagonists. In order to know more the difference in melittin action between GABAergic and glycinergic transmission, we examined the effect of melittin (1 μM) on GABAergic and glycinergic spontaneous IPSCs (sIPSCs) recorded under various conditions by applying the whole-cell patch-clamp technique to SG neurons of an adult rat spinal cord slice. Melittin superfused for 3 min increased the amplitude and frequency of glycinergic but not GABAergic sIPSC in a nominally Ca²⁺-free solution. The enhancement of GABAergic but not glycinergic transmission was reduced in extent by nicotinic (mecamylamine, 20 μM), muscarinic ACh-receptor antagonist (atropine, 1 μM) or α₁-adrenoceptor antagonist (WB-4101, 0.5 μM). These results indicate that glycinergic transmission is enhanced by a direct action of melittin at glycinergic synapses while GABAergic transmission enhancement produced by melittin is mediated by nicotinic and muscarinic ACh receptors, and α₁-adrenoceptors. It is suggested that PLA₂ activation modulates nociceptive transmission by enhancing each of GABAergic and glycinergic transmission in a different manner. [J Physiol Sci. 2008;58 Suppl:S134]

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Galanin, a 29/30 amino acid neuropeptide, is thought to play a pivotal role in various physiological functions including nociception. Galanin and its receptors exist in spinal dorsal root ganglion and spinal dorsal horn neurons. Although intrathecal administration of galanin results in a biphasic modulation of nociceptive behavior such that galanin at low and high doses produces nociception and antinociception, respectively, cellular mechanisms for this result have not been elucidated yet. By use of the whole-cell patch-clamp technique, we investigated the effects of galanin on excitatory synaptic transmission in substantia gelatinosa (SG) neurons of rat spinal cord slices. Superfusing galanin for 2 min dose-dependently produced an outward current at -70 mV in a range of 0.03-1 μM (EC₅₀: 0.078 μM) in about 50% of SG neurons tested. This effect was mimicked by M617, a selective galanin receptor 1 (GalR1) agonist. Galanin also dose-dependently enhanced the frequency of sEPSC without a change in the amplitude at low doses at which galanin produced a minimal outward current; this effect was maximal at about 0.03 μM and resistant to tetrodotoxin. These results indicate that galanin at lower doses enhances the spontaneous release of L-glutamate from nerve terminals while in addition to this presynaptic effect, galanin at higher doses produces a membrane hyperpolarization, possibly by activating GalR1s in the SG; these actions could contribute to at least a part of the behavioral effects of galanin. [J Physiol Sci. 2008;58 Suppl:S134]

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Tissue inflammation triggers either enhancement of nociceptive responses to mild noxious stimuli (hyperalgesia) or eliciting of pain in response to innoxious stimuli (allodynia). This hypersensitivity has been considered as sensitization of peripheral terminals of primary afferent or change in excitability of neurons in the spinal dorsal horn in activity dependent manner. In this study, how natural sensory synaptic inputs are transmitted from peripheral inflamed tissues to the superficial spinal dorsal horn was examined using in vivo patch-clamp recording techniques from substantia gelatinosa (SG, lamina II of the spinal cord) neurons and intracellular recording techniques from dorsal root ganglion neurons. In inflamed rats one to 4 days after inflammation, SG neurons exhibited spontaneous EPSCs with large amplitudes. The frequency of large amplitude of spontaneous and pinch-evoked EPSCs was much higher in inflamed than naive rats. The frequency of large amplitude of spontaneous EPSCs was decreased by topical application of NSAIDs to the inflamed skin. Although spontaneous firings were rarely observed in Aδ and C fibers in naive rats, about 50% of Aδ and C fibers generated APs spontaneously in inflamed rats. The population of C fibers having low mechanical threshold was increased in inflamed rats compared with naïve rats. The increased excitatory inputs in the SG due to sensitization of mechano-sensitive Aδ and C fibers may have an important role for the induction or development of plastic changes of spinal noxious circuits. [J Physiol Sci. 2008;58 Suppl:S134]

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The ascending pathway from the dorsal horn to the laterocapsular part (CeLC) of the central amygdala via the nucleus parabrachialis (PB) plays an important role in the pain-related negative emotion. We have already demonstrated that excitatory PB-CeLC transmission is potentiated in a manner independent of NMDA receptor components in rats with chronic neuropathic pain persisting for a week (Ikeda et al., Pain, 2007). Here we analyzed the course of the PB-CeLC transmission during the first post-operational week to reveal the establishment process of this potentiation. The L5 spinal nerve of young Wistar rats was ligated unilaterally and the paw withdraw threshold was assessed with von Frey filaments. We recorded excitatory postsynaptic currents evoked by PB afferent stimulation in the CeLC at various post-operational days (D) in the brain slices. The tactile allodynia of the hindpaw ipsilateral to the ligation appeared at D0.25 and lasted to D7. In contrast, the PB-CeLC transmission was potentiated in two steps: early (D0.25-1) and late (D2-7) phases. The former was observed in the bilateral CeLC whereas the latter was observed only in the contralateral CeLC. The potentiation at both phases remained even in the presence of NMDA receptor blockers. These results indicate that the side-specific synaptic potentiation in the CeLC resulting from enhanced nociceptive inputs via the spinal cord-PB-CeLC pathway, which is essentially contralaterally projecting, takes place at 1.5-2 days after the onset of persistent pain. [J Physiol Sci. 2008;58 Suppl:S134]

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The activation of presynaptically silent synapses is one of mechanisms underlying learning and memory. To test if this mechanism is involved in the PKC-dependent potentiation of mossy fiber (MF)-CA3 synapse in the hippocampus, exocytosis at individual MF bouton was investigate. We used a TV-42 transgenic mouse line that expresses synaptopHluorin (SpH), one of optical probes measuring exo-endocytosis, specifically in the hippocampal MF boutons. The mouse was deeply ether-anesthetized and the brain was removed. The hippocampal slices were made at 400 μm. Individual large MF boutons were identified under confocal microscopy and the activity-dependent changes of SpH fluorescence were measured. We found that the individual MF boutons are variably responsive to PKC. Among them some presynaptically silent boutons became active. It is suggested hat PKC facilitates a transition of presynaptically silent synapses to be releasable. This transition may occur by switching non releasable active zones to releasable active zone. All animal procedures were conducted in accordance with the guiding principles of NIH. [J Physiol Sci. 2008;58 Suppl:S135]

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Short-term synaptic plasticity shapes the postsynaptic response to bursts of impulses and is crucial for encoding information in neurons, but the molecular mechanisms are unknown. Here we show that activity-dependent modulation of presynaptic Cav2.1 channels mediated by neuronal Ca²⁺ sensor proteins (CaS) induces synaptic plasticity in cultured superior cervical ganglion neurons. A mutation of the IQ-like motif in the C-terminus that blocks Ca²⁺/CaS-dependent facilitation of the P/Q-type Ca²⁺ current markedly reduces facilitation of synaptic transmission. Deletion of the nearby calmodulin-binding domain, which inhibits CaS-dependent inactivation, substantially reduces depression of synaptic transmission. These results demonstrate that residual Ca²⁺ in presynaptic terminals can act through CaS-dependent regulation of Cav2.1 channels to induce short-term synaptic facilitation and rapid synaptic depression. Activity dependent regulation of presynaptic Cav2.1 channels by CaS proteins may therefore be a primary determinant of short-term synaptic plasticity and information-processing in the nervous system. [J Physiol Sci. 2008;58 Suppl:S135]

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Spinocerebellar ataxia type3 (SCA3, Machado-Joseph disease) is an inherited neurodegenerative disorder caused by an expansion of glutamine codon repeats of ataxin-3 gene. Expansion of polyglutamine repeats (polyQ) results in the misfolding of the ataxin-3 protein, thereby conferring a toxic gain of function. Previously, Yanagi and our group reported a new GTPase, CRAG that facilitates degradation of polyQ aggregates through the ubiquitin-proteasome pathway in cultured cells. To examine the potential of CRAG expression for treating SCA3, we produced transgenic mice (polyQ mice) expressing human ataxin-3 with an expanded polyglutamine stretch in Purkinje cells. The model mice showed poor dendritic arborization of Purkinje cells, a markedly atrophied cerebellum and severe ataxia. We analyzed the functional abnormalities of the cerebellum by patch-clamp. Synaptic transmission in Purkinje cells was substantially affected, and persistent multiple innervations of Purkinje cells by climbing fibers were observed in polyQ mouse. Furthermore, induction of long-term depression (LTD) at parallel fiber and Purkinje cell synapses was impaired. Despite of such severe phenotypes, cerebellar ataxia of polyQ mice was markedly improved after lentivector-mediated expression of CRAG in Purkinje cells. In this presentation, we show rescue of the electrophysiological properties in polyQ mutant cerebellum treated with CRAG. [J Physiol Sci. 2008;58 Suppl:S135]

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We previously showed that, in cultured rat hippocampal slices, repetitive induction of LTP (Long-Term Potentiation) by exposure to glutamate at 24 h intervals produced a long-lasting enhancement in synaptic strength accompanied by synaptogenesis. We call this phenomenon RISE to discriminate it from conventional single LTP. During the development of RISE, the component of Ca²⁺-permeable AMPA receptor (CP-AMPAR) in population EPSP increased transiently and the gene/protein expression of GluR1 subunit of AMPAR was enhanced in the corresponding period. Immunohistochemical analysis also showed an increase in the number of GluR1-immunopositive puncta. Therefore, it is suggested that the transiently expressed GluR1 subunit would be distributed preferentially to the newly produced synapses, forming CP-AMPAR composed of GluR1 subunit. In the present study, we examined the distribution of GluR1 during and after RISE establishment using immunoelectron microscopic technique, focusing especially on the location of GluR1 immunoreactivity and its spatial relationship to presynaptic structure. In preliminary examinations, GluR1 immunoreactivity increased predominantly in the denderitic shafts rather than in the dendritic spines in the early phase of RISE development. [J Physiol Sci. 2008;58 Suppl:S135]

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Fos B, one of the immediate early genes, belongs to the Fos family of transcription factors. Fos B has been regarded as a marker for chronic cellular activation since its activity lasts for more than days in contrast to Fos activity which lasts for hours. We reported that bilateral adrenalectomy (ADX) increased markedly the FosB/delta FosB immunoreactivity in the corticotropin-releasing hormone neurons in the PVN and vasopression neurons in the SON. In order to demonstrate that glucocorticoid suppresses expression of FosB/delta FosB immunoreactivity, the effect of glucocorticoid supplementation was examined in these nuclei following ADX. Male Wistar rats were used. The number of FosB/delta FosB immunoreactive neurons increased significantly in the PVN and SON following ADX in comparison with the sham-operated controls. The increase in the FosB/delta FosB immunoreactivity was observed at 24 h after ADX in the PVN and SON. In the PVN, the increase lasted till day 4 and subsided by day 7 following ADX, whereas in the SON, it lasted till day 2 and subsided by day 4. Dexamethasone (0.1 mg/kg/day) supplementation suppressed the enhanced FosB/delta FosB immunoreactivity in both the PVN and SON. Thus it was demonstrated that glucocorticoid suppresses FosB/delta FosB immunoreactivity in the PVN and SON in rats. Physiological meaning of the phenomena is under investigation. [J Physiol Sci. 2008;58 Suppl:S136]

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We have previously reported that chronic administration of not only corticosterone but also aldosterone facilitates spatial memory in adrenalectomized (ADX) rats. In the present study we investigated whether impairment effects on spatial memory occur after continuous blockade of MR, using a Morris water maze procedure to assess spatial memory abilities in ADX rats. The effect of continuous MR blockade was studied following oral administrations of aldosterone dissolved in daily drinking fluid (2.5 μg/ml) with or without injection of MR antagonist spironolactone(50 mg/kg, s.c.) twice a day for 10 days. All rats learned to locate the platform position during the training, as indicated by decreasing escape latencies. However, rats given chronic injection of spironoractone significantly decreased time spent in the platform quadrant during probe test. Furthermore, chronic spironolactone injection increased initial latency to cross the platform location. These findings suggest that continuous blockade of MR impairs spatial memory retrieval but not spatial memory learning. Therefore, continuous MR activation may be involved in spatial memory retrieval. [J Physiol Sci. 2008;58 Suppl:S136]

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We recently reported the cross-regulation of signaling between platelet-activating factor (PAF)- and ACTH-mediated steroidogenesis. PAF and ACTH act mainly through those receptors accompanied by the activation of protein kinase (PK) C and PKA, respectively. In the present study, since the cortisol (F) synthesis in adrenal fasciculate cells is in part via a calcium/calmodulin-dependent process, we studied the cross-regulation of F secretion among PAF, ACTH, and calcium ionophore A23187 in perfused guinea pig adrenal gland. 1) The infusion of 10 μM A23187 for 5 min significantly stimulated F secretion. The secretory response of F to repeated infusion of A23187 was not reproducible and second infusion only slightly increased F secretion. 2) To confirm PAF and A23187 cross-regulation, 10 nM PAF was perfused for 45-50 min before 10 μM A23187 and vise versa. PAF after perfusion with A23187 resulted in potent suppression. By contrast, the cortisol response to A23187 after PAF was unaffected. 3) To confirm ACTH and A23187 cross-regulation, 100 pg/ml ACTH was perfused for 50-55 min before 10 μM A23187 and vise versa. The cortisol response to ACTH after A23187 was unaffected. Similarly, the cortisol response to A23187 after ACTH was slightly suppressed. These results implicate that cross-regulation among processes activated by PAF receptor-PKC, ACTH receptor-PKA, and calcium/calmodulin might function in the multifactorial regulation of adrenal steroidogenesis. [J Physiol Sci. 2008;58 Suppl:S136]

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Mothers are attracted to the body odor of newborn infants. In addition, we have previously reported that the body odors of newborn infants exert effects on moods in postpartum 4–5 days mothers, but little is known about its reason. In the present study, we examined whether their hormonal status influenced the effects of the body odors of newborn infants. The body odors were collected from the 3-day-old infants and 1-month-old infants from their undershirts. Four–five days postpartum mothers (exp. 1) and 1-month postpartum mothers (exp. 2) were exposed to odors of a part of the undershirt with a) control odors, b) their own infant body odors or c) other infant body odors. We used the visual analog scales (VAS) to assess the effects of infant body odors on postpartum moods and collected blood samples to measure their hormonal status. This study was approved by the ethics committee of Nagasaki University. The newborn infant body odors significantly decreased anxiety, depression and fatigue scores, whether infant odors may be originated from their own infants or other infants (exp. 1). However, there were no significant effects of the other infant odors on 1-month postpartum mothers (exp.2). These results suggest that the calming effects on postpartum mother are involved in their sensitivity to the olfactory cue, so that we measured their hormonal status to explore the difference between postpartum day 4-5 and 1-month. [J Physiol Sci. 2008;58 Suppl:S136]

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The terminal nerve (TN)-gonadotropin releasing hormone (GnRH) neurons are suggested to release GnRH peptides from widely branched neural processes and the somatodendritic regions, depending on their firing activity, and the released GnRH is suggested to exert their neuromodulatory action on GnRH receptors located on various target neurons.To address questions on how electrical activities of the TN-GnRH neuronal somata affect GnRH release from various neuronal compartments, we established the primary culture of TN-GnRH neurons, which will enable simultaneous recordings of various physiological signals from various compartments of a single TN-GnRH neuron.The whole brain of an adult freshwater fish (dwarf gourami) was dissected out, and the TN-GnRH neurons were sucked out from the brain with the aid of a suction pipette and plated on a coverslip in culture medium. The isolated TN-GnRH neurons could be cultured up to two weeks. During the culture, the neurons grew both axon- and dendrite-like neurites. Although the cultured neurons had relatively shallow resting membrane potentials and had no spontaneous discharge, unlike the neurons in situ, they showed subthreshold spontaneous membrane potential oscillations and could be induced to fire in phasic or tonic patterns. In addition, stimulus-induced exocytotic events could be observed at the soma and neurites by using fluorescent dye, FM1-43. Therefore, isolated culture of TN-GnRH neuron will open up a wide range of possibilities for studying cellular mechanism of the exocytosis and generation of spontaneous firing activity of peptidergic neurons. [J Physiol Sci. 2008;58 Suppl:S137]

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A variety of neurotransmitters and neuromodulators affect the activity of neurons synthesizing gonadotropin-releasing hormone (GnRH) in the hypothalamus, whereby regulate the release of gonadotropins from the anterior pituitary. Among them are prostaglandins which are synthesized by cyclooxygenages (COX) and are thought to be indispensable for the ovulatory surge of gonadotropins. The aim of the present study was to examine the effect of inhibition of prostaglandin synthesis on hypothalamic GnRH neurons prior to the expected ovulatory surge of gonadotropins. Ovariectomized rats were primed with estradiol benzoate (20 ;μg, sc) 3 days before the experiment, and were treated with progesterone (1 mg, sc) at noon on the day of experiment to reliably induce the gonadotropin surge. COX inhibitors, indomethacin (arylalkanoic acid class of NSAID; 8 mg/kg), flurbiprofen (2-Arylpropionic acid class of NSAID; 1mg/kg) or the vehicle was injected iv at noon. The rats were perfused with formaldehyde under deep anesthesia at 1700h. The brains were fixed and processed for double immunofluerescent staining against GnRH and c-Fos. The COX inhibitors significantly decreased the number of GnRH-immunoreactive cells in the hypothalamus. The result was in marked contrast to the effect of lipopolysaccharide which did not affect GnRH cell numbers but reduced c-Fos positive cells among them. These results suggest that prostaglandins may control GnRH synthesis in the hypothalamus. [J Physiol Sci. 2008;58 Suppl:S137]

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The brain weight is only 2% of the whole body weight, but it contains around 20% of the whole body cholesterol pool. Choresterol and its derivatives are identified to be the natural ligand for retinoic acid receptor-related orphan nuclear receptor (ROR)α, which is widely expressed in brain. Thus, RORα may control brain lipid metabolism, although its function in brain has not yet been fully understood. Apo AI is the major apolipoprotein in cerebrospinal fluid. Although Apo AI gene has native ROR response element (RORE), the role of RORα on Apo AI transcription in brain has not yet been clarified. Thus, we have investigated the effect of RORα and serum on Apo AI transcription. RORα mediated transcription on Apo AI RORE was activated with serum-contained medium, whereas such effect was not observed without serum, indicating that serum may contain a ligand for RORα. In addition to the role of RORα on this gene expression, it activates transcription through thyroid hormone (TH) receptor (TR), which is essential for normal brain development. Thus, we used a native TH response element, malic enzyme (ME) promotor, to examine the effect of RORα and found that TR action was augmented by RORα. Taken together, our results indicate that RORα contributes to sustain brain function in several ways; by lipid metabolism through RORE, and by augmenting TR-mediated transcription on TRE. [J Physiol Sci. 2008;58 Suppl:S137]

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Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase. Cdk5 and its activators, p35 and p39, are enriched in neuron and regulate neurotransmission, synaptic plasticity and neural development. Recently studies have shown that a high Cdk5 activity is also detected in pancreatic beta cells and Cdk5 regulates glucose-stimulated insulin secretion. A number of very recent studies showed single-nucleotide polymorphisms in cdk5 regulatory subunit-associated protein 1-like 1 (CDKAL1) gene in type 2 diabetes mellitus. CDKAL1 shares considerable protein domain and amino acid homology with cdk5 regulatory subunit associated protein 1 (Cdk5RAP1) gene, a known inhibitor of cdk5 activation. In the present study, we produced transgenic mice overexpressing dominat-negative Cdk5 in the pancreatic beta cells. Cdk5 activity in the pancreatic islets of the mice was 48% of that of wild-type islets. The mice showed higher HbA1c than wild-type mice in the age of 6-weeks and 12-weeks. Intraperitoneal glucose tolerance test (IPGTT) revealed the glucose-stimulated insulin secretion was impaired in the transgenic mice. Moreover, the number of pancreatic beta cell was decreased in the transgenic mice through the development. These results suggest that Cdk5 may be involved in the development of pancreatic islets, and the transgenic mouse may be a new model of the patients with type 1 diabetes mellitus. [J Physiol Sci. 2008;58 Suppl:S137]

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The forebrain osmosensitive area called the anteroventral third ventricular region (AV3V) possesses gamma-aminobutyric acid (GABA) receptors (-R) that might cooperate with glutamatergic (Glu)-R shown by us to be involved in the osmotic ADH secretion. However, roles of the GABA-R in regulating ADH and related factors in the absence or presence of hyperosmolality remain unknown. The present study aimed to explore this issue through experiments in awake rats implanted chronically with indwelling vascular and brain cannulae. Infusion sites in the brain were determined by histological inspections. AV3V application of bicuculline (Bic), a GABA(A)-R antagonist (Ant), but not phaclofen, a GABA(B)-R Ant, elicited marked rises in plasma ADH, osmolality (Osm), glucose, arterial pressure (AP) and heart rate, without causing significant changes in plasma electrolytes. These effects of Bic were prevented by preadministering a GABA(A)-R agonist muscimol (Mus), or a Glu-R Ant MK-801 or NBQX. When infused intravenously 2.5 M NaCl augmented plasma ADH, Osm, sodium and AP gradually with time. The responses of ADH and AP were abolished by AV3V application of Mus, whereas in the absence of the osmotic load the Mus treatment affected none of the variables monitored. From these results, we concluded that the osmotic load may inhibit AV3V GABAergic activity to promote ADH secretion through potentiation of local Glu activity. [J Physiol Sci. 2008;58 Suppl:S138]

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The temporal resolution for measuring of vasopressin (VP) release from neurohypophysial nerve terminals is relatively poor. We describe a fibre optic approach for monitoring VP release from the neurohypophysis in response to physiological stimuli, in vivo. Transgenic rats expressing a VP-enhanced green fluorescent protein (eGFP) fusion gene (Ueta et al., 2005, Endocrinology, 146: 406-413) were used. A dual optrode consisting of two optic fibres was constructed; one fibre coupled to a 488nm Ar laser was used for excitation and the other was used for signal detection. The optrode was stereotaxically placed immediately dorsal to the neurohypophysis during bolus intravenous injections of hypertonic saline (HS; 0.1ml, 3M NaCl) and sodium nitroprusside infusion (SNP; 100μM; 100μl/min for 5 min) to unload baroreceptors. HS-induced a rapid increase in fluorescence (40.2±3.2%; n=5, P<0.05) signal lasting approximately 40 s while infusion of SNP, to induce a sustained fall in arterial pressure, was associated with a stimulus-locked increase in fluorescence (19.5±1.5%; n=3). Neither bolus injections nor infusions of saline (0.9%) caused a change in fluorescence. Post-hoc histological analysis confirmed a change in fluorescent signal was detected to HS (n=5) and SNP (n=3) only when the optrode was positioned above the neurohypophysis. We concluded that optrode recording permits detection of putative VP release from the neurohypophysis in vivo. [J Physiol Sci. 2008;58 Suppl:S138]

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BDNF (brain derived neurotrophic factor) is known as a critical player of neural development, synaptic plasticity and a putative rescue factor for neuronal disorders. Recent studies suggest that BDNF also has potential regulatory roles in peripheral tissues. We found that BDNF and TrkB-T1, the truncated form of high affinity BDNF receptor TrkB, expressed in matured adipocytes. Moreover BDNF dramatically increased the expression in white adipose tissue (WAT) in obese mouse. Here we report the novel regulatory role of BDNF in adipokine expression in adipocytes. BDNF suppressed mRNA expression of atherosclerogenic adipokine PAI-1 (plasminogen activator inhibitor-1) in matured adipocytes, both of WAT in vivo and 3T3-L1 adipocytes in vitro. Our data revealed that dephosphorylated form of FoxO1 existed in the nucleus and promoted PAI-1 mRNA expression by binding to the FRE/IRE sequence in the promoter and enhancer region. In contrast, BDNF increased the phosphorylation of FoxO1 in PI3-K/Akt and MEK/ERK1/2/S6K dependent pathways and translocated it from the nucleus to cytoplasm, then suppressing PAI-1 mRNA expression. Akt activated by PI3-K phosphorylated Thr24 and Ser256 of FoxO1, and S6K activated by MEK/ERK1/2 phosphorylated the Ser256. siRNA for TrkB inhibited Akt and ERK1/2 phosphorylation in 3T3-L1 adipocytes in response to BDNF. These results suggest that BDNF/TrkB-T1 plays an important role in PAI-1 expression in adipocytes. This pathway may be a target for the therapy of metabolic syndrome. [J Physiol Sci. 2008;58 Suppl:S138]

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Recently, we have cloned and characterized a coactivator activator (CoAA) using a Sos-Ras Yeast Two-hybrid Screening System and TR-binding protein (TRBP)-C-terminus as bait. Interestingly, a shorter splicing variant is also produced from CoAA gene, named coactivator modulator (CoAM), which functions as a transcriptional suppressor. A series of protein-RNA binding studies revealed that CoAA or CoAM binds to RNA. However, these bindings are not specific. Since transcriptional regulations are mainly activated by histone acetyl transferase (HAT) complex, we examined the effect of CoAA/CoAM on the HAT activity. CoAA activated the HAT activity in the presence of CREB-binding protein (CBP), however, CoAM did not. Thus, we presumed the suppression by CoAM may be related to other than nuclear event. To investigate the location of CoAA or CoAM, immunofluorescent studies were performed. Interestingly, CoAA was located in the nucleus, whereas CoAM is in the cytoplasm. These results suggest that the location of CoAA/CoAM may determine the transcriptional activity, possibly by interacting with cofactor(s) essential for chromatin remodeling. In summary, CoAA/CoAM determines the transcriptional activity by a novel mechanism. [J Physiol Sci. 2008;58 Suppl:S138]

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In the pituitary, estrogen induces lactotroph proliferation and prolactin (PRL) secretion, and long term exposure to estrogen leads to prolactinoma formation. Dopamine (DA) is a well-known inhibitory agent on lactotrophs and affects estrogen-induced lactotroph proliferation. However, the molecular mechanism of the inhibitory action of dopamine is still not clear. In this study, we examined whether DA influences transcriptional activity of estrogen response element (ERE), a target sequence for activated estrogen receptor, in lactotrophs in primary cuture. To determine the lactotroph-specific ERE activity, we developed an adenovirus-mediated reporter assay system in which PRL promoter drives expression of an ERE reporter gene via Cre/loxP system. We found that estrogen-stimulated ERE activity in lactotrophs was inhibited by bromocriptine (BC) as well as DA treatment. Same results were obtained using a combination of discontinuous Percoll gradient-enriched lactotrophs and an ERE reporter assay without Cre/loxP system. The DA action was through D₂ dopamine receptor activation because the BC-induced inhibition was reversed by treament with a selective D₂ dopamine receptor antagonist and a D₁ dopamine receptor agonist failed to inhibit the ERE activity. The BC-induced inhibition of ERE activity was not accompanied by a decrease in ERα protein and mRNA expression, suggesting that the recruitment of coregulators is negatively influenced by DA. These results indicate that there is an inhibitory crosstalk between ERE and D₂ dopamine receptor activation in pituitary lactotrophs. [J Physiol Sci. 2008;58 Suppl:S139]

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Microinjection of angiotensin II (ANG II) into the subfornical organ (SFO) caused drinking in ovariectomized (OVX) female rats that were treated with either propylene glycol (PG) vehicle or estradiol benzoate (EB). The amount of water intake elicited by the ANG II injection was significantly greater in the PG-treated than in the EB-treated animals. In both groups, previous injections of either dizocilpine (MK801), a N-methyl-D-asparatate (NMDA) antagonist, or 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX), a non-NMDA antagonist, into the median preoptic nucleus (MnPO) resulted in the significant attenuation of the drinking response to ANG II, whereas similar injections of saline vehicle into the MnPO was without effect. These results show the involvement of glutamatergic projections from the SFO to the MnPO in the elicitation of drinking response ANG II activation of the SFO, and suggest that the elicitation of drinking response may be mediated through both NMDA and non-NMDA receptor mechanisms in the MnPO. Our data further imply that the circulating estrogen may act to attenuate the glutamatergic SFO inputs to the MnPO. [J Physiol Sci. 2008;58 Suppl:S139]

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X-box binding protein 1 (XBP1) is the transcription factor induced in response to endoplasmic reticulum (ER) stress, which is provoked by the overproduction of unfolded or misfolded proteins, leading to the induction of chaperones, ER-associated degradation factors, or other factors. Growth hormone (GH) is known to have many physiological roles, including the induction of XBP1 gene expression. In this study, we have examined the effect of GH on the XBP1 gene expression, using the liver of hypophysectomized rats.The intravenous administration of GH increased XBP1 mRNA (2.5 Kb and 2.0 Kb) transiently and dose-dependently. The XBP1 mRNA was increased in the adipose tissues, while the increase of 2.5 Kb mRNA was liver-specific. The active form of XBP1 is derived from spliced molecule of the mRNA, and RT-PCR revealed that both unspliced and spliced mRNAs were increased by GH administration. GH administration induced the mRNA of GADD45A, one of the factors activated in response to ER stress, but not the mRNA of CHOP, inducer of apoptosis.These results indicate the possibility that GH has a facilitatory role of unfolded protein response to relieve liver cells from ER stress. [J Physiol Sci. 2008;58 Suppl:S139]

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X-box binding protein 1 (XBP1) is the transcription factor activated in response to ER stress. The XBP1 gene expression in the rat liver is sexually dimorphic and increased by growth hormone (GH). We have examined whether the sexual dimorphism is due to sex steroids or GH secretory pattern.The XBP1 mRNA level of the liver in the normal male rat was higher than that in the normal female. It decreased to the female level after hypophysectomy (HPX) or orchidectomy (OCX). The mRNA level was not influenced by HPX or ovariectomy (OVX) in female rats. To determine the role of sex steroids, rat GH was infused continuously with Alzet miniosmolar pump and estradiol (E2) or dihydrotestosterone (DHT) was given for 7 days to the rat with HPX and OCX. The XBP1 mRNA levels were sustained at the female level after these treatments. To examine the effect of GH secretory pattern, GH was administered every 3-h-bolus (male like) or continuously (female like) for 24-h to the rat with HPX and OCX. XBP1 mRNA level increased 1- and 2-h after the last bolus of GH, and reached to the normal male level. It did not change with continuous infusion. It has been shown that GH secretory pattern changes to male like pattern, when the OVX rat is treated with DHT. The XBP1 mRNA level increased in the OVX female rats treated with DHT, but did not reach to the normal male level.These results indicate that the sexually dimorphic expression of the XBP1 mRNA in the rat liver depends on the GH secretory pattern, but not on the sex steroids. [J Physiol Sci. 2008;58 Suppl:S139]

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On mammalian spermatozoa, capacitation is the essential process to fertile to the egg. Capacitated spermatozoa are generally acrosome reacted and are hyperactivated. Melatonin regulates seasonal reproductive functions. On previous studies, both of positive effects and negative effects of melatonin on sperm motility were reported.In the present study, effects of melatonin on sperm motility were examined. From results, melatonin enhanced hyperactivation of hamster spermatozoa. Effective concentrations were from 1pM to 1uM. Moreover, melatonin regulated hyperactivation through MT1 receptor. [J Physiol Sci. 2008;58 Suppl:S140]

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The brain initiates activation of the gonadotropin-releasing hormone (GnRH) system at puberty onset. Since GnRH neurons are suggested to show a plasticity during postnatal development, remodeling of GnRH neurons may be involved in the onset of puberty. The present study was done by attempting to block plasticity in the GnRH neuronal network, by interfering with the synaptic delivery of GluR1 whose trafficking is critical in synaptic plasticity and thus necessary for memory. To this end, GluR1-C-tail, a dominant negative mutant of GluR1, was expressed in the POA of female rats at day 21 using lentivirus. GluR1-C-tail has been shown to act by competing with GluR1 for synaptic delivery during synaptic plasticity. As the result, the onset of puberty, by checking viginal opening, was significantly delayed. After that, the estrous cyclicity was checked by viginal smear and found that the rat injected with GluR1-C-tail exhibited a regular estrous cycle. However, the surge of luteinizing hormone (LH) secretion in the rat injected with GluR1-C-tail was significantly attenuated, compared to the control rat. Injection with GluR1-C-tail in mature rats had no effect on the surge of LH secretion. The present study suggests that GluR1-mediated plasticity occurs at the time of puberty and is important to maintain surge of LH secretion in adults. [J Physiol Sci. 2008;58 Suppl:S140]

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The gonadotropin-releasing hormone (GnRH) neurosecretory system constitutes the final common pathway in the neuroendocrine control of reproduction. However, the mechanisms of pulsatile and cyclic GnRH release remain poorly understood. To understand the synchronous GnRH neuron activity, which may be base of pulsatile and cyclic GnRH release, we monitored intracellular Ca²⁺ oscillations in GT1-7 cells and native GnRH neurons. Removal of extracellular Ca²⁺ abolished Ca²⁺ oscillations, whereas thapsigargin (inhibitor of ER Ca²⁺-ATPase) had no effect. Nifedipine (L-type Ca²⁺ channel blocker), SNX-482 (R-type blocker) and ω-conotoxin-GVIA (N-type blocker) abolished Ca²⁺ oscillations, indicated that Ca²⁺ oscillations was brought by an influx of Ca²⁺ through the voltage-gated Ca²⁺ channels. GnRH receptor (G-protein coupled receptor) antagonist abolished Ca²⁺ oscillations, suggested that Ca²⁺ oscillations were modulated by GnRH autocrine. Pertussis toxin enhanced Ca²⁺ oscillations, whereas H-89 (protein kinase A inhibitor) abolished Ca²⁺ oscillations. A high [K⁺]_o enhanced Ca²⁺ oscillations in frequency and TTX (voltage-gated Na⁺ channel blocker) abolished Ca²⁺ oscillations. TEA and 4AP (voltage-gated K⁺ channel blocker) enhanced Ca²⁺ oscillations. These results suggest that Ca²⁺ oscillations in GnRH neurons are mediated by voltage-gated K⁺ channel activity affected by GnRH receptor and pertussis toxin -sensitive G protein. [J Physiol Sci. 2008;58 Suppl:S140]

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Cbl-interacting protein of 85 kDa (CIN85) is a multiadaptor protein containing three Src homology 3 (SH3) domains, a proline-rich region and a coiled-coil domain. We have previously shown that CIN85 is involved in regulation of ligand-induced endocytosis of epidermal growth factor (EGF) receptors. To analyze the biological function of CIN85 in vivo, we generated mutant mice deficient in the expression of CIN85. We found that the number of offspring cares of CIN85 knockout (KO) mother mice was less than that of a wild type. Some offspring were cannibalized and neglected by a mother soon after birth. The average number of offspring cares after 5 days of the birth was 7.1 in a wild type, and 2.4 in the mutant. To clarify the cause of this difference, we examined the brain of CIN85 KO mice. The mutant mice displayed absence of the corpus callosum (commissure of the cerebral hemispheres) and increased dopamine levels in the striatum. The low-nursing ability in the KO mice may be partly due to such abnormalities in the brain by loss-of-function of CIN85. [J Physiol Sci. 2008;58 Suppl:S140]

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