The goldfish retinal ganglion cells (RGCs) can survive and regenerate their axons after optic nerve injury. In a previous paper (Devadas et al., Neurosci. Res, 2001) we showed an increased activity of nitric oxide synthase (NOS) revealed by NADPH-diaphorase (ND) staining in the goldfish retina after optic nerve injury. The increase of ND activity was mainly localized in the ganglion cell layer (GCL). In the present study, to further elucidate molecular involvement of NOS in the optic nerve regeneration, we cloned a 490 bp cDNA fragment for goldfish neural NOS (nNOS) with a PCR teqhnique. Levels of nNOS mRNA and protein increased in the RGCs 5 days and peaked at 20-30 days, and then gradually returned to the control level by 60 days after injury. The period of 20-30 days after nerve injury corresponds to the axonal elongation stage in the goldfish optic nerve regeneration process. In retinal explant culture study, dibutyryl cyclic GMP (200-400 μM) and nitric oxide (NO) donor promoted neurite outgrowth, whereas ETPI, a nNOS inhibitor (10-40 μM) and soluble guanylate cyclase inhibitor (ODQ) dose-dependently suppressed it from adult fish RGCs. Furthermore, a specific PKG inhibitor, Rp-8-Br-PET cGMPs significantly inhibited the neurite outgrowth. The data all together indicate that NO-cGMP system is activated thereby leading to the promotion of optic nerve regeneration in the goldfish retina after injury. [J Physiol Sci. 2007;57 Suppl:S140]

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The optic nerve contains a small number of efferent fibers. The efferent fibers, terminal nerve originating from the olfactory bulb, travel along with the optic nerve and terminate in the inner plexiform layer of the retina. After optic nerve transection, the majority of afferent regenerating axons reinnervated the tectum 3-5 weeks by WGA-HRP tracing study. However, it has not been reported when the efferent fibers regenerate after axotomy. In this study, we investigated the recovery time of the efferent fibers in goldfish and zebrafish visual system after optic nerve transection. The efferent fibers were identified by their immunoreactivity to gonadotropin-releasing hormone (GnRH) and FMRFamide (Phe-Met-Arg-Phe-NH₂ ). In control fish retina, localization of GnRH and FMRFamide signals was observed in the nerve fiber layer, and in the border of the inner nuclear and inner plexform layer. These signals of GnRH and FMRFamide were lost by optic nerve lesion. Furthermore, the expression of mRNA of GnRH receptor also disappeared from retina by 10 days after axotomy. The mRNA expression of GnRH receptor was recovered 20 days after lesion in the retina, whereas the expression of GnRH and FMRFamide was recovered 80-90 days after axotomy. This regeneration of efferent fibers was longer than that of afferent fibers in optic nerve. [J Physiol Sci. 2007;57 Suppl:S141]

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As superoxide (·O₂⁻) and hydroxyl radical (·OH) have been implicated in the pathogenesis of Parkinson disease, free radical scavenging and antioxidants have attracted attention as way to prevent progression of this disease. We examined the effects of eugenol, an essential oil extracted from cloves, on 6-hydroxydopamine (6-OHDA)-induced dopamine reduction in the mouse striatum. Eugenol administration 3 days before and 7 more days following one intracerebroventricular 6-OHDA injection prevented the reduction of striatal DA and its metabolites. Eugenol administration for 3 days reduced the increase of thiobarbituric acid-reactive substances (an indicator of lipid peroxidation) induced by ferric ion and increased glutathione and L-ascorbate in the striatum. Eugenol did not change the levels of catalase, glutathione peroxidase, or superoxide dismutase-like activities. Eugenol is known to have ·O₂⁻ and ·OH scavenging activities in vitro. These results suggest that eugenol prevents 6-OHDA-induced DA depression by preventing lipid peroxidation directly and indirectly (via stimulation of glutathione and L-ascorbate generating systems). The effects of eugenol treatment in this model suggest its possible usefulness for the treatment of Parkinson disease. [J Physiol Sci. 2007;57 Suppl:S141]

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Spinal cord injury (SCI) results in gliosis and limited cellular regeneration. Multipotent progenitor cells have significant potential for nervous system regeneration. However, little is known about adult neural stem and progenitor cells. In the present study, we focus on oligodendrocyte. For this purpose, we choose NG2 proteoglycan, which is a marker of oligodendrocyte progenitors.SD rats were divided into a SCI group (n=25) and a sham-operated group (n=5). In the injury group, laminectomy was performed at Th11-12 and contusive compression injury was created by applying a weight of 30 g for 10 min. Rats were sacrificed at 24 h, and 1, 4, 8 and 12 weeks post-injury. Frozen 20μm sections of tissue 5 mm rostral and caudal to the epicenter of injury were prepared. Immunoistochemistry was performed using antibodies against NG2, GFAP and 3CB2. At 4 weeks after injury, NG2-positive glial cells arose from below the pial surface as bipolar cells with processes extending throughout the entire white matter. NG2 expression peaked at 4 weeks after injury, showing a 7-fold increase compared to the 24 h after injury. The NG2-positive cells with processes which increased in the white matter of the spinal cord were GFAP-positive and also co-localized with 3CB2 antigen. These results suggest that NG2 positive cells which derived from subpial layer, may have some lineage to RG and astrocyte progenitor cells after SCI in adult rodents. [J Physiol Sci. 2007;57 Suppl:S141]

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The current study examines 3CB2, radial glia marker, expression after intracerebral hemorrhage (ICH). Animal protocols were approved by the Committee of Kagawa University. Adult male SD rats received an intracaudate injection of 100µl autologous whole blood under general anesthesia. Animals were sacrificed for single and double labeling immunohistochemistry to identify which cells express 3CB2, and for Western blotting to quantify 3CB2 expression. By immunohistochemistry, 3CB2 immunoreactivity was present in large numbers of astrocytes, surrounding the hematoma from day 3 to 1 week after ICH. After 1 month, 3CB2 immunoreactivity was co-localized with a precursor neuronal marker (TUC-4). By Western blot analysis, 3CB2 was strongly expressed at day 3 (p<0.05) and 1 week (p<0.05), and expression persisted for at least 1 month (p<0.05). The ICH-induced 3CB2 expression in astrocytes may reflect an early response of these cells to injury, while the delayed expression in neurons might be a part of the adaptative response to injury leading to recovery of functions. [J Physiol Sci. 2007;57 Suppl:S141]

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Glycans of glycoproteins play many important roles, such as cell-cell interaction and cell surface recognitions. There are two ways to obtain the glycans from glycoproteins, enzymatic digestions and hydrazinolysis. We have been using an enzymatic method using glycopeptidase-A to prepare glycans from the P0 and the PASII/PMP22 glycoproteins in peripheral nerves, and determined their structures containing sulfated glucuronic acids (S-GlcA). The S-GlcA, known as HNK-1 carbohydrate epitope, is found by immunological observations in many adhesion glycoproteins in CNS and PNS, including N-CAM, MAG, L1, P0 and PMPII/PASII. and is postulated to be associated with cell adhesion, cell migration, and brain memory functions. I focused on other adhesion glycoproteins and this time preferred hydrazinolysis to prepare glycans from total brain proteins. Many types of glycans were obtained, however; glycans containing S-GlcA could not be found. A question arose whether hydrazinolysis is applicable to the glycans containing S-GlcA. The proteins, including P0 and PASII/PMP22, from PNS were subjected for the hydrazinolysis, almost all the glycans containing S-GlcA were degraded, whereas other sulfated glycans and sialylated glycans were recovered in expected amounts. It indicates that hydrazinolysis is not applicable for the glycans containing GlcA or S-GlcA. This must be one of the reasons why the structures of almost all glycans containing S-GlcA have not been reported so far, although the immunological observations tell us there are many glycoproteins containing the HNK-1 epitopes. [J Physiol Sci. 2007;57 Suppl:S142]

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Ciliary neurotrophic factor (CNTF) is abundantly expressed in Schwann cells in adult mammalian peripheral nerves, but not in neurons. After periheral nerve injury, CNTF released from disrupted Schwann cells is likely to promote neuronal survival and axonal regeneration. In contrast to expression restricted to Schwann cells in vivo, we observed abundant CNTF mRNA and protein expression in adult rat dorsal root ganglion (DRG) neurons in dissociated cell culture. At later stages (5 and 7 days) of culture, CNTF immunoreactivity was detected in both neuronal cell bodies and growing neurites. These results suggest that CNTF is synthesized and transported to neurites in cultured DRG neurons. Since we failed to observe CNTF expression in DRG neurons in contact with satellite Schwann cells or in explant culture, disruption of neuron-Schwann cell interactions, rather than effects of the culture itself, may be responsible for production of CNTF in the neurons. [J Physiol Sci. 2007;57 Suppl:S142]

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p38 Mitogen-activated protein kinase (p38 MAPK) is one of the MAP kinase families, and supposed to play significant roles in survival of mature neuronal cells. In developing mouse brain, however, the localizations and roles of p38 remain unclear. In the present study, we performed Western blot analysis and immunohistochemical analysis of developing mouse brain using antibody against p38 MAPK. p38 MAPK was preferentially expressed in the brain from embryonic 11th day (E11) to E18 rather than that from postnatal 8 weeks. In the E10-E14 brain, most of the p38 MAPK-positive cells were located in the subventricular zone, and were positive for nestin, but not beta III tubulin. To ascertain the roles of p38 MAPK on early embryonic brain cells, the cultured cells were exposed to selective chemical inhibitors of p38 MAPK (SB202190 or SB203580) for 7 days. These inhibitors increased the number of neurosphere, and intracellular amount of ATP. In addition, similar results were obtained by using nestin-positive neural progenitor cells generating from seceondary neurosphere. These results suggest that p38 MAPK could involve in the proliferation and/or survival of neural progenitor cells in developing mouse brain. [J Physiol Sci. 2007;57 Suppl:S142]

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Hippocalcin (Hpca) is a member of the neuronal calcium sensor protein family and is exclusively expressed in hippocampal neurons. Hpca-deficient (Hpca^−/−) mice display a defect in cAMP response element-binding protein (CREB) activation associated with impaired spatial and associative memory. Here we examined the involvement of Hpca in the extracellular signal-regulated kinase (ERK) cascade leading to CREB activation, because application of PD98059, a broad ERK cascade inhibitor, have resulted in similar levels of CREB activation in Hpca^−/− hippocampus. N-methyl-D-aspartate (NMDA)- and KCl-induced phosphorylation of ERK was significantly attenuated in Hpca^−/− hippocampal slices, as was ionomycin-induced phosphorylation of ERK, whereas forskolin- and TPA-stimulation yielded indistinguishable levels of ERK phosphorylation in both wild type and Hpca^−/− slices. In an in vitro reconstitution assay system, recombinant Hpca affected neither Raf-1 protein kinase activity using recombinant MEK-1 as a substrate nor MEK-1 kinase activity using ERK2 as a substrate. Activation of Ras by NMDA and KCl stimulation of hippocampal slices showed no obvious changes between the two genotypes; however, phosphorylation of Raf-1 was significantly lower in Hpca^−/− slices. These results suggest that Hpca plays an important role in the activation of Raf conducted by Ras. [J Physiol Sci. 2007;57 Suppl:S142]

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We recently reported that the high intracellular stability of the N-terminus-deleted human tyrosine hydroxylase (TH) type 1 mutants might be generated by the loss of a PEST (proline, glutamate/aspartate, serine and threonine) motif located in the N-terminus sequence of Met¹-Lys¹². However, it is not clear whether only the PEST motif can affect the intracellular stability of human TH type 1 (hTH1) proteins. This study was performed to confirm a role of the PEST motif on the stability of hTH1. Wild-type hTH1 and the 6 mutants (del-12, del-22, del-32 and del-42, in which the first 12, 22, 32 and 42 amino acid residues deleted, respectively; del-Pro2, the Pro² residue deleted; del-Ala11, the Ala¹¹ residue deleted) were expressed in AtT-20 mouse neuroendocrine cells in order to clarify how deeply the PEST motif is involved in the intracellular stability of hTH1 protein. The western blot analyses revealed that the mutants del-22, del-32, del-42 and del-Ala11 were much more stable than wild-type in the cells. These results indicate that the PEST motif is critical in regulating the intracellular stability of hTH1 protein. Moreover, it has been reported that the phosphorylations of TH at serine residues, Ser¹⁹, Ser³¹ and/or Ser⁴⁰ induce the change in the tertiary structure of the N-terminus of TH protein. Therefore, in this report, we also suggest that the PEST motif and the phosphorylations can affect the intracellular stability of hTH1 protein, synergistically. [J Physiol Sci. 2007;57 Suppl:S143]

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A loss of inhibition in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. It involves a trans-synaptic reduction in the expression of the potassium-chloride exporter KCC2(Coull. et al. Nature 2003). We have prepared a rat experimental model of trigeminal neuropathic pain produced by a chronic constriction injury to the infraorbital nerve. By means of in situ hybridization histochemistry, we found that KCC2 mRNA was downregulated 1 to 3 weeks after ligation of the infraorbital nerve and it recovered after 4 weeks in the spinal trigeminal nucleus (Sp5). The time course of KCC2 mRNA downregulation and recovery was well correlated with the decrement and recovery of pain threshold to the mechanical stimuli. The KCC2 mRNA downregulation and recovery was also correlated with the increase and recovery of pain response. The result suggests that the KCC2 downregulation may play a role in the trigeminal neuralgia by impairing GABA inhibition which could be crucial for the analgesic action at the Sp5. [J Physiol Sci. 2007;57 Suppl:S143]

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We showed that endogenous vasopressin increases the vasopressin (AVP) and oxytocin (OT) mRNA production in the paraventricular nucleus (PVN) of the hypothalamus after central salt loading in conscious rats. However, as compared to our previous results, there is a conflict between the expression of Fos-like immunoreactivity (FLI) and the mRNA (AVP and OT) in the PVN. That is, endogenous AVP decreases the FLI expression but increases the AVP and OT mRNA after central salt loading. In this study, we evaluated the FLI expression in the PVN after drawing a distinction between magnocellular and parvocellular parts using single and double immunohistochemical staining methods. Endogenous AVP prominently suppressed the FLI expression in the parvocellular parts, but no difference was observed in the magnocellular parts. However, the ratio of AVP- to FLI-positive neurons in the magnocellular parts of the PVN was decreased after pretreatment with AVP V₁ receptor antagonist, OPC-21268. We concluded that the distribution of the hybridization signal (AVP and OT) and FLI in the PVN might not conflict each other after central salt loading. [J Physiol Sci. 2007;57 Suppl:S143]

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To measure dopamine (DA) in the brain in conscious rats, selective biosensor was developed with three layer membranes: iron-exchange membrane, enzyme-containing membrane and antioxidant-containing membrane. Electrochemical (500 mV between electrodes) detection of oxidative current of membrane permeable substances was carried out. DA selectivity was confirmed in vitro as follows. Oxidative current was not found in additions of Glu, Ach, GABA, 5-HT, Adr or NA but in DA treatment with three-layer biosensor, although it was detected in 5-HT, Adr, NA or DA treatment with no membranes. In addition, administration of tyrosine, l-dopa or DA metabolites (DOPAC, HVA, 3-MT) did not induced oxidative current with this biosensor, indicating the selective detection of permeable DA. We next investigated whether DA-selective probe detect the changes of DA content in vivo. Methamphetamine (3.0 mg/kg) was given to the adult rats and DA level in the striatum was monitored with this probe. Oxidative current was gradually increased from 10 min after the administration, exhibiting that real-time changes of DA level was monitored in vivo. Data suggest that real-time change of DA was measurable in vitro and in vivo with a biosensor that has relative high-sensitivity to DA. [J Physiol Sci. 2007;57 Suppl:S143]

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We participate in the project to establish the technique to diagnose a nerve disease by the injury of the shake of infancy at the early stage. The purposes of our research is acquiring the method of detecting the injury to the brain by the diffusion weighted image with the Fractional anisotropy (FA) image and the Trace index (TI) In this presentation, we show the initial data with adult mouse acquired in establishment and the image of the infant rat.In the FA image of the mouse brain, a high numerical value was shown in corpus callosum (0.65±0. 03), and about twice the numerical value was shown to each region of the cerebral cortex (e.g. M1:0. 34±0. 01 and S1Tr:0. 32±0. 01). Moreover, the difference between the regions was not remarkable in the cerebral cortex. However, numerical value at the dentate gyrus (0.25±0. 01) was significantly low compared with molecular cell layer and pyramidal cell layer (Mol:0. 38±0. 05,Py:0. 28±0. 03). In the TI image, the numerical value of the corpus callosum was the same to the cerebral cortex area. Dentate gyrus (0.87±0. 01 10⁻³mm²/sec) showed that numerical values were lower than others (Mol: 1.07±0. 02,Py1.03±0. 02) in the hippocampus. On the other hand, infant age rat's FA image almost showed the description pattern similar to images of mouse. The numerical value of FA indicated a constant value at any stage of development in the corpus callosum, and a significant difference by the day age could not be confirmed. [J Physiol Sci. 2007;57 Suppl:S144]

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We used reverse transcription-coupled PCR to produce a high-resolution temporal map of fluctuations in mRNA expression of 112 genes during rat central nervous system development, focusing on the cervical spinal cord. We have used an established RT-PCR protocol (4) to measure the expression of 112 genes in CNS development. Cervical spinal cord tissue was dissected from triplicate animals or litters (Sprague-Dawley albino rats), in accordance with National Institutes of Health guidelines, from embryonic days 11 through 21 (E11-E21; determined by crown-rump length), postnatal days 0-14 (P0-P14), and adult (P90 or adult). Gene-specific primers were designed from GenBank sequences by using the OLIGO software (National Biosciences, Plymouth, MN). RNA isolated from tissue samples by using RNAstat 60 (Tel-Test, Friendswood, TX) was adjusted to 200 ng/l according to absorption at 260 nm, before RT-PCR (Perkin-Elmer GeneAmp RNA PCR kit, Applied Biosystems); PCR involved preheating a mixture of Taq antibody (TaqStart, CLONTECH), primers, cDNA, and PCR components to 97C for 90 sec before amplification.The PCR cycle was 30 sec at 95C (dissociation), 45 sec at 60C (annealing), and 60 sec at 72C (extension). Amplification was within the exponential range (4). [J Physiol Sci. 2007;57 Suppl:S144]

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Intracellular Cl⁻ concentration ([Cl⁻]_i) in immature neurons is higher than that expected for a passive distribution, resulting in that GABA renders immature neurons depolarizing. The higher [Cl⁻]_i in immature neurons is thought to be attributed to the uptake of Cl⁻ mediated by NKCC1 (Na⁺, K⁺-2Cl⁻ cotransporter). Thus, a dysfunction of this transporter could affect synaptic development through a GABA_A receptor-mediated pathway. To test this possibility, we examined the effects of a Cl⁻ -uptake inhibitor on the development of synaptic activities of rat neocortical neurons in culture. Chronic treatment with bumetanide at 10 μM during the culture diminished the amplitude of synaptically-driven rhythmic depolarizing potentials (RDPs) in neurons and also decreased the frequency of sIPSCs but not of sEPSCs. Chronic treatment with bumetanide decreased vesicular GABA transporter (VGAT)-immunopositive particles without affecting paired-pulse ratio of evoked IPSCs (eIPSCs), indicating a decrease in the number of functional GABAergic synapses. These results suggest that the uptake of Cl⁻ by NKCC1 affects the development of inhibitory synapses by promoting a depolarizing GABA-mediated response. [J Physiol Sci. 2007;57 Suppl:S144]

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Group I metabotropic glutamate receptors (mGluRs) are coupled to phosphoinositide hydrolysis and are thought to modulate neuronal excitability by mobilizing intracellular Ca²⁺. Difference in Ca²⁺ mobilization among subclasses of the receptors has been reported and this difference may cause various neuronal modifications. In hippocampal interneurons, several subclasses of mGluRs including 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 determined. In present study, effects of type I mGluR agonist, DHPG on intracellular Ca²⁺ concentrations ware examined in hippocampal nonprincipal cells. Calcium imaging was performed using acute slices of rat hippocampus. Both transient and oscillatory intracellular calcium elevations were detected after DHPG administration. The calcium elevations were partially inhibited by mGluR1 specific antagonist, CPCCOEt or mGluR5-specific antagonist MPEP. Electrophysiological recording was also performed. GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) were recorded from CA3 pyramidal neurons, by patch-clamp recording method. Perfusion of DHPG facilitated GABA_Aergic spontaneous IPSCs. Degree of contribution of mGluR1 and mGluR5 mediated signal transduction to the IPSC suppression was also determined. [J Physiol Sci. 2007;57 Suppl:S145]

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Adenosine is a neurotransmitter to modulate synaptic transmission in the CNS. Currently, four adenosine receptors, A₁, A_2A, A_2B and A₃ receptor, have been cloned and characterized. Several lines of evidence have indicated that adenosine improves neurological deficits in spinal cord injury through activation of A₁ receptor. Nonetheless, much less is known about the functional role of other adenosine receptors in the spinal cord. In the present study, we examined whether activation of A_2A receptors affects synaptic transmission in spinal motoneurons by using the whole-cell patch-clamp technique. Bath application of an A_2A receptor agonist, CGS21680 (1 μM), increased sEPSC frequency in about a half of neurons recorded at a holding potential of -70 mV. In the presence of bicuculline (20 μM), strychnine (2 μM), CNQX (10 μM) and APV (50 μM), no synaptic activity could be observed at a holding potential of -50 mV in all neurons examined. Under this condition, superfusion of CGS21680 (1 μM) induced an inward current in about 70% of neurons examined. These results indicate that the activation of presynaptic A_2A receptors enhances glutamate release onto spinal motoneurons, and the activation of postsynaptic A_2A receptors directly depolarizes the majority of spinal motoneurons. Thus, the activation of A_2A receptors in spinal motoneurons may aggravate neural damages in spinal cord injury, in contrast to adenosine A₁ receptors. [J Physiol Sci. 2007;57 Suppl:S145]

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Application of dopamine (DA) induces a slow Na⁺ current response in the identified neurons of Aplysia ganglia under voltage clamp. This type of response is produced by the activation of trimeric G-protein sensitive to cholera toxin. We previously reported that the Na⁺ current response to DA is facilitated by the activation of monomeric G-protein Aplysia Rho which is closely homologous to RhoA, RhoB or RhoC in mammalian neuron. In the present study, we investigated possible effector molecules located at the downstream of the Rho in facilitation of the DA-induced response. The Na⁺ current response to DA was gradually depressed after intracellular injection of α-synuclein, an inhibitory protein for phospholipase D (PLD). Furthermore, extracellular application of butan-1-ol, which is known to inhibit PLD activity, significantly depressed the DA-induced current response, whereas butan-2-ol, an inactive isomer of butan-1-ol, had no significant effect appreciably. These results suggest that the facilitating effect of Rho on the DA-induced Na⁺ current response may be mediated by the activation of PLD. [J Physiol Sci. 2007;57 Suppl:S145]

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Effect of increase in rate of superfusion of the medium on the EPSC evoked by stimulation of Schaffer collateral was studied in CA1 pyramidal neuron of rat hippocampal slice under whole-cell voltage-clamp. Increase in superfusion rate of the medium slowly and markedly augmented the amplitude of EPSC without changing those of both AMPA-induced current and IPSC evoked by stimulation of striatum oriens. Paired pulse ratio of the EPSC decreased during the superfusion and Ca²⁺-dependence curve of the EPSC amplitude obtained by changing the [Ca²⁺]_o showed marked increase in maximum response without changing the ED₅₀ in the fast superfusion. Fast superfusion produced rightward shift of the maximum peak in the amplitude distribution histogram of the evoked EPSCs in low [Ca²⁺]_o, indicating increase in quantal size of the transmitter release. Furthermore, application of DPCPX, an antagonist for A₁ receptor, also augmented the EPSC amplitude, and the fast superfusion did not cause additional augmentation of the EPSC amplitude in the presence of DPCPX. These results suggest that major cause for the augmenting effect may be disinhibition of glutamate release from the presynaptic terminal due to increase in clearance of adenosine normaly existing nearby the synapse and inhibiting the transmitter release. [J Physiol Sci. 2007;57 Suppl:S145]

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We examined the effects of dopamine (DA) on the excitatory postsynaptic potential (EPSP) in neurons of the rat dorsolateral septal nucleus (DLSN) by conventional intracellular recording and voltage-clamp methods. A continuous application of DA (1 μM, for 30-60 min) produced a gradual increment of the amplitude of the EPSPs (149 ± 19% of control, n=8). The long-lasting enhancement of the EPSP was mimicked by SKF 38393 (10 μM), a D1 type receptor agonist, but not by quinpirole (10 μM) and PD 168077 (3 μM), D2 type and D4 receptor antagonists, respectively. The SKF 38393-induced enhancement of the EPSP was blocked by SCH 23390 (10 μM), a D1 type receptor antagonist, but not by sulpiride (5 μM) and L-741742 (10 μM), antagonists for D2 type and D4 type receptors, respectively. SKF 38393 increased the amplitude of miniature EPSPs without changing their frequencies. SKF 38393 enhanced the exogenous glutamate-induced responses in DLSN neurons. The ratio of the paired-pulse facilitation of the EPSP was not altered by SKF 38393. Application of dibutyryl cyclic AMP (1 mM) or forskolin (10 μM) mimicked the effect of DA on the EPSP. The SKF 38393-induced enhancement of the EPSP was blocked by H-89 (10 μM), an inhibitor for protein kinase A (PKA) but not by calphostin C (100 nM), an inhibitor for PKC. These results suggest that DA enhances the EPSP by increasing evoked release of glutamate via the postsynaptic D1 receptors-PKA pathway in rat DLSN neurons. [J Physiol Sci. 2007;57 Suppl:S146]

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Increase in the temperature from 36 to 40°C produces a membrane hyperpolarization associated with a decrease in input resistance and depresses the excitatory postsynaptic potential (EPSP) of CA1 neurons evoked by a stimulation of Schaffer collaterals in rat hippocampus. We examined whether the activation of adenosine A₁ receptor contributes on those heat-induced responses in CA1 pyramidal neurons using the intracellular recording techniques. Parasaggital slices of the hippocampus (400μm thick) were obtained from male Wistar rats (130-360g). The slices were superfused with oxygenated artificial cerebral spinal fluid (ACSF) at 2ml/min. The temperature of the ACSF was initially kept at 36°C for 30min. Increasing the temperature to 40°C rapidly produced a membrane hyperpolarization associated with a decrease in input resistance regardless the presence of the DPCPX (1μM), an A₁ receptor antagonist. DPCPX (1μM) blocked the rapid depression of the EPSP induced by the temperature increase to 40°C, which reached a peak within 6min. Application of adenosine (10μM) depressed the amplitude of the EPSP by more than 90% of control at 32°C. However, in the presence of the adenosine (10μM), the EPSP was not depressed by the temperature increase from 36 to 40°C. These results suggest that endogenous adenosine contributes to the heat-induced depression of the EPSP via the A₁ receptors, not to the reduction of the membrane excitation in CA1 neurons. [J Physiol Sci. 2007;57 Suppl:S146]

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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 Kv1.1 channels abundantly and receive a few large end-bulb synapses, while Kv1.1 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 action potentials along the tonotopic axis; the amplitude is smaller in high-CF cells (20 mV) than in low-CF cells (50 mV). Furthermore, the difference was still present under the block of K⁺ currents by DTX. These results may indicate that the process of generating action potentials is also specialized along the tonotopic axis, which may contribute to the precise information processing in NM neurons. In this study, we evoked orthodromic and antidromic spikes under the whole-cell slice-patch recordings, and examined the properties of action potentials along the tonotopic axis in NM of the chick (P2-7). We further evaluated the distribution of Na⁺ channels immunohistochemically, and explored the site of action potential generation. [J Physiol Sci. 2007;57 Suppl:S146]

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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 accuracy 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 accuracy of CD in NL. GABAergic inputs for NL arise mainly from the superior olivary nucleus (SON) and partly from interneurons. SON neurons increase their activity with the increase of stimulus intensity, but poorly hold timing information. Thus, SON neurons are thought to have a role in gain control of NL neurons. On the other hand, interneurons have been proposed to possess a different role from SON neurons, however details are not known. In this study, we examined the difference between SON neurons and interneurons, in their roles for CD in NL. [J Physiol Sci. 2007;57 Suppl:S146]

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Homer1a/Vesl-1S is an activity-inducible member of the scaffold protein family Homer/Vesl, which is known to link metabotropic glutamate receptors (mGluRs) to endoplasmic calcium release channels such as inositol-1,4,5-triphosphate receptors (IP3Rs) or ryanodine receptors. Homer 1a is regarded also to regulate such Homer-mediated coupling. It has been reported that Homer 1a is involved in short-term synaptic transmission. However, it is still unclear whether Homer 1a takes part in induction of long-term depression (LTD), a form of long-term synaptic plasticity. To examine the roles of Homer 1a in inducing LTD in the rat visual cortex, Homer 1a protein was injected by diffusion from whole-cell patch pipettes. The results revealed that Homer 1a injection suppresses LTD dependent on mGluRs, but not that dependent on N-methyl-D-aspartate receptors (NMDARs). Then, induction of endogenous Homer 1a by electroconvulsive shock (ECS) was attempted. Homer 1a thereby induced was also able to reduce LTD magnitude significantly. Reduction of LTD expression after ECS was recovered by injection of anti-Homer 1a antibodies. These results suggest that endogenously induced Homer 1a may serve as a regulator of mGluR-mediated long-term synaptic transmission. [J Physiol Sci. 2007;57 Suppl:S147]

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The effect of zinc on glycinergic postsynaptic currents (sIPSCs) was studied by using the whole-cell patch-clamp technique in mechanically dissociated rat spinal dorsal horn neurons. Zinc reversibly and concentration-dependently increased sIPSC frequency. Low concentration of zinc potentiated the sIPSC amplitude, while high concentration of zinc inhibited the amplitude. The zinc-induced synaptic potentiation was abolished in the absence of extracellular Ca²⁺ or by the addition of Cd²⁺, suggesting the involvement of Ca²⁺ influx into the nerve terminals. The zinc action was also inhibited in the presence of tetrodotoxin, suggesting that zinc causes depolarization of the nerve terminals. In the slice preparation, zinc potentiated the evoked IPSC amplitude and decreased the paired pulse ratio. These results suggest zinc modulates glycinergic transmission in the spinal cord and plays an important role in the pain transmission. [J Physiol Sci. 2007;57 Suppl:S147]

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Dendritic spines are multiple functional units that receive most of excitatory inputs in central nervous system. We show here that a novel drebrin-binding protein is involved in the regulation of dendritic spine formation. Contrary to its predominant localization in the neculei of non-neuronal cells, this protein was localized primarily in nucleus and dendritic spines in neurons. Hence, we named it Spikar for its unique intracellular localization in spine and karyoplasm. Since drebrin A is a neuron-specific F-actin binding protein that regulates spine function and morphology, we analyzed whether Spikar plays a role in the dendritic spine formation. Transfection of shRNA expression vector into cultured hippocampal neurons resulted in ∼20% decrease in endogenous Spikar expression. When neurons were transfected at 3 days in vitro (DIV) and analyzed at 16 DIV, the neurons expressing Spikar shRNA (Spikar-KD neurons) showed lower densities of dendritic filopodia and dendritic spines than control cells. However, the spines formed in Spikar-KD neurons showed normal immunoreactivities of synaptic proteins. Miniature excitatory postsynaptic currents (mEPSCs) significantly reduced in Spikar-KD neurons without affecting the mEPSC amplitude. These results suggest that Spikar is involved in the dendritic spine formation but not in the functional maturation of the spines. [J Physiol Sci. 2007;57 Suppl:S147]

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Midbrain substantia nigra pars reticulata (SNr) is the major output nucleus of basal ganglia, which is important for motor regulation. SNr consists mostly of GABAergic neurons, of which spontaneous firing rate is among highest in the brain, implying that demand for energy sources such as oxygen and glucose is very high. Indeed, we have previously shown that SNr activity is extremely sensitive to hypoxic conditions and under severe hypoxia SNr activity is suppressed quickly, resulting in protection against seizure propagation. In contrast, moderate lowering of extracellular glucose concentration in acute slice facilitates SNr activity. A similar sensitivity for lowering glucose was originally found in lateral hypothalamus, but the underlying mechanism is still uncertain. Here we show by using acutely dissociated SNr neurons that the glucose sensitivity is produced via post-synaptic mechanisms. We also found that dopamine, glycine, and insulin had modulatory effects on SNr activity in addition to well established neurotransmitters in the SNr, GABA and excitatory amino acid. Evidence has accumulated showing that increased SNr activity inhibits locomotion and REM, and is related to rhythmical movements such as of the jaw and limb. Thus, SNr neurons may have a role as fuel sensors which inform energy status for coordinating motor activity and for protecting brain in emergency, and various neuromodulators can regulate the sensor property. [J Physiol Sci. 2007;57 Suppl:S147]

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We have previously reported that a phospholipase A₂ (PLA₂) activator melittin enhances excitatory and inhibitory synaptic transmission in substantia gelatinosa (SG) neurons of adult rat spinal cord slices. The present study examined a detail of a difference in pharmacology between the melittin-induced enhancements of glycinergic and GABAergic spontaneous inhibitory postsynaptic currents (sIPSCs) recorded at 0 mV by using the whole-cell patch-clamp technique. The enhancements of the frequency and the amplitude of GABAergic but not glycinergic sIPSC produced by melittin (1 μM) superfused for 3 min were largely reduced in extent in the presence of a Na⁺-channel blocker tetrodotoxin (0.5 μM). The effects of melittin on glycinergic sIPSCs were also resistant to a non-NMDA receptor antagonist CNQX (10 μM) while being depressed to a great extent by a PLA₂ inhibitor 4-bromophenacyl bromide (10-50 μM). These effects of melittin on glycinergic sIPSC amplitude and frequency were dose-dependent with EC₅₀ values of about 0.7 μM. These results indicate that PLA₂ activation results in enhancing GABAergic and glycinergic transmission in a different manner in such that the former but not latter effect is possibly mediated by the enhancement of excitatory transmission by PLA₂ activation which is followed by the release of neuromodulators such as acetylcholine and norepinephrine involved in enhancing GABAergic transmission. [J Physiol Sci. 2007;57 Suppl:S148]

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Extracellular ATP elicits a variety of physiological activities through the activation of ionotropic P2X and metabotropic P2Y receptors. Recent findings have indicated that spinal cord injury is associated with prolonged purinergic receptor activation, which results in excitotoxicity of spinal motoneurons. However, it is unknown whether purinergic receptor activation can modulate synaptic transmission in spinal motoneurons. By use of the whole-cell patch-clamp technique, we investigated the effects of extracellular ATP on motoneurons of rat spinal cord slices. Bath application of ATP or ATPγS, a metabolically stable ATP analog, produced an inward current at a holding potential of -70 mV in about 30% of neurons examined. When applied repeatedly at a time interval of 10 min, ATPγS produced similar responses with almost the same amplitude. In the presence of TTX, ATPγS also induced an inward current without any significant decrease in amplitude. The ATPγS-induced inward current was mimicked by 2-methylthio ADP, an agonist for P2Y₁, P2Y₁₂, and P2Y₁₃ receptors, but not by α,β-methylene ATP, BzATP, UTP, or UDP. The ATPγS-induced inward current was significantly suppressed by PPADS, a purinergic receptor antagonist, or MRS2179, a selective P2Y₁ receptor antagonist. These results indicate that extracellular ATP directly excites a subpopulation of spinal motoneurons by the activation of P2Y₁receptors. Thus, P2Y₁ receptor antagonists may constitute a new medication for the treatment of spinal cord injury. [J Physiol Sci. 2007;57 Suppl:S148]

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We have reported that there exist regional differences of propofol effects on GABA-mediated inhibitory synaptic transmission in vitro studies (Ishiguro M et al, JPS 56 suppl; s165, 2006). In this study, we aimed to investigate the relation between electrical stimulus intensity and inhibitory postsynaptic currents (IPSCs) without propofol in CA1 pyramidal cells (CA1-PCs) and dentate gyrus granule cells (DG-GCs) in rat hippocampal slices. The monosynaptic IPSCs were evoked by electrical stimulation of GABAergic interneurons and recorded from CA1-PCs and DG-GCs by a whole cell patch-clamp technique. The electrical stimuli (0.1ms pulses of 0.5-5mA) were delivered using bipolar stainless wire electrodes placed within 250 μ m of the recording pipette and near the somatic regions of the CA1-PCs and DG-GCs. IPSCs were evoked at various voltage command steps from -120 to +40mV (holding potential of -60 mV). By increasing the stimulus intensity, the amplitude of IPSCs in CA1-PCs and DG-GCs were enhanced in a double sigmoid manner with a notch at approximately 60% of the maximum stimulus intensity, as well as the decay time constant of IPSCs. There is no clear difference in the enhancement patterns of IPSC amplitude and decay time constant between CA1-PCs and DG-GCs.These results suggest that in CA1-PCs and DG-GCs, neural mechanisms for IPSCs evoked at the lower stimulus intensity (< c., 60%) might be different from those evoked at the higher intensity. [J Physiol Sci. 2007;57 Suppl:S148]

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Few softwares performing neuron tracing and 3D reconstruction from serial sections are available, but they are rather expensive. We have used the 3D reconstruction system developed originally in 80's. It is still working, but quite slow in performance. We developed a new Windows-based tracing software "NeuroShape" based on the same concept, and compared usability. The conventional bright-field microscope, video camera and digital linear scale are reused. A personal computer is linked to the camera and the scale via USB interfaces. The software is newly coded with Ruby, the interpreted scripting language. Microscopic view is shown overlaid on a video image captured. Users then trace the neuron structures specifying XY coordinates by mouse operation and Z coordinate by microscope focusing. Shrinkage correction is also available. To obtain 3D view, the virtual reality modeling language (VRML) is used. We reconstructed 3D models of neurons using the old and new system.Reconstructed images were almost the same and the new system "NeuroShape" saved lot of time for error correction. Final 3D views were shown interactively with nearly real-time response. We could build the system with few expense and stress the usefulness for exploring the 3D structure of neurons. [J Physiol Sci. 2007;57 Suppl:S148]

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The afferent fibers in the vagus nerve transmit visceral information encoded as varying firing frequency to the second-order neurons in the solitary complex (SC). We have demonstrated that efficiency and frequency-dependency of the synaptic transmission from the primary afferent fibers in the solitary tract (TS) to distinct types of second-order neurons (the nucleus of the solitary tract, NTS, and the dorsal motor nucleus of the vagus nerve, DMX) critically depends on the type of postsynaptic neurons, but mostly through differences in the presynaptic mechanisms. Here we analyzed the mechanism underlying distinct types of transmission by evaluating the effects of manipulations that affect release probability on the paired-pulse ratio (PPR) of EPSC amplitudes evoked by two TS stimuli separated by 100 ms in the brainstem slices from young Wistar rats.. Reducing [Ca ²⁺]_o from 2.0 to 0.5 mM significantly increased the PPR of TS-NTS synapses from 0.4±0.2 to 1.1±0.2 (n=6; mean ± SD) but did not significantly affect that of TS-DMX synapses (type I-DMX neurons, from 0.7±0.2 to 0.7±0.2; n=8; type II-DMX neurons, from 0.4±0.1 to 0.7±0.3; n=8). Whereas the PPR of TS-NTS synapse was significantly increased by adenosine (100 μM) to 0.5±0.2 (n=7), that of TS-DMX-type II synapse was not affected (0.5±0.1 with adenosine). These results suggest that distinct presynaptic mechanisms are involved in the distinct short-term plasticity of the SC synapses depending on the function of each postsynaptic target. [J Physiol Sci. 2007;57 Suppl:S149]

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Despite molecular identification of a large number of "presynaptic" proteins, their functions remain largely undetermined. To address this issue, we developed a novel method to directly evaluate the effect of gene silencing by RNA interference on the central neurotransmission in situ. In anesthetized young Wistar rats, either of (1) synthetic siRNA against the gene coding adenosine A1 receptors (Adora1), (2) siRNA of random sequence or (3) vehicle, was introduced to the nodose ganglion (NG) by electroporation. Introduction of siRNA labeled with Cy3 revealed an introduction efficiency of ∼85% of the nodose ganglion neurons. The number of cells was not significantly affected by introduction. Three to nine days after siRNA delivery, adora1 expression in the NG decreased by >80% of non-treated NG. The presynaptic inhibition by adenosine of the transmission from primary afferents to the second-order neurons, as evaluated by electrophysiological recording in the brainstem slice preparation, was markedly attenuated on 11 to 13 days after siRNA delivery. This approach provides a promising strategy for the analysis of the functional role of presynaptic proteins in the brain synapses. [J Physiol Sci. 2007;57 Suppl:S149]

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Hirasawa and Kaneko (2003) proposed a pH-mediated feedback mechanism from horizontal cells (HCs) to cone photoreceptors to interpret the formation of receptive field surrounds in the retinal neurons. To verify the idea that the depolarized HCs release proton, we measured the pH of the external surface (pHo) of HCs isolated from the carp or goldfish retina by a fluorescence ratio imaging technique using a pH-sensitive lipophilic dye, 5-hexadecanoylaminofluorescein. When HCs were depolarized by kainate or high extracellular K⁺, pHo was shifted to acidic side. Electrophysiological and imaging experiments showed that bafilomycin A1,a specific inhibitor of vacuolar type H⁺-ATPase (V-ATPase), suppressed depolarization-induced shift of pHo.Localization of V-ATPase was examined by immunohistochemistry using an anti V-ATPase antibody. In isolated HCs immunoreactivity was observed on the cytoplasmic membrane. In retinal slice preparations, anti-V-ATPase immunoreactivity was found at the cone terminals and on the surface of GAD-positive external HCs.These results suggest that HC dendrites can be the active sites of proton extrusion supporting the proton hypothesis for the feedback from HCs to cone photoreceptors. [J Physiol Sci. 2007;57 Suppl:S149]

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Burst firings of intermediate gray layer (SGI) neurons in the superior colliculus (SC) are important for generation of saccadic eye movements. Although GABA_B receptors (GABA_BRs) are abundantly expressed in the SC, the role of these receptors for the burst generation has been unclear. In the present study, we examined this issue using whole-cell recordings in brain slice preparations obtained from GAD67-GFP knock-in mice in which GABAergic neurons express GFP fluorescence. In the presence of a GABA_AR antagonist gabazine, single electrical stimulation of the superficial gray layer (SGS) evoked burst EPSCs in non-GABAergic SGI neurons. Addition of a GABA_BR antagonist CGP52432 (CGP) significantly increased the duration but not amplitude of the burst EPSCs, indicating that synaptically released GABA activates GABA_BRs. When CGP was locally puff-applied to the SGS, but not to the SGI, the burst EPSCs duration was also prolonged, suggesting that the site of CGP action was SGS. Consistent with these results, CGP also prolonged the duration of burst firings in non-GABAergic SGS neurons. Moreover, in the presence of antagonists for ionotropic glutamate receptors and GABA_AR, local repetitive stimulation which mimicked GABAergic burst firings induced CGP-sensitive slow outward currents in some non-GABAergic SGS neurons. These results suggest that GABA_BRs localized in the SGS function as a negative-feedback mechanism for the burst responses in the SC. [J Physiol Sci. 2007;57 Suppl:S149]

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The type-1 metabotropic glutamate receptor (mGluR1) is required for motor learning and synaptic plasticity in cerebellar Purkinje neurons (PNs). Stimulation of parallel fibers (PFs) is known to elicit two distinct pathways via mGluR1α, an activation of G_q pathway and a direct activation of non-selective cation channels observed as a slow excitatory postsynaptic current (sEPSC). Cerebellum consists of ten lobules in mice and some functional differences between lobules have been reported. In this study we focused on the difference of sPSC between lobules in sagittal section of postnatal 15-17 days mice. We recorded from PNs by whole-cell patch clamp at -70 mV in the presence of blockers of AMPA and GABA_A receptors. By applying tetanic stimulation (100 Hz 10 times) to PFs, we observed a transient inward current in lobule 9. The inward current reached a peak at 716 ± 126 ms (mean ± s.e.m) after stimulation and the amplitude was 190 ± 55 pA (n=5). In lobule 10, sEPSC was slower (1078 ± 178 ms) and smaller in amplitude (139 ± 64 pA), we also observed a faster transient outward current in lobule 10, but not in lobule 9. The time to peak was 272 ± 14 ms, and the amplitude was 44 ± 7 pA (n=5). Taken together, it was shown for the first time that PF stimulation-evoked slow PSCs of PNs in lobules 9 and 10 differ from each other. [J Physiol Sci. 2007;57 Suppl:S150]

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Many physiological studies have been made to elucidate the properties of microcircuitly in the visual cortex. However, the spatio-temporal properties of the cortical network are not studied well. We investigated the signal propagation pattern induced by electrical stimuli in the mouse visual cortex slice, using a 60 channel multielectrode array combined with the current source density analysis. When the current stimulus was applied to layer 4, which receives inputs from LGN, current sink propagated vertically to layer 2/3, and then horizontally in layer 2/3. The horizontal propagation was also observed in layer 4. The maximum amplitude of the sink was observed at dorsolaterally adjacent recording electrode to the stimulus position of layer 2/3. In contrast, stimulation to layer 2/3 induced a strong excitatory synaptic transmission within layer 2/3 and the maximum current sink was observed at the dorsolaterally adjacent electrode, but diminutive sink was observed at the medially adjacent one. These results suggested that the layer 4 stimulus induced excitatory synaptic transmissions within layer 4 neurons, which innervated to layer 2/3. The layer 2/3 stimulus induced the synaptic responses mainly within layer 2/3, and then, the weak signal was propagated to other cortical layers. The layer 2/3 neurons might be tightly coupled each other with excitatory synapses. [J Physiol Sci. 2007;57 Suppl:S150]

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Phospholipase C (PLC)-related inactive protein-1 (PRIP-1) is known as an inositol 1,4,5-triphosphate [Ins(1,4,5)P₃] binding protein, which plays an important role in GABA_A receptor trafficking and function. However, little is known about its role in the functional properties of GABA inhibitory postsynaptic currents (IPSCs) in the barrel cortex. Therefore, we studied the role of PRIP-1/2 in the modulation of GABA IPSCs in layer II/III pyramidal neurons of barrel cortex using PRIP-1 and -2 knockout (PRIP-DKO) mice. The mean frequency, amplitude and 10-90% rise time for GABAergic miniature IPSCs showed no difference between wild-type and PRIP-DKO mice. However, the half decay time in PRIP-DKO mice was significantly shorter than that in wild-type mice. Furthermore, the maximum amplitude of GABA IPSCs induced by puff application (2 sec duration) of 200 μM GABA in PRIP-DKO mice was significantly smaller than that in wild-type mice at high pressures, and the 10-90% rise time and half decay time of GABA IPSCs in PRIP-DKO mice were significantly shorter than those in wild-type mice. Interestingly, an after-response was observed at the offset of puff application only in PRIP-DKO mice. Taken together, these results suggest that PRIP-1/2 may play an essential role in desensitization and resensitization in GABA_A receptors. [J Physiol Sci. 2007;57 Suppl:S150]

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In comparison with the barrel cortex, the primary gustatory area in the insular cortex is thought to have much less distinct functional columns. In order to better understand the inter-columnar information processing in which GABA_A IPSPs are thought to be involved, we examined the differences in the spatiotemporal pattern of excitation spread between the barrel and insular cortices, using voltage-sensitive dye imaging. The excitation evoked by stimulation of layer IV of the barrel cortex spread upwardly into layer II/III, where the excitation spread horizontally into neighboring columns partially, but leaving the layer V/VI unexcited. By contrast, the excitation in layer IV of the insular cortex spread upwardly into layer II/III as well as into layer V/VI downwardly, while the horizontal spread in layer II/III was much less prominent. An application of bicuculline prolonged the time course and enlarged the extent of horizontal spread in layer II/III across the neighboring columns fully, while it just prolonged the time course of vertical spread in the insular cortex, leaving the horizontal spread almost unchanged. These results suggest that the involvement of GABA_A receptors in the lateral inhibition in layers II/III is much more prominent in the barrel cortex than in the insular cortex. In the insular cortex, the sensory information arising from thalamus would be forwarded bi-directionally to the superficial and deep layers, and the reciprocal inhibition between the two layers within a same column is remarkable. [J Physiol Sci. 2007;57 Suppl:S150]

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In the developing brain, synapses are formed once in excess and trimmed off later (pruning). It is under debate whether the pruning of synapse depends on the synapse's activity or inactivity. Here we show that a long-lasting retardation of synaptic development occurred after the repeated activations of metabotropic glutamate receptor (mGluR) in the dissociated culture of the rat hippocampus, and thereby we suggest that the synaptic pruning depends on the synapse's activity using mGluR. We applied a group I mGluR agonist (DHPG) to the hippocampal neurons of 14 days in vitro, which are still in the process of synaptic development. We found that the culture exposed 3 times to DHPG had a significantly smaller number of synapses in the following weeks, though the number of neurons in the culture was unaffected. This effect was not produced by a single exposure to DHPG and was blocked by a mGluR antagonist (MCPG). This effect was most prominently seen in the proximal dendritic region of glutamatergic neurons. In GABAergic neurons, no significant effect was found in both proximal and distal dendritic regions. These results suggest that repetitive activation of mGluR causes retardation of synaptic development in immature neurons as well as the matured slice culture reported previously, and the retardation shows dendritic region-specificity. [J Physiol Sci. 2007;57 Suppl:S151]

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In the stable culture of hippocampal slice, repeated inductions (≥3 times) of LTP by exposures to glutamate (Glu) at regular intervals (3-24h) lead to a slowly-developing (requiring ∼1w to develop) long-lasting (lasting ≥3w) enhancement of synaptic transmission efficiency coupled with an increase in the number of synapses. We call this phenomenon RISE (Repetitive-LTP Induced Synaptic Enhancement) to discriminate it from conventional single LTP. Electron microscope (EM) examination shows that the number of synapses with small postsynaptic density (PSD) increases transiently during the developing phase of RISE, indicating ongoing synaptogenesis. Recently we reported that field EPSP in the culture after repeated Glu-exposures contained a component susceptible to Joro-Spider Toxin (JSTX), a selective blocker of Ca²⁺-permeable AMPAR (CaP-AMPAR) in this period. Here we report that the appearance of CaP-AMPAR is not only a result of RISE but also a cause of its establishment. When we applied JSTX for a limited period (24h) during the developing phase of RISE (4d after the 1st Glu-application), RISE was not established, when examined 14d later. When we examined the culture treated with JSTX after the repeated Glu-exposures by EM, the increase in the number of small-PSD synapses was abolished, although other morphological features including the occurrence of perforated synapses were unaffected. These results suggest that Ca²⁺-influx through CaP-AMPAR is required for the growth and maintenance of new synapses. [J Physiol Sci. 2007;57 Suppl:S151]

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We showed the development of rat's corticospinal (CS) innervation consists of at least two phase. The first stage peaks at postnatal day 7 (P7), when CS terminals are distributed in the whole spinal gray. However, the CS terminals in the ventral side are eliminated until P10. After this elimination, "the second wave" of CS innervation arrives: CS terminals again increase in the ventral and also dorsal side. This second wave quickly occurs from P12 and completes until P18. In this study we investigated which level of CS projection produces the second wave. A small amount of biotin dextran was injected at one site in the sensorimotor cortex and animals were fixed after desired survival time. Serial sagittal or horizontal sections were obtained from the cortex to cervical cord and transverse sections from C5 to C8. Axonal arborization was reconstructed by camera lucida drawing on serial longitudinal sections through the pathway of CS tract as well as transverse section at C5 to C8. In the longitudinal sections, the number of branches derived from the dorsal funiculus at P18 did not increase compared with P10. The number of axons in the medullary pyramid showed no difference between P7 and P14. Therefore it is unlikely that CS neurons not involved in the first wave newly participate in the second wave. On the other hand, the number of branching in the gray matter at P18 was much larger than that at P10. These suggest that the branches give rise to the second wave are generated within the spinal gray matter of the same segment rather than in the more proximal axons. [J Physiol Sci. 2007;57 Suppl:S151]

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D-tubocurarine (d-TC) was used to block the synaptic transmission of frog neuromuscular junction. A recently discovered avidity of IgG for d-TC and also for other quaternary ammonium compounds (Tsuji et al 2003) was used to visualize the d-TC bound to the nicotinic acetylcholine receptor (nAChR) by means of an immunochemical reaction (immunoperoxidase). Regularly disposed transmembrane rod-like structures were observed through the postsynaptic membrane and interpreted as a staining of the interior side of the nAChR. Reference: Tsuji, S. et al (2003) Binding avidity of immunoglobulin G for acetylcholine. Biomed Res 24: 217-221 [J Physiol Sci. 2007;57 Suppl:S151]

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I have suggested that G_βγ subunits dissociated from G_q/11 linked with M₃-receptors reduce the GABA release through an effect on the release machinery, when M₃-muscarinic ACh receptors at the presynaptic terminal of a putative striato-nigral projection fiber are activated. In order to get some more evidences on this hypothesis, I analyzed the effects of Calphostin C (1 μM), an inhibitor of protein kinase C, ω-Conotoxin GVIA (1 μM), a selective N-type Ca²⁺ channel blocker and BAPTA-AM (30 μM), a membrane permeable Ca²⁺ chelator of intracellular store. All these drugs reduced the amplitude of IPSC. Muscarine significantly inhibited these reduced IPSCs in the solutions with the drugs. Thus it was again indicated that the activation of protein kinase C, the Ca²⁺ influx to the presynaptic terminals and the Ca²⁺ release from intracellular store sites at the presynaptic terminals were not the main mechanisms of the muscarinic inhibition of IPSC. This could insist on the justice of the hypothesis mentioned above. Muscarine also reduced the amplitude of the mono- and poly-synaptic glutamatergic EPSCs onto SNr GABA neurons evoked by the electrical stimulation to subthalamic nucleus (STN). This result suggested that muscarine inhibited the glutamatergic EPSCs onto not only SNr GABA neurons but also STN neurons, since it have been reported that the polysynaptic EPSC onto SNr GABA neurons was generated the recurrent glutamate synapses in the STN. [J Physiol Sci. 2007;57 Suppl:S152]

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A comprehensive survey of auditory network formation was performed in the brainstem of the chicken embryo using voltage-sensitive dye recording. Intact medulla/brainstem preparations with the auditory nerve attached were dissected from E5.5 to E8 chicken embryos, and responses evoked by nerve stimulation were recorded optically. In the medulla of E7 and E8 embryos, we identified four response areas, corresponding to the ipsilateral Nucleus magnocellularis (NM) and Nucleus angularis (NA), and the ipsi- and contralateral Nucleus laminaris (NL). The optical responses consisted of a fast spike-like signal followed by a long-lasting slow signal, which reflected the sodium-dependent action potential and glutamatergic EPSP, respectively. In NM, NA and NL, the EPSP-related slow optical signals were detected from some E6 and all E7 and E8 preparations, indicating that functional synaptic connectivity in these nuclei arises by the E7 stage. In the pons of E7 and E8 embryos, we identified two additional response areas, which evidently correspond to the ipsi- and contralateral Nucleus lemnisci lateralis (NLL), the higher-order nuclei of the auditory pathway. Furthermore, we detected optical responses from the contralateral cerebellum, which possibly correspond to transient projections observed only during embryogenesis. The present study demonstrates that the basic functional circuits related to auditory function are established in the chicken embryo at earlier stages than previously reported. [J Physiol Sci. 2007;57 Suppl:S152]

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During development, correlated neuronal activity plays an important role in the establishment of the CNS. We previously reported that a widely-propagating correlated neuronal activity, termed the depolarization wave, is evoked by various sensory inputs. In the present study, we examined whether the depolarization wave occurs spontaneously in the embryonic rat CNS, and if so, where it originates. In E15-E16 rat embryos, spontaneous optical signals appeared in association with the rhythmic discharges of cranial motoneurons, and propagated widely with similar characteristics to the evoked depolarization wave. At E15, the spontaneous wave mostly originated in the cervical to upper lumbar cords. At E16, the wave was predominantly generated in the lumbosacral cord, although a wave associated with the second oscillatory burst was initiated in the rostral cord. At E16, a few waves also originated in the rostral ventrolateral medulla and the dorsomedial pons. When the influence of the caudal cord was removed by transecting the spinal cord, the contribution of the medulla and pons became more significant. These results show that the depolarization wave can be triggered by the spontaneous activity of multiple neuronal populations which distribute widely from the pons to the lumbosacral cord, although the spinal cord usually plays a predominant role. This network possibly works as a self-distributing system that maintains the incidence and complicated patterns of the correlated activity in the developing CNS. [J Physiol Sci. 2007;57 Suppl:S152]

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The cholinergic system in the CNS plays important roles in higher brain functions, through muscarinic receptors. The nucleus tractus solitarius (NTS) is known to plays a major role in the regulation of cardiovascular, respiratory, gustatory, hepatic and swallowing functions. Voltage-dependent Ca²⁺ channels (VDCCs) serve as crucial mediators of membrane excitability and Ca²⁺-dependent functions such as neurotransmitter release, enzyme activity and gene expression. ACh injected into the NTS of rat elicits a decrease in arterial pressure and heart rate similar to that seen with activation of the baroreflex. The purpose of this study was to investigate the effects of ACh on VDCCs currents (ICa) in the NTS using patch-clamp recording methods. In 68 of 99 neurons, an application of ACh caused inhibition of N- and P/Q-types IBa in a concentration-dependent manner. Application of a strong depolarizing voltage prepulse attenuated the ACh-induced inhibition of IBa. Pretreatments with AF-DX116 (muscarinic M2 receptor antagonist) attenuated the ACh-induced inhibition of IBa. In contrast, Pretreatments with Telenzepine (muscarinic M1 receptor antagonist) did not attenuate the ACh-induced inhibition of IBa. Intracellular dialysis of the Gi-protein antibody also attenuated the ACh-induced inhibition of IBa. In contrast, intracellular dialysis of and Gs- and Gq/11-proteins antibodies did not attenuate the ACh-induced inhibition of IBa. These results indicate that ACh inhibits N- and P/Q-types VDCCs via Gi-protein beta gamma subunits mediated by M2 receptors in NTS. [J Physiol Sci. 2007;57 Suppl:S152]

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At the calyx of the Held synapse in the auditory brainstem, P/Q-type presynaptic Ca²⁺ currents undergo an activity-dependent facilitation during repetitive activation mediated by neuronal calcium sensor 1 (NCS-1). Of all 4 Ca²⁺-binding sites of NCS-1, we primarily focused on third EF-hand motif because E120Q mutant has a phenotype in synaptic transmission or exocytosis in other preparation. Unlike the wild-type NCS-1, direct loading of NCS-1 (E120Q) that loses Ca²⁺-biniding capability, into the nerve terminal did not induce the facilitation of calcium current in the basal stimulation. When NCS-1 (E120Q) was included in the presynaptic pipette solution, the rise time of presynaptic Ca²⁺ current was not significantly faster than rise times in the absence of NCS-1. The current-voltage (I-V) relationship of presynaptic calcium current measured at 1 ms after the onset of the command pulse had a similar peak at 0 mV in the presence of NCS-1 (E120Q) and in the absence of NCS-1. To evaluate the activity-dependence further, we tested if the NCS-1 (E120Q) may inhibit the calcium current facilitation caused by a short burst of depolarization (100 Hz by 10 stimuli). In control, calcium current caused 60% facilitation, whereas NCS-1 (E120Q) caused only 20% facilitation. These results suggest that the third EF hand motif (calcium binding site) performs as a sensor detecting the alteration of residual calcium concentration at nerve terminals. [J Physiol Sci. 2007;57 Suppl:S153]

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Voltage-dependent K⁺ currents regulate transmitter release by repolarizing the nerve terminal. Among many types of K⁺ channels, high voltage-activated Kv3 channels and low voltage-activated Kv1 channels are abundantly expressed at the calyx of Held nerve terminal in the auditory brainstem of rodents. During early postnatal development, calyceal synapses undergo a variety of morphological and functional changes. Here we asked whether voltage-dependent K⁺ channels in calyceal terminals undergo developmental changes and whether they contribute to functional maturation of this auditory synapse. From postnatal day (P) 7 to P14, the density of Kv1 and Kv3 K⁺ currents increased in parallel and their activation kinetics also accelerated. Simulation of calyx action potential indicated that both changes in the kinetics and density of K⁺ channels contribute to shortening of action potential duration. Selective block of Kv3 or Kv1 channels at both P7 and P15 indicated that developmental changes in Kv3 channels contributes to high-fidelity firing at high frequency, whereas the developmental increase in Kv1 currents stabilizes the nerve terminal thereby preventing aberrant firing of action potentials. We conclude that developmental changes in both Kv3 and Kv1 channels in the nerve terminal contribute to maturation of high-fidelity fast synaptic transmission at this auditory relay synapse. [J Physiol Sci. 2007;57 Suppl:S153]

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