To induce long-term depression (LTD) at hippocampal CA1 synapses, [Ca²⁺]i must sufficiently rise in CA1 pyramidal cells. Each type of calcium entry is believed to have a distinctive role in LTD induction. The present study focuses on roles of voltage gated calcium channels (VGCCs) in LTD induction at Schaffer collateral-to-CA1 synapses in the hippocampal slices. VGCCs are known to be involved in many important cellular processes including synaptic plasticity. In control experiments, the magnitude of LTD varied depending on stimulus frequencies. Also, LTD evoked by any of the tested frequencies (0.5-2Hz) required NMDA receptor activation. T-type VGCCs turned out to have no significant role in LTD induction, since T-VGCC blockade did not change normal LTD induction. When L-type VGCCs were blocked with nimodipine, LTD magnitude was enhanced with low frequency (0.5-Hz) stimulation and was reduced with high frequency (1- or 2-Hz) stimulation. We were particularly interested in the enhancement of LTD by L-VGCC blockade in response to low frequency (0.5 Hz) stimulation, since we had previously observed ryanodine receptor activation could also enhance LTD in response to the same low frequency stimulation. Application of calcium store depleter, thapsigargin, in addition to nimodipine canceled out the LTD enhancement. Hence, intracellular calcium release seemed to play a part in nimodipine-induced LTD enhancement that we observed in a low stimulus frequency range. [J Physiol Sci. 2006;56 Suppl:S168]

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It has been known that proliferating cells exist in the amygdala, but their functions remain to be clarified. On the other hand, the amygdala is known to play essential roles in the emotional learning. Therefore, we examined whether the auditory fear conditioning affects the cell proliferation in the rat amygdala as well as other brain areas. Male SD rats were used. On day 1, the rats of conditioned group received five pairings of tone (20 sec) as the conditioned stimulus (CS) and foot shock (1 sec) as the unconditioned stimulus (US) (CS/US group). The control group rats received only CS (CS group). After the training, rats were injected with BrdU. On day 2, rats were placed in a cage and exposed to the tone and their freezing responses were evaluated. Thereafter, rats were perfused and their brains were removed. Serial 40 μm-thick coronal sections of the brains were cut with a cryostat and BrdU immunohistochemistry was performed. BrdU-labeled cells were quantified in each brain area. There was no statistical difference in the number of BrdU-labeled cells in subventricular zone, septal nucleus, subgranular zone of dentate gyrus, or entire amygdala between two groups. In the basomedial amygdala (BMA), BrdU-labeled cells were more abounding in CS/US group. These results suggest that the cell proliferation in the BMA may be involved in the formation of the auditory fear conditioning. [J Physiol Sci. 2006;56 Suppl:S168]

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In the central nervous system, synaptic efficiency is modifiable in activity-dependent manners. Homer1a/Vesl-1S, an activity-dependently inducible member of the scaffold protein family Homer/Vesl, has been implicated in long-term up-regulation of synaptic efficiency (various forms of LTP), as well as in short-term modification of AMPA receptors (arguably, both up- and down-regulation). It is not clear, however, whether Homer 1a takes part in induction of LTD (long-term depression). The present experiments examined roles of Homer 1a in inducing LTD at a variety of corticocortical synapses in rat visual cortex slices by using whole-cell patch clamp. Homer 1a was injected by diffusion from patch pipettes. With or Without Homer 1a injected, LTD induction was attempted by pairing 1 Hz stimulation with post-synaptic depolarization for 10 min. Without Homer 1a, LTD was induced at synapses between layer IV axons onto layer II/III pyramidal cells, those between layer II/III axons onto layer V pyramidal cells and those between layer II/III axons onto layer VI pyramidal cells. However, at synapses between layer IV axons onto layer VI pyramidal cells, LTD induction was failed. With Homer 1a protein injected, on the other hand, LTD induction was reduced in magnitude at layer II/III-to-layer VI pyramidal cell synapses, but not at the other synapses examined, suggesting a synapse-specific effect of Homer 1a. [J Physiol Sci. 2006;56 Suppl:S168]

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The glutamate receptor δ2 subunit (GluRδ2) is selectively expressed in cerebellar Purkinje neurons (PNs) and is involved in the long-term depression (LTD). However, little is known about the mechanism of its action. Acute expression of the wild-type GluRδ2 in the GluRδ2-deficient PN rescued the LTD induction, suggesting the direct role of GluRδ2 in LTD. To identify the critical region of GluRδ2 necessary for the LTD induction, we constructed and expressed various mutant GluRδ2 proteins in the GluRδ2-deficient PNs. The mutant GluRδ2 possessing the membrane-proximal 21 amino acid residues (AAs) in C-terminal cytoplasmic region rescued the LTD induction, whereas the mutant with membrane-proximal 13 AAs failed. In addition, overexpression of the membrane-proximal 14-20 AAs fused to EGFP suppressed the LTD induction in a wild type PN. These results suggest that the membrane-proximal 14-20 AAs of GluRδ2 plays an essential role in LTD. Then, we identified protein interacting with C kinase 1 (PICK1) as a molecule interacting with the membrane-proximal C-terminal region of GluRδ2 by yeast two-hybrid screening. PICK1 co-localized with GluRδ2 at spines of PNs, and immunoprecipitation assays showed that GluRδ2 bound to PICK1 mainly through the membrane-proximal 14-20 AAs. These results indicate that the membrane-proximal 14-20 AAs of GluRδ2 are essential for both LTD and interaction with PICK1, and suggest that interaction between GluRδ2 and PICK1 might be critical for the LTD induction. [J Physiol Sci. 2006;56 Suppl:S168]

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Brain-derived neurotrophic factor (BDNF), which was first identified as a molecule regulating neuronal survival and differentiation, has a pivotal role in the regulation of synaptic plasticity, especially long-term potentiation (LTP). Recent studies have shown that BDNF is transported anterogradely along axon and stored in the presynaptic terminals. The mossy fiber (MF) terminals, which are axon terminals of the granule cells in the dentate gyrus (DG) of the hippocampus, contain the highest concentration of BDNF in the CNS. These observations led us to hypothesize that BDNF is released from MF terminals and that its release is dependent on the activity pattern. We made a Sindbis virus vector containing a mRNA coding the fusion protein constract of BDNF and Venus, one of green fluorescent protein derivatives, and inoculated it stereotaxically to the DG cells of mouse hippocampus (P14-21). After 2-3 days, MF boutons accumulating BDNF-Venus were identified in the acute slice under confocal microscopy. The activity-dependent BDNF release was measured as a reduction of the fluorescence intensity of the individual presynaptic terminal. We found that the activity patterns as high frequency stimulation and theta-burst stimulation are more effective on the BNDF release than low frequency stimulation. It is suggested that BDNF is released from the MF terminals during induction of LTP. [J Physiol Sci. 2006;56 Suppl:S169]

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Synaptic plasticity is assumed as the cellular basis of memory. Long-termplasticity that lasts for days/weeks has not been fully analyzed, mainlybecause of the lack of model system. We found previously that the repeatedinduction of LTP in cultured hippocampal slice caused a long-lastingsynaptic enhancement accompanied by the formation of new synapses, which wasseparate from LTP itself. We found recently that the repeated induction ofLTD by mGluR activation in the same specimen caused a long-lasting decreasein synaptic strength accompanied by the elimination of synapses. Thus wepropose that these repetition-dependent synaptic changes can serve as themodel system for the analysis of long-term plasticity. Here we addfollowing findings that support this proposal. 1) The synapse eliminationis independent of the means of LTD induction, since LTD induced not only byDHPG (dihydroxyphenylglycine, a class I mGluR agonist) but also by a lowdose of NMDA (N-methyl-D-aspartate, an NMDAR agonist) or by DHO(dihydroouabain, a Na/K-ATPase inhibitor) led to the equivalent synapticelimination, when repeated three times (as monitored by electrophysiologicaland morphological indices). 2) The elimination required protein synthesis,since the application of anisomycin (an inhibitor of mRNAs translation toproteins) suppressed the development of synapse elimination. [J Physiol Sci. 2006;56 Suppl:S169]

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To understand the functional organization of the central nervous system (CNS), it is essential to know how sensory information is processed within the CNS. We have been approaching this topic by following the ontogenetic patterning of neural circuit formation related to the cranial and spinal sensory inputs using multiple-site optical recording techniques with voltage-sensitive dyes. In this study, we surveyed developmental organization of neural networks related the olfactory nerve (N. I) in the embryonic chick forebrain. Stimulation applied to the olfactory nerve elicited excitatory postsynaptic potential (EPSP)-related optical signals in the olfactory bulb from the 7-day old embryonic stage (E7). The EPSP was mediated by glutamate, and NMDA- and non-NMDA-receptor components were identified. In more developed stages, in addition to the responses in the olfactory bulb, another response area was discriminated within the cerebrum, which seemed to correspond to the higher-ordered nucleus of the olfactory pathway. The results suggest that the olfactory pathway is functionally generated at early stages of development when neural networks related to other visceral and general somatic sensory inputs are also in the process of developing. [J Physiol Sci. 2006;56 Suppl:S169]

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In an accompanying study, using a multiple-site optical recording technique with a voltage-sensitive dye, we examined the developmental organization of the olfactory pathway in the embryonic chick forebrain, and showed that functional synaptic transmission in the olfactory bulb was expressed at around E7. It is known that odor stimuli elicit oscillatory events in the olfactory bulb in various species. We found that oscillatory activity was also generated in the chick olfactory bulb during embryogenesis. At early stages of development (E7-E8), postsynaptic response-related optical signals evoked by olfactory nerve stimulation exhibited a simple monophasic waveform that lasted a few seconds. As development proceeded, the pattern of the optical signal became complicated, and oscillatory activity was observed in a later phase of the postsynaptic response. The oscillation was restricted to the olfactory bulb, and this spatial pattern was different from that of the propagating wave activity termed the depolarization wave. We examined spatio-temporal patterns of the oscillatory activity in different stages, and studied its developmental dynamics. [J Physiol Sci. 2006;56 Suppl:S169]

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We have succeeded in reconstructing the corticospinal (CS) synapses in vitro by co-culturing the rat sensorimotor cortex and cervical spinal cord. In this in vitro model of CS projection, CS synapses are formed diffusely throughout the spinal gray matter but later the synapses in the ventral side are eliminated, which is NMDA-dependent (Ohno et al, J Neurosci 2004) with a critical period of 6-11DIV (Ohno & Sakurai, Neuroscience 2005). In order to use the genetically-modified mice to study the underlying molecular mechanisms of this developmental plasticity, we tried to reconstruct the same system using mice. Entire blocks of the brain and spinal cord were taken from P0 C57 BL/6 mice. Coronal slices of the sensorimotor cortex and axial slices of the cervical cord were sectioned (350 μm), and forelimb areas in the cortex was dissected from each section. By recording field EPSP (fEPSP) along 80 μm-interval lattice in the spinal gray matter in response to the stimulation of cortical deep layer, we evaluated spatial distribution of synapse formation quantitatively. Field EPSPs were recorded diffusely throughout the spinal gray matter at 6-9 DIV, then the amplitudes of fEPSPs in the ventral side began to decrease at 10 DIV, and dominated in the dorsal area at 14-15 DIV. CS axon terminals labeled with biocytin anterogradely distributed diffusely throughout the spinal gray matter at 7-9 DIV but the axons terminals in the ventral area were eliminated until 14 DIV. This synapse elimination from the ventral side was blocked by APV, indicating that this process is also NMDA-dependent. [J Physiol Sci. 2006;56 Suppl:S170]

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We have previously shown in rat brain that ABCA2, which belongs to the A subclass of ATP-binding cassette (ABC) transporter superfamily, is predominantly expressed in the cytoplasm of oligodendrocytes but not in GFAP⁺ astrocytes, CD11b⁺ microglia, or NG2⁺ progenitors. In addition, onset of ABCA2 expression in oligodendrocytes coincides with the appearance of myelin segments immunolabeled with myelin basic protein, implying a role of ABCA2 in transport of substances related to myelination processes. Consistently, expression of ABCA2 was detected in S100β⁺ Schwann cells in human and rat peripheral nerve. Unexpectedly, however, ABCA2 also was detected in S100β-weakly positive cells containing number of densely packed, thin axons in peripheral nerve, implying expression of ABCA2 in non-myelinating Schwann cells. Indeed, multiple immunolabeling with ABCA2, S100β, GFAP, and a zinc finger transcription factor Krox20, one of the most reliable makers for myelinating Schwann cells, revealed that ABCA2 is expressed not only in myelinating Schwann cells but also in non-myelinating Schwann cells. As number of non-myelinating axons are thinly wrapped by single non-myelinating Schwann cells, ABCA2 might contribute to transport of lipid components commonly required for surrounding myelinating and non-myelinating axons. [J Physiol Sci. 2006;56 Suppl:S170]

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Recent reports suggest that the adult human brain contains undifferentiated, multipotent precursor cells or neural stem cells (NSCs). In this study, we tried to isolate the NSCs from the surgically dissected hippocampus of the adult human using "Neurosphere" methods and characterize their ability of the proliferation and the differentiation. When the dispersed cells from the adult human hippocampus were cultured in defined medium containing LIF (10 ng/ml), EGF (20 ng/ml), FGF2 (20 ng/ml) and B-27 (2%), the sphere-forming cells which diameter is approximately 200 μm were observed. These cells can be maintained and expanded in this condition for at least 6 months. The population doubling time was approximately 16 days. Immunocytochemical analysis showed that most of cells under this growth condition expressed neural stem cell marker protein, nestin. To induce differentiation, growth factors were removed from the medium. Immunocytochemical analysis showed that many cells expressed neuron marker protein, Tuj1 and astroglial marker protein, GFAP in this differentiation condition. These results suggest that these cells are multipotent NSCs. To improve the efficiency of neuronal differentiation, we are now examining the effects of various soluble factors in the differentiation medium on the neural and astroglial differntiation of the isolated NSCs. [J Physiol Sci. 2006;56 Suppl:S170]

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Activity of the deep cerebellar nuclei (DCN) takes an important role in outputting processed information from the cerebellum. In this study, we first investigated modulatory effects of serotonin (5-HT) receptor on GABAergic synapses in the rat DCN using whole-cell recordings in the cerebellar slices. Both of an endogenous agonist 5-HT and a 5-HT1 agonist 5-CT decreased the amplitude of stimulation-evoked IPSCs (eIPSC) in DCN neurons, and their effect was reversibly abolished by a 5-HT1A and 1B antagonist, cyanopindolol. Further, a selective 5-HT1A agonist 8-OH-DPAT had no effects of the amplitude of eIPSCs. Based on these results, the activation of 5-HT1B receptor is suggested to be responsible for decreasing the amplitude of eIPSCs. In the developing DCN neurons, we next examined developmental changes in both the kinetics of GABAergic postsynaptic currents and the modulatory effects of 5-HT on GABAergic synapses in the rat DCN neurons. At younger stage (around P14), eIPSCs showed slower kinetics and were more susceptible to the 5-HT-induced modulation than those at older stage (around P21). These pre- and postsynaptic parameters showed time-matched changing during development. These results suggest that the information flow from the cerebellar cortex is finely controlled at younger developmental stages, which is important to form the normal cerebellar function in the adult. [J Physiol Sci. 2006;56 Suppl:S170]

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The lateral superior olive (LSO) is the first auditory center that processes differences in the sound level between the two ears. Here we report the developmental changes in metabotropic glutamate receptor (mGluR)-mediated presynaptic inhibition of GABAergic/glycinergic synaptic transmission onto developing rat LSO neurons using conventional whole-cell patch clamp technique. In addition to a developmental switch of MNTB-LSO afferents from GABAergic to glycinergic IPSCs during development, immature MNTB-LSO synapses could release glutamate with GABA/glycine. Bath application of DCG IV, a selective mGluR 2/3 (group II) agonist, greatly reduced IPSC amplitude in neonatal (< P5) with a significant change in the paired-pulse ratio, which was eliminated in the presence of group II antagonist, suggesting that DCG IV acts presynaptic mGluR 2/3 leading to reduce the release probability of GABA and/or glycine release from presynaptic nerve terminals. However, the mGluR-mediated presynaptic inhibition was gradually reduced with postnatal development, in which DCG IV had little effect on MNTB-evoked IPSCs recorded from P16-18 LSO neurons. At P5 LSO neurons, presynaptic mGluR could be activated by endogenous glutamate released from the ipsilateral anteroventral cochlear nucleus (AVCN) afferent, but not from MNTB terminals. Based on these results, the functional roles of presynaptic mGluR in the development of LSO neurons will be further investigated and discussed. [J Physiol Sci. 2006;56 Suppl:S171]

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Neural stem cells (NSCs) are defined as self-renewing, multipotent progenitor cells that give rise to neurons, astrocytes and oligodendrocytes.Using single-cell-based on-chip cell-cultivation system, we analyzed the process of neural differentiation from the NSC and examined the effects of BDNF, which is known to enhance neuronal differentiation.The NSCs were obtained from the striatum of E12.5 nestin-promoter GFP transgenic mice and an individual cell was placed into 32 pairs of agar microchambers connected by microchannels.The NSCs were differentiated by media containing FBS, retinoic acid and forskolin in the presence or absence of BDNF and cell adhesion, neurite outgrowth were examined by recording a series of phase-contrast images and immunocytochemistry for neural marker, TuJ1.We observed that the NSCs attached the microchamber and a part of cells exhibited neuron-like morphology and extended some neurites along microchannels.BDNF increased the rate of neurite outgrowth.It, however, failed to affect cell adhesion.Thus, using this system, we could address the process of neural differentiation from the NSCs in a single-cell-based level and could demonstrate that BDNF had the ability to promote the neural differentiation. [J Physiol Sci. 2006;56 Suppl:S171]

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Neurotrophins and cytokines are involved in neuronal differentiation, synaptic development and plasticity. In neocortical culture, we reported epidermal growth factor (EGF) family (ErbB1 ligands; EGF, TGF-alpha, HB-EGF) down-regulates an AMPA receptor molecule, GluR1. Using neocortical cultures and EGF-administered animals, we electrophysiologically evaluated the effects of the EGF family on synaptic development and plasticity in the GABAergic neurons. In neocortical culture, subchronic treatment with TGF-alpha reduced the expression of GluR1-immunoreactivity in glutamic acid decarboxylase (GAD) 67 immunopositive GABAergic neurons. Whole-cell patch-clamp recording from morphologically identified putative GABAergic neurons revealed decreases both in AMPA currents and amplitudes of mEPSCs by TGF-alpha. Subcutaneous administration of EGF for 14 days in neonatal mice also decreased protein levels of AMPA and NMDA receptors in the frontal cortex. Immunohistochemical study revealed that the decrease in GluR1 levels was relatively specific for the parvalbumin-positive GABAergic neurons. Miniature analyses in cortical slices show that the amplitudes of mEPSCs in the GABAergic neurons decreased significantly, whereas no alteration was observed in the pyramidal neurons. Thus, activation of ErbB1 receptors during cortical development negatively regulates synaptic inputs and plasticity in the GABAergic neurons. [J Physiol Sci. 2006;56 Suppl:S171]

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During development of cerebral cortex the two major types of neurons, pyramidal and GABAergic neurons, migrate to their destination through different routes and extend their dendrites at different timing. Questions arise whether neuronal activity gives any influence on these processes of neuronal development, and if so, whether its influence differs between the two types of neurons. To address these questions, we used neuron culture preparations of the visual cortex of GAD67-GFP knock-in mice, in which GABAergic neurons can be identified by GFP. With time-lapse imaging analysis we observed effects of drugs, which block or enhance neural activity, on the motility and dendritic development of GABAergic and pyramidal neurons. Until 7 days in vitro (DIV) GABAergic neurons moved very quickly, although the movement was not smooth, while pyramidal neurons did not show such high motility. After their motility diminished, GABAergic neurons started to develop their dendrites. An application of tetrodotoxin (TTX) increased the motility of GABAergic neurons and extended the period when such high motility is maintained. The application of TTX and antagonists for ionotropic glutamate receptors until 7 DIV retarded the dendritic development of GABAergic neurons, while did not significantly affect that of pyramidal neurons. These results suggest that GABAergic neurons are more susceptible to neuronal activity than pyramidal neurons and activity may be a factor to stop migration of GABAergic neurons and to start development of their dendrites. [J Physiol Sci. 2006;56 Suppl:S171]

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Epidermal growth factor (EGF) binds to ErbB1 receptor and exerts a neurotrophic activity on midbrain dopaminergic neurons. Here, using EGF administrated animals and midbrain cultures, we investigated endogenous and exogenous ErbB1 activity on the development of dopaminergic neurons. Immunostaining of tyrosine hydroxylase (TH) revealed that the chronic administration of ErbB1 inhibitors (PD153035, ZD1839) to rat neonates prevented dopaminergic neurons from axonal fiber outgrowth and striatal innervation. Further, protein levels of TH and dopamine transporter decreased in the striatum but did not change in the frontal cortex. In midbrain culture, EGF had promoting activities in cell-survival and dopamine uptake. To monitor the development of intrinsic excitability of the dopaminergic neurons we prepared midbrain cultures from TH-EGFP transgenic mice. Whole-cell current-clamp recording from EGFP positive dopaminergic cells revealed that chronic treatment with EGF increased the number of action potentials induced by current injections. Further, daily administrations of EGF in vivo increased AMPA-mediated synaptic responses in the dopaminergic neurons. These findings suggest that ErbB1 ligands such as EGF have a neurotrophic activity for the development of midbrain dopaminergic neurons. [J Physiol Sci. 2006;56 Suppl:S172]

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In the previous study, we showed that the corticospinal (CS) synapses and terminals are distributed in the whole spinal cord at P7 and then eliminated from the ventrolateral side until P10. In this study, we further investigated the development of CS synapses subsequent to the elimination electrophysiologically and morphologically. Field EPSPs of CS synapses were evoked by stimulation of the medullary pyramid and recorded from the lower cervical cord (C7). For anterograde labeling of the CS terminals at C7, biotin dextran amine (BDA) was injected to the sensorimotor cortex. The spinal cord were fixed and sectioned several days after the injection. At the beginning of morphological study we determined the area of sensorimotor cortex projecting to C7. The amplitude of the largest negative peak of the field EPSPs continuously increased until 4 or 5 postnatal week. On the other hand, the spatial pattern of the distribution of the potentials was nearly constant after P10. The terminal distribution was analyzed by counting the number of axons within ventrolateral and dorsomedial area. After a relatively stable period of P10 and P11 following the elimination, the number of the terminals increased again in the ventrolateral area, which was the case with the dorsomedial area. The continuous amplitude increase of largest negative field EPSPs may be an electrophysiological counterpart of CS terminals re-innervation. Thus development of the CS innervation seems to consist of at least two steps. [J Physiol Sci. 2006;56 Suppl:S172]

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Inhibitory synaptic transmission and its modulation in neurons of the area postrema (AP), one of autonomic nuclei in the medulla, were studied using whole-cell patch-electrodes applied to slices from newborn rats. When external saline containing 20 mM KCl was applied from a "Y tube" to AP neurons, which were whole-cell clamped at -10 mV, massive inhibitory postsynaptic currents (IPSCs) were induced. Most of the evoked IPSCs were blocked by bicuculline, indicating GABAergic identity, while the remaining minority of synaptic currents was sensitive to strychnine. When nicotine (5-100 μM) or capsaicin (0.1-1 μM) was applied to AP neurons, robust appearance of IPSCs with GABAergic identity was induced. After blocking action potential generation in the slice with tetrodotoxin (1 μM), nicotine or capsaicin could still induce GABAergic IPSCs. The nicotine-induced presynaptic facilitation was significantly inhibited by mecamylamine, and it was slightly inhibited by dihydro-β-erythroidine and negligibly inhibited by α-bungarotoxin. Interestingly, responses to capsaicin of the synaptic facilitation showed marked desensitization; even after five minutes of rigorous washout, the magnitude of synaptic facilitation by capsaicin was 10-30% of that evoked by the first application. It is concluded that nicotinic receptors, as well as capsaicin receptors (presumably TRPV1), are expressed at GABAergic presynaptic terminals in area postrema neurons and play a distinctive role in controlling excitability of these neurons for their proper function in the autonomic system. [J Physiol Sci. 2006;56 Suppl:S172]

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The neuronal mechanisms underlying the cardiovascular activities of des-acylated ghrelin (DAG) and ghrelin remain unclear. Not only ghrelin but also DAG that is endocrinically inactive form of ghrelin without binding efficacy to growth-hormone secretagogue receptor type 1a (GHSR-1a) on the pituitary grand exerts cardiovascular actions when microinjected into the rat nucleus tractus solitarius (NTS). These responses may be attributable to a receptor(s) other than GHSR-1a. DAG lacks hydrophobic octanoylation at Ser3 from ghrelin but retains hydrophobicity at Phe4Leu5. We studied the cardiovascular effects in the rat NTS of a synthetic peptide with a hydrophobic-to-hydrophilic substitution at Phe4Leu5 to Ala4Lys5 of DAG^(1-10). The intra-NTS microinjection of 80 or 200 pmol/100 nl of the synthetic peptide produced no changes in the rat mean arterial pressure and heart rate. In addition, pretreatment with 200 pmol of the synthetic peptide had no antagonistic effect on the cardiovascular response induced by 80 pmol of DAG or native ghrelin. The synthetic peptide was incapable of evoking hypotensive and bradycardic responses in the NTS. Our results suggest that hydrophobicity at amino acid position 4 to 5 of DAG may be essential to bind a new receptor and to evoke the cardiovascular responses in the NTS. [J Physiol Sci. 2006;56 Suppl:S172]

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The main problem of attention deficit/hyperactivity disorder (AD/HD), one of developmental disorders of children, is difficulty in control or restrain of behavior. An imbalance of dopaminergic and noradrenergic transmission is proposed for neural mechanisms of AD/HD. On adult rats, we have already reported that methylphenidate, the most common therapeutic agent for AD/HD produced a hyperpolarizing response associated with decrease in a membrane resistance by activation of the inward rectifier K ⁺ channels via α₂-adrenoceptor of locus coeruleus (LC) neurons where the most major source of noradrenergic tones in CNS. The spontaneously hypertensive rat (SHR) are often used as a model of AD/HD. Behavioral studies established that SHR displayed hyperactivity, impulsivity, poor stability of performance and poorly sustained attention, when compared with their normotensive Wistar-Kyoto (WKY) control rats. In the present study, the tonic activity of LC neurons of juvenile SHR was examined by using a whole-cell patch clamp technique and compared with WKY. As a result, the resting membrane potential of LC neurons in SHR (-47.7±0.39 mV) was significantly decreased when compared with WKY (-51.4±0.63 mV). Nevertheless, the frequency of spontaneous action potentials of SHR (0.64±0.24 Hz) was significantly lower than that of WKY (2.14±0.53 Hz). These results suggest that noradrenergic spontaneous activity on LC neurons in SHR is reduced compare with WKY. [J Physiol Sci. 2006;56 Suppl:S173]

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It has been reported that the role of norepinephrine (NE) and dopamine (DA) in the brain is closely related to the etiology of attention-deficit/hyperactivity disorder (AD/HD). In this report, by using the HPLC method, the amounts of NE and DA in the locus ceruleus (LC), the medial prefrontal cortex (mPFC), and the striatum (Str) were measured and compared spontaneously hypertensive rats (SHR), an AD/HD model rat, and Wister-Kyoto rats (WKY) as the contrast rat. The results showed that the amount of NE were significantly larger than those in the mPFC and Str of WKY. On the other hand, the amount of DA has proven to be large in Str, and very small in the LC and the mPFC. This is also the case with SHR; the amount of NE and DA in LC, mPFC, and Str shows the same pattern. However, in the LC, the amount of NE in SHR was increased significantly more than that in WKY. The same tendency can be seen in mPFC, however, in Str, the amount of NE did not show any increase, compared with that in WKY. Furthermore, the amount of DA in the LC was increased significantly in SHR, compared with that in WKY, whereas any significant difference could not be recognized in mPFC and Str. These results suggest that the increase of NE and DA in the LC and mPFC of SHR is related to the characteristic behavior in AD/HD. [J Physiol Sci. 2006;56 Suppl:S173]

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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 nucleus of the solitary tract (NTS) and dorsal motor nucleus of the vagus (DMX). The purpose of this study was to examine how the firing frequency affects transmission efficiency at these synapses. EPSCs evoked by the solitary tract (TS) stimulation were recorded from DMX and NTS neurons in the thick brainstem slices of young rats. TS stimulation at various frequencies (0.1–20 Hz) revealed distinct frequency-dependent responses in the EPSC amplitude among different types of neurons recorded. When stimulated at 20 Hz, NTS neurons and low-pass type DMX neurons exhibited marked amplitude reduction (<30% of the first EPSC) within 10 pulses, whereas high-fidelity (hi-fi) type DMX neurons presented only modest attenuation (>40%). These neurons exhibited distinct short-term plasticity as revealed by paired-pulse ratio (PPR) evaluation. Surprisingly, unlike the NTS neurons, PPRs in the low-pass DMX neurons were not significantly affected by changes in [Ca²⁺]_o, suggesting distinct mechanisms for their short-term depression. These results indicate that the transmission efficiency between the visceral afferents and second-order neurons depends largely on the firing frequency mostly through distinct target cell-dependent presynaptic mechanisms, which might result in differential activation of distinct components in the solitary complex network. [J Physiol Sci. 2006;56 Suppl:S173]

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In order to understand the mechanism how the visceral information is transmitted to the brain, it is indispensable to identify the roles played by already-identified molecules in the synaptic transmission between the vagal afferent fibers and the second-order neurons in the nucleus tractus solitarii (NTS). The molecules underlying the regulation of transmitter release at this synapse are synthesized in the cell bodies located in the nodose ganglion (NG). Here we challenged to establish an optimized method for efficient in-vivo gene transfer into the NG neurons and evaluated expression of the gene product in the NG and its centrally projecting axons. In young Wistar rats, electrical pulses optimized for electroporation were delivered to the NG immediately following injection of pCAGGS-EGFP plasmid vector (5 μg/μl; through courtesy of Drs. J. Miyazaki and K. Nakajima). Two days after delivery, the NG was dissected out and fixed in 4% paraforaldehyde. In some cases, acute brainstem slices including the NTS were prepared and examined with a confocal microscopy a few weeks later. A large portion of somata in the NG and a large number of fibers projecting from NG expressed EGFP. EGFP fluorescence was also detected in the solitary tract and presynaptic termini in the NTS in the brainstem slices. This technique might be applicable to analyzing specific molecule function in the transmitter release in the central termini of the visceral afferent nerves. [J Physiol Sci. 2006;56 Suppl:S173]

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To investigate the Ca²⁺ clearance mechanisms in presynaptic terminals, we measured Ca²⁺ dynamics during nerve stimulation in frog neuro-muscular junctions with a low affinity Ca²⁺ dye, Oregon Green 488 BAPTA 6F (K_d=13μM, pH=7.2). During 100Hz tetanus for 4 sec in a normal Ringer's solution (1.8mM Ca²⁺, 0mM Mg²⁺), [Ca²⁺]_i increased in two phases, firstly steep rising (4 μM/sec for 0.2 sec) and secondly slow increasing (0.8 μM/sec). Whenmitochondria were inhibited by rotenone and oligomycin, slow phase disappeared and [Ca²⁺]_i increased monotony with rapid rate. Membrane voltage imaging of mitochondria with TMRE suggested that Ca²⁺ uptake to mitochondria accompanied by depolarization of mitochondrial membrane potential. When tetanus frequency was reduced to 20Hz, [Ca²⁺]_i dynamics did not affected by rotenone and oligomycin, however, CCCP increased [Ca²⁺]_i during tetanus. The effect of rotenone and oligomycin appeared when [Ca²⁺]_i increased more than 0.6 μM during 40Hz tetanus. These results suggest that Ca²⁺ uptake to mitochondria started when [Ca²⁺]_i increased more than 0.6 μM. CCCP had larger effect on [Ca²⁺]_i increase during 40 Hz tetanus than rotenone and oligomycin. These results suggest that CCCP sensitive some Ca²⁺ clearance mechanisms other than mitochondrial Ca²⁺ uptake contributed largely when [Ca²⁺]_i was lower than some μM. [J Physiol Sci. 2006;56 Suppl:S174]

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Recently it has been reported that background noise can improve detection sensitivity of sensory stimuli not only in animals, but also in humans. The present study was designed to examine how human visual perception may be modified by superposition of noise. Twenty-two undergraduate students with normal or corrected to normal vision participated in this study. In a dim chamber, participants observed a signals of a small light spot (white LED) with its intensity modulated by sine waves, usually at a frequency of 1Hz. Random flickering light modulated by white noise was superposed on the signal. Contrast detection threshold was measured with or without noise, using a psychophysical method (the adjusting method). The threshold first decreased then increased as increase in the noise intensity, with the minimum value at around just above the noise-threshold. Thus the increase in the contrast detection sensitivity was found not only at subthreshold, but also supra-threshold intensity of noise. These findings were independent on the signal frequency (1-15Hz). A further experiment replicated these findings using the up-and-down methods to measure the threshold in five participants (3 male and 2 female, aged 20-25 yr). We concluded from these results that a certain amount of noise, even supra-threshold, can improve contrast detection sensitivity in human visual perception. [J Physiol Sci. 2006;56 Suppl:S174]

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It is known that the rod network in the retina behaves as a high-pass filter to electrical signals. In the turtle and toad retinae, it was found that the time to peak of the response was shorter in rods further away from a slit of light. In the tiger salamander retina, it was shown that the voltage responses to square current injection became more transient as they travel through the rod network. In previous studies, the high-pass filtering behavior has been attributed to an inductance element, a hyperpolarization-activated current, or a K conductance activated by Ca. However, biophysical mechanism underlying the high-pass filter is not fully understood. The objective of this study is to analyze the functional roles of individual ionic currents in the temporal filtering properties of rods through computer simulations. A model of the rod photoreceptor network was developed. The model incorporates much of the known parameters in rod photoreceptors, i.e., the phototransduction cascade in the outer segment, membrane ionic currents (I_Ca, I_Kv, I_K(Ca), I_h, I_Cl(Ca)), intracellular calcium system and electrical junctions between rods.In simulation, the temporal filtering properties of the rod was analyzed. The simulated result shows that single rod itself behaves as a high-pass filter. The mechanism underlying the high-pass filter was examined by changing model parameters. The result suggests that I_K(Ca), I_Cl(Ca) and I_h are responsible for the high-pass filtering. The model also well reproduced the experimental observation that the shortening of the time to peak as the signal propagates laterally. [J Physiol Sci. 2006;56 Suppl:S174]

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As a method of retinal prosthesis we newly developed suprachoroidal-transretinal stimulation (STS), in which electrical current passed between scleral electrode and vitreous electrode (Kanda et al. '04). Previously we reported the spatial properties of STS (Kanda et al., PSJ meeting '05), however the recorded neurons with the receptive fields close to the scleral electrode were few. Here, we enlarged the sampling especially within 2° from the scleral electrode and investigated whether STS can activate neurons in the localized retinal area. 105 unit activities of relay cells in the lateral geniculate nucleus were recorded from 12 adult cats. The response probability to STS of various current intensities between 50-500 μA was examined for each unit, and its relationship with the distance from the electrode was analyzed. It was confirmed that the response probability depended on the distance from the electrode. As the intensity decreased, the units with high response probability limited near the electrode. For example, at 100 μA of STS, the units with the probability over 80% were within 2.5° from the electrode. The median of the probability of the units within 1° was the highest among those with further distance when STS of 150 μA or 200 μA was applied. These results suggested that the retinal activation by STS can be localized near the electrode with adequate current intensity. [J Physiol Sci. 2006;56 Suppl:S175]

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Retinal ganglion cells and subsets of retinal amacrine cells generate TTX-sensitive action potentials evoked by light stimulus. It has been reported that voltage-dependent sodium channel (Na_v) α subunits, Na_v1.1, Na_v1.2, Na_v1.3, and Na_v1.6, are expressed in the retinal ganglion cells (Fjell J et al., Mol Brain Res 50:197-204, 1997; Boiko T et al., J Neurosci 23:2306-2313, 2003). However, subtypes of Na_v expressed in retinal amacrine cells have not been identified. To examine the specific Na_v subtypes expressed in the retinal amacrine cells, we applied in situ hybridization on the rat retina with the RNA probes that recognize Na_v1.1, Na_v1.2, Na_v1.3, and Na_v1.6. We found that Na_v1.2, Na_v1.3, and Na_v1.6 were localized in the ganglion cell layer (GCL). Interestingly, Na_v1.1 was expressed not only in GCL, but also in the inner nuclear layer (INL). Cell bodies of Na_v1.1 positive cells in INL were located on the border between INL and the inner plexiform layer. It is probable that these cells are subsets of amacrine cells. Difference in expression pattern of Na_v might reflex functional difference of action potentials between amacrine and ganglion cells. [J Physiol Sci. 2006;56 Suppl:S175]

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Traumatic injury to the optic nerve often causes a rapid loss of vision within several hours and leads retinal ganglion cells to cell death with slower time course. The previous experiments demonstrated the neuroprotective effect of electrical optic nerve stimulation on the retinal cell death over several days or weeks. However, it is not clear whether the electrical stimulation effectively prevents the acute impairment of visual function as observed in the traumatic optic neuropathy. We examined whether the transcorneal electrical stimulation could improve visual function after the optic nerve crush. A screw electrode was secured on the skull over the visual cortex to record visually evoked potential (VEP) in adult rats. VEP elicited by flash stimuli was recorded before and after the optic nerve crush, and after the transcorneal stimulation to estimate the degree of damage and the effect of stimulation in individual animal. Calibrated optic nerve crush reduced the amplitude of VEP significantly in all animals (30% of pre-crush value on average). Transcorneal stimulation given through a bipolar electrode fitted to a contact lens (intensity 500μA, duration 50μsec, 20Hz, 6hours) significantly enhances VEP amplitude (270% of the post-crush value on average) and the enhancement was preserved for one week in most cases. These results suggest that the transcorneal stimulation has a protective effect against acute impairment of visual function by optic nerve crush. [J Physiol Sci. 2006;56 Suppl:S175]

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We recently developed an electrical stimulation method for retinal prosthesis named Suprachoroidal-Transretinal Stimulation (STS, Kanda et al., IOVS 2004). It was shown that a monophasic pulse of STS focally applied between an anode on the sclera and an intravitreal cathode (inward STS) evokes well-localized field potentials in the superior colliculus (SC) of both normal and retinal dystrophic rats. When the outward STS was delivered by reversing the stimulus polarity, the threshold was much higher than that of the inward STS. To avoid the electrochemical retinal damage due to charge imbalances, a biphasic pulse must be used in the STS-based artificial retina. Thus, we examined the SC response to biphasic STS comparing with that to the monophasic STS. In normal rats, Electrically-Evoked Potentials (EEP) to the single biphasic or monophasic STS were recorded from the SC contralateral to the stimulated eye. In response to the biphasic STS the late oscillatory components of EEP were greatly suppressed, although the early component of EEP was smaller in amplitude than that to the monophasic inward STS. Thus, biphasic STS may be beneficial for generating artificial perception with high temporal resolution, as well as being harmless. [J Physiol Sci. 2006;56 Suppl:S175]

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It is commonly believed that orientation selectivity first emerges in the primary visual cortex (V1). In the present study, first, we examined the orientation selectivity of LGN neurons using optimal and non-optimal (in terms of stimulus size and spatial frequency (SF)) grating stimuli in anesthetized cats. We found that although only about 10% LGN neurons showed significantly orientation-biased response to the grating with optimal size and SF, about 90% of LGN neurons exhibited significant orientation selectivity to grating with diameters larger than its classical receptive field (CRF) and SFs higher than the optimal for CRF response. Then, stimulus-size tuning curves were made for responses to stimulation with preferred orientation tested with large stimulus and with orthogonal orientation. These two stimulus-size tuning curves exhibited profile similar to each other under the optimal SF condition. However, high SF grating caused stronger surround suppression for response to the orthogonally oriented stimulus than that to the optimally orientated stimulus. These results suggest that there is orientation tuned surround suppression that is effective around the CRF boundary and its optimal SF is tuned to higher than that of the CRF responses. We should further address how the orientation selectivity of LGN contributes to that in V1. [J Physiol Sci. 2006;56 Suppl:S176]

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Neuronal responses of the primary visual cortex (V1) exhibit stimulus-size tuning property, and they are suppressed by stimulation with grating patches larger than their classical receptive field (CRF). Recently, it has been revealed that size-tuning property of V1 neurons is dependent on stimulus parameters, such as luminance contrast and spatial frequency. In this study, we examined the effect of temporal frequency (TF) of sinusoidal grating stimulus on size-tuning curves of cat V1 neurons. Our results showed that when the TF was higher than optimal, the strength of surround suppression became weak and CRF-size became larger, suggesting that V1 neurons summate visual information from wider region under high TF condition. We also tested the effect of changing stimulus-size on TF tuning curve. Corresponding to above results, large grating made peak and high cut-off of TF-tuning curve higher than those for small grating. Then we examined neuronal responses of the lateral geniculate nucleus (LGN), and obtained basically similar results to those of V1 neurons. These results suggest that neurons in early visual pathway change their spatial integration property according to TF of stimulus, in such a way that neurons integrate wide visual field for fast moving stimulus, whereas localized field for slow stimulus. [J Physiol Sci. 2006;56 Suppl:S176]

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Neurons in the primary visual cortex (V1) change their responses depending on stimulus parameters such as orientation, spatial frequency (SF), size and so on. In this study, we investigated how stimulus size effects on SF tuning property of neurons in V1 and lateral geniculate nucleus (LGN) of cats. First, we found that V1 neurons increased sensitivity to high SF stimuli when small gratings were used and that SF selectivity of V1 neurons was sharpened when large gratings were used, according to the shifts of peak and high cut-off of SF tuning curves. Second, we measured area summation tuning curves under several SF grating conditions, and found that a higher SF stimulus caused a reduction of the receptive field size and an increase of the surround suppression. The same tendency was observed in LGN, which is the main source of excitatory input to V1. This implies that the relationship between SF and area summation properties observed in V1 has its origin in LGN and is modified in the intracortical network. These results suggest how neural circuit in early visual pathway changes its way of information processing and how it reduces redundancy in various visual environments; for small visual objects, neurons increase SF sensitivity to get fine resolution, on the other hand, for large ones, they sharpen SF selectivity to reduce redundancy. [J Physiol Sci. 2006;56 Suppl:S176]

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It is known that orientation tuning of neurons in the primary visual cortex (V1) becomes sharper as the size of stimulus increases beyond the classical receptive field (CRF) (Orban et al., 1979; Chen et al., 2005; Xing et al., 2005). We investigated relationships between the strength of the orientation selectivity and that of the surround suppression in 74 V1 neurons recorded from anesthetized and paralyzed cats. The orientation selectivity became stronger as an increment of surround suppression due to an enlargement of stimulus that covered the CRF and the receptive field surround (SRF). However, the preferred orientation of the neurons was independent of stimulus sizes and did not vary according to a change in stimulus size. Also, both the surround suppression and the orientation selectivity of responses to the stimulus larger than the CRF were significantly stronger in layers II/III than in layers V/VI. We compared our results with predictions of a simple iceberg model where a large stimulus that covered the CRF and SRF equally and linearly suppressed responses to all stimulus orientations. The model, however, overestimated the sharpening of the orientation tuning by large stimulus. These results suggest that surround suppression in V1 exhibits similar to but less-selective orientation tuning than the response to CRF stimulation. Such an effect of surround suppression can be a reason why the stronger orientation selectivity is observed for large stimulus. [J Physiol Sci. 2006;56 Suppl:S176]

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It has been suggested that oxidation of rod outer segment (ROS) membrane lipids of photoreceptor cells may be involved in light-induced retinal degeneration. All-trans retinal is a potent photosensitizer and its role in mediating photodamage, mainly lipid oxidation by producing singlet oxygen, has been suspected for over two decades as shown in studies using ROS membranes or liposomes. Here we demonstrate that all-trans retinal may actually act as a photosensitizer and produce photooxidation in living cells. Exogenous all-trans retinal caused lipid oxidation under UV light (365nm) in bleached bovine ROS membranes in proportion to either the duration of UV radiation or the concentration of retinal. Similar results were obtained from dark-adapted ROS membranes with retinal released from rhodopsin after irradiation with long wavelength light (530nm). Finally, consistent with previous studies which have implicated that accumulated all-trans retinal may cause light-induced oxidation in photoreceptor cells, intact ellipsoid cells, which are able to quantitatively convert all-trans retinal to all-trans retinol, showed significantly less oxidation compare to those without ellipsoid when exposed to UV light after releasing all-trans retinal with 530nm light. UV did not have much effect when there was no 530nm light treatment. These results indicate that all-trans retinal, which is released from rhodopsin by 530nm light, is photosensitizing component in living cells and causes membrane oxidation in living cells. [J Physiol Sci. 2006;56 Suppl:S177]

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To determine whether mouth guard (MG) wearing improves performance capacity of athletes, we examined the effects of MG wearing on dynamic visual acuity (DVA). Subjects were asked to read and answer 3 numerals presented sequentially at random order. The numerals moved left to right at 90 deg/sec on a CRT monitor under two conditions of head motion: 1) stationary state, and 2) voluntary rightward rotation. In each condition, thirty trials with and without MG were carried out and numbers of correct answers (full score; 3 x 30 = 90) were evaluated as DVA score. We measured DVA improvement indexes (= DVA scores with MG minus those without MG) and occlusal forces. In the condition 1, the correlation-coefficient between DVA improvement indexes and occlusal forces was negative (r=-0.74) for nine subjects (20-32 years), whose DVA improvement indexes were >0 in the condition 2. On the contrary, the correlation-coefficient was positive (r=0.76) in the condition 2. These results suggested that the effects of MG wearing on DVA strongly depended on the condition of head motion and the occlusal force. As we recorded simultaneously electromyographic activities of masseter and sternocleidmastoideus, eye movements and head angular velocities, these data will be described to discuss the effects of MG wearing on DVA. [J Physiol Sci. 2006;56 Suppl:S177]

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Monocular deprivation (MD) during an early postnatal period causes a loss of visual cortical response to the deprived eye (Ocular dominance (OD) shift). When MD is combined with inactivation of the visual cortex by muscimol infusion, cortical neurons lose their response to the open eye (reverse OD shift). Although these two forms of OD plasticity differ in direction, it is largely unknown whether their other characteristics are also different. Such information would be useful to clarify mechanisms underlying these two plasticity. The normal OD shift proceeds rapidly and the physiological effect of MD saturates after 48 hours in kittens. Therefore, we examined how rapidly the reverse OD shift proceeds and whether the shift could be induced in adults, to compare the dynamics and age-dependency of two forms of OD plasticity. We have inhibited the visual cortical neurons by infusing muscimol in four-week old kittens and adults. After 3-6 days of MD, OD of cortical neurons was evaluated by single-unit recording. The reverse OD shift was observed after 6-day MD as reported previously. After 3-day MD, however, the reverse OD shift was not observed and the OD distribution remained similar to that of normal animals. In adults, OD distribution in the inhibited cortex remained unchanged even after 7-day MD. These results suggest that the reverse OD shift might reflect a mechanism of developmental plasticity that has a slower time course than the normal OD shift. [J Physiol Sci. 2006;56 Suppl:S177]

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Functional mapping of macaque primary visual cortex with implanted multi-electrode array Naohisa Miyakawa1, David Blake2, Michael Merzenich2, Manabu Tanifuji1 1 Integrative Neural Systems, RIKEN BSI, 2 Coleman Lab., KeckCenter for Integrative Neurosci., UCSFIt has been known that in primates, different levels of visual information are processed at different areas within the cortical visual streams. Much remain unknown on how each level of information is processed within each area, but some evidence show possibility of information represented in a distributed manner within the local network of respective cortical areas (Tsunoda et al. 2001). To understand the function of this local network activity, we have developed a chronically implantable multi-electrode array that is laid out in high-density grid configuration with 350 μm spacing. We implanted the array to the primary visual cortex of macaque monkey, and detected multi-unit activity (MUA) from all electrodes for as long as 3 month. We reconstructed a two-dimension functional response map by visualizing the neuronal activity evoked with grating stimuli of different orientations. The map showed significant stability over the 3-month period, indicating that our array is capable of monitoring cortical network activity with minimum damage to the cortical tissue over this period.We will show our preliminary result of multi-electrode array recording of infero-temporal (IT) cortex neurons evoked by natural image stimuli. [J Physiol Sci. 2006;56 Suppl:S177]

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In the primary visual cortex (V1), a neuronal response to stimulation of the classical receptive field (CRF) is suppressively modulated by the stimulus presented at the receptive field surround (SRF). Using stationary flashes of sinusoidal grating as stimuli, we examined the dependency of effect of SRF stimulation on the orientation-contrast (OC) between CRF and SRF stimuli (Exp.1) and that on the presentation duration (50 ms vs. 500 ms) (Exp.2) in V1 neurons of anesthetized cats. In Exp.1, CRF was stimulated with a flash (500 ms) of the grating patch with optimal parameters and SRF was stimulated with a flash (50 ms) of the grating annulus that was either iso- or cross-oriented to the CRF stimulus orientation. The late (> 80 ms) component of response was suppressed specifically by iso-oriented SRF stimulus, while the early (40–80 ms) component of response was suppressed by SRF stimulus regardless of OC. In Exp.2, the suppressive effect of short (50 ms) SRF stimulation lasted up to the offset of CRF stimulus (500 ms), and its time course of the suppression was compatible with that of long (500 ms) SRF stimulation. In conclusion, the short (50 ms) presentation of SRF stimulus is sufficient to evoke both orientation-nonspecific fast component and orientation-specific slow component of suppressive effect. [J Physiol Sci. 2006;56 Suppl:S178]

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The aim of the present study was to investigate whether under in-vivo condition, temporomandibular joint (TMJ) inflammation alters the excitability of Aβ-trigeminal root ganglion (TRG) neuronal activity innervating the facial skin by using extracellular recording with multibarrel-electrodes. CFA was injected into the rat TMJ. A total of 36 Aβ-TRG neurons responding to electrical stimulation of the whisker pad was recorded in pentobarbital-anesthetized rats. The number of Aβ-TRG neurons with spontaneous firings and their firing rate in TMJ inflamed rats were significantly larger than those in control rats. The firing rates of their spontaneous activity in the Aβ-TRG neurons were current-dependently decreased by local iontophoretic application of a NK1 receptor antagonist (L-703,606) in inflamed, but not non-inflamed rats. The spontaneous activities were increased by iontophoretic application of substance P in both group of rats.The mechanical stimulation threshold of Aβ-TRG neurons in inflamed rats was significantly lower than that in control rats. There were no significant differences on the mechanical stimulation threshold between control and inflamed rats after iontophoretic application of L-703,606. These results suggest that TMJ inflammation can modulate the excitability of Aβ-TRG neurons innervating the facial skin via paracrine mechanism due to SP released from TRG neuronal cell body. [J Physiol Sci. 2006;56 Suppl:S178]

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Newborn's attraction to the breast milk odors is one of the fundamental behavioral responses that human newborns share with other mammalian species, but little is known about the effects of the breast milk odors on stress response. In the present study, we examined whether breast milk odors affect the stress responses to a capillary puncture on the heel (heelsticks) during routine blood draws to screen for phenylketonuria in 5 days-old infants. Forty eight healthy infants were randomly assigned to the following four groups, 1) control group which was exposed to saline as a sham odor, 2) mother's milk group which was exposed to their own mother's milk odor, 3) other mother's milk group which was exposed to other mother's milk odor and 4) formula milk group which was exposed to formula milk odor. To assess the infant distress, their facial expression (grimacing) and crying were recorded by video camera and their body movements were recorded by actigraph. This study was approved by the ethics committee of Nagasaki University. As we expected, infants showed significantly more distress after heelsticks in all groups. However, stress responses to the heelsticks were significantly attenuated by their own mother's milk odor, but neither by other mother's milk nor formula milk odors. These results suggest that the mother's milk odor has not only an attractive effect but also a calming effect on their own infant. [J Physiol Sci. 2006;56 Suppl:S178]

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Mechanism of the pain relief effect of low-power laser (Ga-Al-As diode laser, 16.2mW) irradiation using cultured mouse dorsal root ganglion neurons associated with C-fiber was studied by patch-clamp technique. Bradykinin (BK) and laser stimulations were limited to the process or the cell body (soma) with a separator developed by us. The action potentials of the soma by BK application to the process were reversibly suppressed by the irradiation for 2 min to the process. After the irradiation to the soma was done without BK stimulation, resting potential was depolarized by potential of 2-3 mV, and the frequency of the spike evoked by a depolarization pulse was enhanced, except for the case in which the irradiation was limited to the process. K⁺ channel openings elicited by BK were reversibly suppressed by the irradiation to the soma. Inward current evoked by a depolarization pulse was not suppressed by the irradiation to the soma. BK-evoked inward currents were suppressed by the irradiation to the process. When BK was applied to the process and the irradiation to the soma was done, the action potentials by BK were not suppressed. The results suggest that the increase in the spike frequency by the irradiation to the soma without BK is ascribed to depolarization due to inhibition of K⁺ channel openings, and the suppressive effect of laser irradiation on BK-evoked action potential may come from suppression of the system of receptor-G protein in the process. [J Physiol Sci. 2006;56 Suppl:S178]

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ATP is now known as a substance that is released from injured/stimulated cells to induce or augment pain. In the urinary bladder ATP is released from the endothelium when the bladder is stretched, and thus released ATP stimulates sensory nerve terminals to transmit the stretched state of the bladder to the central nervous system. We hypothesize that the same happens in the skeletal muscle, and measured ATP release upon mechanical stimulation (compression) of the muscle. The extensor digitorum longus muscle was excised from the deeply anesthetized rats, and superfused with Krebs-Henseleit solution. The superfusate was sampled at a rate of 0.8 ml/min and its ATP concentration was measured with Luciferin-Luciferase method. At first we examined ATP release to repetitive application of 20 g force/10 s stimulation, which was used for the study of nociceptor characteristics, with a servo-controlled mechanical stimulator with intervals of 30 min. ATP release was clearly decreased on repetition of the mechanical stimulus. Then we examined stimulus response relationship. The muscle was stimulated five times at 5, 10, 20 and 40 g forces at a rate of 10 g/s. ATP release was increased roughly stimulus-strength dependently in this range. Whether ATP thus released transmits mechanical event to nociceptors is to be studied. [J Physiol Sci. 2006;56 Suppl:S179]

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It is widely accepted that females are more sensitive to nociceptive stimuli than males. In the previous study, we showed that there were sex differences in the response of CNS to formalin-induced nociceptive stimuli by checking the expression of phosphorylated cAMP response element-binding protein (pCREB) as a marker of neural activity. In the bed nucleus of the stria terminalis (BST), the number of cells expressing pCREB in female rats was increased 5 min after formalin injection but not in male rats. In the present study, we determined which cells expressed pCREB in response to formalin injection in adult male and female rats. Rats were injected with saline or 2% formalin into the planter surface of the right hindpaw, and they were killed 5 min after the injection. Their brains were served to immunocytochemistry and the number of cells expressing pCREB and dopamine- and cAMP-regulated phosphoprotein (DARPP-32) in the BST was counted. We found that, in females but not in males, the number of DARPP-32 cells expressing pCREB in formalin-injected rats was significantly greater than that in saline-injected rats. The present study suggests that DARPP-32 cells in the BST are at least in part responsible for sex difference in the response of CNS to formalin-induced nociceptive stimuli. [J Physiol Sci. 2006;56 Suppl:S179]

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Aim of Investigation: Melittin is the main toxin of bee venom. Previously, we have reported that intradermal injection of melittin into the volar aspect of forearm in humans produces a temporary pain and a subsequent sustained neurogenic-inflammation-skin temperature increase. Furthermore, not only subcutaneous melittin but also subcutaneous glutamate produced neurogenic inflammation on the rats' hindpaw. Aim of the present study was to confirm the involvement of glutamate receptors on melittin-induced neurogenic inflammation. Methods: Melittin or glutamate was injected subcutaneously into the hindpaw of pentobarbital-anesthetized rats. NMDA receptor antagonist, MK-801 or AMPA receptor antagonist, CNQX was injected simultaneously with melittin/glutamate. Peripheral glutamate was collected by microdialysis and quantified using HPLC. Skin temperature increase was analyzed using the computer-assisted-thermography for the evaluation of neurogenic inflammation. Results: Microdialysate concentrations of glutamate were increased significantly following subcutaneous melittin injection. Simultaneous MK-801 injection suppressed not only glutamate-induce-neurogenic inflammation but also melittin-induce-neurogenic inflammation. The suppression effects of CNQX were weak. Conclusion: These data demonstrated that glutamate released following melittin injection partially contributed to neurogenic inflammation by activating NMDA receptors on nociceptors. Melittin-induced glutamate release suggested to prolong the melittin-induced neurogenic inflammation. [J Physiol Sci. 2006;56 Suppl:S179]

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Intrathecal administration of baclofen, a selective GABA_B receptor agonist, is known to have an antinociceptive effect on various pain models. In the present study, we investigated effects of baclofen on modality-dependent excitatory synaptic responses of substantia gelatinosa (SG) neurons in the spinal dorsal horn using in vivo patch-clamp recording technique. Adult male Sprague-Dawley rats were anesthetized with urethane. After thoracolumbar laminectomy was performed, patch electrodes were inserted into the SG at the spinal level of L3-L5 and then whole-cell patch-clamp recordings were obtained from SG neurons. Under voltage-clamp conditions, SG neurons exhibited miniature EPSCs.Baclofen decreased the frequency but not amplitude of mEPSCs. Pinch and touch stimuli applied to the ipsilateral hindlimb evoked a barrage of large amplitude of EPSCs. Baclofen also inhibited the amplitude of large amplitude of EPSCs evoked by those stimuli in a dose-dependent manner. On the other hand, the frequency of large amplitude of EPSCs was not affected by baclofen. These inhibitory actions of baclofen were blocked in the presence of CGP55845, a selective antagonist of GABA_B receptor. The present findings suggest that baclofen inhibits both noxious and innocuous mechanical excitatory transmission in the SG through activation of GABA_B receptors on presynaptic terminals. This inhibition of mechanical inputs to the SG may be a possible mechanism for antinociception by baclofen. [J Physiol Sci. 2006;56 Suppl:S179]

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We have shown that conditioning stimulation of the amygdala has an inhibitory effect on nociceptive neurons of the medullary dorsal horn of the rat. The amygdala is considered to be a complex of anatomically different units. The aim of this study was to compare the inhibitory effects of different nucleus within the amygdala. The animals were anesthetized with N₂O-O₂ and 0.5% halothane and were immobilized with pancuronium bromide. A peripheral test stimulus (a single rectangular pulse of 2.0 msec in duration) was applied to the receptive field of nociceptive neurons, and ipsilateral amygdaloid conditioning stimuli to the recording site were trains of 33 pulses (100-300 μA) delivered at 330 Hz. Effective sites for inhibition were widely distributed throughout the amygdala except for the lateral nucleus. This finding is in contrast with our previous data that inhibitory sites were concentrated into the central nucleus in the cat. There were no significant differences in the mean inhibitory effects by the different amygdaloid nuclei, and the inhibitory effect was between 56.6 and 62.4% of the control (n=32). The present results suggest that there are marked species differences with regard to the antinociceptive mechanisms within the amygdala and support the anatomical observations that the efferent fibers of the amygdala originate mostly from the central nucleus that is projection focus from the other amygdaloid nuclei in the rat. [J Physiol Sci. 2006;56 Suppl:S180]

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OBJECTIVE: Increments of local blood flow and vasodilatation induced by acupuncture and moxibustion have been known as axon reflex of unmyelinated afferent fiber receptors such as polymodal receptors. In this study, effects of application of local anesthetic patch on pain threshold of the skin and axon reflex induced by moxibustion were examined. METHODS: Five healthy volunteers with informed consent (ten forearms allocated anesthetic or sham group, double blinded manner) were used and pin-prick pain and heat pain thresholds were measured. The flare reactions were induced by the Kamaya-mini (Kamaya Co.Ltd, Japan), a kind of indirect moxibustion (peak temperature was about 50°C), and the vasodilatation was measured by blood flow meter. A piece of anesthetic (lidocaine 18mg) or sham patch (30.5 x 50 mm) was applied to the skin surface where pain tests and blood flow measurements were done. RESULTS: The mechanical pain threshold (pin-prick) and local blood flow response were significantly reduced (P<0.05, Wilcoxon t-test) 60 min after the application of anesthetic patch, but the heat pain threshold did not change. CONCLUSION: In this study, the flare reaction evoked by moxibustion was blocked by local anesthetic patch without changing heat pain threshold. These results suggest that lidocaine insensitive type receptors might be involved in the signal transduction mechanism of moxibustion, and also suggest that the thermal sensation provoked by moxibustion was conducted through unmyelinated peptidergic fibers such as the polymodal type receptors. [J Physiol Sci. 2006;56 Suppl:S180]

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Spinal actions of intravenous anesthetics such as propofol are less clear. The aim of this study was to investigate the spinal effects of propofol on nociceptive transmission. Adult male rats were used in this study. For in vitro patch-clamp study, a transverse slice of the spinal cord was cut and blind whole-cell patch-clamp recordings were made from substantia gelatinosa (SG) neurons. The half decay time of GABAergic evoked inhibitory postsynaptic currents (eIPSCs) was increased by bath-applied propofol dose dependently. Furthermore, for in vivo patch-clamp study, a rat was fixed in a stereotaxic apparatus after the lumbar spinal cord was exposed under urethane anesthesia. Propofol was systemically injected from left femoral vein, and its effect was evaluated before and after injection of 5 mg/kg propofol under the voltage-clamp mode. Propofol reversibly prolonged decay time of GABAergic spontaneous IPSC in all neurons tested. In the current clamp mode, pinch stimuli applied to the hindlimb elicited a barrage of excitatory postsynaptic potentials, some of which initiated an action potential (AP). Number of APs decreased after injection of propofol in most of neurons tested. These results suggest that systemically bolus injected propofol in clinical dosage reversibly depress noxious transmission at least in part by enhancing postsynaptic GABA receptors in the SG of the spinal cord. [J Physiol Sci. 2006;56 Suppl:S180]

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