In the brain, prior sublethal ischemia (preconditioning, PC) is known to produce ischemic tolerance of neurons to subsequent lethal ischemia. In addition, previous studies have revealed that the function of neuronal and/or astrocytic glutamate (Glu) transporters is reversed during ischemia, and the reversed uptake of Glu especially by astrocytic GLT-1 is involved in the marked elevation of extracellular Glu in the ischemic brain. This study aims at elucidating whether Glu released from astrocytes is responsible for the development of neuronal ischemic tolerance in astrocyte/neuron co-cultures. Brain ischemia was simulated by deprivation of both oxygen and glucose (OGD) from co-cultures. When the activation of NMDA receptors during PC was suppressed by treatment with AP5, PC-induced neuronal ischemic tolerance was significantly depressed. In contrast, exposure of co-cultures to NMDA instead of sublethal OGD (PC) produced ischemic tolerance of neurons. Interestingly, treatment of co-cultures with dihydrokainic acid (DHK), a specific blocker of astrocytic GLT-1, during PC resulted in the significant reduction in the PC-induced neuronal ischemic tolerance. These results suggested that the activation of NMDA receptors during PC was crucial to the development of PC-induced ischemic tolerance of neurons, and this activation was probably caused by the marked elevation of extracellular Glu resulting from the reversed uptake of Glu by astrocytic GLT-1. [Jpn J Physiol 54 Suppl:S157 (2004)]

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Intracellular recordings were made from CA1 pyramidal neurons in rat hippocampal tissue slices. Administration of oxygen- and glucose-deprived medium (in vitro ischemia) produced a rapid depolarization after 5 – 6 min exposure. When oxygen and glucose were reintroduced immediately after the rapid depolarization, the membrane depolarized further and reached 0 mV (irreversible membrane dysfunction) 5 min after the reintroduction. In the present study, we examined the contribution of protein kinase A (PKA) activation to the irreversible membrane dysfunction. In the majority of CA1 neurons pretreated with PKA inhibitor (H89; 1 μM) the membrane was restored to the preexposure potential level after the reintroduction. Pretreatment with adenylyl cyclase inhibitor (SQ22536; 100 μM or Rp-cAMPS; 100 μM) also restored the membrane to the preexposure level. Pretreatment with calmodulin inhibitor (W-7; 50 μM), but not calmodulin kinase II inhibitor (KN62; 10 μM) or myosin light chain kinase inhibitor (ML-7; 10 μM), restored the membrane to the preexposure level. These results suggest that the activation of PKA via calcium calmodulin complex (Ca²⁺/CaM) produces the irreversible membrane dysfunction following in vitro ischemia. [Jpn J Physiol 54 Suppl:S157 (2004)]

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A pharmacological blockade of inhibition, simultaneous application of bicuculline and strychnine, induced a synchronized rhythmic activity (SRA) in the dorsal root. Application of APV or DNQX eliminated this SRA completely, which suggested the existence of glutamate receptors on the dorsal root. Here, the functional significance of glutamate receptors on the dorsal root was investigated in relation to presynaptic inhibition. In the isolated spinal cord preparation from newborn rats (6- 9 days old), the dorsal root (L4 or L5) was split into two or three rootlets. Using suction electrodes, one rootlet was stimulated electrically and primary afferent depolarization (PAD) was recorded from the adjacent rootlet. After simultaneous application of bicuculline and strychnine, PAD became smaller and much longer. This PAD was eliminated by DNQX or GYKI 53655 addition in the presence of APV. After application of bicuculline, strychnine, DNQX and deletion of extracellular Mg ions, the remaining NMDA component of PAD was not affected by addition of ifenprodil (up to 30 micro M), but was eliminated by a further addition of APV(25 micro M). It was concluded that AMPA type non-NMDA receptors and NMDA receptors without NR2B subunit on the dorsal root, contribute to PAD in rat spinal cord, and might play a significant role in presynaptic inhibition. [Jpn J Physiol 54 Suppl:S158 (2004)]

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We identified apamin-sensitive Ca dependent K currents (IK_Ca) and 3 types of voltage dependent K currents in median raphe neurons (MRNSs) of 0- to 24- days old rats. Potassium currents were recorded in MRNs of 150 to 200 μm thick brainstem slices from rats anesthetized with ketamine hydrochloride (1-2 mg) using whole-cell patch clamp method. Recorded neurons were injected with Lucifer yellow and identified as MRNs by use of anti-5HT antibodies conjugated with Texas red. Voltage dependent K currents (delayed rectifier K current- IK_DR, transient K current- IA, 4AP-insensitive transient K current-IKt) were recorded in TTX (1μM) containing Ca²⁺-free solution. IK_DR was sustained by depolarizing voltage commands with a prepulse of -90 to -50 mV (200 ms duration) and sensitive to 20 mM TEA. IA was identified by subtraction of IK_DR from outward currents induced by depolarizing voltage commands with a prepulse of -90 to -150 mV (200 ms duration) and sensitive to 2-5 mM 4-AP. IKt was a slowly inactivating current sensitive to low concentration of TEA (≤3mM) but not to 4-AP. IK_Ca was identified as apamin (300 nM) sensitive outward current. The current densities of these K currents increased during postnatal maturation (0 to 24 days after birth); 40 to 80 A/F (IK_DR), 30 to 100 A/F (IA), 40 to 70 A/F (IK_Ca). The postnatal developmental changes of K currents were coincident with those of spike duration and afterhyperpolarization in rat MRNs. [Jpn J Physiol 54 Suppl:S158 (2004)]

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Whole-cell recordings were made from substantia gelatinosa (SG, lamina II of the spinal cord) neurons of urethane-anesthetized rats and spinal cord slices to analyze excitatory and inhibitory synaptic responses evoked by cutaneous natural and dorsal root stimulation. Under an artificial ventilation, the lumbar spinal cord was exposed and then the animal was placed in a stereotaxic apparatus. Patch electrodes were inserted into the SG and whole-cell voltage-clamp recordings were obtained from SG neurons. In most SG neurons examined, pinch and air stimulation produced a barrage of EPSCs. At a holding potential of 0 mV, air stimuli evoked a barrage of IPSCs. On the other hand, pinch stimulus elicited IPSCs only at beginning and end of the stimulation. The size of receptive field of IPSCs was larger than that of EPSCs in the same neurons. Electrical stimulation of the dorsal roots evoked EPSCs and IPSCs in SG neurons of horizontal spinal cord slices. L5 dorsal root stimulation elicited IPSCs in SG neurons at L2 spinal level without any EPSCs in the same neurons. It is concluded that the extension of inhibitory synaptic pathway is more widespread than that of excitatory circuitry. This extended inhibitory pathway in the spinal dorsal horn may be a possible mechanism for the lateral inhibition produced by innocuous stimulation. [Jpn J Physiol 54 Suppl:S158 (2004)]

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Location of the neuromuscular junction, end plate, of the masseter muscle was estimated from surface electromyograms (EMGs). The EMGs in response to clenching action were recorded from eight sites over the masseter muscle in a monopolar manner where the reference electrode was put on the tip of the nose. Base lines of the EMGs deflected either negatively or positively depending on the recording sites during clenching action, and these occurrences coincided with the action potential bursts. Slow wave and action potential components were separated from the raw EMG with digital filters. Deflection of the slow wave component was most negative over the inferior masseter muscle close to the mandibular ramus, while the polarity of the deflection shifted to positive over the superior part. The magnitude of the action potential component was largest at almost the same site as the most negative deflection of the slow wave component. The phase of the action potential component with reference to the signals for the channel that showed the most negative deflection of the slow wave also reversed at almost the same site as the polarity change of the slow wave component. All of these findings indicated that the slow wave detected by this method is the externally recorded synaptic potential originating at the meuromuscular junction, and that of the masseter muscle is located on the inferior part close to the mandibular ramus. [Jpn J Physiol 54 Suppl:S158 (2004)]

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We have reported that tetanic stimulation (100Hz, 50s) enhanced miniature end-plate potential (MEPP) frequency in Ca²⁺-free Ringer's solution (0.2mM EGTA, 5mM Mg²⁺) at frog neuromuscular junctions. Furthermore, this enhancement was induced by the accumulation of [Mg²⁺]_i in presynapse during tetanus through Ca²⁺ channels. We examined whether this presynaptic activation affected end-plate potentials(EPPs) using conventional electrophysiological techniques and imaging techniques. The nerve-muscle preparation was circulated with Ca²⁺-free Ringer's solution. EPPs were evoked by 0.25Hz stimulation with puffing 0.9mM Ca²⁺ Ringer's solution to the observed synapse locally. Puff was stopped 3 minutes before tetanus, therefore the extracellar condition was Ca²⁺-free during tetanus. After tetanus, EPP amplitude increased to 5 times and decayed exponentially with a time constant of about 140s. EGTA loaded into the terminal had no effect on this plasticity. Imaging techniques indicated that [Ca²⁺]_i did not increased but [Mg²⁺]_i increased in presynaptic terminals during tetanus. When the extracellar concentration of Mg²⁺ increased from 2 mM to 10 mM, [Mg²⁺]_i peak increased, but this plasticity did not change. Therefore, we concluded that this plasticity depend on neither [Ca²⁺]_i nor [Mg²⁺]_i. Casein kinase 2(CK2) inhibitor, 5,6-dichlorobenzimidazole riboside, reduced this plasticity to 60%. CK2 must be partially responsible for this plasticity. [Jpn J Physiol 54 Suppl:S159 (2004)]

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We have previously shown that at least four voltage-dependent Ca²⁺ channel (VDCC) subtypes, N, P/Q, R and L have been identified in the large mossy fiber (MF) terminals in mouse hippocampus (Tokunaga et al., 2004). The possibility was investigated that the expression of VDCC subtypes were not homogeneous among MF boutons. The dextran-conjugates of Ca²⁺-sensitive indicators was injected at the dentate granule cell layer of mouse hippocampal slice and allowed to be accumulated in the MF terminals. The extracellular Ca²⁺ was replaced with Sr²⁺ and the nerve stimulus-dependent Sr²⁺ increments were optically measured under confocal microscopy. The expression of VDCC subtypes were estimated by a specific suppression of the subtype-dependent component of Sr²⁺ increment by ω-conotoxin GVIA (N-type), ω-agatoxin IVA (P/Q-type) and SNX-482 (R-type), respectively. We found that conotoxin and SNX-482 sensitivities were variable among MF terminals whereas agatoxin sensitivity was not. Even two adjacent boutons in the same axon were different in the toxin sensitivities. The MF synaptic transmission was also variable in the sensitivities to conotoxin and SNX-482 but not to agatoxin. Therefore, MF terminals are suggested to be heterogenous in the expression of N- and R-type VDCCs although not in that of P/Q-type. [Jpn J Physiol 54 Suppl:S159 (2004)]

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The neurotransmitter releases from the presynaptic terminals are triggered by the calcium inflow through voltage-dependent calcium channels (VDCCs), which are classified into several subtypes encoded by different genes. In the previous study of mouse hippocampus we have shown physiological evidences that mossy fiber (MF) terminals are not homogenous in the sensitivities to toxins specific to N- and R-type VDCCs. The possibility was investigated that MF terminals are heterogeneous in the expression of VDCC subtype molecules. The MF terminals were labeled by fluorescent dextrans which were injected at the dentate granule cell layer of hippocampal slice. Synaptosomes were obtained from the CA3 region and immunolabeled with antibodies to synaptophysin, N- and R-type VDCC alpha1 subunits. The synaptophysin immunoreactivity was usually co-localized with fluorescent dextrans in the large MF boutons. The N-type and R-type immunoreactivities were observed in cluster with the synaptophysin immunoreactivity in some boutons but was absent in others. Therefore, it is suggests that the MF terminals were not homogeneous in the expression of N- and R-type VDCC at the molecular level. [Jpn J Physiol 54 Suppl:S159 (2004)]

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Oscillation in the concentration of cytosolic Ca²⁺ (calcium oscillation) is known to be caused by repetitive release of calcium from intracellular calcium stores. For further understanding the mechanism of calcium oscillation, we simultaneously measure cytosolic calcium concentration ([Ca²⁺]c) and intraluminal calcium concentration of intracellular calcium store ([Ca²⁺]s), and studied differences of time course between [Ca²⁺]c and [Ca²⁺]s in bullfrog sympathetic ganglion cells. Cells were loaded with two fluorescent calcium indicators, Oregon Green 488 BAPTA-5N for measuring [Ca²⁺]c and Furaptra for [Ca²⁺]s, respectively. Calcium oscillation was induced by the application of caffeine. [Ca²⁺]s decreased rapidly and then increased gradually. After [Ca²⁺]s reached to the threshold level for calcium release which was below the resting level, calcium was released again from calcium stores. Calcium concentration changes in [Ca²⁺]c and [Ca²⁺]s were not just the mirror image and the decaying phase of [Ca²⁺]c was faster than that of [Ca²⁺]s, suggesting that decaying phase of [Ca²⁺]c may be accelerated by efflux of calcium to extracellular space and/or uptake to other cytosolic organ. [Jpn J Physiol 54 Suppl:S159 (2004)]

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The activation of a unique Ca²⁺-induced Ca²⁺ release (CICR) mechanism via β-ryanodine receptors (RyRs) enhances transmitter exocytosis at the frog motor nerve terminals, once it is primed by repetitive Ca²⁺ entry. Hypertonicity is known to increase the basal frequency of miniature end-plate potentials (MEPPs) in frog neuromuscular junctions and also to increase Ca²⁺-activated ryanodine binding to α- and β-RyR purified from skeletal muscle (Murayama et al.,1998). Thus, it is possible that the hypertonicity-induced transmitter exocytosis results from the effect of hypertonicity to prime the mechanism of CICR. We have tested this hypothesis. The time course of priming, subsequent activation and inactivation of CICR was seen as a slow increase and subsequent decrease in the frequency of MEPP during continuous tetanus. The rate of slow rise in MEPP frequency by the tetanus was enhanced in one hour after the addition of sucrose in a low Ca²⁺, high Mg²⁺ solution. Hypertonicity also enhanced the quantal content of end-plate potentials (EPPs) induced by a single or tetanic stimulus. Ryanodine blocked these enhancements in the hypertonic solution. A small hypertonicity-induced transient increase in MEPP frequency during a tetanus even in a Ca²⁺ free, EGTA solution was also blocked by ryanodine. Results suggest that hypertonicity sets the condition for the activation of CICR and resulting enhancement of transmitter release. [Jpn J Physiol 54 Suppl:S160 (2004)]

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The mice lacking alpha1B subunit of N-type voltage dependent calcium channels (VDCC) show manifest hypertension and lack of baroreflex (Ino et. al. PNAS USA, 2001). The caudal part of the nucleus tractus solitarii (NTS) is the primary integrative center of the cardiovascular control. To determine whether modulation of transmitter release in the NTS underlies these autonomic failures, non-NMDA receptor-mediated EPSCs (eEPSCs) evoked by the primary afferent stimulation were recorded in brainstem slices (3-5 wks). Adenosine (100 μM), which we have shown to suppress glutamate release in this synapse through activation of A1 receptors, decreased eEPSC amplitude to 73.3% of the control in the wild type (CaV2.2+/+; 7 of 8 cells tested) and to 72.5% in the heterozygotes (CaV2.2+/-; n=14/21). Adenosine was ineffective in the homozygotes (CaV2.2-/-; n=4). The effect of adenosine was occluded by ω-conotoxin GVIA (CgTX, 3 μM) in all of CaV2.2+/+ neurons and in 33% of CaV2.2+/- neurons, but not in 77% of neurons responding to adenosine in CaV2.2+/-. CgTX exerted no effect in CaV2.2-/-.These results suggest that the coupling between presynaptic G-protein-coupled receptors and N-type calcium channels underlying transmitter release is perturbed by the decrease in the channel protein expression. [Jpn J Physiol 54 Suppl:S160 (2004)]

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Currently, glaucoma is recognized as an optic neuropathy. Selective death of retinal ganglion cells (RGCs) is the hallmark of glaucoma, which is also associated with structural changes in the optic nerve head. Neuroprotection is an important issue in considering treatment options. Latanoprost, a prostaglandin F2α(PGF2α) analog prodrug, and unoprostone, an analog of a prostaglandin metabolite, have been shown to be effective in decreasing intraocular pressure when used alone or in combination with other ocular hypotensive agents. It is thought that a rise in intracellular calcium concentration is an inducement of cell death. Therefore , we investigated the suppressive effects of PGF2α on voltage-dependent Ca currents (ICa). Fast blue (FB) was injected into the superior colliculus of rats (7-12 days old) to identify RGCs under epifluorescence illumination after retrograde transport of FB to RGCs. Retinas were dissected, treated enzymatically, and dissociated with trituration. Effects of PGF2α on membrane currents at -90 mV and ICa were examined by whole-cell patch clamp method. ICa were recorded in the solution containing TTX(0.5μM), TEA (20mM) and 4AP (0.1mM) using an intracellular pipette containing CsCl₂ (135mM). Currents recorded at -90 mV were not affected by PGF2α, whereas PGF2α reduced the peak component of the ICa reversibly by 14%. We suppose that the suppression of ICa by PGF2α may partly concern with therapeutic effects for glaucoma in preventing cell death in RGCs. [Jpn J Physiol 54 Suppl:S160 (2004)]

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The bipolar cell of the vertebrate retina gives rise to graded responses to light stimuli and transmits the responses to the inner retinal circuit. The transmitter release from presynaptic terminal is known to be related to the the calcium concentration at the axon terminal ([Ca²⁺]_i). We conducted computer simulations analyses using a biophysical model to elucidate [Ca²⁺]_i change during light-induced responses.
The software NEURON was used for the simulations. L-type and T-type voltage-dependent Ca²⁺ channels (VDCCs), the plasma membrane Ca-ATPase, the intracellular fixed and mobile buffer and diffusion were incorporated in terms of the Ca²⁺-related machineries. Kinetics of these machineries were estimated from previous studies. In the model cell in which L-type and T-type VDCCs were distributed uniformly over the entire cell, [Ca²⁺]_i increased to about 800 nM from about 80 nM in response to a +20 mV and 500 msec depolarization from -40 mV mimicking the voltage range of light-induced responses. This [Ca²⁺]_i level is thought to be inappropriate for the transmitter release. When L-type VDCC was locally distributed at the tip of the terminal region, which may correspond to the Ca²⁺-domain region, and Ca-ATPase was concentrated at the terminal region exclusive of the Ca²⁺-domain, [Ca²⁺]_i at the Ca²⁺-domain increased to more than 80 μM from about 6 μM in response to the same depolarization. This [Ca²⁺]_i level is though to be appropriate for the transmitter release. [Jpn J Physiol 54 Suppl:S160 (2004)]

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To study how APP is cleaved by BACE1 (β-secretase) and Presenilin1(γ-secretase) during development of mouse retina, we investigated the localizations of BACE1, Presenilin1, and their proteolytic products (APP-NT, APP-CT, and Aβ) at the times of postnatal 4, 7, 11 days and 12 wks immunohistochemically. BACE1 was localized in IPL together with APP-NT at P4 and P7, then not only in IPL but also in OPL at P11, and finally diffused across the retina at 12 wks. From P7 to 12 wks, APP-NT became distributed in the vitread processes of Muller cells. In contrast, both Presenilin1 and APP-CT showed the similar changes in distribution pattern that they were localized in IPL from P4 to P11 but diffused across the retina at 12 wks. Thus APP-NT and APP-CT were processed differently from each other from P7. Consistently with these findings, Aβ was distributed in IPL at P4 and P7 then not only in IPL but also in OPL at P11. Our surveys indicate that BACE1 cleaves APP during P7 and P11 and also suggest that Muller cells take APP-NT in their vitread processes. [Jpn J Physiol 54 Suppl:S161 (2004)]

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Young rats show aversion to the odor which has been exposed with foot shock. We previously reported that the main olfactory bulb (OB) is a critical site for this olfactory learning. There is, however, little electrophysiological evidence on synaptic plasticity in the OB. Using brain slices, we found that 5 trains of tetanic stimulation of the lateral olfactory tract induced NMDA receptor-dependent long-term potentiation (LTP) of field potentials recorded in the granule cell layer of the OB. Although 3 tetani were subthreshold for LTP, application of noradrenaline (NA) paired with the tetani was capable of inducing LTP (NA-LTP). This NA-LTP was partially reduced by the NMDA receptor antagonist, AP5, and completely blocked by the L-type voltage-dependent calcium channel (VDCC) blocker, nifedipine. NA-LTP was blocked by the beta-adrenoceptor antagonist, timolol but not by the alpha-adrenoceptor antagonist, phentolamine. The NA effect on induction of LTP was mimicked by the beta-adrenoceptor agonist, isoproterenol. Moreover, our results showed that paired-pulse facilitation was significantly decreased by application of NA as well as isoproterenol in the presence of the GABAA receptor antagonist, bicuculline. These results indicate that (1) tetanic stimulation-induced LTP in the OB is dependent on NMDA receptors, (2) NA-LTP requires the activation of NMDA receptors and L-type VDCCs, (3) a presynaptic mechanism underlies the effect of NA on LTP, and (4) NA-LTP is induced via beta-adrenoceptors. Taken together with behavioral data, these results suggest a connection between NA-LTP in the OB and olfactory learning. [Jpn J Physiol 54 Suppl:S161 (2004)]

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Previously we developed a co-culture system of cultured accessory olfactory bulb (AOB) neurons with partially dissociated vomeronasal neurons, referred as vomeronasal pockets (VP), to explore mechanisms of specific synapse formation between sensory and first relay station of the vomeronasal system. However, it was unclear whether fibers from VP formed functional synaptic contacts with AOB neurons. To address this issue, we examined the synapse formation between cultured VP and AOB neurons using a calcium imaging technique combined with electrical stimulation.
After 7 days in culture, a few bundles of fibers from a spherical structure of VP extending to the AOB neurons were observed. After 21 days in culture, significant changes in the fluorescence intensity of AOB neurons loaded with a calcium indicator, fluo-4, were observed when VP was electrically stimulated with a bipolar electrode. When a VP was stimulated, some AOB neurons immediately showed transient Ca²⁺ increases. These responses of AOB neurons were reversibly suppressed by the application of 10μM CNQX; the Ca²⁺ transients disappeared in some neurons and diminished in a few neurons. The application of 25μM APV resulted in the suppression of spontaneous Ca²⁺ increases observed repetitively in many cultured AOB neurons and partially affected evoked Ca²⁺ transients. The application of both CNQX and APV completely blocked Ca²⁺ transients evoked by VP stimulation. These results suggest the formation of functional synapses between the VP and AOB neurons in the culture. [Jpn J Physiol 54 Suppl:S161 (2004)]

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Pheromonal memory formation is associated with an increase in the length of the postsynaptic densities, of the excitatory part of the reciprocal dendrodendritic synapses between mitral and granule cells in the accessory olfactory bulb (AOB). Behavioral experiments have implicated noradrenaline (NA) and α-receptors in the AOB in the formation of the pheromonal memory. We have demonstrated, in slice preparations of the AOB, that long-term theta frequency stimulation of the lateral olfactory tract (LOT) induces NMDA dependent LTP at the mitral-to-granule cell synapses and NA facilitates induction of LTP via α2-receptors. To clarify the cellular mechanisms of α2-adrenergic enhancement of LTP, we performed whole cell recordings from visually identified mitral and granule cells. NA and clonidine diminished voltage-gated calcium currents in mitral cells. Both drugs reduced the frequencies of spontaneous and miniature EPSCs without affecting their amplitudes and also reduced LOT-evoked EPSCs in granule cells. In contrast, clonidine had no effect on miniature IPSCs in mitral cells. In granule cells, NA and clonidine decreased plateau depolarization and increased number of action potential firing during theta frequency stimulation. These results indicate that NA inhibits mitral-to-granule cell synaptic transmission via presynaptic α2-receptors, thereby leading to a smaller depolarizing plateau and thus to increased firing probability in granule cells that may contribute to LTP induction during theta frequency stimulation. [Jpn J Physiol 54 Suppl:S161 (2004)]

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To investigate the role of a neuron playing in nervous system, it is important to determine the inputs and outputs of the neuron. So we tried a method to visualize both the input and the output pathways, maintaining neural functions intact. Rat cochlear nucleus (CN) was mainly used because it is easy to be approached and the neural connections are known anatomically. Visualization of outputs is established already by labeling cell bodies retrogradely with fluorescent dye injected in the output area. We confirmed availability of this method by injecting fluorescent beads or dye to inferior colliculus. We further visualized the input pathway to CN, auditory nerve fibers (ANFs), by injecting fluorescent dye DiI to cochlea. CN was sliced 2-4 days after the injection, and ANFs were identified by fluorescence to make contacts with neurons in CN. Functional synaptic contacts were confirmed by electorical stimulation to the fluorescing fiber with a glass micropipette. To investigate whether the identified single fiber was stimulated appropriately, we recorded responses with varying the stimulus intensity and the distance between the fiber and the microelectrode. These responses were also compared with that by stimulating the root of ANFs. A visualized fiber required weaker stimulation to evoke synaptic responses. Similar records were obtained in the medial nucleus of the trapezoid body (MNTB) after injection of DiI to CN. This method should be available in various parts of nervous systems, particularly useful by using multiple dyes of different fluorescence. [Jpn J Physiol 54 Suppl:S162 (2004)]

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Somatosensory and chemical information from the oral cavity is important for feeding behavior. Recently, we proposed that interactions of neural information between gustatory insular (GI) cortex and oral somatosensory (OS) cortex of rats are strengthened age-dependently, suggesting that somatosensory and chemical information is liable to be integrated in adult age than in young age. In the present study, we focused attention on the roles of neural information from the teeth for the development of the reciprocal interaction between GI and OS cortices. All teeth of upper jaw were extracted at 2-3 weeks old, and raised with fluid feeding. Control animals were raised with normal feeding. Brain slices containing both GI and OS cortices were made at 6-8 weeks old, and optical recordings with voltage sensitive dye and field potential recordings were made. In control animals, signals elicited by electrical stimulation propagated along upper layer between GI and OS cortices, and the propagation was reciprocal. The horizontal propagation was dependent on intracellular ryanodine-sensitive calcium release. In animals with losses of teeth, in contrast, signal propagation was restricted within GI or OS cortex. These results suggest that multiple losses of teeth disturbed development of signal interaction between GI and OS cortices. Sufficient neural information from the teeth may be important for development of neocortical function. [Jpn J Physiol 54 Suppl:S162 (2004)]

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In the retina, while it is known that extracellular ATP inhibits ACh release from cholinergic neurons, the types of purinoceptors on the cholinergic neurons have not been examined. We reported that immunoreactivity for P2X2 localized asymmetrically between the ON and OFF pathways in the cholinergic amacrine cells in the mouse retina. In the present study, we recorded the responses to ATP in the cholinergic amacrine cells of the transgenic mouse retina. Both straburst amacrine cells and displaced amacrine cells responded to 100 μM ATP. ATP induced an inward current. However, part of displaced amacrine cells did not respond to 100 μM ATP. Both types of cells did not respond to 100 μM α,β-methylene ATP. Responses to ATP were inhibited by 100 μM PPADS. When cells received GABAergic-IPSP, 100 μM ATP augmented GABAergic IPSP. These results indicate that inhibitory action of ACh released by ATP is mediated by the presynaptic mechanisms. [Jpn J Physiol 54 Suppl:S162 (2004)]

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Purpose: Previously, we demonstrated that transcorneal electrical stimulation(TcES) promotes the survival of axotomized retinal ganglion cells (RGCs) in adult rats. But the mechanism underlying TcES-induced neuroprotective effect was unclear. Electrical stimulation is known to upregulate TrkB and BDNF mRNA in various neurons. We therefore investigated what kind of mRNAs of neurotrophic factors or their receptors were upregulated in the retina after TcES.Materials and Methods: Adult male Wistar rats were used in this study. TcES (100 µA, 20Hz, 1 hour) was applied to the left eye without optic nerve transection. Rats were divided into subgroups according to the terms of survival ranged from 1 hour to 7 days after TcES. The retinas were desected from the enucleated eyes and total RNA was obtained from the retina. RT-PCR analysis was performed to determine the mRNA expressions of the following neurotrophic factors and receptors: BDNF and TrkB ; CNTF and CNTF-receptor ; bFGF and FGF receptor-1 ; IGF-1 and IGF-1 receptor. Results: RT-PCR analysis showed that the expression of IGF-1 mRNA increased at 1 day after TcES and remained elevated over 7 days after TcES. However, mRNAs of other neurotrophic factors and receptors did not change.Conclusion: IGF-1 mRNA was upregulated in the retina after TcES. This result suggests that IGF-1 may play a role in the TcES-induced neuroprotection on axotomized RGCs in vivo. [Jpn J Physiol 54 Suppl:S163 (2004)]

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Retinal ganglion cells express the voltage-dependent Na⁺ channel (Nav) in the soma and axon. Electrophysiological studies showed that some amacrine cells generate action potentials in their dendrites and somata. In addition, it is reported that action potentials propagate in dendrites of ganglion cells. Thus, it is important to elucidate localization of Nav within dendrites and somata of amacrine and ganglion cells in the inner plexiform layer (IPL).
We performed immunohistochemistry on the rat retina using antibodies to pan-Nav (K58/35, Sigma). We observed bright dots and thick process-like structures in the IPL, in addition to immunofluorescence of the soma and axon of ganglion cells. Most of the Nav-immunoreactive thick processes (NavIRPs) were short (10-15 μm) and had no apparent branch. Interestingly they had a terminal-like structure at an end with shape of sphere, ring or bifurcation. NavIRPs was comparable in staining intensity to axon bundles of ganglion cells. To examine whether NavIRPs has connection with AII amacrine cells, we performed double immunostaining with parvalbumin (PV-28, Swant), a marker of the AII amacrine cell. The staining indicated that NavIRPs were not parts of AII amacrine cells. The terminal-like structure of some NavIRPs seemed to be close to somata of AII amacrine cells. It is possible that NavIRPs generate action potential and modulate local neural activity in IPL. [Jpn J Physiol 54 Suppl:S163 (2004)]

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Network properties of gap junction connections between retinal amacrine cells in the inner plexiform layer were evaluated by techniques of microelectrode- or patch clamp- recording, intracellular labeling and electron microscopy. First, amacrine cells were examined with their light-evoked responses to light flashes. Second, cells' voltage-responses against current injection were characterized. Third, coupled networks of the cell populations were visualized by transfer of intracellularly injected Neurobiotin into neighbors. Contacts between amacrine cells were then investigated with high voltage as well as conventional electron microscopy. Cell-type specific, homotypic dendrodendritic connections in the populations were found. Ultrastructural analysis revealed the presence of gap junctions between light-microscopically documented dendritic contact sites, but not in dendrites apart from interconnected sites. These results demonstrate that gap junction connections between amacrine cells are in homotypical manner. Electrophysiological properties of their homotypic gap junctions provide that these amacrine cells can take an advantage in performing lateral interaction in the inner plexiform layer. [Jpn J Physiol 54 Suppl:S163 (2004)]

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AII amacrine cells play an important role in the rod pathway of the mammalian retina, extending bistratified dendrites into the inner plexiform layer (IPL). Recent studies of electrophysiological analysis of AII amacrine cells demonstrated that TTX-sensitive small spikes (10-15 mV in amplitude) were activated at low threshold (~ -55 mV), but the detail is unclear. To investigate functional role of the spike in signal processing, we examined AII amacrine cells in the mouse retinal slice, using whole-cell patch-clamp recording. Spontaneous spike generation of AII amacrine cell was suppressed by puff application of glutamate, the transmitter of the rods, to the OPL where rods and rod bipolar cells (RBCs) make synapses. It is likely that glutamate hyperpolarized the RBC, which is presynaptic to the AII amacrine cell, and decreased its glutamate release. When depolarizing current was injected at a holding potential of -70 mV, repepetive spikes were evoked. Frequency of the spike was dependent on intensity of the injected current. Moreover, puff application of glutamate to the dendrites of AII amacrine cell in the IPL made spike frequency increase in proportion to the concentration of glutamate. To examine the localization of voltage-dependent Na⁺ channels, TTX was applied to the various region of the dendrite and soma of AII amacrine cell. Application of 1 μM TTX to proximal dendrite and/or soma blocked Na⁺ current much effectively than to distal dendrite. Our results indicate that the intensity of glutamate input from RBCs may be coded by frequency of the spikes around the soma of AII amacrine cell. [Jpn J Physiol 54 Suppl:S163 (2004)]

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We observed very different conduction velocities within the receptive field of fast type On-Off transient amacrine cells tightly coupled to each other via gap junctions. The fastest speeds were found in the dorsal area of the receptive fields, on average 5 times faster than those in the ventral area. The asymmetry was similar in the On- and Off- responses, thus being independent of the pathway, indicating the existence of a functional mechanism within the recorded cells themselves. However, the spatial decay of the graded-voltage photoresponse within the receptive field was found to be symmetrical, with the amplitude centre of the receptive field being displaced to the faster side from the minimum-latency location. Around 75% of varicosity-laden polyaxons in neurobiotin-injected cells were directed dorsally from the origin cells. Based on these results, we modelled the velocity asymmetry and the displacement of amplitude centre by adding a contribution of an asymmetric axonal feedback. Due to the asymmetry in the conduction velocity, the time delay of a light response is proposed to depend on the origin of the photostimulus movement, a potentially important mechanism underlying direction selectivity within the inner retina. [Jpn J Physiol 54 Suppl:S164 (2004)]

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Horizontal cells (HCs) have larger receptive field than the dendritic field, due to gap junctional coupling between the same type of cells forming a syncytium. The receptive field profile of the cells is described by an exponential decline of light response amplitude as a function of distance from a recording electrode when a slit of light is used, the space constant of this exponential decay being proportional to (g_s/g_m)^1/2 (where g_s: gap junctional conductance, g_m: plasma membrane conductance). Effects of pH on H1 type HCs in isolated carp retinae were examined in order to investigate its suppression of transmitter release of glutamate and gap junctions of this syncytium. Acidification of Ringer's solution containing 10 mM HEPES from pH 7.6 to 7.3 hyperpolarised its resting membrane potential, decreased dye diffusion of intracellularly-injected Lucifer Yellow to adjacent cells, and increased the space constant of the receptive field. Effects of alkalinisation of Ringer from pH 7.6 to 7.9 were opposite. These results indicated that the acidification induces a larger decrease in g_m than in g_s. This suggests that higher concentration of proton in the Ringer would strongly suppress the transmitter release of glutamate from cones to HCs via Ca-channels at the cone terminals much more than the gap junctional channels of H1 HCs. [Jpn J Physiol 54 Suppl:S164 (2004)]

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We previously reported that survival of rat retinal ganglion cells (RGCs) after transection of the optic nerve (ON) was significantly enhanced by electrical stimulation (ES) to the ON stump (square pulses: 50 μA×50 μsec, frequency: 20 Hz, stimulation time: 2 hours). We here examined whether 20 Hz was optimal frequency for the ES, and whether ES delivered before the ON cut also enhanced survival of axotomized RGCs. After RGCs of adult rats were pre-labeled with Fluorogold^TM applied to the bilateral superior colliculli, the left ON was transected intraocularly and ES was delivered before or after the ON cut. Seven days after the transection, the average rates of RGCs survival were determined by dividing the mean densities of labeled RGCs in the left retina by that in the right intact retina. With ES after the ON cut, we confirmed that ES at 20 Hz was effective for RGC survival: the survival rate of RGCs was 74 ± 7%, which was significantly higher (p<0.01) than sham operation (59 ± 5%). But ES at either 10 or 50 Hz was not effective. Concerning ES before the ON cut, we found ES at 300 μA was also effective for RGCs survival: the survival rate was 74 ± 6%, which was higher than sham operation (59 ± 5%, p<0.01). Additionally, when ES were delivered 3-hour before the ON cut, the effect went off. These results indicate that 20 Hz is optimal frequency for ES of 50 μA, and that it is important for RGCs survival that the ES is delivered just before or immediately after the ON cut. [Jpn J Physiol 54 Suppl:S164 (2004)]

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[Purpose] We previously reported that electrical stimulation (ES) to the sectioned optic nerve (ON) enhances the survival of adult cat retinal ganglion cells (RGCs) a week after axotomy. This effect was, however, limited to the RGCs on the area centralis (AC). Here we report the optimal current intensity and the effect of ES on each RGC type. [Methods] RGCs were labeled retrogradely with DiI in advance. Immediately after left ON transection, ES of 20 Hz square pulses with 300 μsec duration was applied for 2 hours via a cuff electrode attached to the ON stump. The current intensity was varied at 0.5, 1.0, 3.0 or 5.0 mA. After a week survival, the survival rate of total RGCs was calculated as the ratio of total number of surviving RGCs in the transected retina to that in the paired intact one. To examine the survival rate of each RGC type, each RGC was classified into alpha, beta or other cell by intracellular injection of Lucifer yellow. [Results] The 3.0 mA ES improved the survival rate of the total RGCs as well as that of RGCs in the AC, whereas 1.0 mA ES did only in the AC. The 5 mA ES did not improve the survival rate. The ES of 3.0 mA promoted the survival of beta cells, but not that of alpha cells. [Conclusion] The current intensity of 3 mA is optimal to promote the survival of axotomized adult cat RGCs. This ES is effective to beta cells that are important to the central vision. [Jpn J Physiol 54 Suppl:S164 (2004)]

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Nipradilol (NPR), anti-glaucoma drug, is a donor of nitric oxide (NO), blocks alpha and beta adrenoreceptors, and promotes axonal regeneration of axotomized retinal ganglion cells (RGCs) in cats. I examined either its NO supply or blocking the receptors promotes axonal regeneration of cat or rat RGCs. The left optic nerve (OpN) was cut 40-80 min after NPR- or blocker-injection. A peripheral nerve was sutured to the cut end. After 4-6 weeks, regenerated RGCs (R-RGCs) were labeled with different fluorescent dyes injected into the graft at 10 mm or 20 mm separately. RESULTS: An injection of NPR, 10(-7) M, increased numbers of regenerated RGCs (R-RGCs) into 13,884 (average, N=4) 6 week after the transplantation, comparing 3,470 (N=8) in no injected (control) retinas. Average ratio of R-RGCs with >=20 mm axon in R-RGCs with 10-20 mm axon, which implies axonal growth rate, was 83% in injected retinas, higher than in control retinas, 65%. Sodium nitroprusside (10(-5) M) injection increased numbers of R-RGCs into 6,585, 1.9 fold. None of adrenoreceptor blockers, plazosin (alpha-1), timolol (beta), or ICI-118,551 (beta-2) increased the number of R-RGCs nor 20R/10R ratios. Supporting this, R-RGCs numbers were not increased when carboxy-PTIO, NO-scavenger, was injected prior to NPR. NPR increased R-RGCs by 1.76 fold (4 wk) and 1.46 (5 wk) in the rat eye, indicating it has smaller effect on axonal regeneration of rat RGCs. CONCLUSIONS: Axonal regeneration of cat RGCs is promoted mainly by NO provided by NPR at very low concentration. Smaller effect of NPR on axonal regeneration of rat RGCs may be attributed to fewer population of beta cells. [Jpn J Physiol 54 Suppl:S165 (2004)]

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Photic information is transmitted via the retinohypothalamic tract to the suprachiasmatica nucleus (SCN) in the hypothalamus, where circadian rhythms are generated, but the retinal photopigment which mediates circadian entrainment has remained elusive. Recently, melanopsin, an opsin-based photopigment, was localized to the retinal ganglion cell (RGC) layer of rodents and primates. Furthermore melanopsin containing RGCs provide photic information to the SCN revealed by the retrograde labeling. It is well known that phospholipase C (PLC) is distributed in the retina. In order to examine the phototransduction mechanisms of light information absorbed by melanopsin containing RGC immunohistological study was carried out for mouse. Double staining with one of the antibody of PLC subtype, β-1, -3, -4 and that of melanopsin (cordial gift from Dr. Provencio) revealed that some of the positive RGC to antibody of β-4 was also positive to that of melanopsin and that co-immunoreactivity of PLC β-3 and melanopsin was observed at some biplar cell and horizontal cell. This suggested that neurons in innerneuclear layer also have possibilty to sense light by itself in addition to RGC. [Jpn J Physiol 54 Suppl:S165 (2004)]

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Activity-dependent plasticity of ocular dominance in the primary visual cortex is one of the best-studied examples of plasticity in the critical period of developing brain. Although a variety of molecules are suggested to contribute to the plasticity, it is still not clear why visual cortex expresses high plasticity only in a short period called critical period. Recently it was reported that extracellular signal-regulated kinase 1/2 (ERK 1/2) is necessary in the ocular dominance plasticity. If ERK 1/2 is involved in the mechanism of this plasticity, visual deprivation would change the activity of ERK 1/2 during the critical period. To test this possibility, we examined effects of monocular deprivation on the phosphorylated ERK 1/2 proteins in the rat visual cortex by immunohistochemistry. Phospho-ERK 1/2 immunopositive cells were found mainly in layer 2/3 of the visual cortex. We found a significant decrease in number of phospho-ERK 1/2 immunopositive cells in animals deprived of vision. The reduction of phospho-ERK 1/2 immunopositive cells were more prominent in the cortical area receiving more inputs from the deprived eye. Moreover similar changes were observed in adult rats. These observations suggest that ERK 1/2 activation might contribute to plasticity in young rats, but its activation by visual inputs is not a critical period-specific event. The timing of critical period might be regulated by other mechanism. [Jpn J Physiol 54 Suppl:S165 (2004)]

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Extracellular recordings from inferotemporal (IT) cortex showed that neurons for which the optimal stimuli are the same are clustered in a columnar organization. This result is consistent with the optical imaging experiments showing that a visual stimulus elicits activation of the localized spots in IT cortex. However, it is also known that the object selectivity of neighboring cells is not necessarily the same. Thus, our understanding of the columnar organization in IT cortex is only partial.
Here, we identified the spots activated by one or some of the stimuli among 18 object stimuli with optical imaging, and recorded multiple unit activities (MUAs) from these spots. For pairs of MUAs, we calculated correlation coefficients of responses to these 18 visual stimuli.
The results were as follows:(1) In upper layers, the pairs of MUAs within the spots showed high correlation coefficients supporting the view that neurons with similar object selectivity are clustered together. For the pairs in deeper layers, however, the correlation coefficients were low. Thus, the clustering of neurons with similar object selectivity may not extend down to whole cortical layer. (2) The correlation coefficients dropped down for pairs of MUAs that were separated more than 300 um across the cortical surface. This result suggests that the clustering of neurons with similar object selectivity doesn't spread over the area distant more than 300 um. In spite of these results, the reason why previous experiments showed difference in object selectivity for neighboring cells will be discussed. [Jpn J Physiol 54 Suppl:S165 (2004)]

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Orientation selectivity of cortical cells can be modified by restricting early visual experience (orientation plasticity). We have studied the orientation plasticity by use of cylindrical lens goggles, which were chronically mounted to kittens for restricted exposure to a single orientation. Optical imaging of intrinsic signals elucidated that a remarkable over-representation of the experienced orientation was induced in the visual cortical areas 17 and 18 in kittens reared with the goggles. In order to examine the temporal profile of the orientation plasticity during development, we mounted the goggles for vertical orientation exposure to kittens with different ages for two weeks. We conducted optical imaging in the visual cortex of the goggles-reared kittens to examine how orientation preferences of responsive pixels were distributed in areas 17 and 18. Using the over-representation index characterizing the dominance of the experienced orientation over the other orientations, we evaluated the level of plasticity as a function of the age of the kitten when goggle-rearing was started. The results indicated that the over-representation of the experienced orientation was most prominent in kittens reared with goggles during postnatal 3 to 4 weeks and declined by 8 weeks of age. We also used electrophysiological and histochemical analyses to confirm the findings obtained by optical imaging. The sensitive period profile of the orientation plasticity may be similar to that of the ocular dominance plasticity, suggesting that different functional columns share common mechanisms for their developmental plasticity. [Jpn J Physiol 54 Suppl:S166 (2004)]

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Live hair cells isolated from organ of Corti are easy to identify by their typical shape, while these cells in organ of Corti are hard to identify due to the presence of millions of non-hair cells. Here we report that live hair cells in organ of Corti show a remarkable capability to accumulate fluorescent FM1-43, and that this property is useful to find live hair cells not only in isolated organ of Corti but also in tissue-cultured ones. Cochlea dissected from newborn rats (2-7days) was exposed to FM1-43 and was cut in several pieces. These pieces were embedded in a thin collagen matrix and cultured in a Neurobasal medium. When viewed under a fluorescence microscope, outer and inner hair cells were clearly distinguished as cells stained with FM1-43. When the hair cells were killed with 4% PFA, FM-dye was quickly lost from the cells, indicating that FM-dye stained only live hair cells. FM1-43-stained live hair cells were present in organ of Corti cultured for 15 days in the medium without addition of any neurotrophic factor. The row-alignment of hair cells was still maintained in the culture. By contrast, gentamycin, which is known to be toxic to hair cells, eliminated FM1-43-stained hair cells within 48hour. These data indicate that FM-dye is useful to examine life and death of cochlear hair cells in culture. [Jpn J Physiol 54 Suppl:S166 (2004)]

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Utilizing slice preparations of GFP-GAD67 knock-in mice, in which GABAergic neurons are specifically labeled with GFP fluorescence, we studied physiological and morphological characteristics of GABAergic neurons of IC by whole-cell patch clamp-recording and biocytin-intracellular-staining techniques.
GABAergic neurons of IC majorly fell into two distinct firing types;1) sustained type neurons and 2)transient type neurons. Sustained type neurons showed regularly repetitive discharge pattern in response to a long depolarization current pulse (200ms) and transient type neurons showed spike discharges just at the onset of current pulse. Most of neurons of both types showed depolarizing sag in response to hyperpolarization current pulse, which were blocked by 0.1mM ZD7288,Ih blocker. Some of sustained type neurons changed its discharge pattern when they were hyperpolarized before injecting current pulse . Most of sustained type neurons showed large AHP ,which were blocked by 0.1mM Cd²⁺.Morphologically, sustained type neurons are a heterogeneous group, distributed in all regions of IC and including neurons of various cell soma sizes, dendritic field sizes and orientations ,and some of them innervated broadly in IC with many axon collaterals. Some of transient type neurons showed hump after single spike, which were blocked by 0.1mM Ni⁺. They were found only in the dorsolateral region of IC ,having highly oriented dendritic fields and regionally innervating within the cortices of IC. The functional significance of these neurons will be discussed. [Jpn J Physiol 54 Suppl:S166 (2004)]

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Animals utilize a difference in sound arrival time between two ears (interaural time difference: ITD) for the sound source localization along the horizontal axis. In birds, ITDs are first calculated in neurons of nucleus laminaris (NL) by detecting the coincidence (CD) of binaural synaptic inputs. Previously, we showed in the brain slice preparation of the chicken that CD is improved by the EPSP acceleration due to the activation of low-threshold K⁺ currents and hyperpolarization-activated cation currents; being acute enough to encode physiologically relevant ITD of the chicken (<0.2ms). Because ITD sensitivity of behaving chicken is not yet reported, we examined the characteristic frequency and ITD sensitivity of NL neurons in anesthetized chicken (posthatch 1-6 days). We made extracellular recordings by utilizing a tungsten electrode inserted into a glass pipette, and the recording site was marked by a fluorescent dye (DiI) filled in the pipette. Pure-tone or white-noise stimuli of 70ms duration were presented with earphones, and their ITDs were changed systematically. We found that the response of NL neurons varied periodically with ITDs, and the cycle matched that of the stimulus tone. The acuity of ITD detection, evaluated with the maximum slope of ITD function, was almost equivalent to the acuity of CD observed in the in-vitro study. We will further examine the relationship between the ITD sensitivity and the characteristic frequency of NL neurons. [Jpn J Physiol 54 Suppl:S166 (2004)]

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PURPOSE: The F-box protein S-phase kinase-associated protein 2 (Skp2) positively regulates the G1-S transition by controlling the stability of several G1 regulators, such as the cell cycle inhibitor p27. The mechanisms that regulate the expression of p27 in the inner ear have not been elucidated. The purpose of this study is to evaluate the role of Skp2 by regulating p27 during development of mammalian auditory system. MATERIALS AND METHODS: We examined the expression of Skp2, in combination with the expression of p27, a proliferating cell marker Ki67, and a differentiated hair cell marker myosin VIIa, in the developing inner ear of mice by immunofluorescence histochemistry. RESULTS: Our data showed that the expression of Skp2 in the auditory epithelia and neurons at an early stage of cell proliferation in the inner ear development. During the differentiation processes of inner ear development, the onset of p27 expression was observed and was accompanied with the down-regulation of Skp2 expression in the auditory epithelia. In addition, an alteration of the expression of Skp2 and p27 in the greater epithelial ridge and spiral ganglion neurons appeared after the differentiation of hair cells. CONCLUSIONS: These findings suggest that Skp2 is inversely expressed with p27, and plays a crucial role in the development of mammalian auditory system. [Jpn J Physiol 54 Suppl:S167 (2004)]

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Multi-tonotopic fields have been implicated in the temporal cortex of the rat. To reveal anatomical framework for corticofugal modulations by multi-tonotopic fields including the primary auditory area that largely corresponds to area Te1, we examined corticothalamic projections by making small injections of biocytin in the cortex. Best frequency (BF) was determined by epicortical recording of field potentials evoked by pure tones under anesthesia. The rostral portion of area Te1 and the cortical region (area Te2d) dorsal to the caudal end of area Te1, which both exhibited high BF, projected to the ventral zone of the ventral division of the medial geniculate body (MGV). On the other hand, the caudal portion of area Te1 and the rostroventral margin of area Te1 (area Te1v), which both exhibited low BF, projected to the dorsal zone of the MGV. The results suggest that tonotopically comparable subfields in the primary and secondary auditory areas in the cortex exert synergistic modulations on thalamic information processing through topographic projections that converge to a specific part of the MGV. By contrast, collateral projections to the thalamic reticular nucleus (TRN) terminated in different zones of the TRN in each pair of the cortical regions. Putative corticofugal inhibition via the TRN may operate in different parts of the MGV unlike putative corticofugal excitation through the converging direct projections to the MGV. [Jpn J Physiol 54 Suppl:S167 (2004)]

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Recent studies on the human auditory cortex suggest that processing of spectral and temporal information differs between the left and the right hemisphere. In this study, processing of temporal information in the left and right auditory cortices of guinea pigs was investigated using repeated sounds and optical imaging with a voltage-sensitive dye (RH795). Guinea pigs were anesthetized with ketamine (80mg/kg) and xylazine (40mg/kg). Tone or noise trains were presented at different repetition rates (4, 6, 8, 10, 12, 14 Hz) and at 75 dBSPL. Neural activities were recorded from multiple auditory fields (primary: AI, dorsocaudal: AII, ventroanterior: VA and ventroposterior: VP) of both hemispheres. At the repetition rates of 4 and 6 Hz, the neural activity followed well to each sound and sometimes oscillated at the rate of the repeated sound. At 8 and 10 Hz, the neural activity showed one large peak followed by small peaks and at 12 and 14 Hz, it showed one large peak only. Repetition rate transfer functions (RRTF) were low-pass showing a sharp drop-off in evoked activity per tone above 6 Hz. Synchronization to tone trains was better in field AI than in other fields but synchronization to noise trains was the best in field AII. In the same animals, the cut-off frequencies of RRTF in the left cortex were higher than those in the right cortex. We discuss the temporal processing in the left and right auditory cortices of guinea pigs. [Jpn J Physiol 54 Suppl:S167 (2004)]

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Flavoprotein autofluorescence is an endogenous signal that might be applicable for functional brain imaging, but has not been applied for this purpose as widely as intrinsic signal. This technique is especially useful for imaging mouse cortical activities, because the skull of mice is transparent enough to visualize brain autofluorescence via the intact skull. In the present study, therefore, we tried to record neural activities in the auditory cortex in anesthetized mice with this technique. C57BL/6 mice (5-7 week old, both sexes) were anesthetized with urethane (1.6 g/kg, i.p.). The skin covering the skull was incised, and the temporal muscle over the right auditory cortex was removed. The exposed surface of the skull was covered with liquid paraffin to prevent drying and to keep the skull transparent. Autofluorescence responses in the auditory cortex were elicited by sound stimuli (5-20 kHz for 500 ms), and recorded with a cooled CCD camera attached to an epifluorescence binocular microscope. The responsive areas in the primary auditory cortex exhibited a tonotopy map, which was compatible with previously reported electrophysiological studies. The brain imaging via the intact skull is expected to be useful for investigating neural plasticity, since it can be achieved without surgical damages on the brain. Therefore, we are now investigating modification of the tonotopy map after sound exposure with various parameters. [Jpn J Physiol 54 Suppl:S167 (2004)]

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Although auditory neurons are selective for absolute pitch, we easily perceive relative pitch. How can this be achieved? Previously, we investigated the cue used by rats to discriminate between amplitude-modulated (AM) sounds, and found that the relationship regarding the AM depth of sound stimuli was important for the discrimination. In the present study, we investigated the cue used by rats for sound pitch discrimination at different bandwidths. Water-deprived rats were exposed to 5 kHz (S-) and 10 kHz (S+) pure sounds, and licking a spout during the presentation of 10 kHz sound (S+) was rewarded with water, while licking during 5 kHz (S-) was not. After the rats were trained with this manner in two consecutive days, the performances in discriminating between 10 kHz (S+) and 20 kHz (S-), or 10 kHz (S-) and 20 kHz (S+) were evaluated, and the former performance was better than the latter, indicating that the rats used absolute pitch for discriminating S+ and S-. However, if similar experiment was performed using 7 kHz, 10 kHz and 14 kHz sounds, the learning was dependent on relative pitch. These results suggest that absolute pitch was used as the cue to discriminate sound pitch at broad bandwidth while relative pitch was used at narrow bandwidth. Preliminary experiments with bilateral auditory cortex ablation suggest that sound pitch discrimination using relative pitch might be dependent on the auditory cortex while sound pitch discrimination using absolute pitch might not. [Jpn J Physiol 54 Suppl:S168 (2004)]

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How the duration of sounds is processed in the core and belt fields of the guinea-pig auditory cortex was studied by optical imaging with a voltage-sensitive dye (RH795). The animals were anesthetized with ketamine (80 mg/kg) and xylazine (40 mg/kg). Tone and noise bursts with 5-ms onset and offset ramps were presented at different durations (10, 15, 30, 60, 100, and 200 ms) and at 50-70 dBSPL by a loudspeaker located contralateral to the recording cortex. Neural activity was recorded from the core (AI and AII) and ventroanterior belt (VA) fields. Responses of AI, AII and VA to tone or noise bursts consist of a phasic-on excitatory and a following inhibitory response. In AI and AII, the amplitude and duration of the response did not change when the duration of the tone and noise bursts was changed. The duration of the response to the noise burst was shorter than that to the tone burst. In VA, the amplitude and duration of the response increased significantly when the duration of the noise burst was increased. The increase of amplitude and duration was observed for the noise with more than 100 ms and more than 30 ms durations, respectively. The response in VA to the tone burst, however, showed no change in amplitude and duration when the duration of the tone burst was changed. We discuss the differences in integration of temporal and spectral information between the core and the VA field. [Jpn J Physiol 54 Suppl:S168 (2004)]

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Sound source discrimination was evaluated by sound localization performance and event-related potentials (ERP) with normal subjects using two sound sources in a dark semi-anechoic room. Non-target and target noise burst stimuli were presented by a loudspeaker in front of the subject and a lateral one, respectively. The angle between the two loudspeakers was set between 2.4 and 27.6 degrees. Localization performance were measured by the changes in direction of face from non-target loudspeaker toward target one by using an electromagnetic tracking instrument. ERP were measured by oddball test with the same acoustic stimulating system. The localization performance showed the changes in face angle were rather accurate to point loudspeaker locations. The amplitude of the ERP components that were different between target and non-target ERP were measured as a function of the angle between two speakers. The peak-to-peak amplitude between negative wave with the latency of about 150ms and positive waves with the latency of about 250ms increased as the degree of angle between two loudspeakers increased. The relationship between performance angles and difference ERP amplitude was proportional. These results indicate that the ERP represents the perception of the angles of the two sound sources. [Jpn J Physiol 54 Suppl:S168 (2004)]

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In avian, there are good evidences for hair cells to regenerate after damages. It is recently demonstrated morphologically that some mammalian vestibular hair cells regenerate. However, it is not clear any details of functional regeneration. Here we report physiological evidences of functional regeneration in vestibular hair cells after damage by gentamicin in cultured rat maculaes. The mechano-electrical transduction (MET) was monitored as Ca²⁺ responses by loading fura-2 and by applying mechanical stimulation to the hair bundle. In the control, the percentage of functioning samples was decreased gradually with the day of culture and no MET was found after 2 weeks. Paradoxically, in the sample after damage, MET was found even after 2 weeks. This means that MET re-appeared in 2 weeks indicating functional regeneration of hair cells. To facilitate regeneration, we applied steroid hormones, then the percentage of functioning samples was increased. Hair bundles' loss and regeneration were confirmed morphologically, by Nomarsky optics and by scanning electron microscopy from the same sample. Phalloidin stained samples were also investigated. In the control, number of hair bundles was gradually decreased during culture. However, the number gradually increased in 2 weeks after acute hair bundle loss by gentamicin. In the area hair bundles were completely lost, some short hair bundle like structures emerged 2-3 weeks later. This may indicate that mammalian vestibular hair cells regenerate morphologically and functionally. [Jpn J Physiol 54 Suppl:S168 (2004)]

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Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates ion transporters and channels in addition to second messenger systems. Previous reports indicated that PIP2 is expressed in hair bundles, the mechanically sensitive organelle of hair cells. Whether PIP2 regulates mechanoelectrical transduction channels of hair cells has hitherto not been investigated. Therefore, we have characterized the distribution of PIP2 in hair bundles and determined a physiological role of PIP2 in transduction. According to our immunocytochemical data, hair cells from bullfrog sacculi expressed PIP2 in hair bundles, except on basal part of the hair bundles. This labeling disappeared when hair cells were treated with PI4-kinase inhibitors, phenylarsine oxide and quercetin, which block PIP2-replenishment. Reciprocal distribution of PIP2 and the lipid phosphatase Ptprq suggests why PIP2 is absent from stereocilia bases. Our whole-cell patch-clamp recording data from isolated hair cells show that adaptation of transduction currents became significantly slower when PIP2 was depleted with PI4-kinase inhibitors. In addition, in the bullfrog utricle, the central striolar hair cells, which adapt more rapidly to maintained hair bundle displacement, have much less Ptprq than peripherally located extrastriolar hair cells, which do not adapt or adapt only very slowly. These results suggest that differences in adaptation rate could arise from variations in the concentration of PIP2 in hair bundles. [Jpn J Physiol 54 Suppl:S169 (2004)]

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Extracellular ATP has been recently demonstrated to act on presynaptic P2X receptors to modulate synaptic transmission in CNS. In this study, we examined a role of P2X receptors in modulating excitatory synaptic transmission in dorsal horn neurons by using patch-clamp recordings from spinal cord slice preparations. P2X receptors were highly expressed in central terminals of both superficial and deep laminae neurons. Most presynaptic P2X receptors of lamina II neurons were not affected by bath application ofαβmATP, but activated and facilitated glutamate release by focal application ofαβmATP. In addition, these effects by focal application ofαβmATP were sensitive to PPADS and TNP-ATP. In contrast to lamina II neurons, bath application ofαβmATP activated presynaptic P2X receptors of most lamina V neurons and largely enhanced glutamate release for a relatively longer period. Moreover, presynaptic P2X receptors in most lamina V neurons were sensitive to PPADS, but resistant to TNP-ATP. These results indicate that distinct subtypes of P2X receptors are expressed in central terminals of primary afferents innervating superficial and deep dorsal horn neurons, and modulate glutamate release in a different manner. These distinct roles of presynaptic P2X receptors may serve to understand the various sensations mediated by P2X receptors. [Jpn J Physiol 54 Suppl:S169 (2004)]

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The capsaicin receptor, TRPV1, is a sensory neuron-specific ion channel that serves as a polymodal detector of pain-producing chemical and physical stimuli. It has been reported that inflammatory mediators such as ATP and bradykinin phosphorylate TRPV1 in a PKC dependent pathway. Two serine residues in the cytoplasmic domains of TRPV1, Ser⁵⁰² and Ser⁸⁰⁰ were found to be involved in phosphorylation of TRPV1 by PKC. We made an antibody gainst phosphorylated Ser⁸⁰⁰ of TRPV1 because Ser⁸⁰⁰ is known to function as a substrate only for PKC-dependent phosphorylation. We examined the amount of phosphorylated TRPV1 upon treatment with PMA using the antibody in Western blot analysis and immunostaining. We detected the increase in the amount of phosphorylated TRPV1 upon PMA stimulation in both HEK293 cells expressing TRPV1 and cultured rat DRG neurons. [Jpn J Physiol 54 Suppl:S169 (2004)]

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Prostaglandin E₂ (PGE₂) is one of the inflammatory mediators that play important roles in pain sensation and hyperalgesia in inflamed tissues by exciting or sensitizing nociceptors, and inhibition of PGE₂ production by cyclooxygenase blockers is a popular way to treat pain and inflammation. The capsaicin receptor TRPV1 is a nonselective cation channel exclusively expressed in sensory neurons and activated by various stimuli known to cause nociception in vivo such as capsaicin, protons, heat and some lipids. We examined the interaction between TRPV1 and PGE₂. In behavioral analysis, intraplantar injection of PGE₂ produced thermal hyperalgesia in wild type mice. However PGE₂-induced thermal hyperalgesia completely abolished in TRPV1-deficient mice. PGE₂-induced potentiation of capsaicin-activated current responses was observed in mouse dorsal root ganglion neurons. These data suggest that TRPV1 is necessary for the development of PGE₂-induced thermal hyperalgesia in vivo. [Jpn J Physiol 54 Suppl:S169 (2004)]

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Bradykinin (BK) produced in damaged and inflamed tissues causes pain and hyperalgesia through the excitation and sensitization of nociceptors. BK shifts down the threshold temperature of a nociceptive heat transducer TRPV1 to below the skin temperature, suggesting that BK-induced pain is a result of TRPV1 activation at the skin temperature. To confirm this hypothesis, we examined the contribution of TRPV1 in BK-induced pain, by comparison of nociceptive responses induced by BK between wild-type (WT) and TRPV1 knockout (KO) mice.
When BK was injected into the plantar skin of the hind paw, incidence of the animals that displayed pain-relating licking behavior and total period of licking were significantly reduced in KO mice compared to wild-type (WT) at a lower dose of BK (0.1nmol), while no difference was observed at the highest dose applied (1nmol). This suggests that TRPV1 is involved in BK-induced pain sensation in vivo, but its contribution is not essential at high dose of BK. Single fiber recording from an isolated skin-saphenous nerve revealed that BK excited nociceptive C-fibers in a dose-dependent manner in KO mice, and no difference was observed in the incidence of BK-sensitive C-fibers and the amplitude of excitation in both mice. In addition, increase of intracellular Ca²⁺ level by BK was observed in almost same percentage of cultured dorsal root ganglion neurons derived from KO and WT mice. These results suggest partial contribution of TRPV1 to BK-induced pain. [Jpn J Physiol 54 Suppl:S170 (2004)]

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