To investigate whether neurons with different morphology have different inputs arose from primary afferent in substantia gelatinosa (SG) of the spinal dorsal horn, we performed whole cell recordings with patch pipettes filled with neurobiotin in a horizontal slice preparation of adult rat spinal cord with an attached dorsal root. Dorsal root stimulations revealed that most neurons recorded had evoked inhibitory postsynaptic currents (eIPSCs) that were mediated by GABAergic and/or glycinergic interneurons as well as monosynaptic and/or polysynaptic evoked excitatory postsynaptic currents (eEPSCs). Based on the dendritic arborization, there were at least four cell types (large islet, small islet, vertical and radial cell), and their morphological features were associated with characteristic inputs. Large islet and small islet cells received excitatory monosynaptic inputs by C-fiber, while radial and vertical cells received excitatory monosynaptic inputs by both A-δ and C-fibers. Aδ-fiber mediated eIPSCs were observed in all cell types. C-fiber mediated eIPSCs were not seen in large islet group although observed in other three groups. These results suggest that complex excitatory and inhibitory neuronal circuits originated from primary afferent are responsible for the processing of nociceptive information in SG. [Jpn J Physiol 55 Suppl:S161 (2005)]

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The parabrachial nucleus (PB), the secondary taste relay, bilaterally projects to the parvicellular part of the posteromedial ventral thalamic nucleus (VPMpc), the thalamic relay, with ipsilateral dominance in rodents. However, the termination patterns in the VPMpc have not been consistent with distributions of electrophysiologically-identified taste neurons in the VPMpc. The present study examined precise termination patterns from the PB in the VPMpc using electrophysiologically-guided injections of wheat germ agglutinin-conjugated horseradish peroxidase in rats. Following tracer injections into the PB taste responsive regions, anterograde labels were bilaterally distributed in the VPMpc with ipsilateral dominance. Dense labels were seen rostrally in the central portion and caudally in the ventral portion of the VPMpc but labels were sparse or lacking in the caudodorsal area and peripheral portions of the nucleus. Dense labels were found in a slightly more lateral region on the contralateral side than on the ipsilateral side. The label distribution in the rostral VPMpc was similar to distributions of electrophysiologically recorded taste neurons in previous reports. The findings generally confirmed the previous anatomical reports, further revealed precise projection patterns from the PB taste area to the VPMpc subregions and explained well distributions of taste neurons in the VPMpc. [Jpn J Physiol 55 Suppl:S161 (2005)]

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Cochlear endolymph has a highly positive potential of ∼+80 mV. This so-called endocochlear potential (EP) is essential for hearing and maintained by K⁺-circulation from perilymph to endolymph through cochlear lateral wall. Gastric H⁺,K⁺-ATPase is reported to occur in stria vascularis and spiral ligament of the lateral wall. However, its precise distribution and physiological roles are still unknown. In this study, we therefore examined the localization and function of the H⁺,K⁺-ATPase in mammalian cochlea. RT-PCR analysis identified that gastric α1 but not colonic α2 subunit of H⁺,K⁺-ATPase dominates in cochlea. The immunolabeling using an antibody specific for the α1 subunit revealed that the H⁺,K⁺-ATPase is strongly expressed in intermediate cells of stria vascularis, the tissue which plays a central role in formation of EP. The clear immunoreactivity was also detected in types II, IV, and V fibrocytes of spiral ligament and those of spiral limbus, all of which are involved in cochlear K⁺-circulation. Finally, vascular perfusion of a specific blocker for gastric H⁺,K⁺-ATPase, SCH28080, dramatically reduced EP. These results strongly suggest that gastric H⁺,K⁺-ATPase in cochlear lateral wall positively regulates the K⁺ circulation and is crucial for generation of positive EP. [Jpn J Physiol 55 Suppl:S162 (2005)]

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PURPOSE. To evaluate the performance of a new multi electrode array, the "silicon (Si) microprobe electrode array chip", as a recording device of neural activity, we recorded light-induced responses from the carp retina. The Si microprobe array, each with a few microns in diameter and controlled length, was fabricated on an integrated circuit (IC) chip, by selective Si epitaxial growth, based on a vapor-liquid-solid (VLS) method. METHODS. The isolated retina from a carp was placed with its ganglion cells layer side down over the Si probes. Retinal responses elicited by full-field flashes (white LEDs), were recorded using the Si microprobe electrodes. For comparison, light responses were also simultaneously recorded with a metal electrode. The changes in amplitude of retinal responses were obtained by stimulating the slit-shaped light at different locations. RESULTS. Graded responses were recorded by full-field flashes. The waveform of recorded responses indicated the signals of a typical electroretinogram (ERG). Furthermore, the amplitude of the ERG response was decreased when the slit-shaped light was moved away from the recording electrode. The results suggest that the Si microprobe electrode array chip allow direct multi-recordings of spatial distribution profiles of local ERG. The chip can be applied to retinal experiments as a new recording tool. [Jpn J Physiol 55 Suppl:S162 (2005)]

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Histamine is thought to deliver itch sensation via H1 receptors on primary sensory neurons. Our previous studies showed that H1 receptor-expressing dorsal root ganglion neurons (about 15-20%) are unmyelinated in the guinea pig. However, these may not be peptidergic and capsaicin-sensitive neurons. The profiles of H1 receptor-expressing neurons in the guinea pig are partly incompatible for the physiological data of histamine-sensitive neurons in the rat. In this study, we examined the expression of H1 receptor in the rat primary sensory neurons by immunohistochemistry and transcriptase-polymerase chain reaction (RT-PCR). The immunoreactivity for H1 receptor was seen in 20-30% of the dorsal root ganglia. These were also labeled by isolectin-B4, which is a marker of unmyelinated sensory neurons. Furthermore, half of these neurons were positive to substance P and TRPV1, a capsaicin receptor. A positive signals of H1 receptor mRNA in rat dorsal root and trigeminal ganglia were clearly detected by RT-PCR, but were weaker than those of GAPDH mRNA. On the other hand, H1 receptor mRNA signals in the guinea pig sensory ganglia were more intense than those of GAPDH mRNA. A few of vagal afferent neurons (about 5% of nodose ganglion neurons) were also immunoreactive to H1 receptors in the rat. No expression of H1 receptor mRNA was seen in the guinea pig nodose ganglion in our previous study. These results suggest that the expression pattern of histamine H1 receptor in primary sensory neurons of the rat is partially different from that of the guinea pig. [Jpn J Physiol 55 Suppl:S162 (2005)]

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Peripheral afferent denervation induces reorganization of somatotopic maps in primary somatosensory cortex. In the present study, we investigated somatotopic map plasticity after tail amputation using transcranial autofluorescence imaging of mouse cortical activities. Neonatal mice were anesthetized with ether, and the tail was cut at the base. Mice were anesthetized with urethane 8-13 weeks after tail amputation, and the skull were exposed and covered with clear acrylic resin for keeping the transparency of the skull. Cortical images of green autofluorescence in blue light were recorded through the skull. Neural activities in the primary somatosensory cortex were elicited by vibratory stimulation applied to the contralateral hindpaw or tail. In control mice, an activity-dependent autofluorescence increase was observed in the hindpaw; tail base and tail tip regions, which were adjacent to each other in the posterio-medial direction in the somatosensory cortex. In the tail-amputated group, the tail base region shifted toward the region corresponding to the tail tip in control mice. Furthermore, the tail base and hindpaw regions were expanded toward the border between them. Similar results were observed in mice, which received tail amputation at 8 weeks old. These results clearly indicate that somatotopic map was reorganized after tail amputation and the plastic changes were visualized using transcranial autofluorescence imaging of mouse cortical activities. [Jpn J Physiol 55 Suppl:S162 (2005)]

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Learning is partly attributed to the functions of the neocortex. To investigate the roles of auditory cortex (AC) in a sound discrimination behavior, we investigated sound frequency discrimination by rats with or without AC lesion. Previously we reported that normal rats depend on relative difference for discriminating sound frequencies at narrow bandwidth (0.5 octave), while they depend on absolute frequency at wide bandwidth (1 octave). Now we report that AC lesion rats exhibited almost normal discrimination behaviors at both bandwidth between the rewarded (S+) and unrewarded (S-) sounds. After they learned to discriminate between 10 kHz (S+) and 5 kHz (S-) tones, however, they failed to discriminate between 20 kHz (S+) and 10 kHz (S-), while naïve and sham-operated rats could discriminate it. These results indicating that AC might have a critical role to re-learn the discrimination paradigm. The roles of AC were further tested with associative discrimination test, using 10kHz sound as S+ and 10kHz sound plus light as S-. In this test, AC lesion rats failed to discriminate between S+ of single modality and S- of multi modalities while normal rats performed well. The present results strongly suggest that AC works as an auditory interface to the neocortex, which are responsible for flexible learning or association of sensory information with mixed modalities. [Jpn J Physiol 55 Suppl:S163 (2005)]

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Somatosensory cortical circuits are modified in an activity-dependent manner. Somatosensory information is serially processed by the primary (S1) and secondary (S2) cortices. In the present study, we investigated activity-dependent modification of the functional connections between S1 and S2 in rat cortical slices. Neural activities were elicited by repetitive electrical stimulation, and visualized using green autofluorescence of flavoproteins in blue light. The stimulation applied in S1 produced neural responses spread into S2. However, when S2 was stimulated, the neural activities were not invaded into S1. We also investigated the properties of the functional connections with field potential recordings and patch clamp recordings from pyramidal neurons. The anisotropic propagation of neural activities demonstrated using autofluorescence imaging was confirmed by the electrophysiological experiments. Activity-dependent changes in neural activities were observed in the barrel cortex one or two weeks after trimming the contralateral whiskers. In these slices, supragranular neural activities after stimulation of infragranular layers were clearly suppressed. Furthermore, neural activities elicited by S1 area near the border between S1 and S2 were spread into the adjacent barrel cortex area rather than into S2. These results suggest the activity-dependent modification of the barrel cortex circuits and functional connections between S1 and S2. [Jpn J Physiol 55 Suppl:S163 (2005)]

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The fish retinal ganglion cells can successfully recover their axons after optic nerve transection. We searched molecules to trigger the initiation of optic nerve regeneration during the early stage of this process with molecular cloning technique. By using differential hybridization between normal and axotomized retinas, we cloned a full-length cDNA for goldfish purpruin, a secretory 22 kDa retinol-binding protein from the retinal cDNA library. The levels of purpruin mRNA was upregulated in the photoreceptor layers 2-5 days after optic nerve injury. The retinol-binding protein with retinol showed a priming action on neurite outgrowth in the goldfish retina (Matsukawa, et al., 2004). In the present study, in order to test transcriptional regulation of prupurin gene, we do cloning of cDNA and genomic DNA for zebrafish purpurin using the goldfish cDNA probe. The amino acid sequence of zebrafish purpurin cDNA was 94% homology to that of goldfish. We further investigated purpurin mRNA levels in the zebrafish retina by Northern blot hybridization following optic nerve injury. The cellular localization of this mRNA was observed by in situ hybridization. After screening of zebrafish genomic DNA library using goldfish purpurin cDNA, we clone a 10 kbp zebrafish genomic DNA. From the data of sequence analysis, the genomic DNA had six exons and exon-intron boundaries. We are now in progress to do luciferase reporter assay for analysis of promoter regions in the 5’upstream of coding regions of prupurin gene. [Jpn J Physiol 55 Suppl:S163 (2005)]

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The limbic system including the amygdala is responsible for learning using reward or punishment, and receives auditory inputs partly mediated by the auditory cortex (AC). We have reported that discrimination learning of sound sequences requires the presence of AC. Cortical neurons in AC are well activated by complex sound stimuli, which consist of harmonic overtones and have several formants. In the present study, we investigated possible roles of AC in discrimination leaning of complex sounds with multiple poles in the frequency spectrum. Water-deprived rats were trained to discriminate between two sounds of different spectral patterns. Licking a spout during presentation of one of the two (S+) was rewarded with water while the other (S-) was not. Either S+ or S- was presented randomly in a trial, which was repeated every one minute for 12 hours on consecutive 4 days. Percentage of trials in which rats licked the spout was calculated separately for S+ and S-, and test performance estimated as the difference was clearly increased during the test in control rats. To investigate the roles of AC, electrolytic lesion was bilaterally produced in AC. In the rats with AC lesions, test performance was significantly attenuated for discrimination learning between sounds with three or four poles in the frequency spectrum, while no significant effect of the lesion was found for discrimination between the stimuli with a pole or between pure tones. These findings indicate that AC plays a critical role in discrimination learning between sounds of complex spectrum patterns. [Jpn J Physiol 55 Suppl:S163 (2005)]

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It has been shown that pH of the invaginating synaptic cleft of the cone terminal is controlled by the membrane voltage of hirozontal cells, low in the dark and high during surround illumination (Hirasawa & Kaneko, 2003). High pH enhances the amount of L-glutamate released from the cone terminal, resulting in the formation of the antagonistic surround component of receptive field of bipolar cells and the higher-order neurons in the visual system. By an imaging technique, we measured the pH of the immediate surrounding space of a horizontal cell (pH_o) isolated from the carp retina to study the mechanisms producing the pH change. Horizontal cells were stained with pH-sensitive dye, 5-hexadecanoylaminofluorescein (H110), and the ratio of the fluorescent emission of 530 nm excited by 490 nm to that excited by 440 nm light was calculated. Cells were superfused with a Ringer solution buffered with 28 mM NaHCO₃. Depolarization of the horizontal cell by bath-application of 100 mM K⁺ solution ( [nearly equal] 100 mV depolarization) lowered pH_o by 0.21±0.05 pH unit. The amount of pH change was related monotonically to the amount of depolarization. Since the cleft of the invaginating synapse of cone terminals is a space where communication to the open extracellular space is much restricted, the pH change of the cleft will be larger than the open space. It will probably result in suppression of transmitter release from the cone terminals. [Jpn J Physiol 55 Suppl:S164 (2005)]

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In the present study, we investigated the roles of the efferents and centrifugal afferents in cell survival in the adult rat main olfactory bulb (MOB). Rats underwent unilateral transection of these central connections at the posterior end of the MOB. The effects of the transection were examined by morphological and histochemical methods. In the MOB ipsilateral to the surgery, many degenerating cells were present in both the mitral cell layer (MCL) and GCL at 3 days after surgery. The numbers of degenerating cells in the ipsilateral MOB were 6.4-fold greater in the MCL and 65.8-fold greater in GCL compared to the contralateral MOB. We also obtained essentially the same results by using TUNEL assay (the MCL, 11.8-fold; the GCL, 22.6-fold). Furthermore, the degenerating and TUNEL-positive cells in the ipsilateral MOB were located predominantly in the deep, rather than the superficial, GCL (p<0.0001 for both). Immunohistochemistry for activated caspase-9 further supported the occurrence of apoptotic cell death in the mitral and deeply located granule cells. These results indicate that not only axotomized mitral cells, but also deeply located granule cells that were not directly injured, underwent apoptosis after transection of the central connections, and suggest that the roles of the central connections in cell survival differ among granule cells according to their depth in the GCL. [Jpn J Physiol 55 Suppl:S164 (2005)]

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Objective: To establish an adequate experimental model of the chronic muscle pain. Methods: A total of 24 male rats were allocated to four groups. Eccentric exercise [EEG], exercise under ischemia [EIG], 3% λ-carrageenan injection [CIG; 0.1 ml, i.m.] and acidic saline (pH 4) [AIG; 0.1 ml, i.m.] groups. These procedures were done to the gastrocnemius [GS] muscle and pain thresholds were measured by von Frey filament testing [VFT] and Randall-Selitto test [RST] over the GS muscle for 6 weeks after the conditioning stimulation. Results: In the EEG, the threshold of RST of exercised side were the lowest on the 1st day after exercise and returned on 7th day. No influence was observed in the contralateral side. In the EIG, however, the lowest thresholds appeared 2 days after the stimulation and it prolonged for 21 days. The VFT thresholds were not changed significantly in the EEG and EIG. On the other hand, in the CIG and AIG, the thresholds of VFT were the lowest on the 1st day after the injection and then returned to the baseline on the 4th week. However, the thresholds of RST were not changed. Conclusion: In the present study, repeated eccentric exercise under the ischemic condition induced the elongation of the muscular hyperalgesia for three weeks and no cutaneous hyperalgesia was observed. On the contrary, the other procedures of chemical injections induced long-lasting cutaneous hyperalgesia, but no change was detected by the RST. These results suggest that the eccentric exercise under the ischemic condition might to be a useful experimental model of the chronic muscle pain. [Jpn J Physiol 55 Suppl:S164 (2005)]

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Flavoprotein autofluorescence signals are intimately coupled with neural activities, and are applicable for functional brain imaging. The transparent skull of mice allows trans-cranial autofluorescence imaging of cortical activities in mice. In this study, cortical plasticity induced by monocular deprivation in the mouse visual cortex was investigated with this technique. C57bl/6 mice were anaesthetized with urethane. An increase in autofluorescence in the visual cortex was elicited by light stimuli at the timing of ON- and OFF-responses. Field potential recordings were performed to confirm, whether underlying neural activities were reflected by the autofluorescence signals. Evoked potentials after light stimuli were observed inside but not outside the autofluorescence responsive area. Autofluorescence signals were about 10 times larger in amplitude and faster in timecourse than intrinsic signals, which have been widely used for functional brain imaging. Effects of monocular deprivation for 4 days in the critical period were evaluated a few weeks after the deprivation with autofluorescence imaging. Monocular deprivation selectively diminished the autofluorescence responses to light stimuli applied to the deprived eyes in the binocular zone but not in the monocular zone of the visual cortex. These results indicate that cortical plasticity induced by monocular deprivation can be investigated with trans-cranial autofluorescence imaging in the mouse visual cortex. [Jpn J Physiol 55 Suppl:S164 (2005)]

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Recent studies have shown that multiple glutamate receptors are expressed on peripheral primary afferent terminals, and they may contribute to peripheral nociceptive signaling. However, little is known about the action of subcutaneous glutamate on the nociceptors in humans. Thus, glutamate was injected subcutaneously into the forearms of healthy volunteers with or without Lidocaine Patch in order to investigate whether the subcutaneous glutamate induces neurogenic inflammation following the excitation of nociceptors. Glutamate caused severe pain immediately after the injection on the subjects without Lidocaine Patch. The intensity of pain expressed by VAS peaked within 5 minute, and gradually decreased, but slight pain was maintained for 30 minutes. Skin temperature on fingers, monitored by thermography, led to a temporary decrease immediately after the injection. On the other hand, skin temperature around the injection site, started to increase about 3 min after injection, reached a peak in 20-25 min and then decayed gradually. Pretreatment with Lidocaine Patch for 60 minutes did not alleviate glutamate-evoked pain sensation, but attenuated skin temperature increase around the injection site. These data demonstrate that subcutaneous injection of glutamate activates nociceptors, and that Lidocaine Patch blocks glutamate-evoked neurogenic inflammation in humans. [Jpn J Physiol 55 Suppl:S165 (2005)]

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To investigate the integration process of motion involved in the dorsal stream and shapes involved in the ventral stream, we examined the neuronal activities of the area MT in the shape from motion (SFM) condition, in which shapes were made visible by dots within invisible contours of shapes moving coherently in one direction. Monkeys performed a shape discrimination task and a direction discrimination task. In the shape task, one of 4 shapes in the white solid condition was presented centrally as a sample stimulus, and after a delay period the same shape was presented peripherally as a target with a distracter presented on the opposite side. Two shapes were defined by moving dots against static dots during this period. Moving directions were selected from the preferred and null directions of the recording cell. Monkeys were required to select the same shape as the sample stimulus. In the direction task, moving dots were presented during the sample period and monkeys were required to ignore the shape difference and to select the same direction of movement as the sample stimulus during the discrimination period. The physical conditions during the discrimination period were identical between two tasks. About 30% of MT neurons showed significantly greater response in the shape task and about 20% showed significantly greater response in the direction task. These results may suggest the involvement of the area MT in the shape discrimination under the SFM condition. [Jpn J Physiol 55 Suppl:S165 (2005)]

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We investigated interhemispheric interactions between bilateral somatosensory cortices through activity-related hemodynamic signals and their dependence on stimulus time lag. We measured optical intrinsic signals (586 and 610 nm) in rat somatosensory cortex evoked by electrical pulses to the contralateral hindpaw (test stimuli, TS) while delivering electrical pulses to the ipsilateral hindpaw or homotopic cortical regions of the contralateral hemisphere (conditioning stimuli, CS) under α-chloralose anesthesia. The responses to CS-TS were normalized by the responses to TS without CS. RESULTS: (1) Optical responses to either hindpaw CS or TS were observed not only in the contralateral somatosensory cortex but in homotopic regions of the ipsilateral hemisphere with the magnitude of approximately 1/10 of contralateral responses. The cortical CS elicited optical signals bilaterally in the same manner. (2) Hindpaw CS-TS responses were 92 ± 12, 78 ± 23, 56 ± 27, 83 ± 8 and 107 ± 9% at 80, 60, 40, 20 and 0 ms lag time, respectively. The optical responses were significantly suppressed at 40 ms and slightly augmented at 0 ms. (3) Cortical CS-TS responses were highly suppressed at 20 ms and still suppressed at 0 ms. DISCUSSION: The results probably reflect the manner of interhemispheric neural interactions. The earlier suppression in the cortical CS-TS paradigm than in the hindpaw CS-TS paradigm may be explained by neural transmission time from the hindpaw to the contralateral cortex, suggesting interactions via interhemispheric callosal connections. [Jpn J Physiol 55 Suppl:S165 (2005)]

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Neurotrophins (NTs) have been revealed essential for the survival of spiral ganglion neurons (SGNs). However, little is known about whether they are necessary for growth of fibers from these SGNs toward the brain. Here we report that either brain derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) are crucial for fibers growth from SGNs in culture of newborn rat cochlear explants in serum-free medium. Moreover, we introduced a new image processing technique to analyze the effects of NTs. This new technique clarified the difference between BDNF and NT-3 in quantitative manner. SGNs were dissected from cochlea of postnatal day 2-4 Wistar rats, were cut to approximately 200 μm in diameter, and were placed on a cover glass coated with poly-L-lysine in a 35 mm dish. The SGN-explants were grown in serum-free medium supplemented with neurotrophin (nerve growth factor (NGF), BDNF, or NT-3) for 3 days. The preparations were fixed with 4% paraformaldehyde and were immunostained against neurofilament (160 kD). The total neurite length per cell was estimated and the dose dependency of neurotrophin was compared with each condition. Significant effect on neurite elongation was observed with either BDNF (≥2 ng/ml dose) or NT-3 (≥10 ng/ml), while the neurite growth was poor with NGF. [Jpn J Physiol 55 Suppl:S165 (2005)]

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It has been well known that one of most rapid and efficient method for complete paralysis in whole arm is to inflate a tourniquet, which is positioned on the arm proximal to the axilla (Goodwin et al., 1972). This method may have direct effects on the distal motor organs, but also on the central neuronal system. Somatosensory evoked potentials (SEP) is one of useful techniques to detect the influence on the somatosensory system. However, there are very few previous studies which focused on the influence of arterial occlusion on SEP to median nerve stimuli. In the present study, we investigated the SEP to median nerve stimuli during the arterial occlusion.Ten right-handed volunteers (seven men and three women, 19-22 years old) with no history of neurological or other disease participated in the study. The SEP to right median nerve stimuli delivered at a rate of five per second was recorded during rest (control condition) and during vascular occlusion at a pressure of 250 mmHg applied by at a tourniquet to the proximal end of the upper arm (experimental condition). Each length of control and experimental conditions was 120 s. Although no significant change was observed in the amplitudes of Erb’s potential and N13, that of N20 significantly decreased during arterial occlusion (p<0.01) with the enhanced sensitivity of the thenar eminence muscle to median nerve stimuli. These results suggest that the transient, arterial occlusion around the upper arm will induce attenuation of the function in the median nerve. [Jpn J Physiol 55 Suppl:S166 (2005)]

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Amiloride-sensitive epithelial Na⁺ channels (ENaCs) are proposed to be involved in salt taste transduction. Electrophysiological studies in C57BL/6 (B6) mice demonstrated that responses to NaCl are inhibited by amilolide in the choda tympani (CT) but not in the glossopharyngeal nerve, suggesting a lack of amiloride sensitivity (AS) in the posterior tongue. The AS also differs among inbred mouse strains. Unlike B6 mice, 129P3/J (129) mice showed almost no amiloride inhibition of NaCl responses even in the CT. In this study, using B6, 129 mice and their F₂ hybrids, we examined possible relationships of the AS with mRNA expression levels in fungiform papillae (FP) and single nucleotide polymorphisms (SNPs) of three subunits of ENaC (α,β,γ). The mRNA expression levels of each ENaC subunit examined using RT-PCR analysis were similar in the B6 and 129 strains. Sequencing detected three SNPs in the α-subunit. One of these SNPs resulted in an amino acid substitution, R616W, near the predicted 2nd transmembrane domain in the 129 strain. No SNPs were found in sequences of β- and γ-subunits. Electrophysiological and sequencing analyses in F2 hybrids indicated that there was no relation between the AS and the α-subunit SNP (R616W). These results suggest that neither expression levels of the three ENaC subunits, nor the SNP in the α-subunit participate in the mouse strain differences in the AS. [Jpn J Physiol 55 Suppl:S166 (2005)]

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Flavoprotein autofluorescence is intimately coupled with neural activities and applicable for functional brain imaging. Autofluorescence imaging is especially useful for observing mouse cortical activities, because the skull of mice is transparent enough to visualize cortical autofluorescence via the intact skull. In the present study, we investigated neural activities in the auditory cortex of anesthetized mice with this technique. C57BL/6 mice 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 various sound stimuli at 5-20 kHz for 500 ms. Sound stimuli were applied as pure tones or amplitude-modulated (AM) tones with a sawtooth wave at 20 Hz. Cortical responses were recorded with a cooled CCD camera attached to an epifluorescence binocular microscope. Neural activities elicited by sound stimuli at 5-20 kHz exhibited a tonotopic map in the auditory cortex. Although AM sounds modulated by a fast-rise sawtooth wave and a slow-rise sawtooth wave have the same spectral patterns, the neural activities elicited by the former were larger than those elicited by the latter, indicating neural activities in the auditory cortex are strongly influenced by the temporal profile of sound stimuli. [Jpn J Physiol 55 Suppl:S166 (2005)]

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Fish retinal ganglion cells can survive and regrow their axons after optic nerve transection. The regenerating optic axons reach to the optic tectum 1 month and thereafter the visual function recovers 3-4 months after axotomy. Therefore, we followed up spike activities of ganglion cells in the carp retina during optic nerve regeneration. The spike activity of ganglion cells (both types of ON and OFF cells) suddenly declined by less than 20% of the control value in frequency 5 days after nerve injury. The spike frequency at 50 days after axotomy was 50% of the control and it was recovered to control level at 100 days after axotomy. In contrast, the area-dependency of ganglion cells to various spots of central light stimuli (0.3 mm-4 mm in diameter) recovered a little bit more later. The area-dependency of ganglion cell response could never be seen 5-50 days after optic nerve transection. The normal area-dependency of ganglion cell response could be only seen just 100 days after optic nerve injury. On the other hand, horizontal cell response (S-potentials) in the carp retina did not change both in amplitude and in area dependency during this long period (0-100 days) after optic nerve transection. These results strongly indicate that the recovery of area-dependency of ganglion cells in the carp retina is just well correlated with that of visual function from the optic nerve injury and horizontal cell activities in the outer retina are not affected during this period. [Jpn J Physiol 55 Suppl:S166 (2005)]

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Olfactory cues appear to guide the Japanese toads (Bufo japonicus) to migrate to the spawning site in the breeding season, in which large responses in electoro-olfactogram (EOG) are usually observed. Olfactory receptor neurons (ORNs) in the toads collected throughout a year were labeled by antiserum to olfactory marker protein (OMP), a marker for mature ORNs. The number of ORNs started to increase at a beginning of winter torpid period (December), reached a maximum in the breeding period (mostly February) and gradually decreased during the foraging period (April–October). In the winter torpid period the ORNs with small round-shaped cell body were densely distributed in the basal layer of the olfactory epithelium. In the breeding period the ORNs with large cell body appeared in the intermediate layer, while the ORNs became sparse in the basal layer. These results suggest that the ORNs migrate to the intermediate layer to mature in the breeding period. EOG to odor stimulus (isoamyl acetate) was recorded in the toads of the breeding and foraging periods. The threshold of odor response in the breeding period was about 10 times lower than that in the foraging period. Moreover, in the breeding period maximum odor responses were elevated to the level of about 7 times as large as those in the foraging period. Higher odor sensitivities of the toads in the breeding period may be induced by the maturation of ORNs and concomitant increase in the number of those neurons. [Jpn J Physiol 55 Suppl:S167 (2005)]

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Unlike the mammals, the fish optic nerve can regenerate after nerve transection. Although the regenerating optic axons in goldfish can reach to the tectum 4-6 weeks after axotomy, the visual function restores just 4-6 months later. GAP-43 which is a 43 kDa growth associated protein, is localized in the growth cone. In order to study level and cellular localization of GAP-43 mRNA in the goldfish retina and tectum during optic nerve regeneration, we got a 639 bp cDNA fragment for goldfish GAP-43 by PCR amplification. Using this cDNA fragment, Northern blot and in situ hybridizations were performed in the goldfish retinas and tecta following optic nerve injury. In the retina, levels of GAP-43 mRNA started to increase 3-5 days, peaked 6.5-fold 10-20 days and rapidly decreased 4-fold by 30 days after nerve injury. The 4-fold increase of GAP-43 mRNA levels lasted 3-4 months and then gradually declined to the control level 5-6 months after injury. The change of GAP-43 mRNA levels was only localized in the ganglion cells. The levels of GAP-43 protein showed a similar expression pattern to that of the mRNA. In the tectum, the levels of GAP-43 protein increased 2.2 fold for a long time (20-100 days) after injury. The change of GAP-43 protein levels was localized in the tectal plexiform layers. These results strongly indicate that the time course of GAP-43 expression is well correlated with that of the recovery of fish visual function from the optic nerve injury. [Jpn J Physiol 55 Suppl:S167 (2005)]

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Unlike the mammals, fish optic nerve can regenerate after optic nerve transection. The regenerating optic axons reinnervate the tectum and finally the visual function completely restores. It is very important to quantitatively score the functional regeneration of fish visual system. We developed a computer image processing system for quantification of moving zebrafish behavior. The system was consisted of a computer and two CCD cameras with an interface board. The image of moving zebrafish in an aquarium was captured at 30 frames/sec. We applied this system to evaluate the optic nerve regeneration after nerve injury in zebrafish. Two visually guided behaviors, chasing and schooling were followed up for 100 days after axotomy. The ratio of chasing in which one fish chases the other was almost reduced in a pair of two blind fish with optic nerve transection. The recovery of chasing ratio was 80 days after axotomy. In development of zebrafish, the chasing ratio equivalent to adult fish could be seen 60 days after hatching. The schooling behavior in which many fish swim in a group was analyzed in both normal (10 fish) and mixed (9 normal fish and 1 blind fish) groups. We made a quantification to detect a different behavior of both groups. Thus, we can quantify the complex behavior of zebrafish from single to many and from embryo to adult using this system. Our image processing system is a powerful tool in the fields of neurobiology of zebrafish. [Jpn J Physiol 55 Suppl:S167 (2005)]

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A subset of rodent taste cells is known to generate action potentials in response to taste stimuli. Some of these cells express molecular markers for cell types that may transmit taste information to gustatory nerve fibers. The present study investigated the possible role of spiking taste cells on gustatory sensory transmission. Taste responses of spiking taste cells in isolated mouse fungiform taste buds were examined and compared them with those of chorda tympani (CT) nerve fibers innervating fungiform papillae. Response selectivity among four taste stimuli (NaCl, HCl, saccharin, quinine) was evaluated as the entropy value of the breadth of responsiveness of each taste cells and fibers. The mean entropy value was not significantly different between fungiform taste cells and CT fibers, indicating that the range of responsiveness of spiking taste cells may be close to that of innervating axons in mice. The cluster dendrogram obtained from data on response patterns of individual mouse fungiform taste cells to four taste stimuli resembled that of CT nerves. Proportions of taste cells predominantly responding to one of four taste stimuli were similar to those of nerve fibers. These results indicate corresponding groupings of taste cells and nerve fibers in mice. This consistency in response characteristics of taste cells and nerve fibers suggest that taste cells generating action potentials may play a major role on transmission of taste information to gustatory nerve fibers. Supported by JSPS Grants-in-Aid 15209061 (YN). [Jpn J Physiol 55 Suppl:S167 (2005)]

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Previous molecular cloning studies provided evidence that several molecules were specifically involved in the taste signal transduction. T1r3 is a component molecule of receptor which responds to sweet and umami substances. Gustducin (Ggust) is a G protein α-subunit and Trpm5 is a cation channel, both of which participate in the signal transduction for sweet, umami and bitter tastes. In the present study, taste responses of the chorda tympani nerve (CT) were compared among mice knocked out each of the taste related genes (T1r3-, Ggust- and Trpm5-KO mice). Compared to those in wild type mice, responses to sweet substances in these 3 KO mice were diminished but not fully abolished. Further analysis on the residual sweet responses using gurmarin (Gur), a inhibitor of sweet responses, revealed that in T1r3 KO mice, responses to sucrose and glycine were clearly inhibited by Gur, whereas no such Gur inhibition was observed in responses to maltose and glucose. In Trpm5 KO mice, CT responses to all sweet compounds tested were more or less inhibited by Gur. In contrast, in Ggust-KO mice no Gur inhibition on sweet responses was observed. These results suggest the possibility that Ggust may play a crucial role in the Gur-sensitive transduction pathway for sweet taste responses in mice. [Jpn J Physiol 55 Suppl:S168 (2005)]

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Clinically, it is known that chronic pain develops following soft tissue injuries without a clear nerve lesion. In this study, we tried to evaluate effects of cast limb-immobilization on the sensory and musculoskeletal system of the rat. Adult male Sprague-Dawley rats, weighting 300-400g, were used. Left hindlimb was immobilized by plaster casts for two weeks: the hip, knee and ankle being flexed in the standing position. We measured the following items bilaterally: 1. von Frey tests (VFTs) at the plantar surface and at the medial surface of the calf, 2. pressure pain threshold (PPT) of the calf using a push-pull gauge, 3. the maximum thickness of the calf to evaluate muscle atrophies, 4. the hindpaw thickness and circumference to evaluate the edema, 5. range of motion (ROM) of the hip, knee and ankle, 6. skin temperature of the hindpaws. Pain behaviors evaluated by VFTs and PPT increased bilaterally (ipsilaterally predominantly) and lasted for more than 8 weeks after cast-off. In addition, a bilateral decrease of the calf thickness and ipsilateral reduction of ROMs and increases in the circumference, thickness and skin temperature of the hindpaws were observed in the immobilization side up to 4 weeks after cast-off.It is noteworthy that a simple limb-immobilization caused pain behaviors, which lasted long after recovery various signs of peripheral tissue dysfunction. The animal model developed in this study is valuable for further investigation of chronic pain. [Jpn J Physiol 55 Suppl:S168 (2005)]

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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 pCREB as a marker of neural activity in the accumbens nucleus (ACB) and the bed nucleus of the stria terminalis including the lateral (BSTL) and the medial subdivisions (BSTM). In the ACB and the BSTL, 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 examined time-course changes in the number of cells expressing pCREB in adult male rats and female rats at proestrus. Rats were injected with saline or 2% formalin into the planter surface of the right hindpaw, and they were killed 10, 30, and 120 min after the injection. Their brains were served to immunocytochemistry and the number of cells expressing pCREB in the ACB, BSTL, and BSTM was counted. In all areas examined, the number of cells expressing pCREB in formalin-injected rats was not different from that in saline-injected rats at any time point, irrespective of the sex. It is suggested 1) that the ACB and BSTL are activated only during acute phase and not during later phases of behavioral responses to nociceptive stimuli in female rats and 2) that the ACB and BSTL are refractory to nociceptive stimuli through all phases in male rats. [Jpn J Physiol 55 Suppl:S168 (2005)]

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Cyclic nucleotides are key soluble molecules that mediate diverse functions of biological systems. They are, generally, produced by activities of G-protein-mediated enzymatic cascade. In case of olfaction, AC-cAMP system is densely and locally distributed in the ciliary membrane where olfactory signal transduction takes place. Generally, the activity and temporal dynamics of the enzyme and cytoplasmic cAMP concentration ([cAMP]i) are indispensable information. But, sensory cilia express fine structure (200 nm), which has actually limited any experimental manipulations for long period of time. Here, we show a concept to estimate [cAMP]i within such a fine structure. The activities of CN-gated membrane channels were monitored, while [cAMP]i was freely manipulated through the UV-photolysis of intracellulary-loaded caged compounds. Since in this condition production of cAMP is proportional to the intensity of light, we could obtain the relation between [cAMP]i and the membrane current. Therefore, [cAMP]i could be estimated inversely from the appeared current size. Using such logics and techniques, we monitored ligand (odorant)-activated adenylyl cyclase activities and its temporal dynamics within a fine cilia. The present study confirmed experimentally that G-protein-mediated enzymatic activity increases monotonically with time after the ligand stimulation, as has been estimated from the previous theoretical work based on the thermo-dynamical modeling, mostly done on the rod photoreceptor cell. In addition, we calculate the actual value for the cAMP production rate. [Jpn J Physiol 55 Suppl:S168 (2005)]

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We used functional fMRI with healthy human volunteers to study how the processing of odor information is influenced by preceding notification of odors. Seventeen right-handed volunteers with normal olfactory function estimated by T&T olfactometry participated in the study. At 4l/min pressurized clean air was directed continuously to plastic tubings connected to the mask on the nose with an aspiration tubing. Seven odor exposures using 13.5% of isovaleric acid at the highest scale of olfactometer was delivered for 8 seconds each with 32 seconds interruption by sorenoid-operated valves. Images were acquired by 1.5-T MRI. The imaging data were analyzed using FEAT. Significant acitivation was found in the cingulated gyrus, hippocampus, thalamus, external orbito-frontal (eOFC) and entorhinal (Ecx) cortices well-known as the brain regions involved in olfactory processing, when subjects were stimulated without notification. Only 1 subject (1 male and 8 female; age mean = 24) recognized the odor unpleasant. Seven subjects (4 male and 3 female; age mean = 23) notified of unpleasant odor before scanning, however, showed significant activation in the insula, the globus pallidus, eOFC, and ECx. All subjects showed unpleasantness to the odor. These results suggest that notification facilitates olfactory processing by activation of the brain region involved in emotion. [Jpn J Physiol 55 Suppl:S169 (2005)]

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Suprachoroidal-transretinal stimulation (STS), which we have developed as a less invasive electrical stimulating method for an artificial retina, was reported to elicit well localized field responses in the superior colliculus of retinal dystrophic rats (Kanda et al., 2004). In this study, we analyzed the spatial properties of STS evoked single-unit activities, which were recorded from the relay cells of the lateral geniculate nucleus (LGN) in anesthetized cats. A stimulating platinum electrode (diameter: 100 μm) was placed on the sclera with local lamellar resection, and a platinum wire (diameter: 0.2 mm) was inserted into the vitreous as the reference electrode. We obtained sixty-three single-unit responses of LGN relay cells to a biphasic pulse (duration: 0.5 ms) of STS. The latencies of their initial responses were ranged from 5-15 ms. The response probability of each unit depended on both stimulus intensities and the distance (D) between the retinal position of the receptive field and the stimulating electrode. With 200 μA of stimulus intensity, the response probabilities of the units were over 80% in the case of D less than one degree in visual angle, decreasing drastically to 10% more than three degrees. Thus, it is suggested that STS can stimulate the retina focally and be suitable for artificial retinas. [Jpn J Physiol 55 Suppl:S169 (2005)]

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The retinal ganglion cells(RGCs) of adult rat fail to regenerate and become apoptotic cell death after optic nerve injury. On the contrary, fish RGCs can survive and regenerate their axons after injury. In our previous study, we compared rat and goldfish RGCs after optic nerve injury with respect to the cell death and survival signals. A survival signal, Akt and a pro-apoptotic Bad were rapidly phosphorylated 6-8 fold by 3-5 days after goldfish optic nerve injury. Subsequently, an anti-apoptotic Bcl-2 was increased thereby maintaining the optic nerve regeneration. Furthermore, inhibition of the PI3K by wortmannin dose-dependently suppress neurite outgrowth in culture system. On the other hand, Akt and Bad phosphorylation levels in the rat retina were rapidly decreased 2-3 days after optic nerve injury. It is known that insulin-like growth factor I (IGF-I) is an upper stream growth factors of PI3K/Akt pathways and induces differentiation neural retina and inhibits caspase-3-dependent apoptosis in rat retina. In the present study, IGF-I induced neurite outgrowth and the effect was inhibited by wortmannin in the adult rat retina. To determine the link of IGF-I to PI3K/Akt system, we are now in progress to measure the levels of IGF-I in the goldfish and rat retinas before and after optic nerve transection. [Jpn J Physiol 55 Suppl:S169 (2005)]

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AII amacrine cells (AIIs) play a crucial role in the rod pathway of the mammalian retina: they receive glutamatergic input from rod bipolar cells (RBCs), and also send sign-conserving output at distal dendrites via gap junctions and glycinergic sign-inverting output at proximal dendrites. AIIs generate TTX-sensitive spikes. To investigate functional role of the spike, we performed whole-cell patch clamping of AIIs in the mouse retinal slice preparation. Local application of glutamate to the dendrites of AIIs increased spike frequency in dose-dependent manner. When depolarizing current was injected, repetitive spikes were evoked. Frequency of the spike was dependent on intensity of the injected current. Normalized spike frequency was plotted against the mean membrane potential (MMP) for both glutamate application and current injection experiments. Data were fitted with Boltzmann function to estimate V_f1/2 (MMP at which spike frequency was the half-maximum). V_f1/2 for current injection was similar to that for local application of glutamate to the dendrites (about -50 mV). Thus, it is likely that spike frequency of AII is dependent on excitatory glutamatergic input from RBCs. To examine the localization of voltage-gated Na⁺ channels (Na_vs) within AII, 1 μM TTX was applied. Application of TTX to the proximal dendrite and the soma blocked Na⁺ current more effectively than that to the distal dendrite, indicating that Na_vs mainly localize around the soma. Localization of Na_vs at the soma and the proximal dendrites may be important for effective release of glycine in signal processing. [Jpn J Physiol 55 Suppl:S169 (2005)]

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Although spinal anesthesia using local anesthetics has been often conducted in surgery of the leg and lower abdomen, the mechanism of their pharmacological action in the neuronal network of the dorsal horn has not been fully understood. We intended to analyze the effects of local anesthetics on the local neural circuitry of the dorsal horn. Because an optical recording technique using a voltage-sensitive dye is suitable for the analysis of multi-point neural activities, we investigated the effects of lidocaine and bupivacaine on the dorsal horn neuronal network function. Spinal cord slices of juvenile rats were stained with a voltage-sensitive dye. Neural activity in the dorsal horn elicited by electrical stimulation of the dorsal roots were analyzed using a high-speed optical recording system. Both lidocaine and bupivacaine suppressed neural excitability in a dose-dependent manner with preferential depression of post-synaptic activity. These pharmacological actions were topographically different within the dorsal horn. The depressant effect of bupivacaine was greater and more long-lasting than that of lidocaine. We conclude that synaptic blockade is the major action of local anesthetics in the neuronal network of the dorsal horn in vitro. [Jpn J Physiol 55 Suppl:S170 (2005)]

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While the relation of convergence eye movement and lens accommodation is well established, the roles played by pupillary near reflex in near response are controversial. We tested this contribution by analyzing eye and pupil movements evoked by ordinary and three-dimensional (2D and 3D) movies with known disparity and luminosity. Eye and pupil movements in both eyes were continuously monitored by a newly-developed video-based dual eye/pupil meter. Ten subjects watched 2D/3D movies in dark room (10 lux) with polarized glasses. The movies were presented on an 80 inch screen by two liquid crystalline projectors with polarized filters to provide binocular disparity field-sequentially. Pupillary responses while watching 2D and 3D movies were almost coherent. Then pupillary reflexes were mainly dependent on change in the luminance of the movies (pupillary light reflex). However, small 2D/3D differences were still detected. Convergence eye movements, measured as the difference between right and left eye while subjects were watching 3D movie, were dependent on the magnitude of the disparities, although small movements were still elicited by 2D movies. Based on analyses of the relation among small differences in pupil between 2D/3D conditions, convergence movements, and luminosity and disparity of the image, we discuss the roles of the pupillary near reflex in the near response. [Jpn J Physiol 55 Suppl:S170 (2005)]

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Spectral envelopes are important for vowel recognition. Psychophysical studies have previously showed that the sharpness of formants can contribute to the vowel quality. However, it is unclear how cortical neurons process this parameter. In this study we investigated this issue in primary auditory cortex (A1) neurons of awake cats. Stimuli were the multi-frequency tones with a spectral prominence. The spectral peak frequency was always located at the cell’s best frequency, while the prominence-amplitude was shifted systematically. Pure and two tone stimuli were used to delineate excitatory and inhibitory subfields of the frequency response field (FRF). The cell’s responses to different prominence-amplitudes were investigated with reference to the spectral location of the prominence relative to cell’s FRF. We found that A1 cells showed preference of higher prominence-amplitude when the sound energy was on a portion of FRF including moderate inhibitory subfield, while the same A1 cell could show preference of lower prominence-amplitude when the sound energy was on a portion of FRF including negligible inhibitory subfield. The results suggest that A1 can discriminate the sharp-prominence from the dull-prominence when the prominences have the same spectral peak frequency; and the prominence preference is originated from the shift of balance of the spectral excitation and inhibition. The present study has provided physiological data supporting the interpretation that the vowel perception is processed in A1 by analysis of the spectral shape cues. [Jpn J Physiol 55 Suppl:S170 (2005)]

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Neurons in the cochlear nucleus (CN) receive primary auditory signals from the spiral ganglion neurons in the inner ear and transmit secondary signals to the higher auditory neurons in the medulla and thalamus. Little is known about the mechanisms how the CN neurons grow their fibers and find the higher order neurons during development. We report here that growth of fibers from the CN neurons changed dependence on BDNF in a few days after the birth. CNs were dissected from embryonic and newborn rats and cultured them in Neurobasal Medium (Gibco). Monolayer,culture of embryonic rat CN neurons represented two types of neurons, large long-axon neurons and small bipolar ones. Neurotrophins (NGF, BDNF or NT3) caused little changes in the growth of these neurons. Explants of small sections from embryonic CNs in collagen matrix extended thousands of thin fibers without addition of neurotrophins in the growth medium. By contrast, explants of CNs from day5 and day8 extended fibers in a BDNF-dependent manner. Only short fibers were grown when NGF or NT3 were added to the medium. Altogether, CN neurons changed their neurotrophin dependence, from no-dependence to BDNF-dependence, in a short period after the birth. [Jpn J Physiol 55 Suppl:S170 (2005)]

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Sensitization in spinal cord is believed to underlie the induction of abnormal pain sensitivity. Recently, purinergic receptors are shown to play important roles for hyperalgesia and allodynia. By optical recording of neuronal excitation in rat spinal cord slices with voltage dye, we analyzed actions of purinergic receptor agonists in the superficial dorsal horn. Excitation evoked by dorsal root stimulation of C fiber-activating strength was facilitated after the application of a P2X receptor agonist abmATP and a P2Y receptor agonist UTP. The long-term potentiation by abmATP was significantly inhibited by glial metabolism inhibitor monofluoro-acetic acid, evidence suggesting that abmATP acts via glial cells. In contrast, UTP facilitated presynaptic excitation recorded by anterograde labeling of primary afferent fibers with voltage dye. The facilitation was not observed in the presence of D-AP5 and CNQX, where excitatory synaptic transmission from afferents was blocked. UTP therefore acts on presynaptic elements via postsynaptic neurons. These results indicate that P2X and P2Y receptors sensitize pain pathway in the dorsal horn by acting two distinct mechanisms. Supported by the Grant-in-Aid from JSPS and a grant from Yazaki Memorial Foundation to KM. [Jpn J Physiol 55 Suppl:S171 (2005)]

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In WGA-HRP tracing study of goldfish optic nerve, the majority of regenerating axons arrived at the tectum 3-5 weeks after optic nerve transection. On the other hand, it is unknown when the centrifugal fibers from nervus terminalis reinnervate to the retina after axotomy. To investigate the recovery of these efferent fibers, we used goldfish and zebrafish visual system by immunohistochemical and WGA-HRP tracing studies. The efferent fibers express FMRF amide (Phe-Met-Arg-Phe-NH₂ ) that is a molluscan cardioexcitatory peptide, and gonadotropin-releasing hormone (GnRH). In the control retina, FMRF amide and GnRH immunoreactivity were colocalized in the inner nuclear and the nerve fiber layer. In the optic nerve, their positive staining could be sparsely seen a dotted-like appearance. After optic nerve transection, the immunoreactivity of FMRF amide and GnRH in the retina disappeared by 20-30 days, while their expression gradually recovered 40-50 days after axotomy. This regeneration process of efferent fibers was roughly correlated with the regeneration time of afferent fibers of optic nerve. We also studied the efferent fibers of optic nerve in various animal species. [Jpn J Physiol 55 Suppl:S171 (2005)]

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To investigate the relationship between the degree of masticatory efficiency and the physical properties of a swallowing food bolus, we measured the texture of a food bolus immediately prior to swallowing during mastication of test food. Eighteen adult volunteers participated. Masticatory efficiency of each participant was obtained using the sieving test reported by Manly and Braley (1950), and they were divided into two groups (high efficiency group, H group; low efficiency group, L group). Each participant masticated three types of test food (rice cake, RC; peanuts, P; soft biscuit, SB) prepared in 5g units. The texture of a food bolus collected from the oral cavity immediately prior to swallowing was measured by texture profile analysis. Three texture parameters (hardness, adhesiveness, cohesiveness) were obtained from the stress strain curve. Hardness of all three types of food bolus in the L group was significantly higher than that in the H group. Adhesiveness of the P bolus in the L group was significantly (p<0.05) lower than that in the H group. In contrast, adhesiveness of the RC bolus in the L group was significantly (p<0.05) higher than that in the H group. On the other hand, adhesiveness and cohesiveness of the SB bolus did not differ significantly between the two groups. These results suggest that the degree of masticatory efficiency may clearly influence the physical properties of a food bolus at the initiation of swallowing during mastication of solid food. [Jpn J Physiol 55 Suppl:S171 (2005)]

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Sensory input via the pharyngeal branch of the glossopharyngeal nerve (GPN-ph), which innervates the pharyngeal region and the superior laryngeal nerve (SLN), which innervates the laryngeal region are very important for eliciting reflex swallowing. Our previous study found that nitric oxide (NO) was involved in reflex swallowing from the pharynx. It is known that NO is produced by the activation of NMDA receptor. In this study, we investigated whether NMDA-NO pathways modulate initiation of reflex swallowing from the pharyngo-laryngeal region. Urethan-anesthetized rats were used. To evoke swallowing, the GPN-ph and the SLN were stimulated by repetitive electrical stimulation (10-20 μA, 10-20 Hz, 1.0 ms). The latency for the first swallow and the time interval between swallows were measured. L-NNA, a non-selective inhibitor of NO synthase (NOS), and 7-nitroindazole, a selective inhibitor of neural NOS extremely reduced occurrence of swallowing evoked by the GPN-ph, whereas swallowing evoked by the SLN was little changed. Furthermore, MK-801, an antagonist of NMDA receptor, suppressed reflex swallowing evoked by the GPN-ph and by the SLN. NMDA restored the suppression of reflex swallowing by MK-801. These results suggest that NMDA-NO pathways are involved in the neural mechanism of initiation of reflex swallowing from the pharynx. [Jpn J Physiol 55 Suppl:S172 (2005)]

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Previous studies have shown that the jaw-opening reflex (JOR) is modulated during mastication. However in these studies, analyses have been carried out for limited period within the masticatory sequence (i.e. from food intake to just before swallow). Our aim is to study if the modulatory pattern of the JOR is consistent throughout the masticatory sequence in awake rabbits. The masticatory sequence was divided into three masticatory periods (preparatory, rhythmic-chewing and preswallow periods) based on the jaw movement patterns and the activity pattern of the jaw muscles. Electromyographic activity of the jaw-opening muscle (Digastric) and the jaw movement trajectories were recorded. Repetitive electrical stimulation (single pulse, 0.2 ms duration at a rate of 1 Hz, 1.2 times the threshold) of the inferior alveolar nerve was conducted before (for control response) and during mastication to evoke the JOR. The amplitude of the JOR was normalized to the control response and the modulatory pattern of the JOR was compared among the masticatory periods. The JOR was generally suppressed during the rhythmic-chewing and the preswallow periods; the suppressive effect was phasically weakened in the middle part of the jaw opening during the rhythmic-chewing period. On the other hand, the reflex was not only suppressed but also facilitated during the preparatory period. The results suggest that the modulatory pattern of the jaw-opening reflex is different among the masticatory periods. [Jpn J Physiol 55 Suppl:S172 (2005)]

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The locomotory behavior of Caenorhabditis elegans consists of four simple events, forward and backward movements, omega-shaped turns and rests. The wide variety of behaviors of this worm is achieved through a combination of these simple locomotions. To gain insight into the neuronal mechanisms regulating this locomotion, we analyzed the locomotory behavior of C. elegans over a long time period. In this study, we revealed the existence of at least two distinct behavioral states–pivoting and traveling–in the forward locomotion of C. elegans in the absence of food. Pivoting is characterized by pronounced directional switching and resulting in short-duration forward movement, whereas in the traveling state, forward movement is of longer duration. Pivoting has occurred when we transferred a well-fed worm to unseeded plate, and then transition to traveling has taken place, successively. We examined the function of chemosensory neurons in this behavioral alteration by laser-ablation. Duration of forward movement was shortened by the loss of any one of the six chemosensory neurons (AWA, ASE, ADF, ASH, ASI, and ADL). On the other hand, loss of AWC, ASK or AFD chemosensory neurons lead extension of duration of forward movement. Similar to the case for chemosensory neurons, ablation of AIA, AIY and AIB, AIZ interneuron resulted in short- and long- duration forward movement, respectively. These results suggested that C. elegans regulate their locomotion using multiple chemosensory and interneurons having opposed functions. [Jpn J Physiol 55 Suppl:S172 (2005)]

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It is well known that head moves rhythmically and coordinately with mandible during masticatory movement. The present study investigated whether the head-neck behavior can also be influenced by the jaw movement during the production of voice. Using a video-scanning method, the head-neck and jaw motions were analyzed in 16 healthy adults phonating two types of syllables, /ata/ and /aka/ in Japanese. The subject was asked to phonate 10 times in each syllable following after the sample voice. The order of syllables was randomized. The voice via a microphone was converted into LED signals and recorded with the motion signals from the LEDs placed around the facial and neck regions. The head-neck movement was divided into two hinge movements, the head movement around an atlanto-occipital joint and the neck movement around a cervical-spinal joint, respectively. The head extension and the neck flexion synchronized for the jaw opening were observed in all subjects. The motion wave of 500 ms period before the starting point of the vocal signal was selected and averaged 10 times for analysis. The amounts of movement were compared between the phonation of /ta/ and /ka/. There was a higher degree of correlation between the head extension and the jaw opening than that between the neck flexion and the jaw opening. The neck flexion and the jaw opening were started at about the same time, while the head extension was always later than these motions. From these findings, it was supposed that the head behavior was closely related to the jaw movement, while the neck did not depend on it during voice production. [Jpn J Physiol 55 Suppl:S172 (2005)]

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Our recent study has shown that simian saccadic adaptation is accelerated by preceding adaptation and that this facilitatory effect is nullified by repetition of zero-error movements. This suggests that a memory of learning is erased by such movements. In the present study, we examined effects of zero-error movements on the metrics of saccades. Using intrasaccadic target step (ISS) paradigm, we decreased the gain of saccades to 10 deg target steps in one horizontal direction (1st adaptation). We reversed ISS to bring the gain back to ∼1.0 (recovery), after which we required the animal to make zero-error saccades for 30 min (zero-error block). Then we reversed ISS again to induce another gain decrease (2nd adaptation). Saccades with sizes of 9.1-10.0 deg were sorted into 0.1 deg bins, and the duration and peak velocity of saccades having a similar amplitude were compared. For a given amplitude, the duration decreased during 1st adaptation and was still shorter in the early zero-error block than in preadaptation block. However, by the late zero-error block, the duration returned to a preadaptation level. Duration then decreased in subsequent 2nd adaptation as in 1st adaptation. Correspondingly, the peak velocity showed opposite changes. Saccades in non-adapted direction did not exhibit similar changes. Results are consistent with our previous suggestion that repetition of zero-error movements erases the memory of preceding learning and returns the saccadic system to a preadaptation state. [Jpn J Physiol 55 Suppl:S173 (2005)]

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Tongue movements participate in oro-facial motor functions including chewing, sucking, swallowing, speech and respiration. During mastication and swallowing, tongue plays an important role of squeezing foods to make food bolus, and transporting them from the oral cavity to the pharynx. However, up to now our knowledge as to the functional role of tongue is still limited due to the technical difficulty to record the dynamic activity of tongue muscles. To this end, we recorded EMG activity of extrinsic tongue muscle in healthy subjects. In addition, tongue pressure against the hard palate as well as EMG activity in the submental muscle and laryngeal movements were also recorded. The subject was instructed to swallow test foods or water with different hardness. The same tasks were also tested in different postures which were upright and body reclined at 30 degrees. Patterns of tongue muscle activity and tongue pressure varied among the test foods and postures. When the subject swallowed hard food, tongue muscle activity and pressure were larger and the duration was longer as compared with soft food or water swallowing. When compared among the postures, the amplitudes tended to be larger as the body reclined. Contrary to these changes, the submental muscle activity hardly changed regardless of conditions. The results suggest that human tongue activity may be modulated by the peripheral inputs and/or subject’s posture during swallowing function. [Jpn J Physiol 55 Suppl:S173 (2005)]

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Motoneurons (MNs) are the output neurons from the central nervous system and their activities cause muscles to contract. In addition to their peripheral axons MNs have central collaterals that contact Renshaw cells (RCs) that provide recurrent inhibition to MNs. It has been a general notion that acetylcholine is the only transmitter released from MN synapses both peripherally and centrally. In this study, we re-examined the physiological nature of the MN-RC synapse using glutamic acid decarboxylase-green fluorescence protein (GAD67-GFP) knock-in mouse neonates (Tamamaki et al. J Comp Neurol (2003) 467:60-79). We performed whole cell recordings from visually identified GABAergic neurons in the lumbar ventral horn using isolated spinal cord preparations. Patched neurons were identified as RCs as electrical stimulation of the ventral root evoked short latency (<5 ms) excitatory postsynaptic currents (EPSCs). These EPSCs were reduced to about 35% of control by blocking nicotinic receptors with mecamylamine or d-tubocurarine. Antagonists for ionotropic glutamate receptors, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and D-(-)-2-amino-5-phosphonopentanoic acid (AP5), further reduced the EPSC to 10% of control. Furthermore, by using immunohistochemical methods, we found vesicular glutamate transporter 2 (VGLUT2) colocalized with MN axon terminals. These results indicate that mammalian spinal MNs, in addition to acetylcholine, co-release glutamate to excite RCs. [Jpn J Physiol 55 Suppl:S173 (2005)]

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Introduction: The purpose of this study is to clarify the projection to forelimb motoneurones from medulla oblongata in adult mice.Methods: The results were obtained from 15 mice (BALB/C) with age of 2-4 months. The animals were anaesthetized with a mixture of midazolam and fentanyl. Corticofugal fibres were stimulated in the ipsi- and contralateral pyramids at 0.5mm rostral to the obex. Results: The termination of the corticospinal fibres in C7 was assessed by recording the extracellular field potentials. The area with negative field potentials is covered the dorsal horn, lamina V, lamina VI and the medial part of lamina VII. Intracellular recording have been made from 30 forelimb motoneurones in the C6-C7 segments. The pyramidal EPSPs had latencies of 3.5 to 5.8 ms, which suggest minimally a trisynaptic linkage. Small pyramidal EPSPs could still be evoked after corticospinal transection in the rostral C2 segment. The EPSP amplitude increased almost twice 0.5 to 1.0 mm dorsal to the pyramid (the reticular formation) and the latency shortened to 2.3 ms. In the contralateral MLF, about 1.5 mm dorsal to the pyramid, the EPSP amplitude remained rather unchanged, but the latency decreased to 1.3 ms. Discussion: The major termination of the corticospinal fibres in the mouse is in the dorsal horn and medially in the intermediate layers, but not laterally in the intermediate layers and not in the motor nuclei. A reticulospinal pathway may provide for the fast and strong excitatory input to forelimb motoneurones in the mouse. [Jpn J Physiol 55 Suppl:S173 (2005)]

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We have developed internal capsule (IC) hemorrhage model rats by injection of collagenase (1.4 μl of 7.5 U/ml in saline) into the striatum near the IC, which had a small hematoma but relatively bigger motor dysfunction caused by rapid degeneration of myelin and axon in the IC within the first 24 hours after the lesion. To investigate whether iron was involved in pathological mechanism in early-phase after intracerebral hemorrhage, an iron chelator, clioquinol (CQ: 100 mg/kg/day orally) was daily administrated to IC model rats from 7 days before (pre-treatment) or from 6 hours after (post-treatment) the lesion to 7 days after the lesion. Behavioral test showed that pre-treatment of CQ significantly reduced the motor dysfunction 7 days after the lesion as compared with vehicle-treatment. However, post-treatment of CQ have not a significance but a tendency to reduce the motor dysfunction after the lesion. Retrograde labeling of neurons in sensorimotor cortex at 14 days after the lesion by Fluoro-Gold injection (corticospinal tract at C3-C4 level) showed that there were more labeled neurons in pre-treatment group (n=4; 57.2 ± 16.5% of contralateral side, 0.7 mm anterior to the bregma) compared to those in vehicle-treatment group (n=6; 14.7 ± 12.7%). Data suggested that inhibition of iron accumulation by clioquinol treatment attenuated motor dysfunction by protecting the damage of corticospinal tract after ICH, probably mediated by reduction of hydroxyl radical from catalytic chain reaction by iron. [Jpn J Physiol 55 Suppl:S174 (2005)]

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