TRPV2 (transient receptor potential channel vanilloid subfamily 2) was shown to receive noxious thermal stimuli more than 52°C, and to be expressed in a subgroup of myelinated sensory afferents. However, TRPV2 mRNA signals have been detected in many peripheral tissues of the rat in subsequent studies. We further investigated the expression of TRPV2 in rat peripheral nervous system by immunohistochemistry and transcriptase-polymerase chain reaction (RT-PCR). TRPV2 immunoreactive neurons seen in dorsal root ganglion (DRG) did not contain Parvalbumin, which is included in proprioceptive sensory neurons, and fluoro gold, a retrograde tracer, injected into dorsal column nuclei. TRPV2 immunoreactive neurons were also observed in nodose ganglion (vagal sensory neurons) and superior cervical ganglion (postganglionic sympathetic neurons), but these neurons were rare. On the other hand, many TRPV2 immunoreactive neurons were seen in intermuscular (Auerbach's) and submucosal (Meissner's) plexus of the intestine. Some of these neurons were positive to Calbindin D-28k, a marker of intrinsic afferent neurons in enteric nervous system. An analysis by RT-PCR also showed a gene expression of TRPV2 in the intestine. These findings show that TRPV2 is expressed not only in somatic and visceral afferent neurons, but also in enteric afferent and efferent neurons. [Jpn J Physiol 54 Suppl:S170 (2004)]

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It is possible that nociceptive stimuli evoke neurotransmitter release not only from the primary afferent terminals in the dorsal horn but also from the peripheral terminals via axon reflex. Recent studies have shown the presence of glutamate receptors on peripheral terminals of nociceptors. Thus, evoked pain sensation and axon reflex was evaluated in order to investigate whether the subcutaneous injection of glutamate excites the nociceptors.

Sodium glutamate was injected subcutaneously into the forearms of healthy adults who gave their informed consent prior to participation. Glutamate caused severe pain immediately after the injection and the peak pain was observed within 5 minutes. Although the intensity of pain expressed by VAS has gradually decreased, slight pain was maintained for 30 minutes. The skin temperature monitored by thermography started to increase about 3 min after injection, reached a peak in 10-20 min and then decayed gradually. The area of the temperature-increase almost disappeared in about one hour.

Sodium glutamate injection into the hindpaw of the pentobarbital-anesthetized rats also produced a significant skin temperature increase. Subcutaneous glutamate also produced extravasation and paw edema. The volume of edema remained unchanged for more than one hour.

These data demonstrate that subcutaneous injection of glutamate evokes pain sensation and axon reflex. It is suggested that peripheral glutamate would create a vicious circle where pain is maintained if glutamate is released from peripheral terminals via axon reflex following nociceptive stimulus. [Jpn J Physiol 54 Suppl:S170 (2004)]

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Enhanced excitation of primary afferent neuron is well known in peripheral nerve injury. On the other hand, pathomechanisms of lumbar radiculopathy is still unclear. Therefore, we investigated the electrical properties of dorsal root ganglion neurons in lumbar radiculopathy rat in use of whole-cell patch clamp recordings. Left L5 spinal root of male Sprague-Dawley rats was ligated tightly in lumbar root constriction group. To compare the excitability of DRG neurons from root constriction and sham operated rats, we recorded action potentials of the neurons. Repetitive discharge of action potential was observed by application of long pulse of current injection (1000ms). Root constriction group exhibited decreased threshold current, more depolarized resting membrane potential, and longer duration of action potential. Most of sham group DRG neurons (85%) exhibited single spike by the long current injection. In contrast, 60% of root constriction group DRG neurons exhibited multiple spike pattern. The incidence of multiple spike pattern was significantly higher in root constriction neurons than in sham neurons. Enhanced excitation of DRG neurons observed in the present study was similar to that in peripheral nerve injury model. [Jpn J Physiol 54 Suppl:S170 (2004)]

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Many clinical studies have indicated the usefulness of Neurometer in the quantification of nerve dysfunction in patients. It is reported that the three sine-wave pulses at 2000-, 250-, and 5-Hz produced by the device selectively stimulate Aβ-, Aδ- and C afferent fibers, respectively. However, there are few electrophysiological studies showing the selectivity. Intracellular recordings were made from dorsal root ganglion (DRG) neurons and action potentials (APs) elicited by the sine-wave pulse were analyzed. L4-6 DRGs were isolated from 7-8-week-old rats together with a proximal dorsal root having a length of 7-12 mm. Antidromic stimulation at the central end of the dorsal root was performed with a suction electrode to classify recorded neurons into one of three groups, Aβ-, Aδ-and C-type, on the basis of a combination of values of the axonal conduction velocity, duration of AP and threshold stimulus intensity. After the identification of cell type, the dorsal root was stimulated with Neurometer. Aβ-fibers recorded were activated by all the three sine-wave stimuli. Ad-fibers were excited by 250-Hz and 5-Hz stimuli. The frequencies of APs elicited in these A fibers were higher at 250-Hz than other stimuli. On the other hand, C-fibers were only activated by 5-Hz stimuli. These results indicate that Neurometer makes possible to examine the selective activation of the peripheral nerves that differ in diameter. The present study in DRG may provide a basic knowledge for evaluating an effect of analgesic drugs and a change in pain sensation in human. [Jpn J Physiol 54 Suppl:S171 (2004)]

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Clinical muscle tenderness such as stiff neck and lumbago is often treated with massage, stretch, or vibration; however, the effectiveness of these procedures and their action mechanisms remain unclear. In the present experiment, we evaluated the effects of vibration stimulation on pain in exercise-induced muscle soreness (DOMS) model. The subjects were 13 healthy volunteers (9 males and 4 females), with ages ranging from 19 to 24 years (mean 20.9 years). The non-dominant upper arm was exercised with a load (2 kg for women and 3 kg for men) until exhaustion, and 3 sets with the same load were done with a 5 min rest period between sets. Eight subjects received vibration at 25 Hz with amplitude of 8.0 mm to exercised muscle (biceps brachii) 1 day after exercise and another were served as control. The present exercise protocol induced DOMS 1 day after exercise. Pain magnitude expressed in numerical rating scale (NRS) was increased upon the active movement of the arm and in the full extension position 1 day after exercise, but not increased in the full flexion position. Vibration given 1 day after exercise decreased NRS in full extension position only (p<0.01). Pressure pain threshold measured by a push-pull gauge was decreased 1 day after exercise but not changed by vibration. These results suggest that DOMS is composed of several factors and vibration can modify only a part of it, and that through peripheral mechanism. [Jpn J Physiol 54 Suppl:S171 (2004)]

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The effect of local ischemia on the development of muscle pain produced by eccentric exercise was examined in lightly anesthetized rats. A total of 16 rats were allocated to the exercise group (EG) and the ischemic exercise group (IG). For the ischemia, the femoral artery and vein were tied with a thin thread before the exercise. Repetitive eccentric exercise was produced by manual extension of the gastrocnemius (GS) muscle during tetanic contraction elicited by electrical stimulation of the tibial nerve. The development of muscle pain was determined by mechanical withdrawal thresholds (MWT) by Randall-Selitto test to the GS muscle. The focal electrical stimulation (constant current pulses of 1ms in duration) of the GS muscle was successively stimulated at C-fiber strength (10 train pulses, interval: 0.1-1Hz) and flexion reflex activity was recorded from the biceps femoralis muscle. In the EG, the MWT was the lowest value on 1st day after the exercise and then increased gradually to the baseline level on 7th day. On the other hand, in the IG, the MWT was the lowest value on 2nd day after the exercise and then increased gradually to the baseline level on 14th day. The wind-up, the increment of C-fiber responses to the successive stimuli, at 0.5 or 1Hz was only observed on 2nd day after the exercise in the IG. In the present study, the local ischemia elongated the muscle pain until 14th day after the exercise and induced the wind-up phenomena on 2nd day after the exercise. These results suggest that the local ischemia might be a possible cause of development of the chronic muscle pain. [Jpn J Physiol 54 Suppl:S171 (2004)]

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The present study was designed to elucidate the effect of chronic inflammation on spinal dorsal horn nociceptive neurons with advancing age. Foot temperature, thickness and dorsal horn neuronal activities were studied in the aged and adult rats with complete Freund's adjuvant (CFA) inflammation. The changes of foot temperature and thickness were much higher and longer lasting in aged rats than those of adult following CFA injection. One hundred fifty nociceptive neurons were analyzed in the aged (29-34 month old) and adult (9-12 month old) rats. Nociceptive neurons were classified as wide dynamic range (WDR) and nociceptive specific (NS) neurons according to their responsibility to mechanical stimulation of the receptive fields. One hundred twenty four WDR neurons (aged: 59, adult: 65) and 26 NS neurons (aged: 13, adult: 13) were identified. NS neurons from the inflamed adult rats showed significantly higher responses to noxious stimulation. On the other hand, WDR neurons from both inflamed adult and aged rats and NS neurons from the inflamed aged rats did not show higher responses to mechanical stimulation. Background activity of WDR neurons from the adult rats was significantly higher than naive, whereas that of aged rats and NS neurons were not. The afterdischarge followed by noxious mechanical stimulation was significantly larger in WDR neurons in both adult and aged rats, whereas no significant differences were observed in NS neurons. These suggest that chronic peripheral inflammation induces differential effects on pain perception in aged and adult rats. [Jpn J Physiol 54 Suppl:S171 (2004)]

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To study the functional patterns of spinal projections of primary afferent fibers, EPSCs and IPSCs evoked by the electrical stimulation of single dorsal root were examined in the substantia gelationosa (SG) neurons at the lumbar segmental level in horizontal slices of the adult rat spinal cords using a blind whole-cell patch-clamp technique. In voltage-clamp mode, most of the EPSCs elicited by L5 dorsal root stimulation (DRS) were recorded from L3 to L6. SG neurons in L4 and L5 received Aδor C fiber-mediated EPSCs, or both, whereas the majority of SG neurons in L3 and L6 received only C fiber-mediated EPSCs (11/14; 78% in L3 and 13/18; 72% in L6). Aδ- and C-fibre-mediated IPSCs were recorded from L2 to S1. In current-clamp mode, L5 DRS evoked EPSPs and IPSPs, some of which were accompanied by action potentials (APs). The Aδ-fiber-mediated APs were almost limited within L5 level (40% of SG neurones showed AP in L5, and 5% in L4), while the C-fiber-mediated APs were distributed over two spinal cord segments from L5 to L4 (40% in L5, and 35% in L4). Furthermore, the spreaded inhibitory inputs were considered to be functional because the L2-DRS-evoked APs were suppressed by L5-DRS-evoked IPSPs in 45% of the L2 SG neurons tested. These findings suggest that the excitatory projection field of C fibers spreads more rosrocaudally than that of Aδ fibers and the differences between excitatory and inhibitory projection field extent in the SG may be related to the inhibitory modulation of nociceptive transmission. [Jpn J Physiol 54 Suppl:S172 (2004)]

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The potential role of nicotinic acetylcholine receptors (nAChR) in modulating numerous physiological actions has been recently elucidated. The nAChRs are considered as novel therapeutic targets for learning, development and aging as well as analgesia. However, the antinociceptive action of nAChRs in spinal cord is yet to be confirmed. In order to determine which subtypes of nAChRs are involved in antinociception, we examined the roles of nAChRs in modulating inhibitory synaptic transmission in dorsal horn neurons by using patch-clamp recordings from spinal cord slice preparations. Bath application of nicotine enhanced GABAergic/glycinergic IPSCs in both superficial and deep spinal dorsal horn neurons. When β4 nAChRs agonist cyticine was applied, GABAergic/glycinergic IPSCs were significantly increased in superficial dorsal horn neurons, but failed to be increased in deep dorsal horn neurons. On the other hand, when we applied α4β2 nAChRs agonist RJR-2403 or α7 nAChRs agonist Choline, IPSCs were not increased in superficial dorsal horn neurons, but largely increased in deep dorsal horn neurons. These results indicate that distinct subtypes of nAChRs are expressed in inhibitory interneurons of spinal dorsal horn and facilitate GABA/glycine release. These specific nAChRs expression in spinal inhibitory interneurons may contribute to the unserstanding of the mechanism of nicotine-induced antinociception. [Jpn J Physiol 54 Suppl:S172 (2004)]

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[Objectives] The aim of the present study was to elucidate the functional role of the paratrigeminal nucleus (Pa5) neurons in trigeminal pain and autonomic system.
[Methods] <Immunohistochemical study> The left vagal nerve was transected and Complete Freund's adjuvant (CFA) was injection into the left temporlomandibular joint (TMJ) at 2 days after injection of Fluorogold (FG) into the left parabrachial nucleus (PBN). The rats were anesthetized with sodium pentobarbital and perfused at 3 days after CFA injection. The effect of vagal nerve transection on the expression of Fos and/or FG-labeled neurons in the PBN and Pa5 was studied.<Electrophysiological study> The single neuronal activities were recorded from the Pa5 responsive to noxious stimulation of the face. The single neurons were classified as WDR and NS neurons. In addition, the effect of vagal or sympathetic nerve stimulation on the Pa5 neuronal activities was precisely analyzed.
[Results] A large number of Fos protein-like immunoreactive cells were expressed in the bilateral Pa5. The Fos/FG double-labeled neurons were increased in the ipsilateral Pa5 after the vagal nerve transection. A number of nociceptive neurons in Pa5 was modulated by electrical stimulation of the vagal or sympathetic nerve stimulation.
[Conclusions] These findings suggest that the Pa5 neurons would be involved in the transmission of the trigeminal nociceptive information to the Pa5 neurons in correlation with autonomic function. [Jpn J Physiol 54 Suppl:S172 (2004)]

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It has been accepted that the descending system from the nucleus locus coeruleus (LC)/nucleus subcoeruleus (SC) plays a significant role in spinal nociceptive processing. The present study was designed to examine modulation of nociceptive processing in the caudal part of the trigeminal sensory nuclear complex, the trigeminal subnucleus caudalis which is generally considered to be involved in the relay of oral-facial nociceptive information. Experiments were performed on anesthetized Sprague-Dawley rats. Electrical stimulation was delivered at a stimulus intensity below 100μA in the present study. Stimulation at sites inside the LC/SC produced a reduction of both spontaneous activity and responses of subnucleus caudalis neurons to somatic input, especially nociceptive input. Increasing stimulation frequency in the LC/SC resulted in an increase in inhibitory effects on nociceptive responses of subnucleus caudalis neurons. At three of nine sites outside the LC/SC, electrical stimulation was effective on descending inhibition. A significant difference in the inhibitory effects was observed when the inhibitory effects were compared between sites of stimulation inside the LC/SC and three effective sites of stimulation outside the LC/SC. These findings suggest that nociceptive processing in the subnucleus caudalis is under the control of the descending modulation system from the LC/SC. To understand the effects of repetitive stimulation with high frequency on fine unmyelinated LC/SC fibers, the existence of recurrent collateral excitation in the LC/SC may be considered. [Jpn J Physiol 54 Suppl:S172 (2004)]

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In order to clarify the involvement of the primary afferent neurons for an induction of the neuropathic pain in the trigeminal region, we developed the neuropathic pain model in the SD rats with chronic constriction nerve injury (CCI) of the inferior orbital nerve (ION). Two ligatures were applied to the ION of the rats through the oral cavity. Mechanical allodynia was observed at 3 days after ION-CCI and lasting more than 14 days. We could not observe thermal allodynia in the rats with ION-CCI. At 3, 5, 7 and 14 days after application of two ligatures around the ION, single unit activities were recorded from ION under general anesthesia with 1-2% halothane. C, Aδ and Aβunits were defined on the basis of the conduction velocity calculated from the distance between recording and stimulation sites and antidromic latency from the brain stem. These three types of units were recorded at the early stage of the ION-CCI. The longer time was passed after the ION-CCI, the more frequently Aδ units were recorded. The highest spontaneous activity was recorded at 3 days after CCI during observation period. This high frequency firing was progressively decreased after that, but that was still higher than naïve at 14 days after the CCI. The mechanical and cold responses of the Aδ units were also highest at 3 days after the ION-CCI and progressively decreased after that. These suggest that the abnormal primary afferent firing observed in the rats with ION-CCI would be involved in neuropathic pain in the trigeminal region. [Jpn J Physiol 54 Suppl:S173 (2004)]

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In the present study, the two types of the inferior alveolar nerve (IAN) injured models were developed as follows; the IAN transection model (T model) and the IAN removed model in which a part of the IAN was taken out in 1mm length (R model). The time-course changes in GAP-43 expression of the trigeminal ganglion (TRG) neurons and the jaw opening reflex (JOR) occurrence were studied in the rats with IAN injury. The following results were obtained.1. The strong expression of the GAP-43 was observed in the T model at 3 to 60 days after IAN transection. On the other hand, the GAP-43 expression was increased at 3 to 30 days and decreased after that in the R model.2. The JOR could not be elicited by IAN stimulation until 7 days after IAN transection and could be observed in the 40% of the T models at 14 days and 100% of them at 60 days after that.3. JOR threshold was gradually decreased in the T model after IAN injury.4. The number of large diameter TRG neurons with GAP-43 positivity was significantly smaller in T and R models at 7 to 60 days after IAN injury as compared with that of naive. 5. The number of GAP-43 positive small diameter TRG neurons was significantly larger in T and R models after IAN injury as compared with naive rats except that in R model at 7 days after nerve injury. The number of them was gradually decreased both in T and R models. These results suggest that the regeneration mechanism was different on the basis of the type of nerve injury and the small diameter primary afferent neurons would be dominantly involved in the regeneration of the injured nerve. [Jpn J Physiol 54 Suppl:S173 (2004)]

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It is widely accepted that there is a sex difference in nociceptive responses. For example, females are more sensitive to formalin-induced nociceptive stimuli than males. In the present study, we examined whether there was a sex difference in the brain responses to formalin-induced nociceptice stimuli or not, by checking the expression of pCREB in cells of the accumbens nucleus (ACB) and the bed nucleus of the stria terminalis (BST) including the lateral and medial subdivisions. Adult male and female Wistar rats were injected s.c. with saline or 2% formalin dissolved in 50 μl of saline into the planter surface of the right hindpaw. They were killed by an over dose of pentbarbital sodium 5 min after the injection and served to immunohistochemical processing. We found that the number of pCREB expressing cells in both sides of the ACB and BST in male rats injected with formalin was not different from that in male rats injected with saline. In contrast, the number of pCREB expressing cells in both sides of these areas of female rats injected with formalin was significantly greater than that of female rats injected with saline. In saline-injected rats, there was no significant sex difference in the number of pCREB expressing cells. These results show that more areas in the forebrain are activated by formalin injection in female rats than in male rats, suggesting a sex difference in the brain response to nociceptive stimuli. [Jpn J Physiol 54 Suppl:S173 (2004)]

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Propofol is known as one of the short acting (i.v.) anesthetics. However, detail mechanism of the anesthetic effect of propofol is still unknown. It is very important to know which areas of CNS are activated by the propofol administration for clarifying the neuronal mechanism of its anesthetic effect. We introduced sodium pentobarbital (Nembutal) as a positive control drug. Three stages of anesthetic levels were defined as light, middle and deep anesthetic stages from the frequency of the wave forms of the EEG activity during intravenous infusion of propofol or Nembutal. The large spikes were exposed on the small waves at the middle stage during propofol (9.95mg/kg/20min.) or Nembutal (43.2mg/kg/66min.) infusion. When the anesthetic level is in deep, almost no clear waves were recorded during propofol (32.75mg/kg/32min.) or Nembutal (59.7mg/kg/68min.) infusion. A large number of Fos protein-LI cells was expressed in the medullary dorsal horn, area postrema, parabrachial nucleus, inferior colliculus, PAG, lateral hypothalamus and basolateral Amygdara after noxious heat stimulation of the face during middle or deep stage after propofol or Nembutal treatment. The strong depression of Fos protein-LI cells in the all areas of CNS was observed at deep stage after Nembutal treatment. On the other hand, no clear change in Fos expression was observed at the deep stage during propofol treatment. These date suggest that the propofol may induce anesthetic effect without any action on the pain pathway in CNS. [Jpn J Physiol 54 Suppl:S173 (2004)]

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The A beta, A delta and C fibers are suggested to be selectively stimulated by sinusoidal current stimulation (SCS) at 2k, 250 and 5Hz, respectively. However no direct electrophysiological evidence has been reported. In this study, we examined the characteristics of the sensations and the afferent discharges evoked by the different frequencies of SCS by use of microneurography.Ten healthy volunteers with informed consent were used. A pair of surface gel electrodes (20x25 mm) was put around the middle finger for SCS. Subjective detection threshold and the characteristics of sensations elicited by the three different frequencies of SCS were recorded. Then a tungsten microelectrode was inserted to the medial nerve and unitary discharges of the afferents were recorded, and their conduction velocities and response to SCS were investigated.The mean of detection threshold (T) at 2k, 250 and 5Hz SCS were 1.8, 0.9 and 1.0 mA, and their major sensations provoked at 2T were pressing, tingling and pulsing, respectively. In microneurogram, the A beta fibers were clearly activated by 2kHz SCS and regular discharges about 30Hz appeared at twofold threshold intensity of firing. The A beta fibers were also activated relatively higher intensity of SCS at 250 or 5Hz. The activities of A delta and C fibers were not recorded. These results suggest that each frequency of SCS induced different sensation at relatively low intensity which may activate the afferent fibers in different way. [Jpn J Physiol 54 Suppl:S174 (2004)]

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Reportedly experimental systemic inflammation produced by injecting complete Freund's adjuvant into the tail of rats (AI rats) increased the level of nerve growth factor (NGF) many fold above normal level in the inflamed tissue and DRGs. Enhanced retrograde transport of NGF to DRGs would lead to a rapid and large increase in the production of neuropeptides such as substance P (SP) and calcitonin gene-related peptide (CGRP), resulting in hyperalgegesia. However, little is known about the time course of changes in NGF and neuropeptides levels during development of persistent inflammation. Therefore we examined changes in these substances at different time points after inoculation of adjuvant. No NGF immunoreactivity (IR) was found in any specific structures in normal rat L4-6 DRGs, but found in AI rat DRGs 7 and 14 days after adjuvant inoculation. NGF-positive cells composed 9% of DRG neurons and were of small and medium size. After 21 days NGF-IR was not detected in any DRGs. Similar to NGF, percentage of SP and CGRP-positive cells were increased in AI rat DRGs after 7 and 14 days, but after 21 days it returned to normal. Trk-A receptor for NGF also showed a similar change as SP and CGRP. However, hind paw swelling begins at nearly 14 days after inoculation and reaches its peak at 21 to 28 days. This controversy between the time course of inflammatory sign and that of changes in NGF and neuropeptides raises a question what maintain inflammatory sing and hyperalgesia when the level of NGF and neuropeptides returned to normal. [Jpn J Physiol 54 Suppl:S174 (2004)]

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Our previous report of the decreased mechanical withdrawal threshold in the eccentrically contracted muscle of rats, and the increased number of c-Fos immunoreactive neurons in the superficial dorsal horn of the spinal cord at L4 after compression to that muscle demonstrated the existence of mechanical hyperalgesia (tenderness) in the exercised muscle of rats. The purpose of this study is to examine whether the mechanical response of C-fiber sensory receptors of the muscle is altered after exercise. Rats were given eccentric contraction (ECC) by electrical stimulation of the common peroneal nerve innervating the extensor digitorum longus muscle 2 days before the electrophysiological experiment. Activities of single nerve fibers (mechanically sensitive but not stretch sensitive ones whose conduction velocity was less than 2.0 m/s) were recorded from the muscle-nerve preparations in vitro. Net discharge rate of C-fiber receptors during a ramp mechanical stimulation (0-20 g in 10 sec) was significantly higher in the ECC preparation than the control (CTR) (ECC (n=33): 5.5 ± 3.2 Hz (mean ± SD), vs CTR (n=13): 3.0 ± 2.6 Hz, p<0.01, un-paired t test). In addition, the threshold of the mechanical response was significantly lower in the ECC preparation (ECC: 5.2 ± 3.1 g, CTR: 8.7 ± 4.8 g, p<0.05). These changes in muscle afferents might be related to the tenderness in delayed onset muscle soreness after exercise. [Jpn J Physiol 54 Suppl:S174 (2004)]

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Electrophysiological recordings of slowly adapting type I mechanoreceptive (SA I) units innervating the rat hairy skin were performed using an in vitro skin-nerve preparation. The skin was placed with the epidermis-side up in a organ bath. Outskirts of touch dome were incised at a depth of about 200μm so that a test solution blowed upon these incisions percolates through the dome. Caffeine (1-10 mM) blowed from a tube increased the responses of SA I unit to a constant mechanical indentation of 100μm in a dose-dependent manner. Application of 10 mM caffeine increased the response of SA I units by 45-105% (mean=73%, n=9) of the value in the absence of caffeine and shifted stimulus-response relation curves upward without changing the indentation threshold of 12-25μm. 10 mM caffeine did not almost induce spontaneous discharges, unlike the findings in rat sinus hair type I units. On the other hand, solitary Merkel cells freshly dissociated from the rat footpad skin were investigated with whole-cell configurations of the patch clamp technique. At a holding potential of -30mV, extracellular application of caffeine (10mM) produced a TEA-sensitive outward transient current, lasting several seconds, with an increase of conductance in many cells tested. In some Merkel cells, 10 mM caffeine produced an outward transient current followed by an inward sustained current, lasting a minute or longer, with a decrease in membrane conductance. [Jpn J Physiol 54 Suppl:S174 (2004)]

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Many people are suffering from muscle pain, but research for its mechanism is lacking. One reason for this is absence of appropriate methods to evaluate it. One conventional method to measure muscle pain in an awake subject is to apply pressure through the skin. Its inevitable complication is to affect the skin thus may cause undesirable pain. It would be desirable to use the optimum shape of the pressure probe that gives maximum stress to the muscle with a minimum skin stress. Since the effective stress at the muscle is not observable, it is necessary to establish a quantitative model to predict it. Toward this goal, we developed 3-D finite-element model of human forearm. Four layers (the skin, subcutaneous tissue, muscle and bone) were defined and assumed to be linear elastic materials. Two kinds of cylindrical pressure probes, with diameters of 2 and 10 mm, were constructed. Forty g load was applied for the former and 1000 g load for the latter, so that both probes would give pressure of 124.6 mN/mm² to the contact area. A pressure with 2 mm-probe resulted in the maximum principal stresses of 1250 and 2.93 mN/mm² at the epidermis and the muscle, respectively, while that with 10 mm-probe gave 1470 and 47.0 mN/mm2 for those tissues. The maximum principal stress of the epidermis was 427 times larger than that of the muscle in the 2 mm-probe, while the factor was 31.3 times in the 10 mm-probe. These results indicate that the larger probe is more appropriate for measuring muscle pain by transcutaneous pressure. [Jpn J Physiol 54 Suppl:S175 (2004)]

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The patch-clamp method has been applied to a wide variety of preparations. We also developed this method from in vitro and in vivo rat spinal cord preparations to understand the processing of sensory transmission. To address the sensory integration further, we performed in vivo patch-clamp recordings from somatosensory cortex neurons and analyze synaptic responses evoked by hindlimb stimulation. A rat was fixed in a stereotaxic apparatus under anesthetic (urethane or ketamine). Cranium of the rat was removed partially and surface of cortex was exposed. After making a bath with a plastic cup on a cranium hole, the surface of the brain was perfused continuously with Krebs solution. Under this condition, firstly, extracellular unit recordings were carried out for identification of the cortex that received inputs from the contralateral hindlimb. Patch-clamp recording electrodes were inserted into the location at where the unit firing frequency increased. Under the voltage-clamp mode at a holding potential of -70mV, spontaneous EPSCs were observed. The EPSCs frequency was increased by brush stimulation to the contralateral hindlimb. Under the current-clamp mode, the stimulation increased the frequency of action potential. These results indicate that the sensory information is transmitted not only to the spinal cord dorsal horn but also to the sensory cortex in rats anesthetized with anesthetics, suggesting a possibility that the sensory inputs are well preserved under the treatment with certain anesthetics. [Jpn J Physiol 54 Suppl:S175 (2004)]

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Recent molecular biological studies revealed many molecules concerned in taste reception. However, there is little evidence for direct involvement of these molecules at the cellular level. Here, we investigated taste responses of receptor cells generating action potentials and mRNA expression of taste related genes in these cells at the same time. Using loose patch technique, we recorded taste responses of receptor cells of mouse fungiform papillae. Two types of NaCl responding cells exist: one is amiloride-sensitive and the other amiloride-insensitive. In some cells, responses to MSG were enhanced when MSG was mixed with IMP. Responses to sweet substances in some cells were suppressed by apical treatment of gurmarin and recovered after apical application of β-cyclodextrin. About 60% of taste cells responded to one of four taste stimuli tested and the entropy value presenting the breadth of responsiveness was 0.207 ± 0.253, which was close to that for the nerve fibers. These results suggest that taste cells generating action potentials have response characteristics to taste stimuli that are comparable to those for nerve fibers. At the end of recording, the cell was withdrawn from a taste bud and examined mRNA expression of taste related genes by RT-PCR. Our preliminary data indicate that a taste cell responding to sweet stimuli expressed T1R3 and α-gustducin mRNA. Thus, this technique might be useful to examine molecular expression in receptor cells responding to taste stimuli. [Jpn J Physiol 54 Suppl:S175 (2004)]

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Mouse taste buds in fungiform papillae consist of ~50 cells. These taste bud cells (TBCs) expressed various neurotransmitter receptors, such as 5-HT and ATP receptors on their basolateral membranes. We investigated the functional expression and distribution of ATP receptors in single taste buds by monitoring [Ca²⁺]_in with a Ca²⁺-sensitive dye, Fura-2 AM, in situ. Four taste buds out of eight contained ATP-sensitive TBCs. The application of 100 μM ATP to basolateral membranes increased [Ca²⁺]_in in 4.5 ± 1.7 TBCs (mean ± SD, n = 4) an ATP-sensitive taste bud. Such ATP-sensitive TBCs preferably occurred in the central part of each taste bud. Under in-situ whole-cell clamp conditions, TBCs were depolarized and fired action potentials in response to the application of 100 μM ATP to their basolateral membranes. Although the application of 5-HT also increased [Ca²⁺]_in, the ratio of 5-HT-sensitive taste buds to all taste buds examined was 0.35 (n = 62), the number of 5-HT-sensitive TBCs was 1.3 ± 0.8 (n = 22) a 5-HT-sensitive taste bud, and 5-HT-sensitive TBCs preferably occurred in the peripheral part of taste buds. So far, ATP and 5-HT receptors never coexist on the same TBCs, though they occurred in the same taste buds. These results showed that the expression of ATP and 5-HT receptors was differentially regulated. It appears that both ATP and 5-HT are used in a paracrine system in taste transduction mechanism and in forming cell-networks, though ATP- and 5-HT-releasing sites remain to be identified. [Jpn J Physiol 54 Suppl:S175 (2004)]

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Mouse taste bud cells (TBCs) expressed various voltage-gated channels, including inward rectifier K⁺ channels, on their basolateral membranes to generate receptor/generator potentials and action potentials. Although tight junctions among TBCs prevent the free diffusion of taste substances from the receptor membranes to the basolateral membranes of TBCs, a little amount of taste substances may permeate tight junctions and modify taste transduction mechanism. In reverse, changes in ion channel currents test the permeation of taste substances. We investigated the permeability of tight junctions as the changes of inward rectifier K⁺ channel currents in magnitude (I_ir) and in activation potential (E_ir) under in-situ whole-cell clamp conditions. Their basolateral membranes were irrigated with a physiological saline containing 5 mM K⁺. The application of 500 mM KCl to the receptor membrane increased I_ir and shifted E_ir to the positive direction. Since E_ir was equal to the equilibrium potential of K⁺ on the basolateral membrane, we calculated K⁺ concentration there by the shift. The increase in K⁺ concentration on the basolateral membrane thus calculated was 4.3 ± 4.8 mM (mean ± SD, n = 23). KF (500 mM) less effectively increased K⁺, indicating that gap junctions were less permeable to F⁻ than Cl⁻. The application of 100 mM CsCl to the receptor membrane decreased I_ir without shifting E_ir. The estimated Cs⁺ concentration on the basolateral membrane was ~0.3 mM (n = 2). These results showed that tight junctions were low-conductance, ion-selective barriers. [Jpn J Physiol 54 Suppl:S176 (2004)]

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It is proposed that amiliride-sensitive epithelial Na⁺ channels (ENaCs) are involved in taste signal transduction for Na⁺ salts. Previous studies in C57 BL mice demonstrated that responses to NaCl are inhibited by amiloride in the choda tympani (CT) nerve but not in the glossopharyngeal (IXth) nerve, suggesting lack of amiloride sensitivity (AS) in the posterior tongue innervated by the IXth nerve. The AS also differs among inbred mouse strains. BALB mice showed very weak AS even in the anterior tongue. In this study, by using in situ hybridization and semiquantitative RT-PCR techniques, we examined expression of three subunits (α,β,γ) of ENaC in the fungiform papillae (FP), circumvallate papilla (CP) and tongue epithelial tissue without taste papillae (ET) in C57BL mice before and after CT nerve crush. The results demonstrated that, in intact mice, signals for α subunit were clearly detected in some spindle-shape cells in both FP and CP, whereas those for β and γsubunits were detected in FP, but only slightly in CP. After nerve crush, signals for three subunits in FP were observed in a few taste cells at 2 weeks and clearly detected at 4 weeks. These results together with those reported by previous studies suggest that expression patterns for the three subunits may contribute to AS and the tongue regional difference in AS. With regard to the strain difference in AS, we are examining the gene polymorphism for each subunits of ENaC in C57BL and BALB strains. [Jpn J Physiol 54 Suppl:S176 (2004)]

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The solitary tract nucleus (NTS), the primary gustatory relay, predominantly ipsilaterally projects to the pontine parabrachial nucleus (PB), the secondary relay. But, Williams et al. (1996) indicated a significant contralateral projection. The present study examined a pathway of the contralateral projection and a distribution of axon terminals in the contralateral PB with an anterograde neuronal tracing method using wheat germ agglutinin-conjugated horseradish peroxidase. After recording taste responses in the rostral portion of the NTS with a glass pipette containing a tracer solution, the tracer was injected electrophoretically. Following tracer injection we found anterograde labelings in the bilateral PB. Labeled axons connecting the bilateral PB were observed in the rostral medullary velum. Anterograde labelings in the contralateral PB were distributed symmetrically to those in the ipsilateral PB. However, they were quite sparse compared with dense labelings in the ipsilateral side. Further, in the ipsilateral PB very dense labelings were seen in areas abutting dorsally and ventrally to the brachium conjunctivum, where gustatory neurons have been reported to be located, but such denser labelings were not found in a diffuse distribution of the labelings in the contralateral side. The findings indicate that NTS gustatory neurons contralaterally project to the PB through the rostral medullary velum and suggest that the contralateral projection is not significant in gustatory information processing. [Jpn J Physiol 54 Suppl:S176 (2004)]

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Leptin, released from adipose tissue, is known to inhibit obesity by regulating food intake. Our recent studies have revealed taste nerve fibers in the mouth which respond to long-chain fatty acids. In this study, we investigated the effect of leptin on taste responses, particularly on the responses to fatty acid (oleic acid) and sweet substances (sucrose) in fatty and normal Wistar rats. The nerve activities were recorded from the whole nerve bundle or pauci-fiber bundles of the pharyngeal branch of the glossopharyngeal nerve (GP nerve) and the chorda tympani nerve (CT nerve). Oleic acid was applied to the pharyngolaryngeal region and sucrose was applied to the anterior part of the tongue. Oleic acid elicited vigorous discharges in the pharyngeal branch of the GP nerve in both fatty and normal Wistar rats. After intravenous administration of leptin (10 ng/ml, 3 ml/kg), the response to oleic acid was gradually decreased in normal rats, whereas the response in fatty rats was not changed. The response to sucrose in the CT nerve was also decreased by leptin in normal rats, but it was not changed in fatty rats. These results are the first findings indicating the existence of a leptin-suppression-system on the taste response to fatty acids and sweet substances in normal Wistar rats, and suggesting that such systems are lacking in fatty Wistar rats. [Jpn J Physiol 54 Suppl:S176 (2004)]

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Using MEG we located the primary gustatory cortices (PGC) at the ventral end of the central sulcus (cs) and the transition between the parietal operculum and insula (area G) because of the fastest activation (Kobayakawa, et al, 1999), though MEG can detect activation only inside sulci. This study aimed to examine the extent of the PGC by fMRI using a computer-controlled tastant-delivery system. 12 volunteers (21-33 years old, right-handed), without dysgeusia, participated in the study. The tongue tip was continuously flowed with fluids during the recording session, consisting of at least 5 alternating control and stimulation periods separated with a small air. The stimulation period supplied 16 trials of stimulations, each consisting of 1 M NaCl (ca 1s) followed by 3 water rinse (ca 1s) separated with an air bubble, and the control period supplied water in the same sequence as stimulation. BOLD signals were detected by 1.5 T Siemens MRI scanner (Magnetom Vision) with EPI protocol, and processed using SPM 99. Signals were collected for 5 stimulation periods 9 s after the onset of stimulus period. In all subjects significant activations (1% uncorrected) were found at area G on one or both sides of the hemisphere, and in 58% at the rholandic operculum and cs. In some cases activation continued from area G to the frontal operculum. [Jpn J Physiol 54 Suppl:S177 (2004)]

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It is now fairly well established that olfactory signal transduction is mediated by cytoplasmic cAMP. Up to this point, however, dynamics of cytoplasmic cAMP has been still a matter of speculation. This is mainly because that olfactory cilia have very fine structure which has limited applicable experimental protocols. In the present work, we used caged substances to estimate the dynamics of cytoplasmic cAMP, while the membrane response was monitored by whole-cell recording method. Experiments were carried out in the presence and the absence of Ca influx which causes drastic changes in the waveform of the current responses. Mainly, it is known that Ca ion has two effects on the olfactory system; feedback to the CNG channel and opening of Cl channel.Through the experiments using brief pulses of UV light, intensity-response relations were found to be fitted by the Hill equation with high cooperatively (Hill coefficient, n, of 5) in the presence, and with low cooperativity (n=2) in the absence of Ca influx. The rising phase of the current was fitted also by the Hill equation with n of 5. From such observation, it was expected that the current developing phase represents the time course for the [cAMP]i increase. To support this idea, the time course of the rising phase became less steep when the Ca influx is removed. It is therefore likely that [cAMP]i increases almost linearly with time. Also, the present work reconfirmed a classical concept that Ca ion contributes to signal amplification in olfactory signal transduction. [Jpn J Physiol 54 Suppl:S177 (2004)]

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In the subventricular zone (SVZ), neurogenesis occurs continuously, even in the aged brain. To explore the roles of newly generated cells migrating from the SVZ in olfactory functions, bromodeoxyuridine-immunoreactive (BrdU-ir) structures in the sagittal section of the main olfactory bulb (MOB) of young and old male rats were studied. BrdU labeling indicated that the density of BrdU-ir cells in the MOB of young rats was higher than that of old rats. In young rats, the density of BrdU-ir cells at the region near the rostral end of the accessory olfactory bulb (AOB) was higher than that at the region distant from the AOB. The present study indicates that newly generated cells are differently distributed in the MOB of young and old male rats. [Jpn J Physiol 54 Suppl:S177 (2004)]

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To investigate the role of glutamate receptors in the synaptic transmission between dendrites in the AOB, evoked synaptic currents were measured from mitral cells in slice preparations prepared from 23- to 36-day-old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step from -70 mV was applied to a mitral cell under the whole-cell configuration. Under control conditions, the voltage step evokes GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs). We have demonstrated that, in Mg²⁺-free solution, the NMDA receptor antagonist D,L-APV significantly blocked the IPSCs. The previous studies also demonstrated that an agonist for group II metabotropic glutamate receptors (mGluR2/mGluR3), DCG-IV, suppressed dendrodendritic inhibition whereas the mGluR2/mGluR3 antagonist LY341495 enhanced it.
In the present study, in order to conduct further investigation on the role of mGluR2/mGluR3 in the synaptic transmission, the IPSCs were recorded from mitral cells prepared from mGluR2 mutant mice. Genetic ablation of mGluR2 markedly impaired the effects of DCG-IV and LY341495 on the IPSCs, respectively. The present results provide the further implication that mGluR2, as well as NMDA receptors, plays an important role in reciprocal transmission between mitral and granule cells in the mouse AOB. [Jpn J Physiol 54 Suppl:S177 (2004)]

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The purpose of this study is to investigate whether NO modulates initiation of reflex swallowing. Recent studies have revealed that nitric oxide synthase (NOS) is located in brain stem regions involved with swallowing. However, little is known regarding the contribution of NO to reflex swallowing. Urethan-anesthetized rats were used. To evoke swallowing, the pharyngeal branch of the glossopharyngeal nerve (GPN-ph) and the superior laryngeal nerve (SLN) were stimulated by repetitive electrical stimulation (10-80μA,10-30Hz,1.0ms). The latency for the first swallow and the time interval between swallows were measured. N-nitro-L-arginine (L-NNA), an inhibitor of NOS, L-arginine (L-Arg) and sodium nitroprusside (SNP), NO donors, were intravenously administrated. L-NNA extremely reduced occurrence of swallows evoked by the GPN-ph, whereas swallows evoked by the SLN were only slightly changed. On the other hand, L-Arg and SNP facilitated reflex swallowing. These results indicate that NO modulates reflex swallowing and plays an important role in the initiation of reflex swallowing especially evoked by the GPN-ph. [Jpn J Physiol 54 Suppl:S178 (2004)]

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The aim of the present study was (1) to investigate how the tongue reflex is modulated during wakefulness and sleep and (2) to examine if modulation of the tongue reflex activities during active sleep is related with respiratory phases or rapid eye movements in the freely moving rabbit. Electromyographic (EMG) activity of tongue muscle was recorded in the genioglossus (tongue-protruder, GG) muscle, and the GG reflex was evoked by electrical stimulation of the inferior alveolar nerve. The current intensity of stimulus was set at 1.5 times threshold to evoke the reflex to evaluate the modulatory mode of GG reflex. Peak to peak amplitude of EMGs in the reflex was compared among wakefulness and sleep stages.GG activity showed a wide variation, some had respiratory-related activity and others did not. However, in all animals, the reflex was suppressed during sleep, and the inhibition was significantly stronger during active sleep than that during quiet sleep. In some cases, the reflex was completely suppressed during active sleep. Furthermore, during this sleep stage, the reflex amplitude during inspiratory phase was larger than that during expiratory phase. On the other hand, the rapid eye movements during active sleep were not related to the changes in the reflex activity during active sleep. Not only basic but also clinical studies have paid attention to loss of tone in upper airway muscles including tongue muscles during sleep. The neuronal mechanisms controlling tongue muscle activity in sleep were discussed. [Jpn J Physiol 54 Suppl:S178 (2004)]

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Neuronal activities in the red nucleus (RN) neurons during jaw movements were investigated in urethane anesthetized rats. Jaw movements were induced by mechanical stimulation applied to the hard palate and incisive papilla or by repetitive electrical stimulation of the primary jaw motor area. The RN neurons increased, decreased or no changed neuronal activities during mechanically induced jaw movements. The increase type neurons were located in the dorsolateral region of the RN. No neuronal activities that related to phase of jaw movements (jaw-opening and jaw-closing phase) and that were changed by passive jaw opening, and by pressure stimuli applied to the teeth, tongue, gingiva, superficial masseter and temporalis muscles were observed. The RN neurons showed increase, decrease or no changes in their firing patterns of activities to electrical stimulation of the primary jaw motor area. The increase type and decrease type neurons were mainly located in the dorsal and ventral region of the RN, respectively. These results suggest that the RN neurons are involved in control of jaw movements. [Jpn J Physiol 54 Suppl:S178 (2004)]

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Tongue movements participate in oro-facial motor functions including chewing, sucking, swallowing, speech, respiration and so on. Although previous studies reported the coordination of jaw and tongue movements to clarify the patterns of tongue muscle activity, our knowledge as to the functional role of tongue muscles during chewing and swallowing in humans is still limited due to the technical difficulty to record the dynamic activity of tongue muscles. In this study, we recorded the EMG activity of left tongue protruder (genioglossus, GG), jaw-closer and supra- and infra-hyoid muscles during command swallow and natural mastication of test foods as well as rhythmic experimental open-close jaw movements in healthy subjects. For GG recording, the electrode assembly was fabricated on a dental acrylic resin. During command swallow, the subject was required to swallow test foods in different postures which were upright and head inclined w/ and w/o body reclinced to test the effects of head and body postures on the tongue activity. The activity pattern of GG muscle was different among the postures. During experimental rhythmic open-close jaw movements, all the muscles recorded were well-coordinated while the coordination was not observed in the case of natural mastication. Particularly, the GG activity did not always show the rhythmicity. The results suggest the variability of human tongue activity during mastication and swallowing. [Jpn J Physiol 54 Suppl:S179 (2004)]

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To investigate the effect of masticatory performance on the masticatory behavior, the masseter muscle activity during mastication of test food was measured in adult participants. Eighteen adult volunteers participated. Masticatory efficiency of each participant was obtained using the sieving test reported by Manly and Braley, and they were divided into two gropes (high efficiency group, H group; low efficiency group, L group). Three types of test food (rice cake, RC; peanuts, P; soft biscuits, SB) were prepared in 5g units. Electromyographic activities (EMG) of the masseter muscle were recorded using bipolar surface electrodes. The amplitude of EMG in the first five chewing cycles (E stage) and the final five chewing cycles (L stage) was averaged and compared statistically (Student's t-test). Significant difference in the number of chewing strokes until swallowing during mastication of RC, P or SB between two groups was not obtained. The masseter muscle activity at the E stage during mastication of RC, P or SB in the L group was significantly lower than that in the H group. In the H group, the masseter muscle activity at the E stage during mastication of the three test foods was significantly higher than that at the L stage. In contrast, the activity at the E stage during mastication of P was significantly lower than that at the L stage in the L group. These results suggest that the degree of masticatory efficiency may influence the masseter muscle activity at the E stage during mastication. [Jpn J Physiol 54 Suppl:S179 (2004)]

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Ionotropic glutamate receptor δ2 subunit (GluRδ2) is specifically expressed in cerebellar Purkinje neurons (PNs). GluRδ2 knockout mice (δ2-/-) show the impairment in synaptic regulation and motor coordination. To elucidate how the GluRδ2 deficiency affects motor coordination, we have analyzed their eye movements and found that δ2-/- showed abnormal spontaneous and reflexive eye movements. In δ2-/-, eyes moved continuously with 1Hz and 10 Hz oscillatory components even if no stimulus was applied. δ2-/- showed a large phase delay in the optokinetic response and a high gain in the vestibulo-ocular reflex. Simultaneous recordings of the spontaneous eye movement and the activity of a floccullar PN revealed that PNs in δ2-/- tended to generate several forms of spike activities presumably induced by climbing fiber inputs. These activities showed the 10 Hz periodicity and correlated to the spontaneous eye movement. Abnormalities in spontaneous and reflexive eye movements were reduced by destruction of inferior olive nuclei by administration of 3-acetylpyridine. These results suggest that altered climbing fiber activities are related to abnormalities in the spontaneous and reflexive eye movements in GluRδ2 knockout mice. [Jpn J Physiol 54 Suppl:S179 (2004)]

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Convergence eye movements are evoked by approaching visual target and feed-back controlled with its retinal slip. Prediction of target motion improves convergence phase lag in human. In cat, conditionally learned or predicted convergence eye movements are found before the onset of target motion after training with combination of alart signal and target motion. The lateral supurasylvian area (LS) is one of extrastriate visual corteces in cat. Neurons sensitive to a visual target moving in depth are found in LS. Microstimulation in LS elicites convergence eye movement. LS cortical lesions decrease occurance of predicted and amplitude of visually-guided convergence. Amplitude of visually-guided convergence was decreased after injection of muscimol into LS central visual area while predicted convergence was not affected. To investigate the role of LS in these two sort of convergences, we recorded 67 approach neurons in alert cats (n=6). Necessory surgical procedures were done under Nembutal anaesthesia, movement of both eyes were recorded with magnetic coil method, a tungsten-in-glass microelectrode was used for microstimulation and unit recording. Activity of 9 neurons preceeded the predicted convergence and were related to dynamic parametres of convergence. 7 out of 9 neurons were also related to visually-guided convergence. Focusing on these convergence related neurons, we will discuss the role of the LS in controlling visually guided or programmed convergence eye movements. [Jpn J Physiol 54 Suppl:S179 (2004)]

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Rapid coordinated eye - head movements, called saccadic gaze shifts, displace the line ofsight from one location to another. A critical structure in the gaze control circuitry is themidbrain's superior colliculus (SC), which drives gaze saccades by relaying corticalcommands to brain stem eye and head motor circuits. We have proposed that the SC lieswithin a gaze feedback loop and generates an error signal specifying gaze position-error(GPE), the distance between target and current gaze positions. We investigated thisfeedback hypothesis, in cat, by briefly stopping head motion during large (around 50°) gazesaccades made in the dark. In the caudal SC, a cell's firing frequency gradually increased to a maximum that just preceded the optimal gaze saccade encoded by the cell's position. In "brake" trials the activity-level just preceding a brake-induced gaze plateau continued steadily during the plateau and waned to zero only near the end of the corrective saccade. The duration of neural activity was stretched to reflect the increased time to target acquisition, and firing frequency during a plateau was proportional to the plateau's GPE. By comparison in the rostral SC, the duration of saccade-related pauses in fixation cell activity increased as plateau duration increased. The data show that the cat's SC lies in a gaze feedback loop and that it encodes GPE. [Jpn J Physiol 54 Suppl:S180 (2004)]

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Pontine short-lead burst neurons (SLBNs) carry the final output signal of the horizontal saccade generator. Our previous study has shown that these SLBNs receive glycinergic inhibitory input during contraversive (off-direction) saccades and that this inhibition does not originate from omnipause neurons (OPNs). In the present study, we examined a possible contribution of inhibitory input to the formation of burst activity of SLBNs for ipsiversive (on-direction) saccades, activity that determines the size and velocity of movements. Using three-barrelled micropipettes, we recorded extracellular spike activity of SLBNs and applied iontophoretically a glycine receptor antagonist, strychnine, in the alert cat. Off-line analysis calculated parameters of neuronal activity and of eye movements. Inspection of the relation between the mean burst firing rate and the mean horizontal velocity of saccades revealed an increase in burst activity after drug application. We tested statistically whether the regression lines of this relation for pre- and postapplication data were different. No significant difference was found in the slope of the regression line, but the y-intercept was significantly larger after application in the majority of neurons. Results indicate that, during on-direction saccades, SLBNs receive an inhibitory input of non-OPN origin that significantly reduces the effect of a concomitant excitatory drive, thereby shaping the final saccadic signal. [Jpn J Physiol 54 Suppl:S180 (2004)]

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Omnipause neurons (OPNs), located near the midline of the pons, discharge tonically at a high rate during fixations and stop firing during saccades in all directions. We have previously reported that the pause of OPN firing is caused by postsynaptic inhibition whose time course closely parallels that of saccadic eye velocity. To clarify the neurotransmitter that mediates the saccade-related inhibition, we studied effects of strychnine, a glycine receptor antagonist, and bicuculline, a GABA_A receptor antagonist, on the firing pattern of OPNs in alert cats. Using multibarrel micropipettes, we recorded extracellularly spikes of single OPNs before and after iontophoretic application of the antagonists. Application of strychnine shortened the duration of saccadic pause; the initiation of pause relative to saccade onset was slightly delayed and the resumption of tonic discharge relative to saccade end was markedly advanced. Application of bicuculline had no significant effect on the duration and timing of the pause, but increased tonic firing rate during fixation. These results suggest that inhibitory inputs causing saccadic pause of OPNs are mainly glycinergic. Tonic activity of OPNs for the duration of fixation might be regulated by GABAergic inhibition. [Jpn J Physiol 54 Suppl:S180 (2004)]

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We have shown previously that saccadic adaptation occurs faster when repeated after a short interval, a phenomenon we call facilitated adaptation. In the present study, we attempted to delimit where in the oculomotor system the saccadic motor learning might be facilitated. To this end, we investigated how adaptation of horizontal or vertical saccades affected subsequent adaptation of oblique saccades in rhesus monkeys. Saccadic adaptation was induced by intrasaccadic step (ISS) of the target. After the gain of horizontal or vertical saccades was substantially reduced by backward ISS (control adaptation), we used forward ISS to increase the gain back to ~1.0. Then we changed the target step direction by 45 deg and subjected oblique saccades to a similar backward ISS (test adaptation). The rate of gain change was estimated as a slope of a regression line fitted to the initial 100-200 saccades. Analyses of gain change rates indicated that preceding adaptation of horizontal or vertical saccades facilitated adaptation of oblique saccades. Further analysis revealed that the horizontal and vertical components of oblique saccades exhibited different gain change rates. When preceded by adaptation of horizontal saccades, horizontal component adapted faster than vertical component. Similarly, preceding vertical saccade adaptation exerted a larger facilitatory effect on vertical component. The facilitation thus appears to be dominant in the learned component. These results imply that saccadic adaptation facilitation may occur at a structure that controls the component gain of saccades. [Jpn J Physiol 54 Suppl:S180 (2004)]

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Visual inputs to the superior colliculus were investigated by recording field potentials in different layers of the superior colliculus following electrical stimulation of the optic chiasma and photo-stimulation with flash light in anesthetized (isoflurane 1.5 - 2.0%) and immobilized macaque monkeys (n = 3). Electrical and photo-stimulation induced a slow negative field potential in the superficial layers (depth 0 - 1000 μm from the dorsal surface) which was reversed in the intermediate and deep layers (depth 1000 - 2000 μm) at the latency of 6 - 8 ms and 38 - 46 ms, respectively. Sharp negative field potentials composed of multiple spike activities overrode the slow negative field potential at the depth of 400 - 800 μm. When bicuculline (10 mM, 2 μl) was injected into the intermediate and deep layers, a negative field potential was induced in these layers with a latency of approximately 13 ms (electrical stimulation) and about 45 ms (photo-stimulation), which lasted for 100 - 150 ms. When bicuculline was injected into the superficial layer, the negative field potential in the superficial layer was enhanced but the field responses in the intermediate and deep layers were unchanged. These results have suggested that visual inputs induce strong activation of intermediate and deep layer neurons in the superior colliculus when these layers are released from GABA_A receptor mediated inhibition. [Jpn J Physiol 54 Suppl:S181 (2004)]

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The smooth pursuit system moves the eyes in space accurately to track slowly moving objects of interest while compensating for visual inputs from the moving background. In daily life, smooth-pursuit is performed by coordination of eye and head movements (i.e. gaze-pursuit). To examine the effects of visual background and contribution of head movement in head-free pursuit, in this study we examined smooth gaze pursuit in a young Japanese monkey that was trained to pursue a target moving on the vertical screen at 0.5Hz (±20°). Eye and head movements were recorded by the search coil method. Body movements were restrained and care was taken to minimize the weights of head holder during head-free pursuit. Eye-, head- and gaze- (eye+head) gains (re target velocity) were calculated in head restrained and free conditions with or without a random dot visual background. In head-free pursuit, vertical gaze velocity gains with and without a background were 0.75-0.90 and 0.86-0.97, respectively. Head velocity gains were 0.24-0.44 and were similar with or without a background. Upward gaze velocity was significant lower than downward. Eye velocity gains ranged from 0.41 to 0.65. Upward eye velocity gains were significantly lower than downward in both head-free and restrained conditions. Eye velocity in head-free pursuit led target velocity by the mean of 14°, whereas head velocity lagged target velocity by the mean of 34°. No asymmetry was observed in leftward and rightward pursuit with a background. [Jpn J Physiol 54 Suppl:S181 (2004)]

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The smooth pursuit system interacts with the vestibular system to maintain the visual target on the fovea. To investigate whether vestibular inputs contribute to predictive smooth pursuit especially to the timing of its initiation, we measured the latency of smooth pursuit during whole body rotation. Two male Japanese monkeys (Macaca fuscata, K, M) were rotated trapezoidally in the yaw plane (20°/s, ±10°) with a random inter-trial interval. A laser spot was projected onto the tangent screen and moved vertically with a constant delay ranging from 100 to 700ms from chair onset. The delay was given as a block and the monkeys were required to pursue the spot. After this training for 40-60 min, the tracking target was briefly (500-700ms) extinguished ("target blanked") at 80ms after the onset of chair rotation. Latencies of smooth pursuit with or without target blanking were compared with those before training. When the delays of target onset from chair were 100, 300, 500, and 700ms, mean latencies of pursuit of each monkey without target blanking were 87, 208, 352, and 521ms (K), and 88, 134, 365, and 365ms (H), respectively. The latencies of pursuit with target blanking were similar. These results indicate that smooth pursuit was initiated before the onset of target motion with the latencies proportional to the delays used for training even without the presence of the target, suggesting that the monkeys predicted the timing of pursuit initiation with the use of vestibular input. [Jpn J Physiol 54 Suppl:S181 (2004)]

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To investigate neural mechanisms underlying behavioral set-shift, we trained animals to perform an oculomotor set-shift task which required search for a correct pair of saccade targets by trial and error. In this task, a trial began when the animal fixated on a center spot and, after a delay period, four spots were illuminated. Then after another delay period, the center spot was extinguished and served as a GO signal. The animal was required to make saccade to one of four spots. A correct target was shifted between a pair of spots in every trial. The target pair was shifted in block without any instruction. Thus animals were required to search a new correct pair by trial and error. We recorded from the supplementary eye field (SEF), while the animal performed this task. Here we report some task-related neuronal activity that characterizes the SEF. [Jpn J Physiol 54 Suppl:S181 (2004)]

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To understand how the MT/MST areas are involved in vergence tracking, we examined activity of MT/MST neurons during smooth pursuit in frontal planes and vergence eye movements in two head-fixed Japanese monkeys. As a search stimulus, the monkeys tracked a 0.5° stereo spot moving sinusoidally in virtual space created using frame-sequential presentation and shutter glasses. We tested 143 neurons in MT/MST during both frontal-pursuit and vergence tracking. The majority of MT/MST neurons (69%) responded only during frontal-pursuit, 16% responded during both frontal-pursuit and vergence tracking, and the remaining 15% responded only during vergence tracking. Visual response to sinusoidal motion of a stereo spot or optic flow was tested while the monkeys fixated a stationary spot. The majority of neurons tested responded to optic flow (82/132) with large receptive fields, and preferred directions of visual response of the majority of them were opposite to those during tracking. About half of neurons tested also responded to spot motion (22/47). Using disparity step target motion, we examined latencies of neuron discharge. Some of them (10/26) discharged before the onset of vergence eye movements even after subtraction of visual components. These results suggest that MT/MST contains neurons that are involved in the initiation of vergence tracking. Supported in part by Japanese MEXT and NIH grants (EY006069; RR00156). [Jpn J Physiol 54 Suppl:S182 (2004)]

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Recent studies have suggested that physical training after brain insults improves motor functions. The purpose of this study is to investigate the effect of treadmill training on intracerebral hemorrhage (ICH) model rats. Collagenase (1.4 μl of 200 U/ml) was injected into the left striatum in adult Wistar rat. ICH model rats received treadmill training (speed: 9 m/min, for 30 min per day) everyday from 4 day after ICH (D4) to D14. Motor function was evaluated at 24 h, D3, D7, D14, D21, D28, D35 and D42 by four behavioral tests: spontaneous ipsilateral rotation, beam walking ability, forepaw grasp, hindlimb retraction. Each test was scored from 0 (normal) to 3 (severe): total score was 0-12. We found that total score at D14 was low in training-group compared to ICH only (control), indicating that recovery of disturbed motor function was improved by treadmill training. Especially, recovery of forepaw function was improved at D14. The positive effect of treadmill training was detected till D21, but thereafter the score was not decreased further. We further investigated the effect of longer-term training (from D4 to D21) on ICH. Longer-term training had no further recovery of motor function. Data suggest that treadmill training from D4 to D14 could improve the recovery of forepaw motor function. [Jpn J Physiol 54 Suppl:S182 (2004)]

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Hemorrhage around the internal capsule (IC) induced relatively sever motor dysfunction with a relative small brain damage. We tried to make a new model of intracerebral hemorrhage with a small IC hematoma. Rats were injected type IV collagenase (1.4 μl of 1-200 U/ml) into the striatum near left IC. Behavioral function was evaluated for 7 days after the injection with five parameters: spontaneous rotation, hindlimb retraction, beam walk ability, forepaw grasp and methamphetamine-induced rotation. Hematoxylin-eosin staining revealed that 7.5 U/ml is the optimal concentration, because higher concentrations caused too large tissue damage and lower concentrations did not induce motor dysfunction. To confirm this, we labeled in advance primary motor neurons in the sensorimotor cortex with retrograde tracer Fluoro-Gold (FG: 1μl of 2% solution into the corticospinal tract (CST) of C 2-3 level), and then counted labeled-cells in the sensorimotor cortex every 0.5 mm sections (from 2.2 mm rostral to 1.3 mm caudal from bregma). Number of labeled neurons at 0.8 mm caudal to bregma was decreased to 79.7 ± 6.2% (n=4, p=0.016) of that of contralateral side at 14 day after the lesion compared to saline-treated group (n=3, 97.6 ± 2.7%). In addition, FG treatment into CST in C 2-3 after the lesion did not label ipsilateral motor neurons. Data suggest that collagenase injection into an area near IC with 1.4 μl of 7.5 U/ml induced a relative small brain damage with relatively bigger motor dysfunction resulting a good model of IC hemorrhage. [Jpn J Physiol 54 Suppl:S182 (2004)]

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To investigate whether naloxone has the effects on the spinal cord contusion injury, a contusive spinal cord injury (SCI) was made in female Wistar rats with a weight (20 g, 2.5 mm diameter, 50 mm height) after laminectomy at Th 9/10. Naloxone (dissolved in 50% ethanol) was treated using osmotic pump (0.1 mg/day for 7 days, s.c.) that was implanted in the dorsal side of the neck at 4 hours before SCI. Early improvement in BBB score was found in the naloxone-treated group compared with controls. Rats were sacrificed at 1 day, 3 days, 9 days and 2 weeks after SCI, and offered for immunohistochemical staining using a microglial marker (OX-41), oligodendrocyte (OL) progenitor markers (NG2, O4) and degraded myelin basic protein marker (MBP-D). The number of NG2 and O4 positive cells in regions at 10 mm caudal to the lesion was not significantly different between the naloxone-treated group and controls. Number of MBP-D positive cells especially in the ventral white matter at 1 day and OX-41 positive cells at 9 days were significantly decreased in the naloxone-treated groups. In the naloxone-treated group, most of microglial cells showed ramified morphology in the ventral horn at 9 days after SCI. Data suggest that treatment with naloxone facilitated early functional recovery after SCI partly with modification of microglial function. [Jpn J Physiol 54 Suppl:S182 (2004)]

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