The STN is a target for deep brain stimulation (DBS) in the treatment of Parkinson's disease. DBS effects are likely to affect both axons and somata within the STN, but details of effects are not known yet. We report on experiments to study the extent of stimulation effect within the STN. Two monkeys received two chronic recording chambers each. The two chambers were directed at the same STN. One of them was used to carry out electrical stimulation of the STN with a microelectrode (using monophasic stimulation at 1/s, pulse width 50 μs, amplitude < 300 μA), and the other to simultaneously record the neuronal activity in STN with standard extracellular single-unit recording techniques. Major response of STN neurons to the stimulation was a cessation of activity, starting immediately after the stimulation and lasting for 20–70 ms. STN neurons remote from the stimulation site as well as those adjacent to the stimulation site ceased to fire. Inhibitory responses were scattered in a broad area of the STN regardless of the location of stimulation electrode in the STN. Prominent suppressive effects on the activity of STN neurons would result from the electrical stimulation of the STN. [J Physiol Sci. 2008;58 Suppl:S191]

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Accurate reaching toward a visual target is disturbed after the visual field is displaced by prisms, but recovers with practice. When the prisms are removed, errors in the opposite direction are observed (after-effect). We previously reported in monkeys that the after-effect disappeared in 24 hours after a repetition of 250 trials, but remained as large for 72 hours after 500 trials. In the present study, we examined if a gradual increase of visual displacement (Exp 1) and/or a break of one hour (Exp 2) would establish long-lasting after-effects with a smaller number of trials. In Exp 1, we increased displacement from 0 to 15 D in 100 trials, and had the monkey continue the task with the 15 D displacement for additional 100, 200 and 400 trials. Twenty-four hours later, we found significant after-effects in the large-number condition (100+400 trials; p<0.001), but not in the small-number condition (100+100 trials; p>0.05). The results show that the gradual increase did not consolidate after-effects. In Exp 2, we gradually increased the visual displacement in 100 trials and had the monkey continue the task for another 100 trial-block. Then in one condition the monkey took a break of one hour before completing another 100 trial-block with prisms. In the other, the monkey took no rest. With the one-hour rest, we found significant aftereffects (p<0.001) in all five experiments but only in three of five without the rest. The aftereffects were significantly larger in the break condition (p < 0.05). The results suggest that a kind of consolidation process is triggered during the break between the two 100-trial exposure blocks. [J Physiol Sci. 2008;58 Suppl:S191]

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This study examined functional roles played by the dorsal premotor cortex (PMd) and the primary motor cortex (MI) in conditional visuomotor behavior, in which it is necessary to recognize a visual object initially and to choose a motor act associated with it subsequently. For the first time after these two processes, a subject can prepare and execute it. We recorded neuronal activity in the PMd and MI of two macaque monkeys (Macaca fuscata) while they were performing a version of a conditional visuomotor behavior. We found that a group of PMd neurons retrieved conceptual motor information (i.e., a relative position of a target) indicated by a visual object. We also found that an entire process in which the conceptual motor information was transformed into a physical movement was reflected in a population of neuronal activity in the PMd. By contrast, involvement of the MI in these processes was meager. Instead, a great majority of MI neurons was active while a reaching movement was being executed, and the activity mainly reflected the direction of the movement. These results suggest that the PMd is involved in core processes for establishing a physical movement based on information instructed by a visual object, while the MI is mainly involved in executing a planned movement. [J Physiol Sci. 2008;58 Suppl:S191]

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It has been shown that neurons in the PMd and PMv respond to the appearance of visual signals, leading to an idea that these areas are involved in a visually guided motor behavior. To further examine the involvement of these areas in this behavior, we initially trained two monkeys (Macaca fuscata) to perform two visually guided tasks. 1) In a conditional visuomotor task, they were required to determine a forthcoming motor act based on identity of a visual object (i.e., indirect visuomotor processing). 2) In a visually guided reaching task, they were required to reach a visual target (i.e., direct visuomotor processing). We subsequently recorded neuronal activity from the PMd and PMv while they were performing the two tasks, which allowed us to compare response properties of each neuron under distinct modes of visuomotor processing. We found that PMd neurons were more responsive to the appearance of a visual cue requiring indirect visuomotor processing, and that the activity reflected an instructed motor act. We also found that PMv neurons, by contrast, were more responsive to the appearance of a visual target under the direct visuomotor processing, and that the activity reflected visuospatial feature of the target. These results suggest that the PMv and PMd are differently involved in the two modes of the visually guided motor behavior; the PMd and PMv are more involved in the indirect and direct visuomotor processing, respectively. [J Physiol Sci. 2008;58 Suppl:S191]

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Physiological studies revealed that the PMd and area 46d are commonly involved in planning action. Anatomical studies revealed that these two areas are interconnected with cortico-cortical connections. In this study, we examined whether the PMd and area 46d would also receive shared inputs from other frontal cortical areas, and, if this is the case, how neurons projecting to these areas would be distributed. To achieve these goals, we injected two fluorescent tracers into the PMd and area 46d of macaque monkeys. Analyzing location of neurons labeled with each tracer revealed frontal cortical areas containing both neurons projecting to the PMd and to area 46d. These regions were located in the cingulate cortex (area 24, 23), the medial frontal cortex, and the fundus of the superior limb of the arcuate sulcus. We also found that neurons projecting to the PMd or area 46d were distributed less than 500 micrometers in a majority of cases. These results revealed that the PMd and area 46d receive shared inputs from these frontal cortical areas and that neurons projecting to the PMd and area 46d exist in close proximity, suggesting that the local circuits play an important role for the PMd and area 46d to execute information processing in an integrated manner. [J Physiol Sci. 2008;58 Suppl:S192]

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To elucidate functional roles of primary motor cortex (M1) in the control of locomotor movements in primates, we recorded activity of M1 neurons in a freely moving monkey. The animal walked quadrupedally on the treadmill belt moving at various constant speeds. We tested effects of the walking speed on the neuronal discharge pattern. During simple locomotion, almost all neurons (33/34) exhibited task-related modulation. Of these, 23 neurons modulated their discharge phasically, and 8 did phasically and tonically time-locked to the step cycle. The rest modulated tonically. At the speed of 1.0 m/s, mean discharge frequency of task-related neurons was 18.6 ± 2.3 spikes/s. For neurons showing phasic and phasic-tonic modulation, the peak activity occurred once or twice at widely different times during the step cycle. Frequency of the peak activity ranged from 17.4 to 98.4 spikes/s (mean 42.2 ± 4.4 spikes/s). Of the task-related neurons, 21 were tested for the effects of treadmill speed on their discharge patterns. For locomotion at speeds between 0.7 and 1.3 m/s, more than two thirds of neurons (15/21) increased or decreased mean and/or peak discharge frequency along with an increase of the speed. All these results suggest the possibility that output of the primary motor cortex in macaque monkeys directly and/or indirectly acts on spinal circuitries generating a basic pattern of rhythmic activity during simple locomotion in a manner different from that in subprimates. [J Physiol Sci. 2008;58 Suppl:S192]

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To clarify the mechanism of recovery from cortical damage of CNS, we measured regional cerebral blood flow (as an index of brain activity) after lesion of primary motor cortex (M1) using positron emission tomography (PET) with H₂¹⁵O in an adult macaque monkey. After mapping of motor representation in M1 using an intra-cortical microstimulation, we applied ibotenic acid (7μl, 10μg/μl)to its digit region, by which the hand of damaged side was resulted in paralysis. About one month after lesion, the finger dexterity was recovered as the same level as in the pre-lesion stage. In this recovery stage, significant increases in activities in several brain areas including the bilateral premotor cortex were observed compared to the pre-lesion stage in which the contralateral sensorimotor cortex for used hand was mainly activated during the precision grip task. The present result indicates that distributed brain regions take over the function of lesioned M1 for the control of precision grip. [J Physiol Sci. 2008;58 Suppl:S192]

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It has been reported that that discharge of motor units (MU) represented elongating trends in spike interval during voluntary constant-force isometric contraction. To investigate neural mechanisms underlying the elongating trends mechanisms, activity of the single MUs in the vastus medialis muscle during involuntary contraction via tonic vibration reflex was compared with voluntary contractions having almost the same amplitudes and time courses. Subjects performed involuntary or voluntary isometric knee extension with their knee and hip joint angles at 90deg. The action potential of single MU was recorded using non-invasive surface array electrodes. Vibratory stimulation was applied perpendicularly to the patella tendon of knee extensors, which were completely relaxed. The vibration frequencies were 75 and 100Hz, the amplitude was 0.5-0.8mm. It was already reported that MUs were classifiable by their response to vibration with activation based on either a monosynaptic reflex process (MSR) or mono- and polysynaptic process (M-PSR) (Romaigure et al., 1991, Kamo 2007). The MSR MUs discharged at a longer interval without an elongating trend, with similar synchronization between MUs, as compared with that during voluntary contraction. However, M-PSR MUs discharged in much the same way during both types of contraction. These findings suggest that the elongating trend in spike interval during voluntary constant-force contraction is not explainable merely by the decline in muscle spindle discharge.This work was supported by Academic Frontier Project at Japan Women's College of Physical Education,2007 [J Physiol Sci. 2008;58 Suppl:S192]

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During target-reaching movements, humans can make mid-flight adjustments (switching) when the target jumps to new positions. To examine if the primary visual cortex is involved in the adjustment, we have established a method to suppress the visual cortex during the movement. Three normal human subjects, who gave informed consents, participated in experiments. They sat ca. 40 cm in front of a CRT monitor, with their chin supported by a chin rest. A figure-of-eight coil for transcranial magnetic stimulation (TMS) was held over the primary visual cortex as described previously (Kammer et al., 2005), whose position was checked on MRI brain images from each subject (Navigation System, NexStim). When subjects gazed at a 1 cm square target in the center of the screen, sound GO signal was given to reach to the target. After 25 ms from the movement onset, the center target sometimes disappeared, and a new target could appear either transiently (duration<10 ms) or constantly. It appeared 10 cm left or right to, and 2 cm below the central target. TMS was sometimes delivered over the visual cortex 70 or 100 ms after appearance of the new target. Subjects were instructed to reach the new target, and their arm positions were monitored by Optotrak system (NDI). After each trial, subjects reported if and where they saw a new target. In two of the subjects, we succeeded to suppress visual perception during reaching, without disturbing the movement. We further analyzed suppression effects on the arm movement. [J Physiol Sci. 2008;58 Suppl:S193]

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A behavior consists of elementary actions, which must be assembled in a proper sequence in order to successfully achieve the intended behavior. The present study examined how monkeys learn to take and use forceps to pick food. They had already learned to use 'hand-held forceps' to pick up food. We investigated the step of reach-grasp movement toward forceps by comparing responses in tasks of different contexts. (1) When the forceps was placed on a plate with food stuck at its tip, monkeys reached for and grasped the forceps while opening the finger (pre-shaping) and then grasped it as though automatically, and brought the attached food to the mouth, indicating that they had a capability to perform the sensorimotor transformation necessary for grasping forceps. However, (2) when the forceps and food were placed apart each other on a plate to be picked up, they did not execute actions on the forceps; instead, they brought their hand toward the food and mimed to use the forceps for taking food. After the practice under the experimenter's guide, monkeys began to reach out the clenched-hand for the forceps and hooked, rather than grasped, the forceps with the fingers. With further practice, they began to open/close the fingers aiming at the forceps, but this was only after their hand contacted the forceps. Finally they became to reach for the forceps while opening the fingers, that was pre-shaping. The results suggest that the control of grasping involved in tool-use actions is dissociated from that for the inherent behavior. [J Physiol Sci. 2008;58 Suppl:S193]

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Coordinated cyclical movements of ipsilateral elbow and knee or wrist and ankle can be performed more stably when the joints rotate in the iso-direction (e.g. wrist extension coupled to ankle dorsiflexion when forearm is in the prone position) than in the opposite-direction. This holds true regardless of the coupling of actions (e.g. extension-dorsiflexion, extension-plantarflexion), since the same results are obtained whether the forearm position is prone or supine. We investigated whether this direction dependence of the stability of coordinated movements was applicable to the coordinated movements of fingers and toes. Subjects (eyes closed) performed cyclical extension-flexion of fingers and toes in the two direction modes: iso- or opposite-direction, and the two coupling of actions modes: simultaneous (e.g. fingers flexion coupled to toes flexion) or alternate actions (e.g. fingers flexion coupled to toes extension). The stability of the movements was affected by the direction and the coupling of actions independently. In an additional experiment, motor evoked potentials (MEPs) of finger flexor and extensor muscles were elicited while subjects performed cyclical toes extension-flexion movements without activities of hand muscles with forearm prone or supine position. MEPs of finger extensors and flexors were modulated depend on the coupling of actions, not on the direction. In conclusion, the coordinated movements of fingers and toes were affected by both the direction and the coupling of actions, and the latter was supported by the neural coupling of fingers and toes. [J Physiol Sci. 2008;58 Suppl:S193]

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The amplitude of motor-evoked potential (MEP) is increased during action observation and motor imagery. The present study investigated whether the excitability of the corticospinal tract is further increased when subjects observe an action while they image the same action. MEP induced by transcranial magnetic stimulation were recorded from the biceps brachii (BB) and brachioradialis (BR) muscles in four different conditions: (1) relax (control), (2) observation of flexion-extension of the elbow attentively (OBS), (3) imagery of flexion-extension of the elbow (IMAG), (4) observation of flexion-extension of the elbow with imagery of the same action (OBS + IMAG). The amplitudes of MEP in BB during IMAG and OBS + IMAG were significantly increased compared to that during the control. In addition, facilitation of MEP size during OBS + IMAG was significantly greater than that during IMAG. And the degree of increase in MEP size during OBS + IMAG was significantly larger than the sum of the values obtained from OBS and IMAG. However, when the subjects imaged the elbow flexion-extension while they observed different actions, MEP amplitudes were not increased compared to that during only imagery of the flexion-extension of the elbow. For BR, the amplitudes of MEP were not changed among four conditions. These findings suggest that the combination of action observation and motor imagery could enhance the excitability of corticospinal tract, possibly through modulation of the mirror neuron systems. [J Physiol Sci. 2008;58 Suppl:S193]

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Postural balance is maintained by integrating various modes of sensory input, and creating a suitable combination of motor responses to control the position of the center of mass. This study examined the influence of diapers on static and dynamic postural control in the elderly by measuring body sway while standing and wearing diapers. On a force platform, body sway was recorded while standing for thirty seconds with eyes open and closed, and during movement while wearing a diaper, and while standing with eyes open after wearing a diaper for twenty seconds. Subjects wore 5 different diaper samples with different degrees of abdominal contractibility affecting wearing ease, or underwear. Subjective evaluation of each diaper was also noted. There was no difference between young and elderly groups in total length of body sway (LNG) and enveloped area of body sway (ENV) while standing with eyes open and closed. However, LNG and ENV during movement while wearing diapers were significantly larger in the elderly than in the young. On Pearson's analysis, changes in wearing ease correlated significantly with changes in LNG and ENV in both the young and elderly groups. Negative correlations were prominent in the elderly. Furthermore, changes in LNG during upright stance after wearing diapers correlated with changes in wearing ease in the elderly group only. These findings suggest that young and elderly people have similar abilities to maintain postural control under static conditions, but the elderly lose balance more easily while performing routine tasks. [J Physiol Sci. 2008;58 Suppl:S194]

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To understand the functional difference between dominant and non-dominant limbs in human unipedal standing, we examined postural and electromyographic (EMG) responses to galvanic vestibular stimulation (GVS). EMG activities were recorded from muscles of ankle, knee, and hip joints of both limbs. Movement of center of pressure (COP) was recorded by a force plate. For GVS, electric DC current of 0.6 mA were applied for 5 second during standing. Kinematic analysis revealed that GVS induced displacement of head (frontal region), thorax (angulus sterni) and pelvis (spina iliaca anterior superior) positions toward the side of the anode, and the ankle position of the unloaded leg moved in the opposite direction. Resultantly, the COP changed its lateral position toward the anodal side. When the anode was put on the contralateral side to the supporting limb, displacements of the body positions were larger than those when the anode was ipsilaterally set. Correspondingly, GVS evoked activation of supporting limb muscles. When the anode was put on the ipsilateral side to the supporting limb, m. extensor digitorum longus (EDL, ankle evertor) was activated, followed by activation of m. tibialis anterior (TA, ankle flexor and adductor). The anode positioned contralaterally at first activated TA, then EDL. These results were common whether the supporting limb was dominant or not. Differences between dominant and non-dominant limbs are currently investigated in detail. [J Physiol Sci. 2008;58 Suppl:S194]

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Modern humans spend a considerable length of time everyday in a seated position, working, driving, relaxing, etc.. Little is known, however, regarding the neuronal control of human body balance while seated. There are many reports of the neuronal control of body balance while standing, but few studies of the neuronal control of body balance while seated. We examined the functional role of various muscles during upper limb movements while in a seated position. Twelve subjects, between 21 and 49 years of age, volunteered to participate in this investigation. None of the subjects presented with signs of neurologic disorders. Electromyogram (EMG) activities were recorded from the arm, shoulder, back, abdominal, and leg muscles with bipolar surface electrodes. Subjects sat on a chair and performed various upper limb movements at different speeds. We also recorded the force exerted by the back. All data were synchronized using a pulse generator. The results indicated that 1) various shoulder, trunk, and hindlimb muscles control body balance during arm movements while in a seated position, 2) EMG activities (the intensity and timing of EMG bursts) of the shoulder, trunk, and hindlimb muscles depended on the direction and speed of the upper limb movements. The results of the present experiments indicate that neuronal control of the shoulder, trunk, and hindlimb muscles is required to maintain body balance while in a seated position. [J Physiol Sci. 2008;58 Suppl:S194]

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It is known that in the wrist flexors (the flexor carpi radialis (FCR) and ulnaris (FCU) muscles) the afferent inputs from one muscle project to not only the motoneurons innervating the muscle itself, but also to the motoneurons of its synergist. Consequently, such spinal connections should cause some interaction between the reflex responses of the two synergists. However, it is not clear yet how the central nervous system controls the reflex activity of these synergists. The present study shows that the reflex responses (the amount of EMG activity in each muscle) of wrist synergistic muscles were modulated by the radio-ulnar joint angle, which allows a change of the lengths of FCR and FCU muscles while keeping the wrist joint at a constant angle. This result means that the response of the wrist stretch reflex is produced by using the synergists as the muscle length demands, even when a similar reflex flexion is apparently induced by stretch stimulus from a constant wrist angle. In addition, in both the short and long latency reflexes, two factorial ANOVA revealed significant interaction between muscles (FCR and FCU) and radio-ulnar joint angles. Thus, the supra-spinal system for processing long latency reflex seems to mediate the reflex responses of wrist synergists in a similar fashion to that of the spinal reflex system. [J Physiol Sci. 2008;58 Suppl:S194]

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The pedunculopontine tegmental nucleus (PPN) consists of cholinergic and non-cholinergic neurons located at the pontomesencephalic junction. The PPN is involved in the control of postural muscle tone via descending projections to the pontine reticular formation (PRF). Neuroanatomically the PPN receives GABAergic efferents from the substantia nigra pars reticulata (SNr), one of the basal ganglia output nuclei. Here we designed to test whether the GABAergic SNr-PPN projection contributes to the modulation of postural muscle tone. For this purpose intracellular recording was obtained from hindlimb motoneurons in decerebrate cats. Short train pulses of stimuli (3 pulses with 5 ms intervals, 30 μA) applied to the PPN induced a mixture of EPSPs and IPSPs. The EPSPs were induced within 30 ms and the IPSPs consisted of a peak latency of 40-50 ms with a duration of 40-50 ms. The late IPSPs were preferentially observed in both extensor and flexor motoneurons. Microinjection of atropine sulfate (10-20 mM/0.25 μl) into the PRF reduced the amplitude of the IPSPs. Conditioning stimuli applied to the SNr (30-60 μA and 100 Hz) alone did not alter input resistance of the motoneurons. However, it significantly reduced the amplitude of the IPSPs and amplified the EPSPs. Subsequent microinjection of bicuculline (5-10 mM/0.25 μl) into the PPN blocked the SNr effects. These results suggest that the GABAergic output from the basal ganglia controls postural muscle tone by modulating the activity of cholinergic PPN neurons projecting to the PRF. [J Physiol Sci. 2008;58 Suppl:S195]

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Constraint-induced (CI) therapy is a method to promote the use of damaged function with a reduction of the use of undamaged normal function. Recently, CI therapy is known as a useful method to promote motor functional recovery after brain injury. However, the effect of CI therapy on the normal function by forced-disuse has not been clarified yet. To investigate whether CI therapy causes the decrease of normal functional ability (undesirable dysfunction), animals were restricted their one forelimb continuously for one week followed by behavioral assessment and Argyrophil III staining. Staircase test, horizontal ladder test and cylinder test revealed that skilled reaching of forelimb use in staircase, coordination and step in ladder walking movement, and symmetric use of forelimb in cylinder were not changed. Argyrophil III staining to detect early histological damaged state indicated that positive cells were appeared in hippocampus CA1/CA2 pyramidal cell layer bilaterally, while those were not detected in sensorimotor cortex and spinal cord. Data suggest that CI therapy did not show the decrease of normal functional ability, but has a slight effect on histological damage of hippocampus, probably due to stress by continuous restraint. [J Physiol Sci. 2008;58 Suppl:S195]

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This study aimed to understand 1) how supraspinal locomotor driving signals are involved in generation of coordinated quadrupedal locomotion in rabbits, and 2) whether sensory feedbacks are necessary for such locomotor generation. For the first purpose, in decerebrate rabbits with a right hemisection of the upper cervical spinal cord, we observed locomotor movements of fore- and hind-limbs evoked by stimulation to the mesencephalic locomotor region (MLR). Stimulation to the left or right MLR evoked coordinated, rhythmic movements of fore- and hind-limbs on the left side but not on the right side. After additional hemisection of the left caudal medulla, MLR stimulation could no longer evoke any limb movements. For the second purpose, in decerebrate, paralyzed rabbits, we recorded electroneurograms (ENG) from extensor and flexor nerves of the hindlimbs during MLR stimulation. During the stimulation, bilateral hindlimb ENGs exhibited rhythmic activities, but nonalternating bursts of left and right ENGs were not always observed. These findings suggest that locomotor driving signals from the left or right MLR are mediated to both sides of the brainstem, and that the signals descending on one side of the spinal cord would activate central pattern generators (CPG) of fore- and hind-limbs on the same side, coordinately. In addition to the supraspinal signals, sensory feedback signals from active body parts during locomotion would be necessitated to coordinate activities of hindlimb CPGs on both sides. [J Physiol Sci. 2008;58 Suppl:S195]

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Rats with neonatal hemidecortication exhibit fairy normal tactile placing reflex (Barth and Stanfield 1990) and reach-grasp movements (Takahashi et al. 2007) of their contralesional forelimb. The neuronal mechanism for the control of contralesional forelimb movements in these animals remains to be clarified. In the anterograde tracing experiments, we have shown that corticospinal fibers originating from the sensorimotor cortex of the remaining side issued aberrant collaterals to the contralesional spinal gray matter. The present study was designed to investigate whether the aberrant collaterals establish functional connections to contralesional forelimb motoneurons as well as ipsilesional motoneurons in the hemidecorticated rats. In the intact animals, electrical stimulation to the medullary pyramid induced no clear field response in the spinal gray matter nor EPSP/IPSP in forelimb motoneurons on the ipsilateral side to the stimulus. In the hemidecorticated rats, in contrast, stimulation of the remaining pyramid elicited negative field potential in the dorsal horn of bilateral cervical spinal cord. Oligosynaptic pyramidal excitation and/or inhibition were detected in intracellular recordings from motoneurons on both sides. The results indicated that pyramidal neurons in the remaining cortex could transmit signals to contralesional motoneurons via a few synapses. We will also describe the location of the intercalated neurons in the presentation. [J Physiol Sci. 2008;58 Suppl:S195]

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Convergence eye movement is slow adduction of both eyes normally elicited by an approaching visual target as a part of the near response (convergence eye movement, lens accommodation and pupillo-constriction), one of the cerebral reflex. While the most important cue for convergence eye movement is binocular disparity, convergence eye movement before target movements (anticipatory convergence) have been also reported in cats (Toda et al., 2006) and human (Kumar et al., 2002). In this study, we used unseen target to evoke and examine pupil reaction of anticipatory convergence in human. The subjects are nine healthy human (all male, 22 to 64 year-old, five are naiive to the purpose of the experiment). A video eye tracker (30fps) was used to record positions and diameters of both pupils. The visual targets were two LEDs at the front of the subject with distances of 114cm and 24cm. They were alternatively turned on and off in a regular timing (1.6 to 4Hz). The room was darken enough (under 1lx) to make both LEDs were unseen by the subject. Anticipatory saccadic convergence were found in all nine subject and differences between mean onset times of convergence eye movement and pupillo-constriction were significantly elongated in anticipatory convergence than in case of normal convergence. Reflecting functional segregation in the cerebral near response controllers, these results suggest that convergence eye movement system is more directly affected by higher centres corresponding to anticipation. [J Physiol Sci. 2008;58 Suppl:S196]

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The aim of this study was to test whether GABAergic receptors on neurons in dorsal nucleus of the lateral lemniscus (LLD) contribute to the control of swallowing. In acutely decerebrated cats (n=12), swallowing was induced by electrical stimulation (30–80 μA at 10Hz for 10–20 seconds with rectangular pulses of 0.2 ms duration) applied to the superior laryngeal nerve (SLN). Repetitive electrical stimulation (30–50 μA at 50Hz for 10–20 seconds) applied to the LLD facilitated the SLN-induced swallowing. Muscimol, a GABA_A receptor agonist, bicuculline, a GABA_A receptor antagonist, and baclofen, a GABA_B receptor agonist, were microinjected (0.10–0.15 μl, 5 mM) into the LLD through a stereotaxically placed glass micropipette. We observed that an injection of muscimol suppressed the SLN-induced swallowing. However, an injection of bicuculline facilitated the swallowing. An injection of baclofen did not alter the swallowing. Moreover, electrical stimulation applied to the substantia nigra pars reticulata (SNr), a basal ganglia output nucleus, suppressed the SLN-induced swallowing. The SNr-effect was further prevented by injection of bicuculline into the LLD. These results suggest that GABAergic projections from the basal ganglia to the brainstem modulate swallowing through the activation of GABA_A receptors on LLD neurons. [J Physiol Sci. 2008;58 Suppl:S196]

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A bolus of liquid food is usually held and processed in the oral cavity until swallowing. We investigated the influence of liquid bolus viscosity on the duration of the oral stage from food intake to swallowing, and also on the amplitude of the mylohyoid muscle EMG (MH-EMG) at the swallowing recorded by surface electrodes placed on the skin over the mylohyoid muscle, since the MH-EMG indicates the tongue activity when the palate is compressed by the tongue. Eleven healthy volunteers (mean age 25.9) participated. Three liquid samples (SS, starch syrup with a viscosity of 67 Pa s; H, honey with a viscosity of 7.8 Pa s; W, pure water with a viscosity of 0.9 mPa s) were used as test food. Subjects were instructed to swallow 1 ml of each of the three samples placed on the frontal part (F) or middle part (M) of the dorsal surface of the tongue with a syringe. When the samples were placed on the F, the duration of the oral stage was significantly longer for SS than for H and W. But no significant difference in the amplitude of MH-EMG at the swallowing was not obtained among three samples. When the samples were placed on the M, the duration of the oral stage was significantly longer for SS than for H, and the amplitude of MH-EMG at the swallowing was significantly larger for SS than for W. These findings suggest that the viscosity of liquid bolus has a significant effect on the duration of the oral stage, and also that the setting position of the liquid food on the tongue at the food intake has influence on the tongue activity at the swallowing. [J Physiol Sci. 2008;58 Suppl:S196]

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It is known that we can enhance the athletic performance with voluntary clenching of the teeth, but the relationship between the clenching pattern and motor activity is not fully understood. To investigate the effects of occlusal pattern on motor activity, we examined the effects of thickness of occlusal plate and occlusal contact point on the modulation of soleus H-reflex. The subjects were 12 male and 5 female (aged 18 to 35) with normal dentition. First we examined the stimulation threshold displaying the maximum amplitude of the H-reflex in each subjects, and performed two experiments; 1) the recording of the H-reflex with clenching of four different plate (thickness; 2, 4, 6, 8 mm), 2) the recording of the H-reflex with clenching at bilateral, unilateral and frontal side. There was no significant correlation between the amplitude of the H-reflex and the thickness of occlusal plate (p >0.05). The amplitude induced by clenching at habitual mastication side was larger than that by clenching at non-habitual mastication side and frontal side (p <0.01). And there was no significant difference between the amplitude by clenching bilateral side and that by clenching habitual mastication side (p >0.05). These results suggest that the equal bilateral clenching is important for stabilization of motor activity. [J Physiol Sci. 2008;58 Suppl:S196]

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The aim of present study is to clarify the neuronal mechanisms of an inhibition of swallowing reflex following capsaicin injection into the whisker pad region and masseter muscle in the anesthetized rats. We analyzed the number of swallowing reflexes induced by intraoral water application and the effect of capsaicin injection into the whisker pad region or master muscle (1,10,30mM) on occurrence of swallowing reflex was precisely analyzed. The pERK immunohistochemistry in the trigeminal spinal nuclei (Vi/Vc and Vc) and the nucleus of solitary tract (NTS) was also carried out in capsaicin-treated rats.In summary:1) The number of swallowing reflexes following capsaicin injection into the whisker pad region or masseter muscle was decreased in dose dependent manner. 2) The pERK-LI cells in Vi/Vc-Vc and NTS neurons were significantly higher in the rats with capsaicin treatment compared with vehicle-treated rats.3) The number of swallowing reflexes following capsaicin (10mM) injection was significantly increased folllowing PD98059 (MEK1/2inhibitor) intrathecal administration. 4) The pERK-LI cells were significantly decreased in PD98059-treated rats.These findings suggest that the ERK phosphorylation in Vi/Vc-Vc and NTS neurons would be involved in an inhibition of swallowing reflex following noxious stimulation of the facial skin or deep muscles. [J Physiol Sci. 2008;58 Suppl:S197]

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To investigate the organization of motor cortical inputs in the external (GPe) and internal (GPi) segments of the globus pallidus, we examined the responses of the GPe/GPi neurons to the stimulation of the face, arm and leg regions of the primary motor cortex (M1) and the arm and leg regions of the supplementary motor area (SMA) in a Taiwan monkey, and compared them with the responses in the subthalamic nucleus (STN). Attention was paid to the early excitation in the GPe/GPi evoked by cortical stimulation, because it is mediated by the cortico-STN projection. Neurons responding to M1 stimulation were located in the caudoventral part of the GPe/GPi, while neurons responding to SMA stimulation were found in the more dorsal and rostral part. These responsive neurons were arranged in a somatotopically organized manner. Some neurons (GPe, 35%; GPi, 20%) responded to both M1 and SMA stimulation. In the STN, neurons responding to M1 stimulation and those responding SMA stimulation were found in the dorsolateral and ventromedial parts of the STN respectively, and similar percentage of neurons (30%) responded to both stimulation. These observations suggest the somatotopically organized cortical inputs from the M1 and SMA in the GPe/GPi via the STN in spite of considerable convergent inputs from these cortical areas. [J Physiol Sci. 2008;58 Suppl:S197]

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We found the projection from the cortical masticatory area (CMA),which induced chewing-like jaw movement by repetitive electrical stimulation, to the primary motor cortex of the orofacial area (face-MI) using histologicall methods. However, the functional role of this neural connection is still unknown. The aim of this study is to understand the influence of this intracortical connection on elicitation of the rhythmic jaw movements induced by repetitive electrical stimulation to the CMA. Unanesthetized guinea pigs fixed on a stereotaxic apparatus were used. Jaw movements and masticatory muscles EMG activities were recorded. The stimulation electrode was fixed into the CMA where rhythmical jaw movements were evoked. We examined changes of elicitation of jaw movements before and after muscimol injection that inactivated neurons in the face-MI. After inactivation of the face-MI, the latency of cortically-induced jaw movement was prolonged with the same stimulus intensity. In some cases, rhythmic jaw movements were not evoked. On the next day, cortically-induced rhythmic jaw movements from the same site were the same as before muscimol injection. From these results, it is suggested that the intracortical neural connection from the CMA to the face-MI plays an important role on elicitation of rhythmical jaw movements evoked by repetitive electrical stimulation to the CMA. [J Physiol Sci. 2008;58 Suppl:S197]

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It has been shown previously that Renshaw cells (RCs) are strongly inhibited by sensory stimuli in the cat spinal cord (Wilson et al. J. Neurophysiol. 27, 1063-1079. 1963). However, much remains unknown about the physiological and pharmacological nature of synaptic inputs from spinal reflex pathways to RCs. We performed whole cell recordings from identified RCs in the lumbar ventral horn using isolated spinal cord preparations taken from GAD67-GFP-knock-in mouse neonates (as described in Nishimaru et al. PNAS. 102, 5245-5249, 2005) and examined the evoked responses to electrical stimulation of dorsal roots (DRs). In voltage clamp recordings, electrical stimulation of ipsilateral DR (intensity 1.5-3 times of threshold) of the segment in which the RCs soma is located, evoked a short latency (<10 ms) EPSC followed by a barrage of IPSCs. The evoked response was completely blocked by bath-application of CNQX and AP5 indicating that EPSCs are mediated by ionotropic glutamate receptors and IPSCs are polysynaptically elicited in the RCs. Both PSCs persisted in the presence of mecamylamine, a nicotinic receptor antagonist, indicating that they are unlikely to have derived from motor axon collaterals. Electrical stimulation of ipsilateral DRs in caudal segments and contralateral DRs only evoked long latency (>20 ms) IPSCs. These results indicate that RC activity is differentially modulated by different sources of sensory inputs in the developing mouse spinal cord. [J Physiol Sci. 2008;58 Suppl:S197]

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Daily restricted feeding (RF) with fixed time during daytime causes an advance of circadian rhythm of mouse liver clock gene expression. However, the mechanism of entrainment signal is poorly understood. Here, we examine whether daily administration of various type of nutrition caused entrainment of liver clock gene expression rhythm using Bmal1-luciferase transgenic mouse. Circadian rhythm change of liver bioluminescence was recorded through Lumicycle after 1-6 days of administration of various nutrients. We administered the corn carbon dehydrate, egg albumin or soybean oil after adjustment of each calorie. Among these nutrients soybean oil has most strong effect on phase-shift of gene expression rhythm. Administration of glucose through oral or intraperitoneal caused the phase advance, however the value of change was small. Thus, slow supply of calorie may be important to cause phase shift. We still examine the other nutrients such as amino acids, dextrin and sugar. We also try to find the effect of combination of nutrients on phase-shift of liver clock. [J Physiol Sci. 2008;58 Suppl:S198]

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Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii contains two FAD molecules. One FAD binds to the apoprotein relatively loosely so that it is released from the apoprotein in solution of high ionic strength e.g. 1 M KBr. The other FAD binds very tightly so that it is released only under denaturing conditions e.g. 6 M guanidine hydrochloride. In the absence of the loose FAD, the tight FAD shows normal flavin absorption spectrum, while the holoETF shows unusual spectrum: the valley at about 400 nm is very shallow. It seems likely that the loose FAD shows unusual flavin spectrum and the tight FAD shows normal spectrum in the holoETF. But there is another possibility that the loose FAD shows normal spectrum and the tight FAD shows unusual spectrum due to the environmental change caused by the binding of the loose FAD. To elucidate this subject, we prepared ETF which has 8-NH₂-FAD at the tight site and no FAD at the loose site by denaturation-renaturation method. 8-NH₂-FAD shows largely different spectrum from FAD, so that the spectral change due to the environmental change should be different from that of FAD. In fact, the spectral change upon binding of loose FAD to the ETF with tight 8-NH₂-FAD was strictly the same as that to the ETF with tight FAD. This result indicates that the loose FAD not the tight FAD shows the unusual spectrum in the holoETF. [J Physiol Sci. 2008;58 Suppl:S198]

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In order to measure the diffusion coefficients the recovery time of static magnetic field was evaluated to be less than 1 ms after turning-off of the gradient pulse at the maximum intensity 30 gauss/cm. Then an NMR method was developed to measure simultaneously the diffusion coefficients of several different kinds of simple liquids. Each liquid sample was kept in a different small container arrayed along one axis. The reading gradient was applied along the axis to encode the location of the samples in the signals. The spin echo pulse sequence was used and a pair of the motion-probing gradient was placed before and after the 180 degree pulse. We could get the diffusion coefficient values of acetone, water, methanol, and DMSO, in order according to larger to smaller values. The diffusion MRI study was conducted on a brain sample isolated from a rat and kept in a formalin solution. The best resolution image was about 0.1 mm resolution with the slice thickness 0.5 mm. On the diffusion-weighted images the spin echo signals were attenuated faster on the water molecules having the larger value of diffusion coefficient, such as those in the formaldehyde solution. It made us possible to obtain the brain images on the sample kept in the solution when the larger motion probing gradients were used. [J Physiol Sci. 2008;58 Suppl:S198]

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We have maeasured the orientation of the collagen fibers in articular cartilage by using H-2 double-quantum-filtered (DQF) spectroscopic imaging that depicts exclusively water interacting with oriented structures. Cartilage and bone plugs 7-8 mm in diameter were obtained from fresh bovine femoral lateral and medial condoyles. The size and orientational dependency of the quadrupolar splitting allows us to estimate the oriented structure of the collagen fibers. In the absence of load, a large quadrupolar splitting is observed in the calcified zone (1320 ± 230 Hz) and a smaller splitting in the radial zone (450 ± 80 Hz). The splitting disappears in the transitional zone and reappears in the superficial zone (160 ± 40 Hz). When the cartilage was rotated by 90 degree and was set its surface parallel to the static magnetic field, the splitting of the calcified and deep layers are halved while the splitting of the surface layer is increased. This is an indication that the fibers align perpendicular to the bone at the deep layer and parallel at the surface. Under static loads, the increase of the splitting at the surface and the thickening of this zone continues which indicate flattening of the collagen fibers near the surface. On the other hand, the splitting at the deep layer completely collapses that may be a result of the crimping of the collagen fibers in this layer. [J Physiol Sci. 2008;58 Suppl:S198]

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Equivalent cross-relaxation rate (ECR) imaging (ECRI) is a measurement method that can be used to quantitatively evaluate a change in the structural organization by MRI. The aim of this study is to evaluate utility of ECRI as an in vivo cell functional imaging for living tissues such as liver tissue. Fifteen patients with hepatocellular carcinoma were studied. We adopted the off-resonance technique for preferential saturation of the immobile protons to evaluate the ECR values. The single saturation transfer pulse frequency was employed at the frequency 7 ppm downfield from the water resonance. The ECR value was defined as the percentage of signal loss between unsaturated and saturated images. The ECRI were constructed on the basis of the percentage of ECR. The ECR value measured the whole liver of non-cancer part without hepatocellular carcinoma. We examined a correlation between ECR value and liver function tests such as biochemical tests. The normal organization showed higher ECR value. The liver with hepatitis showed lower ECR value. The liver with liver cirrhosis showed lowest ECR value. A statistically significant difference was obtained the ECR values of each tissue (p < 0.05). Among liver biochemical tests, values for AST, ALT, ChE, albumin, total bilirubin, platelet and ICG were closely related to ECR values (correlation coefficient = 0.57, 0.47, 0.63, 0.68, 0.58, 0.71 and 0.69, respectively). Therefore, ECRI is a potentially useful method for liver function by MRI. [J Physiol Sci. 2008;58 Suppl:S199]

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We previously reported that rat adrenal chromaffin cells contained GABA which may play a paracrine or autocrine role. The fractional study of bovine adrenal medullae indicates that GABA is recovered in fractions with low density (corresponding to 10-15% sucrose) but not those with high density (50-65%), whereas dopamine-β-hydroxylase was detected in both fractions, suggesting that chromaffin granules are heterogeneous in terms of GABA contents. In the present study, this notion was morphologically examined in rat chromaffin cells and PC12 cells by immunocytochemical methods and immunoelectron microscopy. Immunocytochemistry with anti-vesicular GABA transporter (VGAT) antibodies showed dot-like staining in the cytoplasm, which was partially co-localized with chromogranin A-like immunoreactivity, a marker of chromaffin granule. Synaptophysin-like immunoreactivity, a marker of synaptic-like vesicle, was also detected in the cytoplasm and it did not seem to be co-localized with VGAT or chromogranin A-like immunoreactivity. The immunostaining of endogenous VGAT in PC12 cells also partly coincided with that of chromogranin A, but not that of synaptophysin. These results demonstrate that in adrenal chromaffin cell, GABA is accumulated in chromaffin granules but not in synaptic-like vesicles and support that chromaffin granules are heterogeneous: one group of granules contains GABA; the other does not. Fine localization of VGAT in chromaffin cells by immunoelectron microscopy will be shown and more details of GABA-containing vesicles will be discussed. [J Physiol Sci. 2008;58 Suppl:S199]

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Immature cerebellar granule cells (CGCs) express GABA_A receptors in the external granular layer (EGL).However its function has not still been well understood.Evaluation of spatio-temporal distribution of GABA in the developing cerebellum, serve us to clarify the role of GABA_A receptors in EGL.To this end, we have developed the novel method to image the extracellular GABA in acute slices with an enzyme-linked assay system using GABase, which catabolize with simultaneous production of NADPH. This imaging enables us to examine the distribution of extracellular GABA.Intense NADPH fluorescence was observed in the upper part of EGL at P3, whereas it was restricted around the GABAergic Purkinje cells at P8.Whole cell patch-clamp recordings from CGCs in EGL confirmed that CGCs could tonically respond to the extracellular GABA.To evaluate the mechanism of increases in extracellular GABA in EGL, we examined the expression pattern the vesicular GABA transporter (VGAT), which is involved in vesicular GABA release, by immnohistochemistry. We found that VGAT was expressed on Bergman glial fibers in EGL at P3.Taken together, our results suggest that the temporal release in extracellular GABA in EGL, it might be regulated by VGAT. [J Physiol Sci. 2008;58 Suppl:S199]

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Parathyroid cells directly monitor extracellular Ca²⁺ concentration using Ca receptors (CaR). In isolated frog parathyroid cells, high extracellular Ca²⁺ concentration induces a large increase in chloride conductance. In the present study, we investigated how extracellular Ca²⁺ increases parathyroid chloride conductance while recording the whole cell current. High extracellular Ca²⁺ induced an increase of the inward current at -50 mV in a dose-dependent manner. The apparent EC₅₀ for Ca²⁺ was about 6 mM. When either U73122 (an inhibitor of PLC), tetrahydrolipstatin (an inhibitor of DAG lipase), methyl arachidonyl fluorophosphonate (MAFP, an inhibitor of MAG lipase) or eicosateraynoic acid (ETYA, an inhibitor of lipoxygenase) was applied extracellularly, the magnitude of extracellular Ca²⁺-induced current decreased significantly. External MS-PPOH (an inhibitor of epoxygenase) did not affect the current response. Internal dialysis of 2-arachidonyl glycerol (2-AG) induced a Gd³⁺-sensitive inward current, but internal 2-AG ether did not elicit any response. Internal dialysis of arachidonic acid also induced ETYA-sensitive inward current. The results suggest that the arachidonic acid metabolite is involved in the extracellular Ca²⁺-sensing of frog parathyroid cells. [J Physiol Sci. 2008;58 Suppl:S199]

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In fertilized mammalian eggs, long-lasting Ca²⁺ oscillations are generated by repetitive Ca²⁺ releases from intracellular Ca²⁺ stores through the IP₃ receptor/Ca²⁺ channel. Although Ca²⁺ entry from outside is also required to maintain Ca²⁺ oscillations, the regulatory mechanism of the Ca²⁺ influx pathway is poorly understood. In the present study, we tested the inhibitory effect of several Ca²⁺ channel blockers on the sperm-induced Ca²⁺ responses in mouse eggs, in order to characterize pharmacologically the Ca²⁺ entry channel responsible for the maintenance of Ca²⁺ oscillations. The Ca²⁺ oscillations were inhibited by Gd³⁺ or La³⁺ at millimolar concentrations, whereas Ni²⁺, Co²⁺ and SKF96365, an agent known to nonspecifically inhibit store-operated channels, were ineffective at all concentrations tested. The capacitative Ca²⁺ entry (CCE) which was activated in the eggs treated with thapsigargin and measured by the Mn²⁺ quenching technique, was inhibited by Gd³⁺ and La³⁺ at similar concentrations. The CCE was inhibited by SKF96365 as well, contrasting to Ca²⁺ oscillations. The difference in the pharmacological profiles suggested that the Ca²⁺ entry through the pathway other than CCE may be responsible for the refilling of Ca²⁺ stores and the maintenance of Ca²⁺ oscillations in the fertilized egg. [J Physiol Sci. 2008;58 Suppl:S200]

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Previous studies on the nitric oxide (NO) increase at fertilization in sea urchin eggs indicate that the NO increase is Ca²⁺-dependent and attributed to the late Ca²⁺ rise. However, its role in fertilization still remains unclear. To investigate whether the NO increase is related to any ionic changes we made, simultaneous measurements of the activation current, by a single electrode voltage clamp, and NO, using the NO indicator, DAF-FM. Results demonstrated the NO increase occurred at the time of peak current (t_p) which corresponds to peak [Ca²⁺]_i, suggesting that NO has no relation to any other ionic changes besides [Ca²⁺]_i. To ascertain the role of the NO increase, a NO scavenger, PTIO was used to eliminate the NO increase. We attempted to measure O₂ consumption by a polarographic method to examine whether NO regulated a respiratory burst for protection as reported in other biological systems. However, measuring O₂ consumption resulted in an inconclusive output. To explore further, autofluorescence of reduced pyridine nucleotides, NAD(P)H, which is related to O₂ consumption, was measured in controls and when the NO increase was eliminated by PTIO. Surprisingly, PTIO decreased the rate of the fluorescence change in NAD(P)H and the late phase of increase in NAD(P)H was eliminated. PTIO also suppressed the production of H₂O₂ and caused high and weak fertilization envelope (FE). These results suggest that NO increase upregulates NAD(P)H and consolidates FE hardening by H₂O₂. [J Physiol Sci. 2008;58 Suppl:S200]

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Hyposecretion of saliva and consequent dry mouth lead to severe caries and periodontal disease. Therapeutic radiation for head and neck cancer and sialadenitis result in atrophy and fibrosis of salivary glands, but the mechanism is not clear. As a model for dysfunction of salivary glands, we examined the change of gene expression in primary cultured parotid acinar cells. Acinar cells were isolated from rat parotid glands, and were cultured in the medium containing rat serum. Cells attached to the dishes and most of them spread as a monolayer after culture for 48 h. The expression pattern of differentiation markers were changed during the culture. Expression levels of amylase and aquaporin 5, markers of acinar cells, decreased, and cytokeratin 14 and vimentin increased. Claudin-4 and claudin-6, which are expressed in embryonic duct cells, but not in acinar cells, began to be expressed. Addition of Src kinase inhibitor PP1 or PP2 suppressed these changes, suggesting that inhibition of Src kinases suppressed dedifferentiation of acinar cells. PP1 did not affect the expression levels of E-cadherin and occludin. Immunofluorescence microscopy showed that tight junctions were formed both in the presence or absence of PP1. The immunofluorescence signals for claudin-3 and claudin-4 were observed in both cultures, but the intensities of signals were different. These results suggest that inhibition of Src family kinases changes expression pattern of claudins and may influence the paracellular permeability. [J Physiol Sci. 2008;58 Suppl:S200]

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The effect of the immunosuppressant drug FK506 on microsomal Ca²⁺ release was investigated in rat pancreatic acinar cells. When FK506 (0.1-200 μM) was added to pancreatic microsomal vesicles at a steady state of ⁴⁵Ca²⁺ uptake, FK506 caused a dose-dependent and a biphasic release of ⁴⁵Ca²⁺. Almost 10% of total ⁴⁵Ca²⁺ uptake was released at FK506 concentrations up to 10 μM (K_m = 0.5 μM), and 60% of total ⁴⁵Ca²⁺ uptake was released at FK506 concentrations over 10 μM (K_m = 55 μM). The presence of cyclic ADP-ribose (0.5 μM), which is known to modulate the ryanodine receptor (RyR), increased the FK506 (< 10 μM)-induced ⁴⁵Ca²⁺ release. The presence of heparin (200 μg/ml), an inhibitor of the inositol 1,4,5-trisphosphate receptor (IP₃R), resulted in significant inhibition of the FK506 (30 μM)-induced ⁴⁵Ca²⁺ release. These results indicate that two types of FK506-induced Ca²⁺ release mechanisms operate in the endoplasmic reticulum of rat pancreatic acinar cells: a high-affinity mechanism of Ca²⁺ release, which involves activation of the RyR, and a low-affinity mechanism of Ca²⁺ release, which involves activation of the IP₃R. FK506 is known to modulate the RyR or the IP₃R by dissociating FK506-binding protein (FKBP) from the receptor. To determine the involvement of FKBP in the FK506-induced ⁴⁵Ca²⁺ release, the effect of an FKBP antibody on the release was investigated. [J Physiol Sci. 2008;58 Suppl:S200]

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It has been shown that ghrelin is produced in stomach and pancreatic islets, and serves as a physiological regulator of insulin release. However, the ghrelin's signaling mechanisms in islet β-cells downstream of its receptor, growth hormone (GH) secretagogue-receptor (GHS-R), are largely unknown. This study aimed to explore the signaling mechanisms for insulinostatic ghrelin in islet β-cells, with special attention to heterotrimeric G-proteins and K⁺ channels. In rats, systemic ghrelin administration decreased plasma insulin concentrations and this effect was blocked by treatment with pertussis toxin (PTX), whereas stimulation of GH release was unaffected by PTX. In isolated islets of rats, GHS-R antagonist markedly increased glucose-induced insulin release, while exogenous ghrelin suppressed it, both in a PTX-sensitive manner. Ghrelin increased voltage-dependent K⁺ (Kv) currents without affecting ATP-sensitive K⁺ channels in β-cells of rats, and this effect was abolished after PTX treatment. Ghrelin also attenuated glucose-induced action potentials and cytosolic Ca²⁺ increases. The effects of ghrelin on glucose-induced cytosolic Ca²⁺ increase and insulin release were blunted in the β-cells treated with PTX and with antisense oligonucleotide specific for Gα_i2 subunit of heterotrimeric G-proteins. These findings demonstrate that ghrelin attenuates glucose-induced insulin release via PTX-sensitive Gα_i2-mediated activation of Kv channels in β-cells. [J Physiol Sci. 2008;58 Suppl:S201]

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Cell proliferation and migration of vascular smooth muscle (VSM) cells in diabetes mellitus (DM) is promoted possibly by 1) autonomous activation of their stimulatory signal transductions, 2) autonomous inhibition of their inhibitory signal transductions, 3) autocrine stimulation by growth factors or 4) enhanced responsiveness to growth factors. To answer this question, cell proliferation and migration of cultured aortic VSM cells derived from type 2 DM model OLETF rats and from normal LETO rats (OVSMC and LVSMC) was compared in different culture condition.: serum-free DMEM (SFM), 10% FBS-DMEM or SFM containing platelet-derived growth factor-BB (SFM-PDGF). In addition, we examined whether elevation of intracellular cAMP by olprinone hydrochloride (OPN), an inhibitor for type 3 phosphodiesterase (PDE), inhibits cell proliferation and migration. As compared with LVSMC, cell proliferation and migration of OVSMC were significantly increased in SFM and 10%FBS-DMEM, but not in SFM-PDGF. Exposure of OVSMC and LVSMC to OPN (1 mM), dibutyryl-cAMP (3 mM) or dibutyryl-cGMP (3 mM) for 72 hr significantly inhibited PDGF-BB (2 nM)-induced cell proliferation and migration. Intracellular cAMP was significantly increased by OPN in both OVSMC and LVSMC. In conclusion, cell proliferation and migration of VSM cells in type 2 DM might be autonomously enhanced, which could be regulated by elevation of intracellular cAMP and cGMP. [J Physiol Sci. 2008;58 Suppl:S201]

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We previously reported that lidocaine at lower concentrations (1-100 μM) inhibits axonal transport. Here we examined the involvement of Ca²⁺ channels in the lidocaine-induced inhibition of axonal transport in cultured mouse DRG neurons. Clinically relevant concentration of lidocaine (37 mM) was also investigated. Axonal transport of organelles and neurite growth were assessed by video microscopy. Cell membrane disruption was evaluated by trypan blue staining. Duration of experiments was 30 min. Lidocaine (100 μM-37 mM) inhibited axonal transport in a time- and concentration-dependent manner. Incubation in Ca²⁺-free medium blocked the inhibition of axonal transport induced by 100 μM lidocaine, but not by 37 mM lidocaine. The action of lidocaine at 100 μM was blocked by N-type Ca²⁺ channel blocker ω-conotoxin MVIIA, but not by L-type channel blocker nifedipine. These effects on axonal transport correlated with the effects on neurite growth. The percentage of trypan blue-stained cells increased during treatment with 37 mM lidocaine, which was not prevented by Ca²⁺-free medium or ω-conotoxin MVIIA. 100 μM lidocaine had no effect. These results suggest that inhibition of axonal transport induced by lower concentration of lidocaine is possibly mediated by Ca²⁺ influx through N-type channels. However, higher concentration of lidocaine may disrupt cell membrane and then causes the arrest of axonal transport. In addition, when ω-conotoxin is used as an analgesic in combination with lidocaine, ω-conotoxin may reduce neurotoxicity of lidocaine. [J Physiol Sci. 2008;58 Suppl:S201]

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D-Allose, the C-3 epimer of D-glucose, is one of the rare sugars which occurs rarely in nature. D-Allose was shown to have the most potent inhibitory effect among all rare hexoses on cell proliferation of various human cancer cells. In particular, various lymphoma cell lines have been elucidated to have different susceptibility to D-allose. In other words, the proliferation of some human leukemia cell lines was significantly inhibited by D-allose, and some were not. We chose 5 leukemia cell lines, and found that MOLT-4F has the highest susceptibility to D-allose. Then, we investigated the effect of D-allose on MOLT-4F and Daudi cells which possess the highest and lowest sensitivity to D-allose, respectively. We examined three points: (i) alteration of the D-glucose uptake by D-allose; (ii) incorporation of D-allose into cells; (iii) protein or gene expression altered by D-allose. We found that D-glucose utilization was significantly inhibited by D-allose. However the addition of excess amount of D-glucose in the medium could not abolish the inhibition. These results were suggested the important role of this protein in the effect of D-allose. We found that TXNIP gene expression and its protein expression were specifically and markedly enhanced in MOLT-4F cells by D-allose. This may explain the susceptibility of MOLF-4F cells to D-allose. [J Physiol Sci. 2008;58 Suppl:S201]

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Natural killer (NK) cells play a key role in inflammation and tumor regression through their ability to migrate into tissues. CXCL12 is a chemokine that promotes lymphocyte invasion and migration into tissues; however, the mechanism for this process remains incompletely understood. We investigated that localization of MMP-1 and α2-Integrin on the surface of NK3.3 cells stimulated by CXCL12. CXCL12 enhanced the invasion into type I collagen on NK3.3 cells. GM6001 (a brode synthetic MMP inhibitor), TIMP-2 (recombinant tissue inhibitor of metalloproteinases) and Pertussis toxin (PTX: a smoll Gi protein inhibitor) significantly inhibited CXCL12-stimulated enhanced invasion. When NK cells were cultured in the presence of CXCL12, the production of MMP-1 was significantly enhanced compared with the unstimulated NK cells. To localize MMP-1 and α2 integrin subunit on NK-cell surface, NK cells cultured in the presence or absence of CXCL12 were stained with anti-MMP-1 antibody and anti-α2 integrin antibody. Although unstimulated NK cells expressed the majority of the MMP-1 colocalized with α2 integrin subunit around cell surface, stimulation with CXCL12 enhanced MMP-1 aggregation and colocalization with α2 integrin subunit on NK-cell surface. PTX significantly inhibited this aggregation.These results indicate that the CXCL12-stimulated NK cell invated into type I collagen, and this invasion involved in the aggregation of MMP-1 and α2 integrin subunit on NK-cell surface. [J Physiol Sci. 2008;58 Suppl:S202]

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We reported previously that allograft-induced macrophages (AIM) in C57BL/6 (H-2D^bK^b) mice were essential for the rejection of H-2D^dK^d allografts including BALB/c skin and Meth A tumor. Recently, we isolated a cDNA encoding a novel receptor on AIM for the H-2K^d molecule by the expression cloning method using an anti-AIM monoclonal antibody (R12) and H-2K^d tetramer and named it macrophage MHC receptor 2 (MMR2). To know the physiological roles of MMR2, we are trying to generate mouse strains deficient in MMR2 by homologous recombination. In the present study, a targeting plasmid containing a neomycin (neo^r) /thymidine kinase cassette in the MMR2 locus was constructed using genomic DNA fragments derived from Sv129 mouse strain; and the targeting plasmid was introduced into mouse ES cells. To detect homologous recombination and the presence of the neo^r allele, 168 ES clones were screened by PCR analysis of genomic DNA. Among them 4 clones were detected as homologous recombination products in the 3′ locus of MMR2 gene. The recombination of 5′ locus is under investigation. [J Physiol Sci. 2008;58 Suppl:S202]

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Urokinase plasminogen activator (uPA) plays vital roles in various biological processes involving extracellular proteolysis, fibrinolysis, cell migration, proliferation and angiogenesis. Binding of uPA to its receptor uPAR initiates a proteolytic cascade that results in conversion of plasminogen to plasmin. Plasmin, through its own proteolytic function degrades a range of extracellular basement membrane components and activates others such as matrix metalloproteinases. uPA expression can be regulated by several internal as well as external agents. This study focuses on the regulation of uPA gene expression by the polyphenol, curcumin. Curcumin is an active constituent of the rhizome, turmeric that has been used since ancient times to treat skin wounds. As uPA plays a vital role in wound healing processes, we studied the effect of curcumin on uPA expression. Treatment of human fibroblast cells with curcumin caused an upregulation of uPA expression, both at the RNA and protein levels, in a dose-dependent manner. The increase in uPA expression enhanced both fibrinolytic activity as well as rate of cellular migration towards wound region, thus accelerating wound healing. Silencing of uPA by siRNA technique attenuated the cellular migration and wound healing rates. Our results show that curcumin enhances wound healing by upregulating uPA expression. [J Physiol Sci. 2008;58 Suppl:S202]

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3T3-L1 cell line is a well-established and commonly utilized in vitro model to assess adipocyte differentiation. Cell migration of 3T3-L1 preadipocytes was investigated using Dunn chemotaxis assay glass chamber system. Preadipocytes adhered to the surface of the glass and migrated randomly even in the normal Krebs Ringer bicarbonate-hepes solution (pH7.4). When the cells were placed in a concentration gradient of ATP for 60 min at 37°C, migration towards the source of ATP (chemotaxis) was observed with the maximal migration of 80 μm. In contrast, fully differentiated mature adipocytes tightly adhered to the extracellular matrix and appeared to be almost immobilized during the incubation. These results indicate that the extracellular ATP or other possible ligands can act as a chemoattractant for preadipocytes, but not for mature adipocytes. We next tried to use a piece of isolated mouse mesenterium as an extracellular matrix to examine whether chemotaxis activity of preadipocyte was observed on the surface of mouse tissue. 3T3-L1 preadipocytes well adhered to the mesenterium within 60 min and migrated on the membrane. These observations suggest the possibility that preadipocytes translocate their body to the different place from their origin in vitro. [J Physiol Sci. 2008;58 Suppl:S202]

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Calcineurin/NFAT pathway plays important roles in various tissues such as cardiac muscle, neuron and immune systems. Recent studies have shown that the pathway is also involved in keratinocyte growth/differentiation. However, little is known the mechanism in the regulation. In the present study, we showed that a novel splicing variant form of calcineurin A-beta, which was deficient in the auto-inhibitory domain, was predominantly expressed in keratinocytes and had Ca²⁺-independently its activity in the cells. Cyclin G2 was identified as a new target of calcineurin/NFAT pathway in the regulation of the growth of follicular keratinocytes. A cell-permeable NFAT translocation inhibitor (11R-VIVIT) inhibited nuclear import of NFAT1 and consequently reduced the expression of cyclin G2, resulting in induction of hair growth in nude mice. Finally, overexpression of cyclin G2 inhibited 11R-VIVIT-induced keratinocyte growth and hair growth both in vitro and in vivo. [J Physiol Sci. 2008;58 Suppl:S203]

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Enamel is generated by ameloblast derived from dental epithelium and enamel formation requires reciprocal epithelial-mesenchymal interaction. TGF-β1 is one of the signaling molecules known to regulate the cross talk between dental epithelia and mesenchymal cells. However, TGF-β1 effect on the process of enamel formation is little known. We investigated the role of TGF-β1 on calcified extracellular matrix (ECM) by using primary amaloblast culture system. Dental epithelial cells from the area that contained cells of the ameloblast-lineage were prepared from lower incisors of 8-day-old ICR mice. They were divided into 4 groups and seeded onto the collagen-coated culture dishes (10⁴-10⁵ cells/mL) in HuMedia-KG2 (Kurabo Ind. Ltd.) medium. After the cells were grown to 100% confluency, we added TGF-β1 and CaCl₂ to each group, as follows, 1) control, 2) TGF-β1, 3) CaCl₂, and 4) TGF-β1 and CaCl₂. After experiments for 10-14 days, we examined for the calcification of ECM by alizarin staining and stimultaneously isolated mRNA from each group to investigate for the alkaline phosphatase (ALP) activity. As a result, group 3 (with CaCl₂) was the most staining by alizarin. Group 4 (with TGF-β1 and CaCl₂) was weaker staining than group 3, whereas group 1 (control) and 2 (with TGF-β1) were not stained. For the activity of ALP, we confirmed stronger expression in group 3 than in group 1 and 4, while ALP expression of group 2 was the most light. These results indicated that TGF-β1 suppressed the ameloblast-formed calcification of ECM. [J Physiol Sci. 2008;58 Suppl:S203]

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