To investigate the organization of olfactory information in the anterior piriform cortex (aPC) of the guinea pig, we obtained high resolution mapping of odor-induced activation by optical imaging of intrinsic signals. One of possible codes for odor concentrations was shown in the aPC. Amyl acetate, butyraldehyde, butyric alcohol, ether and xylene were used as odor stimuli. Odor-induced cortical activation, which primarily originated in layer II, appeared in a narrow band beneath the rhinal sulcus over the lateral olfactory tract, corresponding to the dorsal part of the aPC (aPCd). Lower concentrations of odorants activated the rostral region of the aPCd, whereas higher ones induced caudally spreading activation, and the site at which neural activation reached its maximum extent depended upon odor concentration. Furthermore, the activated total area in the aPCd increased in proportion to concentrations raised to a power of 0.5 to 0.8. Unit recordings from the aPCd indicated a rostro-caudal gradient in odor-threshold among cortical neurons. These results suggest that the dorsal part of the anterior piriform cortex may be associated with odor concentration. A rostro-caudal gradient in axonal projections from mitral/tufted cells and/or in association fibers may play a key role in odor-concentration coding in the olfactory cortex. [Jpn J Physiol 54 Suppl:S19 (2004)]

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Several gustatory cortices have been identified in macaque monkeys: primary gustatory cortices (PGC: areas G and 3), adjacent cortices (area 1-2 and insula), and higher-order gustatory cortices (HGC) including the precentral opercular (PrCO), orbitofrontal opercular (OFO), and orbitofrontal cortices (areas 12 and 13). To investigate coding mechanism of taste perception, we examined neural activities in the PGC and HGC in the monkey during a salt-water discrimination GO/NOGO task. Most neurons differentially responded to two cues, salt and water. In the reaction time task, the onset latency in the exposed frontal operculum (area 3 and the PrCO) was distributed with two modes, each at short and long latencies, but neurons in the buried part (area G and insula), OFO, and orbitofrontal cortices had long latencies, suggesting different roles of two cortical groups in the task. Analysis in the incidental incorrect trials revealed that some neurons in the HGC yielded activities related to subsequent behavioral responses. In the delayed task and its reversal, the PGC almost exclusively contained neurons signaling the physicochemical nature of the cues and the HGC contained neurons related to the subsequent behavior, attention or short-term memory, as well. Thus, it is indicated that the PGC processes pure gustatory information, whereas the HGC are involved in gustatory perception and other higher-order function. [Jpn J Physiol 54 Suppl:S19 (2004)]

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Analyses of experimental tasks for odorant stimulation and responses in higher center of olfaction, Life Electronics Laboratory, Kansai-center, AIST, Japan.
The present study is to develop the suitable odorant stimulation and to obtain the olfactory active areas using magnetoencephalography(MEG) non-invasively and to clear the first order olfactory center and the more higher olfactory center. The olfactory magnetic fields were measured by a 122-ch whole-cortex biomagnetometer with three different stimulation tasks, individually. In the first experiment the evoked response(latency: 300-400ms) were obtained using the odor stimulation synchronized with subject's respiration and active areas were estimated in the bilateral orbito-frontal areas which were coincident with regions found as the higher olfactory center in monkey. In our second experiment, we applied an odd-ball paradigm using two odors by the different present rates and obtained the more later component such as cognitive response in a few superior temporal or near the insular areas. In the third experiment, we tried to study active olfaction by self sniffing odor. Right hemisphere dominance was found in the orbito-frontal area and the attentive olfactory activity might be emphasized preliminary. These three experimental tasks and results suggest us the idea of mechanism on the higher order olfactory information processing in human. [Jpn J Physiol 54 Suppl:S19 (2004)]

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The gustatory-related regions of the cerebral cortex in human beings have been the issue of argument for a long time since Penfield and Boldrey (1937). Recent development of imaging techniques, however, has yielded various non-invasive methods, e.g., fMRI, PET and MEG, and allows us to measure the cerebral activities of living human subjects without surgical invasion. Among them, MEG has good temporal resolution, and can give a good estimation of the location of the activity much more precisely than EEG, because the magnetic field generated from living brain is free from distortion by the skull. MEG is, therefore, the most appropriate method to investigate the primary gustatory cortex in human.
We investigated the primary gustatory cortex (PGC) using a tactile-free taste delivery system and MEG, and found the activation at the transition between the parietal operculum and insula with the shortest latency. The ventral end of central sulcus was also activated with almost the same latency in the case of NaCl stimulation. The average latencies of activation were about 120 ms for NaCl and 220 ms for saccharin. In the range of 30 mM to 1M NaCl, the magnitude of ECD in the PGC increased significantly in a concentration-dependent manner. The cortical activation for saccharin was suppressed by gymnemate.
These results suggest that the transition area between the parietal operculum and the insula and the ventral end of the central sulcus are the PGC in human, which is posterior to the PGC in subhuman primates. [Jpn J Physiol 54 Suppl:S20 (2004)]

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MscL is the mechanosensitive channel that is ubiquitously found among bacteria. Since MscL open by membrane tension in its purified form, MscL probably senses the tension of membrane directly. Cloning MscL gene revealed that MscL consists of 136 amino acid monomer. Crystal structure of MscL showed that MscL is a homopentamer of subunit that has two transmembrane domain, M1 and M2. The clue to the interaction that stabilizes the closed channel or causes channel opening has been obtained through random and site-directed mutagenesis study. Isolation of mutant channel that opens with little tension showed that mutations in glycines of M1 decrease the threshold. One-by-one substitutions of Gly-22, which resides within channel pore constriction, with all other amino acid indicate that hydrophobic interaction within constriction keep the channel in the closed state. Thus, the exposure of the hydrophobic residues in the constriction probably serves as the energy barrier that should be overcome by membrane tension during gating. On the other hand, mutagenesis study of the residues in the lipid-protein interface showed that the hydrophilic substitution at the periplasmic ends of M1 or M2 causes loss of function. This finding suggests that the residues at the periplasmic ends perceive the negative pressure from the membrane through hydrophobic interaction. Thus, pulling the periplasmic ends of M1 apart from the molecular fivefold axis while holding the cytoplasmic ends of M1 together may cause the tilting of M1 and initiate the channel opening. [Jpn J Physiol 54 Suppl:S20 (2004)]

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Toward understanding physiological events based on ion channel molecules, we systematically surveyed ion channel genes from the recently revealed genome sequence of a tunicate, Ciona intestinalis, which belongs to the protochordates. We found that Ciona contains most sets of voltage-gated channels and transmitter-gated channels that are involved in neuronal signaling. On the other hand, Ciona lacks most of auxiliary subunits such as Nav-beta subunits and MiniK that are important for fine tuning of excitability. Ciona also lacks many channel genes related to sophisticated epithelial ion transport and pain sensation, probably reflecting that the nervous system of noiception and kidney are missing in the protochordate. We also noted that Ciona genome showed unexpectedly large gene diversity in the channel species that are closely related to homeostatic control of ion concentration and body fluid. These include connexins, amiloride-sensitive cation channels, Cl channels and Trp-like channels. We also discovered a novel family of putative membrane proteins that show voltage-dependent gating but no ionic currents. We present that one of these, called CiVsp, contains both modules of voltage sensor and enzyme connected by a short linker sequence, and it exhibits voltage-sensitive modification of enzyme activity through coupling between the two domains in the single molecule. This is the first report of single membrane protein which can transduce electrical signal into chemical signal without requiring ionic flow through membrane. [Jpn J Physiol 54 Suppl:S20 (2004)]

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The vertebrate hindbrain is a segmented structure. The teleost hindbrain contains reticulospinal (RS) neurons that are periodically arranged along the neuraxis. RS neurons sharing a common morphology in adjacent segments are referred to as segmental homologs. The Mauthner (M) cell in the fourth segment (S4) is known to trigger fast escape. MiD2cm in S5 and MiD3cm in S6, serial homologs of the M-cell, are predicted to contribute to this behavior. To investigate whether these homologs are organized as a functional unit for sound-induced escape, we studied the afferent projection from auditory nerve (VIIIn), output firing properties of these neurons and physiological connections among them in adult goldfish. Labeling with different fluorescence tracers showed that the VIIIn projected to nearby the S4-S6 RS neurons. Tone burst and electrical stimulation of VIIIn evoked EPSPs in them. Stepwise depolarization elicited a spike at the onset in the M-cell, but repetitive firing in MiD2cm and MiD3cm. The atypical firing property of M-cell was mediated by dendrotoxin (DTX)-I-sensitive, voltage-gated potassium channels together with recurrent inhibition. The M-cell, but not MiD2cm or MiD3cm, expressed Kv1.2, a DTX-I-sensitive potassium channel subunit. Presynaptic activation of M-cell produced disyaptic IPSPs in MiD2cm and MiD3cm, suggesting inhibitory connection from the M-cell to them. Together, the M-cell and its segmental homologs may sense common auditory information but send different outputs to the spinal circuits and make a hierarchical organization to control adaptive escape behavior. [Jpn J Physiol 54 Suppl:S21 (2004)]

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Neuronal networks generating coordinated rhythmic motor activity such as locomotion are already functional at birth in the mouse spinal cord, suggesting that these neuronal networks develop during prenatal stages. Indeed, as early as embryonic day (E)12.5, periodic spontaneous motor activity could be observed in the mouse fetus (Suzue (1996) Neurosci. Lett. 218:131-134). We examined the neuronal mechanisms of rhythmic motor activity during early fetal period, using isolated spinal cord preparations taken from mouse fetuses at E13. In these preparations, periodic spontaneous bursts (PSB) recorded from lumbar ventral roots were abolished by simultaneous blockade of glutamate, glycine and GABA_A receptors, while they were not abolished by blockade of glutamate receptors, a excitatory device in the matured neuronal network. Bath-application of glycine or GABA evoked burst activity in the ventral roots, suggesting that these neurotransmitters exert excitatory effects during this period to generate PSB. Moreover, whole cell patch clamp recordings in ventral horn neurons revealed that high intracellular Cl⁻ concentration is causing excitation by these amino acids at this stage. Such excitatory synaptic inputs via glycine and GABA_A receptors during the early fetal period may play an important role in development of neuronal circuit. [Jpn J Physiol 54 Suppl:S21 (2004)]

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Animals flexibly adjust their endocrine, autonomic, and behavioral functions in response to ever-changing environments. Here the peptidergic nervous system is believed to play an important role. In this symposium, I introduce our recent studies on an extrahypothalamic GnRH (gonadotropin-releasing hormone) peptidergic neuron system, the terminal nerve (TN) GnRH system, which we suggest is neuromodulatory and is involved in the control of motivational state of the animal. By using a fish model system, the dwarf gourami (tropical freshwater fish) brain, we have demonstrated that TN GnRH neurons project widely in the brain, show characteristic pacemaker activities that are modifiable by multimodal sensory and hormonal inputs. The pacemaker activities are generated by the combination of a TTX-resistant persistent Na⁺ current and a certain kind of TEA-sensitive K⁺ current as well as the conventional TTX-sensitive Na⁺ current. The TN-GnRH neurons also express various kinds of voltage-dependent Ca²⁺ currents (mainly L, N, and R) and several different types of ionotropic as well as metabotropic glutamate receptors, which presumably play important roles in the physiological functions of the peptidergic modulator neurons. We find that solitary nest-building (NB) behavior of the male dwarf gourami in the absence of females can be quantitatively analyzed as a good index of motivated state. We obtained evidence to suggest the involvement of GnRH peptides in the control of this "motivated" NB behavior by analyzing effects of GnRH agonists and antagonists. [Jpn J Physiol 54 Suppl:S21 (2004)]

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In case of development of an effective clinical therapy , it is necessary to establish a model in animals larger than laboratory animals as a pre-clinical assessment. And conventional contusion models required preinjury laminectomy to expose the cord, which changes the condition surrounding the spinal cord. Then, we developed a new SCI model in dogs that uses a balloon and no laminectomy. A balloon catheter was inserted via the intervertebral foramen, into the extradural space in 7 dogs. The balloon was inflated at the L1 level by injection of saline. After 4 h of surgery, a region of high intensity was observed with MR imaging. Basso, Beattie and Bresnahan (BBB) scores of models in which compression was achieved by inflation of the balloon with 1.5 ml saline remained at zero for 6 months after surgery. During electrophysiological studies performed by magnetic stimulation, no hindlimb movement was observed on stimulation beyond the area of contusion. Pathological examination showed the formations of cavities surrounded by scar tissue and that these scarred areas contained high levels of collagen. These findings closely resembled those of clinical cases.In our clinical experience on the recovery of injured peripheral nerves, we realized the importance of the local biological condition for the regeneration. Using this SCI model we are now attempting to several trials and the latest results are presented in this symposium. [Jpn J Physiol 54 Suppl:S22 (2004)]

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Recently, there is an explosive increase in the expectation that the transplanted bone marrow stromal cells (BMSC) may regenerate the damaged central nervous system (CNS) and improve neurological function. Indeed, numerous reports have shown that the transplanted BMSC migrate into the damaged CNS and differentiate into the neural cells, improving neurological deficits in various animal models. However, there still is a considerable gap between their histological findings and neurological behaviors. The authors have run the following experiments for these 3 years in order to explore how the transplanted BMSC improve their neurological findings: (1) Establishment of BMSC transplantation system for mice cerebral infarct or spinal cord injury model, (2) Establishment of autogeneic BMSC transplantation into the rat brain and spinal cord, and (3) Fluorescence bioimaging of the BMSC transplanted into mice cerebral infarct or rat spinal cord injury model.In this symposium, the authors will present the additional results obtained from other ongoing experiments. [Jpn J Physiol 54 Suppl:S22 (2004)]

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The availability of signals to induce the appropriatedifferentiation of the transplanted and/or endogenous neural stem cells(NSCs) as well as the timing of the transplantation are important forsuccessful functional recovery of the damaged CNS. Due to the immediately post-traumatic microenvironment of the spinal cord, i.e. the transient upregulation of cytotoxic inflammatory cytokines including IL-6, the acute inflammatory stage is not favorable for the survival and neuronal differentiation of NSC transplants. Correspondingly, we could demonstrate that the optimal timing of NSC-transplantation is 1-2 weeks after injury, when the IL-6 expression is already downregulated. Furthermore, we could show that the neutralization of IL-6 signaling by the administration of blocking antibody in the acute phase of mouse model of spinal cord injury represents an attractive option for the treatment of spinal cord injury. In spite of these findings using rodents, a pre-clinical study using non-human primates is necessary before NSPCs can beused in clinical trials to treat human patients with spinal cord injury (SCI). Here, we show that human NSPCs grafted into spinal cords of adult common marmosets 9 days after contusion injury survived, differentiated into neurons, astrocytes, and oligodendrocytes, and eventually promotedfunctional recovery, suggesting that NSPC transplantation may be a feasible treatment for human SCI. [Jpn J Physiol 54 Suppl:S22 (2004)]

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The failure of injured axons to regenerate long distances in the adult mammalian CNS leads to permanent paralysis and devastating functional deficits. Although axons do not regenerate beyond the lesion site in adult CNS tissue, they have the ability to regrow for long distances if provided with an appropriate microenvironment. Search for the microenvironment appropriate for axonal regeneration has been the main theme in this field, which includes inhibiting neurite growth inhibitory proteins like NogoA or MAG with antibodies, grafting peripheral nerves or embryonic CNS tissues or transplanting Schwann cells or olfactory ensheathing cells (OECs). However, repaired neural connections with these treatments were small in amount and short in extension, which are considered mostly aberrant and achieve less functional recovery. In contrast, we have achieved rapid and marked functional recovery in adult paraplegic rats by transplanting type-2 astrocytes-rich culture cells. In the present symposium, I am going to discuss on the mechanisms inducing axonal regeneration by these cells. [Jpn J Physiol 54 Suppl:S23 (2004)]

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Localization of sound source by acoustic cues is basing on the time and the intensity differences of sound captured by two ears. The sound source localization in azimuth depends on the resolution of interaural time differences (ITD). Temporal information coded in the auditory nerve fibers is first extracted in the nucleus magnocellularis (NM) and ITD is then calculated in the nucleus laminaris (NL) in birds. We investigated the accuracy of synaptic transmission in NM and estimated the limiting accuracy of coincidence detection in NL by using slice preparations of chicken's brain stem. NM neurons showed a tonotopic differentiation, due mostly to the expression of low threshold K current (LTK, esp. Kv1.1). NM neurons fire with a minimum jitter (10 μs) when driven trans-synaptically. The coincidence of bilateral inputs from NM generates action potentials in NL, and the time window of coincidence detection (CD) was narrow; 340 μs. Accuracy of CD had a strong positive correlation with the time course of EPSP. EPSP time course was shortened by two currents: H current and LTK current. The time course of EPSP became close to or faster than that of EPSC. Assuming the limiting time course of EPSP as that of mEPSC gave a limiting time window of CD as narrow as 150 μs in the chicken. This limiting accuracy of CD was nearly the accuracy achieved by barn owl NL, in which the difference of 1 degree azimuth angle produces 4 μs ITD producing nearly 5% changes of the spike rate. Barn owl and man have the ability of localizing the sound source at 1 degree azimuth angle, most precise among terrestrial animal. [Jpn J Physiol 54 Suppl:S23 (2004)]

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Owls localize sound sources precisely for hunting mice in darkness. The main cue for sound localization in azimuth is the interaural time difference (ITD). The ITD is computed online in the 3rd order auditory relay station, nucleus laminaris (NL). NL neurons serve as coincidence detectors of binaural signals for ITD computation. Although the cellular mechanism for coincidence detection has long been the subject of discussion and modeling, coincidence detection has not been directly observed in vivo. We developed a new technique for intracellular recording from owl's NL neurons in vivo by using coaxial glass electrodes in which one (microelectrode) was inserted into a patch-electrode type capillary. These two electrodes were advanced by two independent microdrives. NL neurons produced small action potentials and sinusoidal oscillatory potentials whose frequency corresponded to the stimulus sound. These sound analogue psps changed their amplitudes with ITD. The number of spikes evoked by sound changed with the amplitude of the sound analogue psps. Carr et al (1993) reported that the initial segment of owl's laminaris neurons was covered with myelin, and they inferred from this anatomical feature that the spike initiation site of the owl's laminaris neuron is not the initial segment but the 1st node of Ranvier. The small spike observed in this study supports their interpretation that the site of spike generation is separated from that of synaptic integration in the owl's NL neurons. [Jpn J Physiol 54 Suppl:S23 (2004)]

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Efferent control of neuronal activity in the central nucleus of the inferior colliculus by the primary auditory cortex was studied using a novel photo-dynamic technique realizing direct and reversible inactivation of neuronal activity in focused areas of the brain in minutes. We used a near-infrared diode laser (wavelength, 830 nm; power density, 1.2-4.8 W/cm²) for the inactivation study. The laser-induced inactivation is thought to be brought about via reversible change of membrane property; hyperpolarization of membrane potential and decrease in membrane resistance. Laser irradiation to the primary auditory cortex through the dura mater reversibly depressed the spontaneous neuronal activity and auditory responses to the acoustic stimuli in the irradiated area within 5-10 minutes. The photo-dynamic inactivation of the primary auditory cortex synchronously changed neuronal activity in the central nucleus of the inferior colliculus; facilitation or inhibition of auditory responses and/or spontaneous (background) activity, and shortening or prolongation of latency period of auditory responses. This study demonstrated patterns in control of neuronal activity in the central nucleus of the inferior colliculus by the primary auditory cortex. [Jpn J Physiol 54 Suppl:S24 (2004)]

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Four band noise vocoded speech sounds were used to understand speech processing system in the central nervous system. Temporal change in frequency (or spectral) information, such as formants and the fundamental frequency, has been believed to be essential components for speech information processing. However, recent studies revealed that four band noise vocoded speech sounds where speech signals were replaced by four bands of noise with preserved ultra-slow amplitude envelope (low pass filtered at 16 Hz) got very intelligible (greater than 80%) after a short training period. Even intonation could be easily discriminated, which is usually discriminated by a change in the fundamental frequency. These facts indicate we start to create plasticity by using central pathways, which were not conventionally used, and match the outputs of the pathways with the existing templates. That is why in the beginning these noise vocoded speech sounds were not intelligible at all. Here psychophysical studies were carried out with an aid of fMRI measurements. The outcome of the present research would contribute to understanding the central system for speech perception and recognition, understanding reference structure in the brain and constructing systems for training perceptually impaired people. This research was supported by Special Coordination Funds, and a grant to RCAST at Doshisha University from the Ministry of Education, Culture, Sports, Science and Technology of Japan. [Jpn J Physiol 54 Suppl:S24 (2004)]

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The age at onset of idiopathic Parkinson's disease is the 6th or 7th decade. The autonomic nervous dysfunctions in the cardiovascular system and control system of body temperature are also associated with the motor symptoms as main symptoms in idiopathic PD. The pathology of the brain is characterized by the degeneration of dopaminergic cells in the zona compacta of substantia nigra and of noradrenergic cells in the locus ceruleus, associated with the intracellular inclusion body so called Lewy body. The patients with early-onset parkinsonism (EOP) with the age at onset younger than 40 years account for about 10 per cent of all patients with PD. In the category of EOP, autosomal recessive juvenile parkinsonism (AR-JP) so called Park 2 is hereditary PD which has most frequent incidence. The clinical characteristics such as diurnal fluctuation of the symptoms, effectiveness by sleeping, levodopa-induced dyskinesia at the early stage, hyperreflexia, absence of autonomic nervous dysfunction, dystonia of the leg and foot, and slow progression are shown in Park 2. In this symposium, we will show the comparison of the clinical features, genetic back-ground, autonomic nervous function, and so on between idiopathic PD and EOP. And we will discuss the difference of brain-stem function between the two types of PD. By the contrast, it will be clarified that the brain-stem nuclei especially the dopaminergic cell group in the ventral tegmental area of the mid-brain have an important role in the central control of autonomic nervous function. [Jpn J Physiol 54 Suppl:S24 (2004)]

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Repetitive transcranial magnetic stimulation (rTMS) is recently assessed as a therapeutic modality for various neuropsychiatric diseases, such as depression and Parkinson's disease. It is thought to have a lasting biological effect analogous to long-term potentiation (LTP) or depression (LTD) at synaptic level, and recent neuroimaging studies in human have shown modification of multiple regions via functional connection to the target. However, there are few studies regarding the effects on neurotransmitter.Dopamine is one of the most important neurotransmitters in the brain and known to play a role in control of movement and reward/emotion system. Recent studies have suggested that it also regulates autonomic system involved in sleep, cardiovascular response or metabolism. These functions are presumably related to activities in mesolimbic DA pathway, which terminates at nucleus accumbens (NAc).We have recently shown the regional effect of rTMS on neuronal activity and DA release revealed by positron emission tomography (PET) in macaque monkey. The motor cortical rTMS have induced long-lasting activity changes not only in motor-related, but also in limbic-related areas, such as orbitofrontal cortex, which is known as a higher center of autonomic system. In addition, motor cortical rTMS induced DA release in ventral striatum including NAc. These findings show rTMS may functionally modify mesolimbic pathway and serve as a therapeutic modality in emotional/autonomic disorders. [Jpn J Physiol 54 Suppl:S25 (2004)]

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The overall changes in heart rate (HR) during 24-hour period has a positive relationship with arterial pressure (AP), suggesting that the changes in HR is controlled not by the arterial baroreflex but by a central feedforward signal (termed central command) coupled with daily activities. It has been reported that the positive AP-HR relationship is lost in idiopathetic Parkinson's disease patients. We hypothesized that the midbrain dopaminergic system may play a role in producing central command for autonomic control of cardiac rhythm. To solve this, we attempted to 1) study the effect of stimulation of the dopaminergic neurons on HR, AP, and femoral or brachial blood flow in anesthetized animals, 2) to determine the effects of lesion of the neurons on the cardiovascular responses during locomotion in conscious animals, and 3) to compare the cardiovascular responses during voluntary handgrip exercise, walking, and postural changes in normal and Parkinson's disease subjects. The stimulation of the midbrain ventral tegmental area (VTA), but not substantia nigra (SN), caused an increase in limb blood flow, indicating vasodilation of muscle blood vessels. VTA lesion blunted the centrally-induced cardiovascular responses at the onset of locomotion. Similarly, we observed attenuated cardiovascular responses during handgrip exercise, walking, and postural changes in idiopathetic Parkinson's disease patients. These results suggest that dopaminergic neurons in the midbrain may produce central commands for the cardiovascular adaptation during exercise. [Jpn J Physiol 54 Suppl:S25 (2004)]

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Arterial pressure (AP) shows circadian variation and changes with vigilance state (sleep/wake). AP and heart rate (HR) decrease during resting period (light phase for rats or mice) in comparison to active period (dark phase). During non-REM sleep, autonomic functions such as AP, HR and respiration are quite stable showing constant values throughout this stage. However, during REM sleep, significant fluctuations in autonomic functions can be observed. AP increases in the transition from non-REM to REM sleep, exhibiting large phasic surges during REM sleep, although the skeletal muscles lose their activity completely. Injection of 6-hydroxydopamine (6-OHDA), which is one of cathecholaminergic neurotoxin, into the ventral tegmental are (VTA) induces the destruction of dopaminergic midbrain-nucleus accumbens (NAc) pathway. We have observed that 6-OHDA injection into VTA causes the attenuation of both circadian variation and fluctuation during REM sleep in AP. Non-dipping type of AP circadian rhythm and a variety of sleep disorders are characteristics of Parkinson's disease. We now therefore hypothesize that the midbrain dopaminergic system, especially midbrain-NAc pathway, may have the important role on the circadian and sleep-related control of AP. [Jpn J Physiol 54 Suppl:S25 (2004)]

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Corticotropin-releasing hormone (CRH) and vasopressin (AVP) neurons, in the paraventricular nucleus of the hypothalamus (PVN), are the principal regulators of the hypothalamic-pituitary-adrenal (HPA)-axis. AVP is primarily a neurohypophysial hormone but is co-expressed in the parvocellular CRH neurons. We sought to characterize the responses of hypothalamic neuroendocrine neurons to stressors and elucidate the implication of brain stem catecholaminergic neurons in the stress response. First, the time course profiles of AVP gene expression, as determined by in situ hybridization using either intronic or exonic probe, were disclosed to be different, depending on the nature of the imposed stressors, in contrast to rather uniform expression profiles of the CRH gene. Second, a large population of both CRH- and AVP neurons became Fos positive in the PVN after hemorrhagic shock, as determined by immunocytochemistry. However, approximately 40% of the tyrosine hydroxylase (TH)-containing neurons in the nucleus of the solitary tract (NTS) became Fos positive, thus, a discrete population of the NTS catecholaminergic neurons were activated after the stress. Third, a mouse line, heterozygous for a mutated TH gene, was introduced to examine whether catecholamine deficiency affects responsiveness of the HPA-axis to stressors. The CRH gene expression patterns were different between the TH (+/+) and TH (+/-) mice following forced swim stress, confirming the implication of the catecholaminergic system in the transmission of stress responses. [Jpn J Physiol 54 Suppl:S26 (2004)]

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Emotional stress can affect food intake. Food intake can also affect emotion. However, the underlying mechanisms of the interaction are unclear. Both emotional stress and food intake are suggested to activate prolactin-releasing peptide (PrRP)/noradrenergic neurones in the medulla oblongata and oxytocin neurones in the hypothalamus. The medullary PrRP/noradrenergic neurones project to hypothalamic oxytocin neurones. An icv injection of PrRP activates oxytocin neurones. An icv injection of oxytocin or PrRP affect emotional responses or food intake. It is thus possible that PrRP and/or oxytocin may play a role in the control of emotional stress responses and/or food intake. In order to determine physiological roles of endogenous PrRP and oxytocin, we examined effects of anti-PrRP IgG and an oxytocin receptor antagonist. An icv injection of the oxytocin receptor antagonist attenuated freezing behaviour in response to conditioned fear and oxytocin release into the blood after noxious stimuli. An icv injection of anti-PrRP IgG increased food intake during the period when food intake was activated. The anti-PrRP IgG injection also attenuated oxytocin release in response to conditioned fear. All these data are consistent with a view that PrRP and oxytocin neurones activated after emotional stress and food intake play a role in the control of emotional stress responses and food intake. [Jpn J Physiol 54 Suppl:S26 (2004)]

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Recently transgenic animals are widely used to understand the physiological role of genes in the field of neuroendocrinology as well as human disease study. Although transgenic mice are widely available, few transgenic rats have not been available because it is difficult to generate transgenic rats. However, most neuroendocrinological and physiological data have been obtained from rats. Thus, we attempted to generate transgenic rats for applying physiological study. It is well known that arginine vasopressin (AVP)-secreting neurons is located in the supraoptic nucleus (SON), the paraventricular nucleus (PVN) and the suprachiamatic nucelus (SCN) of the rat hypothalamus. AVP has not only antidiuretic effects on kidney but also various functions in the central nervous system (CNS) such as stress response and circadian rhythm. To understand the physiological aspects of AVP-secreting neurons in the CNS, we generated the fusion gene of AVP and enhanced green fluorescent protein (eGFP). Finally, we succeeded to make the AVP-eGFP transgenic rats. In the transgenic rats, eGFP was strongly observed in the neurons and fibers of the SON, the PVN and the SCN. The hypothalamo-neurohypophyseal tracts and terminals in the posterior pituitary also expressed eGFP. There was no ectopic expression of eGFP gene in the CNS of transgenic rats. The AVP-eGFP transgenic rats give us new tool to study the physiological role of AVP-secreting neurons in the CNS and dynamics of AVP in the living neurons. [Jpn J Physiol 54 Suppl:S26 (2004)]

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The hippocampus and amygdala play major roles in the elaboration of stress responses to psychogenic stimuli. Damage to select regions of these structures substantially alters the HPA-response to stress: damage to the ventral subiculum enhances stress responsiveness, whereas medial amygdala lesions diminish stress-induced corticosterone secretion. Importantly, neither the ventral subiculum nor the medial amygdala directly innervate paraventricular nucleus (PVN) neurons controlling the stress response. Rather, regulatory information is relayed largely through cell populations in the bed nucleus of the stria terminalis (BST), medial preoptic area (mPOA), dorsomedial hypothalamus (DMH) and the peri-PVN region. Combined tract-tracing and in situ hybridization studies indicate that ventral subicular projections to BST, mPOA, DMH and peri-PVN are predominantly glutamatergic, whereas projections to the same regions from the medial amygdala are GABAergic. The data support the hypothesis that the hippocampus, via the subiculum, inhibits stress responses by glutamatergic excitation of GABAergic PVN-projecting neurons. In contrast, the amygdala, by way of the medial amygdaloiod nucleus, activates the stress response by disinhibition, involving GABAergic inhibition of GABA neurons that in turn project to the PVN. The data suggest that hippocampal and amygdaloid outflow is integrated by forebrain/hypothalamic structures prior to accessing the PVN, and affords an opportunity for limbic information to interface with hypothalamic homeostats prior to activating a stress response. Supported by NIMH grant MH49698. [Jpn J Physiol 54 Suppl:S27 (2004)]

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The monoamine and opioid systems control emotional and addictive behaviors. Transgenic mice lacking monoamine transporter gene or opioid receptor gene have been produced by gene targeting. Behavioral analysis of these mutant animals has highlighted distinct roles of monoamine transporter and opioid receptor and revealed their important roles in emotional behaviors. The examinations of responses to addictive drugs have clarified involvement of monoamine transporter and opioid receptor as molecular targets for emotional behavior. The conditioned place preference (CPP) paradigm and intravenous self-administration are popular behavioral paradigms for reward system. Many of the findings from these studies have been quite surprising; for instance, cocaine retains its rewarding effect in dopamine transporter (DAT) knockout mice indicating a polygenic basis of psychostimulant reward. Such studies are also some of the first attempts to examine gene-gene interactions behaviorally in a specific manner. We have examined extracellular dopamine and serotonin release using in vivo microdialysis in the strains of knockout mice with normal or enhanced psychostimulant reward. Those data have demonstrated the critical roles of monoamine transporter and opioid receptor, and confirmed their involvement in several emotional responses. Ongoing studies therefore help in understanding the molecular basis of emotional behaviors and will contribute to the development of novel therapeutics for mental disorders including drug abuse. [Jpn J Physiol 54 Suppl:S27 (2004)]

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Extensive studies performed in the last 10 years have unveiled a considerable amount of the molecular basis of cell death including apoptosis and some forms of necrosis, which are implicated in various diseases. A change in mitochondrial membrane permeability is one of the most critical events in cell death in mammals because it leads to mitochondrial dysfunction (necrosis) as well as the release of apoptogenic factors, including cytochrome c, into the cytoplasm, which in turn activates apoptosis-driving proteases called caspases. The Bcl-2 family of proteins, consisting of anti-cell death and pro-cell death members, regulate cell death mainly by controlling mitochondrial membrane permeability, although precise mechanism is still to be elucidated. Various models for apoptotic mitochondrial membrane permeabilization have been proposed, including the formation of a protein-conducting pore which consists of either oligomeric pro-apoptotic members of Bcl-2 family such as Bax/Bak or VDAC with Bax (Bak), the formation of transient lipidic pore and physical rupture resulting from either mitochondrial swelling or membrane instability. I will summarize currently proposed models and introduce our recent findings, and discuss how the mitochondrial membrane permeability is controlled during cell death. [Jpn J Physiol 54 Suppl:S27 (2004)]

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We have developed an artificial liposome system that recapitulates intermembrane space protein release from mitochondria induced by Bcl-2 family proteins during apoptosis. Internally loaded fluorescein-dextrans are released in the same manner as in cytochrome c release from mitochondria. We have further characterized the system by using peptides from the BH3 region of pro-apoptotic Bcl-2 family proteins. The BH3 peptides from Bid and Bim activated Bax directly, while those from Bad and Bik showed no direct activation of Bax, but reversed the inhibition of Bcl-xL on Bax/Bid induced membrane permeabilization. These results are in good agreement with recently published observations obtained with mitochondria, supporting the hypothesis that our liposome system is physiological.We have also studied how p53, a tumor suppressor, could induce apoptosis in a transcription-independent manner using the liposome system. We observed that when p53 is over-expressed in the cytoplasm, it induces apoptosis by releasing cytochrome c from mitochondria. We then immunoaffinity purified p53 from UV-treated cells and added to liposomes in the presence of Bax, and it induced dextran release at a similar molar concentration range to that of Bid. We conclude that p53 can act like a BH3-only Bcl-2 family protein to directly activate Bax.The liposome system may be used to study how Bcl-2 family proteins as well as other proteins like p53 regulate mitochondrial outer membrane permeabilization in apoptosis. [Jpn J Physiol 54 Suppl:S28 (2004)]

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It is now well established that the mitochondria play an important role in the regulation of apoptotic cell death by mechanisms which have been conserved during evolution. Thus, it seems that a host of lethal agents target the mitochondria and stimulate their release of cytochrome c and other pro-apoptotic proteins, which can trigger caspase activation and other parts of the apoptotic process. Cytochrome c release is governed by the Bcl-2 family of proteins and occurs by a two-step process, which is initiated by dissociation of the hemoprotein from cardiolipin, the phospholipid that anchors it to the outer surface of the inner mitochondrial membrane. The dissociation of cytochrome c from cardiolipin is promoted by pro-oxidants and inhibited by glutaredoxin-2 and other mitochondrial antioxidant enzymes. Release of the solubilized pool of cytochrome c into the cytosol may then occur by pore formation mediated by pro-apoptotic Bcl-2 family proteins, notably Bax and Bak, or by Ca²⁺-triggered mitochondrial permeability transition. In both cases, there seems to be a role for voltage dependent anion channels (VDAC) in the outer mitochondrial membrane, and our recent findings suggest that VDAC located in the plasma membrane of apoptotic cells may also be important for cell death mediated by the mitochondrial pathway. Taken together, these findings have further emphasized the critical role of the mitochondria in the regulation of cell life and death. [Jpn J Physiol 54 Suppl:S28 (2004)]

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Prior to apoptotic body formation, cells quickly exhibit the apoptotic volume decrease (AVD) in response to stimulation with a death receptor- or mitochondrion-mediated apoptosis inducer. The AVD induction preceded cytochrome c release, caspase activation, DNA laddering and cell death. Pan-caspase blockers failed to abolish the AVD induction. Anion channel blockers abolished the AVD induction and prevented apoptotic biochemical events and cell death. Apoptosis inducers rapidly activated outwardly rectifying Cl⁻ currents that were sensitive to osmotic shrinkage. Thus, it is concluded that activation of volume-sensitive anion channels is involved in the AVD induction which is an early prerequisite to apoptosis. Necrotic cells exhibit persistent swelling until cell rupture. Lactacidosis causes the necrotic volume increase (NVI) and cell death in brain neurons and glial cells. In cultured neuronal and glial cells, lactacidosis in fact induced persistent swelling without following volume regulation. Volume-regulatory anion channels were inactivated by lactacidosis. When lactacidosis-resistant anion channels were introduced by applying the VacA toxin, glial cells exhibited volume regulation after transient swelling under lactacidosis conditions. Pretreatment of glial cells with VacA suppressed necrotic cell death after a lactacidosis insult. Thus, it is concluded that inhibition of volume-sensitive anion channel is involved in lactacidosis-induced NVI and necrotic death in glial cells. [Jpn J Physiol 54 Suppl:S28 (2004)]

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It is a long known paradigm that inorganic ions such as Ca²⁺ play an essential role in control of cell proliferation, death and survival. The universality of Ca²⁺ and other inorganic ions has frequently led to paradoxical difficulty in unraveling correlation of these factors with specific biological responses. Remarkable development in genome/protein science has provided powerful tools to tackle this problem. The recently discovered gene superfamily for transient receptor potential (trp) protein (TRP) and its homologues is an excellent example to discuss this issue. TRP homologues have been demonstrated to form Ca²⁺-permeable cation channels that sense wide aspects of extracellular stimuli: temperature, pH, redox and metabolic stresses, and stimulation of plasmamembrane receptors by humoral factors. The topics of the symposium include the dramatic progress in establishing functional characteristics of TRP Ca²⁺/ cation channels. To gain fundamental understanding on determinants that link TRP channels to specific cellular responses, we will particularly focus on the issue how TRPs transform the above stimuli into signals that regulate activation of transcription factors, cell cycle, and other Ca²⁺-dependent responses, ultimately contributing to control of cell fate and homeostasis. Other channels such as Cl⁻ channels that link to cell death are also an important subject of the symposium. [Jpn J Physiol 54 Suppl:S28 (2004)]

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NMDA receptors mediate activity-dependent, competitive synaptic refinement in the visual and somatosensory cortices. In cerebellar Purkinje cells, climbing fibers and parallel fibers are competitive to structure territorized dendritic innervation, but Purkinje cells lack functional NMDA receptors. Here, we demonstrate that the P/Q-type Ca channel alpha1A, a major Ca channel subtype in Purkinje cells, is crucial for the competitive synapse formation. In the alpha1A knockout mouse, many ectopic spines were protruded from proximal dendrites and somata of Purkinje cells. Innervation territory of parallel fibers was expanded proximally to innervate the ectopic spines, whereas that of climbing fibers was regressed to the basal portion of proximal dendrites and somata. Furthermore, multiple climbing fibers, consisting of a strong climbing fiber and one or a few weaker ones, persisted in the majority of Purkinje cells and were co-wired to the same somata, proximal dendrites, or both. The lack of _lpha1A, therefore, results in the persistence of parallel fibers and surplus climbing fibers, which should normally be expelled from the compartment innervated by the main climbing fiber. These results suggest that a P/Q-type Ca channel alpha1A fuels heterosynaptic competition between climbing fibers and parallel fibers, and also fuels homosynaptic competition among multiple climbing fibers. This molecular function facilitates the distal extension of climbing fiber innervation along Purkinje cell's dendritic tree and also establishes climbing fiber mono-innervation of individual Purkinje cells. [Jpn J Physiol 54 Suppl:S29 (2004)]

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Steroid receptors which include estrogen receptor (ER) a andb, progesterone receptors (PR) A and B, androgen receptor (AR), glucocorticoid receptor (GR), and mineralocorticoid receptor (MR) are ligand-dependent transcriptional factor. With the advent of green fluorescent protein (GFP) and its color variants, the subcellular distribution of many steroid receptors has been found to be much more dynamic than previously thought. These receptors are localized wither in the cytoplasm or nucleus at the unliganded condition, but in all cases, the addition of a ligand leads to almost complete nuclear translocation of the receptors. Upon estradiol treatment ER a and b in the same cell were relocalized to show discrete pattern, and they were localized at the same discrete cluster, suggesting that both subtypes of ERs were bound to the same nuclear sites. In the presence of the estradiol, however, the discrete staining pattern of ER a and b were mostly overlapped with Brg-1, indicating that most of the ERs clusters are involved in the chromatin remodeling machinery. FRAP analysis showed that nuclear ER a and b, PRA and B and, AR are dynamic and mobile, but its mobility was different. Transgenic (Tg) mice in which GFP was expressed under the ER a promoter activity were generated. Ovariectomy caused significant reduction of cell bodies of GFP neurons containing ERa in the medial preoptic area, but not in the ventromedial nucleus. These results suggest that estrogen affects ERa cells at the region specific manner. [Jpn J Physiol 54 Suppl:S29 (2004)]

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Biomolecules assemble to form molecular machines such as molecular motors and cell signal processors and dynamically function in response to the environmental changes in cells. The dynamic behavior of the individual biomolecules at work has been detected using recently developed single molecule measurements both in vitro and in vivo. Single molecules are visualized. Chemical reactions are monitored at the single molecule level. Conformational changes of individual protein molecules are detected. Single molecules are manipulated. These techniques have allowed the measurements of dynamic and kinetic behavior of biomolecules, which was obscured in the average values in previous ensemble measurements. The series of kinetic processes have been measured without separating each steps and synchronizing large number of molecules. It is also possible to monitor the behavior of molecules of interest exclusively in complicated systems containing variety of molecules such as cells. These measurements have demonstrated that the individual protein molecules dynamically change their locations, the interaction with other molecules and conformational and functional states. The dynamic properties of proteins provide flexibility to the system, allowing the systems to effectively respond to the stimuli. Thus the single molecule techniques have been proven to be a powerful tool to understand the unique operation of biomolecules in biosystems. [Jpn J Physiol 54 Suppl:S29 (2004)]

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Reporter assay system that employ firefly and Renilla luciferases is widely used as a conventional and powerful tool for monitoring single gene expression. One important application of the reporter assay system is the simultaneous monitoring of two gene expressions in the same cell population because the expressions of many genes are regulated concurrently by transcription factors. However, even with the current system, more than one gene expression cannot be monitored simultaneously because one of two luciferases must be used as an internal control. To overcome the limitations of the reporter assay system, we have recently developed a novel reporter assay system, tricolor reporter assay system, which consists green- and red-emitting Phrixothirix luciferases as dual reporters and Renilla luciferase as internal control. In this system, activities of green and red luciferases can be calculated simultaneously by devising the emissions with an optical filter. We have successfully employed this system in analyzing the effects of clock gene products CLOCK, BMAL1 and retinoid receptor-related orphan nuclear receptorα (RORα) on the enhancer elements of Per1 and Bmal1 promoters. The results indicate that the RORα regulates bidirectionally Bmal1 (positively) and Per1 (negatively) transcriptions simultaneously in the same cell populations. This system could be easily developed for monitoring four or more gene expressions using additional luciferases and optical filters, and for monitoring real-time rhythmic expression profiles in vivo. [Jpn J Physiol 54 Suppl:S30 (2004)]

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Animal experiments in Japan are regulated by a legal and scientific matrix consisting of the Law for the Humane Treatment and Management of Animals, the Standards Relating to the Care and Management, etc. of Experimental Animals, and voluntary guidelines formulated by the scientific associations concerned following the administrative guidance notified by the Ministry of Education. The Law adopts the self-regulation system for animal experiments. It may look lenient from the social viewpoint. To ensure a convincing self-regulation system, we should prepare written documents on institutional policies and responsibilities for animal experiments as well as institutional guidelines on the care and use of laboratory animals. The guidelines should include a performance-based approach so that well-trained laboratory animal scientists can give research personnel advice and the institutional animal care and use committee can evaluate the planned scientific procedures if they are compatible with animal welfare. The laboratory animal has its specific mission to provide reliable experimental data as a partner of the researcher. Then, what is our responsibility for laboratory animals to accomplish their mission without the force of law but with a moral sense? I would like to discuss the strength and weakness of the self-regulation system based on an international comparison and suggest what the most appropriate system will be to implement our responsibility. [Jpn J Physiol 54 Suppl:S30 (2004)]

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Macaque monkeys, especially Japanese monkeys (Macaca fuscata), have physiological functions close to humans and have been used as important laboratory animals in various fields of biomedical researches including physiology. The Japanese monkeys used in the laboratories have been supplied from the wild source, where monkeys were captured as agricultural pest or from small colonies of bred animals. Thus, no large scale breeding colony has been founded in Japan. However, strong criticism against use of wild animals for laboratory research were raised and Ministry of the Environment decided to restrict the use of captured monkeys for laboratory use. Further, now we need to enhance the quality of laboratory animals for future development of science. From these reasons, we decided to launch the project to establish breeding colonies of Japanese monkeys and applied the project to "National Bioresource Project". The application has been approved and started from the fiscal year 2002. However, when we started to collect founder monkeys for the colonies, the project faced strong protest from animal right groups. In this symposium, I present the current status of the project and discuss about how to solve such social issues and about public relations of animal experiments in general. [Jpn J Physiol 54 Suppl:S30 (2004)]

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Life span and decline of physiological functions in aged animals are regulated by interactions between genes and the environment. Genes have an important role in predisposing elderly individuals to several common and complex disorders caused by environmental risk factors. Recent completion of the Human and Animal Genome Project has opened a new era of studies to identify genes, which regulate the life span, rate of aging, and susceptibility to age-related diseases. Many gene mutations for increasing or decreasing the life span have been identified in model animals including worms, flies, and rodents, as well as in humans. It is of particular interest that mutations in glucose or insulin/insulin-like growth factor-1 signaling pathways consistently extend the life span by slowing the aging process and increasing stress resistance in these species. In addition, studies of mutant animals suggest that the reactive oxygen species in the mitochondria might be the main determinant of life spans. Human genetic disorders, which manifest accelerated aging or premature aging, suggest the importance of genes maintaining chromosomal stability. Genetic analysis of senescence-accelerated mouse strains (SAMP) indicate that several genes contribute to their short life spans. These findings suggest that the aging process may be controlled by many genes and might be decelerated by environment regulation of these genes. [Jpn J Physiol 54 Suppl:S31 (2004)]

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It is well known that skeletal muscle fibers atrophy in response to reduced contractile activity, gravitational unloading, and/or aging. However, the molecular mechanisms responsible for such muscular responses are still unclear. Muscle fiber atrophy induced by gravitational unloading generally is associated with the shift of fiber phenotype toward fast-twitch type. But the soleus muscle in aged rat is composed of more slow-fibers than young rat, although fiber atrophy is induced. Therefore, it is suggested that the fiber size and phenotype are regulated by different mechanisms. Possible mechanisms responsible for the regulation of morphological and/or metabolic properties of skeletal muscle in response to contractile activity or aging, as well as the countermeasures for prevention of muscle atrophy, will be discussed in the symposium. [Jpn J Physiol 54 Suppl:S31 (2004)]

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Aerobic training at moderate intensity such as the lactate threshold was found to be effective in lowering resting blood pressure in hypertensive patients. Moreover, several studies have also reported on the favorable effects of exercise training in elderly hypertensive patients with or without pharmaceutical drug therapy. However, there is the variation in the depressor response to the exercise therapy among the patients. In order to clarify the factor that contribute the variability in the depressor response among hypertensive individuals, we analyzed the data from 122 hypertensive patients who performed same exercise therapy. We obtained the results that variations in the depressor response to exercise therapy were partly determined by baseline MBP, suggesting the possible involvement of other factors, including genetic factors. Thus we are trying to search the link between the genetic factors and the variation in the depressor response. [Jpn J Physiol 54 Suppl:S31 (2004)]

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Sports activities in individuals with disabilities were initially part of the rehabilitation programs, however, at present, these activities include actual sports competitions. Thus, there is a need for investigating exercise physiology and sports science in subjects with spinal cord injuries (SCI). We have been investigating these issues in athletes with SCI who participated in the Oita International Wheelchair Marathon Race (OIWMR) for more than 20 years. We measured muscle strength and volume capacity over 10 years in 100 straight participants in OIWMR. Our results showed that muscle strength remained constant although the volume capacity decreased significantly in these subjects. To identify the factors involved in improvement of the race time in OIWMR, our physiological and kinesiological studies showed that the race time was independent of maximum oxygen uptake and muscle strength and depended on skillful wheelchair propulsion. We also studied changes in heart rate, arm movements, renal function, natural killer cell activities, and muscle enzymes in the same athletes. We also studied sympathetic nerve activities, and endocrine and cardiovascular responses in persons with disabilities during exercise and other stresses in laboratory setting. Our results indicated that physical disabilities do not pose great medical impediment to exercise and sports activities although it is advisable to eliminate any stress stimuli to the paralyzed parts of individuals with SCI. [Jpn J Physiol 54 Suppl:S32 (2004)]

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We examined the effects of protein supplementation on body temperature regulatory capacity after aerobic training in older men. In the previous study, we suggested in older men that the sensitivities of cutaneous vasodilation and sweating at a given increase in esophageal temperature (T_es) did not increase after 18-wk aerobic training, which would be caused by unchanged plasma volume (PV) after training due to an attenuated increase in plasma total protein content (TP_tot) (JAP, 93: 1630-1637, 2002). Recently, we found in older and younger men that the supplementation of protein & carbohydrate (CHO) (3.1 kcal/kg & 0.18 g protein/kg, respectively) immediately after a bout of exercise increased TP_tot and PV measured at 23 h after the bout while that of placebo (0.5 kcal/kg, 0 g protein/kg) did not (FASEB J, 17: A943, 2003). In the present study, we examined the effects of protein & CHO supplementation during aerobic training on TP_tot, PV, and also the sensitivities in older men. Fourteen older men (68±2 (SE) yrs) exercised at 60~70%Vo_2peak, 60 min/day, 3 times/wk, for 8 wks with taking protein & CHO (S, n=7) or placebo (P, n=7) after exercise each day. After 8-wk training, TP_tot and PV in S increased significantly but not in P. Also, the sensitivities of the rises in cutaneous vascular conductance (ΔCVC/ΔT_es) and sweating (ΔSR/ΔT_es) at a given rise in T_es increased significantly in S but not in P. Thus, the supplementation of protein & CHO enhanced the improvement of thermoregulatory capacity after endurance training by increasing PV in older men. [Jpn J Physiol 54 Suppl:S32 (2004)]

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Retrograde signaling from postsynaptic cells to presynaptic neurons is essential for regulation of synaptic development, maintenance, and plasticity. Membrane-permeable molecules, such as gases and lipids, are well known candidates for retrograde signals. Recent studies suggest that secreted molecules also may act as retrograde signals. However, the molecular mechanism of the retrograde-signal release from postsynaptic cells is largely unknown. Here we report that a peptidic retrograde signal functions at neuromuscular junctions in C. elegans and the novel C2 domain protein AEX-1 regulates the signal release from muscle.
aex-1 mutants show several neural defective phenotypes such as reduced Ach transmission and abnormal localization of the synaptic vesicle fusion protein UNC-13. Contrary to these phenotypes, we found that aex-1 is predominantly expressed in muscles and intestine. Our experiments showed that muscle-specific AEX-1 expression rescues some of neural defects including abnormal presynaptic UNC-13 localization, but neuron-specific expression does not, suggesting that AEX-1 regulates neural activities from muscles. Furthermore, in muscles, this signaling pathway requires the AEX-5 prohormone convertase, which processes prohormone maturation, suggesting that a peptide is the retrograde signal in this pathway. AEX-1 protein has a C2 domain and a UNC-13 homology domain, and this protein and its vertebrate homologues appear to regulate exocytosis. Our results indicate the presence of a general mechanism in muscle to modulate neural activities, such as the synaptic transmission efficacy by AEX-1. [Jpn J Physiol 54 Suppl:S32 (2004)]

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Synapses show dynamic changes during development and adulthood, and mechanisms underlying these dynamic changes have been studied intensively. By contrast, it is largely unknown how synaptic strength is maintained stably in mature brain, which must be important for stable information transfer and storage. In the present study, we examined this issue using climbing fiber (CF) - Purkinje cell synapse in the mouse cerebellum as a model. Most Purkinje cells are innervated by single CFs in the adult. CF activation yields large and constant excitatory postsynaptic currents (EPSCs) in an all-or-none fashion, which makes a reliable electrophysiological examination possible. To block neuronal activity locally and chronically within the cerebellum, we implanted a small piece of ethylene-vinyl acetate copolymer (Elvax) containing TTX on the surface of the mouse cerebellum at P24. Three to seven days after the implantation, acute cerebellar slices were prepared and CF synapses were examined electrophysiologically. The amplitude of CF-mediated EPSC was significantly smaller, and glutamate concentration transient at the CF synaptic clefts was lower in the TTX-treated mice than those in control mice. The frequency but not the amplitude of quantal EPSC was reduced in the TTX-treated mice. Similar presynaptic changes were induced by chronic blockade of postsynaptic AMPA receptors by means of Elvax containing NBQX, a selective antagonist of AMPA receptors. These results suggest that presynaptic functions of mature CF synapses are maintained by postsynaptic activity through some retrograde signaling. [Jpn J Physiol 54 Suppl:S33 (2004)]

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High-frequency stimulation produces long-term potentiation (LTP) at inhibitory synapses of layer 5 cells in developing rat visual cortex. Induction of this LTP was prevented by inhibition of postsynaptic G proteins, phospholipase C, inositol trisphosphate receptors, or Ca²⁺ increase, indicating that postsynaptic Ca²⁺ elevation is necessary for induction. On the other hand, the maintenance of LTP requires the activity of presynaptic, but not postsynaptic, cells, albeit at a low frequency. Our analysis has demonstrated that LTP maintenance is mediated by presynaptic Ca²⁺ entries through multiple (P, N and L) types of high-threshold Ca²⁺ channels, which activate Ca²⁺-dependent reactions different from those triggering transmitter release, suggesting that this LTP is expressed and maintained presynaptically. Analysis of the expression site of LTP, using paired-pulse stimulation and coefficient of variation, was consistent with this view. These observations strongly suggest that the production of LTP requires retrograde signals from postsynaptic cells to presynaptic terminals. I will describe the experimental data used to test the involvement of retrograde messenger candidates in the production of LTP at visual cortical inhibitory synapses. [Jpn J Physiol 54 Suppl:S33 (2004)]

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Kainate receptors (KARs) are poorly understood subtype of ionotropic glutamate receptors distributing widely and heterogeneously in the central nervous system (CNS). Recent studies elucidated the roles of KARs in activity-dependent modulation of synaptic transmission at certain synapses in the CNS, including hippocampal mossy fiber synapses where KAR subunits are most abundantly expressed. An important notice of these studies is that these receptors localize not only on postsynaptic but also on presynaptic membrane, and these presynaptic KARs impart an 'presynaptic' associativity to mossy fiber long-term potentiation (MF-LTP), an unusual form of plasticity that is independent of NMDA receptor activation and is expressed presynaptically. Homosynaptic as well as heterosynaptic positive feedback regulation of transmitter release via presynaptic KARs provides a novel way of signaling at the central synapses. To examine the cellular mechanism of activity-dependent modulation by presynaptic KARs, we adopted optical methods to monitor presynaptic Ca²⁺ and voltage transients at the mossy fiber synapses. We revealed that activation of presynaptic KARs elicits prominent depolarization of the presynaptic terminals and causes use-dependent facilitation of presynaptic Ca²⁺ influx by an action potential. Thus, presynaptic KARs at the mossy fiber synapses amplify presynaptic Ca²⁺ rise during repeated stimuli, and thereby affect the threshold of induction of MF-LTP. [Jpn J Physiol 54 Suppl:S33 (2004)]

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Several recent studies including ours have revealed that the activity of the postsynaptic neuron can influence presynaptic function through endogenous cannabinoids (endocannabinoids). In this presentation, we will introduce our recent studies on this phenomenon in the hippocampus. We made paired whole-cell recordings from cultured hippocampal neurons prepared from newborn rats or mice, recorded evoked excitatory and inhibitory postsynaptic currents and examined roles of endocannabinoids in the modulation of synaptic transmission. We have obtained the following results: (1) Endocannabinoids can be released from postsynaptic neurons either by depolarization or by activation of G_q/11-coupled receptors including group I metabotropic glutamate receptors (mGluRs) and M₁/M₃ muscarinic receptors. (2) Postsynaptic depolarization and activation of group I mGluRs or M₁/M₃ receptors work in a cooperative manner to release endocannabinoids. (3) The released endocannabinoids act retrogradely on presynaptic cannabinoid CB1 receptors and suppress the transmitter release. (4) Among three candidates of endocannabinoids (2-arachidonyl-glycerol (2-AG), anamdamide, noladine ester), 2-AG is the most effective in suppressing synaptic transmission. These results indicate that endocannabinoids mediate retrograde signals, by which the postsynaptic neuronal activity influences the transmitter release from presynaptic terminals. Wide distributions of CB1 and G_q/11-coupled receptors suggest that similar endocannabinoid-mediated retrograde modulation may function in various brain regions. [Jpn J Physiol 54 Suppl:S33 (2004)]

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Because of its simple anatomical structure and its smooth movements, the oculomotor system has been regarded as a good model to study the more complex motor control system, such as the skeleto-muscular system. Traditionally, simple stimuli were used to explore the oculomotor system, assuming its simple reflexive operation. However, recent studies have challenged the oculomotor system with more complex stimuli and have revealed a variety of responses. Thus, it has opened the way for us to further study the oculomotor system as a probe to understand the higher integrative function of the brain. This symposium will focus on the sensory, perceptual, and cognitive processes that influence the neural control of eye movements. [Jpn J Physiol 54 Suppl:S34 (2004)]

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The population coding here involves pooling the activities of neurons based on single unit recordings. We are interested in the neuronal mechanism of short-latency vergence eye movements elicited by disparity steps of a large-field random-dot pattern. We simultaneously recorded vergence eye movements and single unit activities in the medial superior temporal (MST) area of monkeys by applying 11 disparity steps. Disparity tuning curves, describing the dependence of the responses on the magnitude of the steps, were calculated both for neuronal and ocular responses, i.e. vergences. Very few MST neurons had disparity tuning curves resembling those of the vergence responses. Yet, when the curves for all the cells recorded from a given monkey were simply summed together they fitted the tuning curves for the vergence responses of the monkey very closely, even reproducing the idiosyncratic differences between the animals. The results suggest that the initial vergence responses associated with disparity steps applied to the large-field patterns are encoded in the summed activity of the disparity-sensitive cells in the MST. Further, the latency data indicate that these neuronal activities in the MST occur early enough to play a role in the generation of the vergence eye movements at short latencies. [Jpn J Physiol 54 Suppl:S34 (2004)]

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The vestibuloocular reflex (VOR) stabilizes retinal image during head motion by counter rotating the eyes with nearly an equal speed. When the VOR is inadequate the image slip on the retina evokes a visually-driven response to reduce the slip. If this situation persists, the VOR recalibrates its characteristics so that it can produce adequate eye movements without the aid of the visual system. This phenomenon is called VOR motor learning. In the laboratory it is induced chronically by magnifying or minifying lenses installed on the animal for several days to months, or acutely by exposing it to the visual stimulus moving in- or out-of-phase with a vestibular stimulus for several hours. It has been known the cerebellar flocculus complex (FL) is necessary for this motor learning, but its functional role has been in debate due to the inconsistent results of flocculectomy on the learned animals. We have addressed this problem by performing single unit recording from FL Purkinje (P) cells before and after acute and chronic vertical VOR motor learning in squirrel monkeys. We showed that the information content encoded in P cell firing changes in parallel with the learning in different ways for acute and chronic cases. We also showed that acutely learned VOR gains decay significantly while chronically learned gains do not. These results with other lines of evidence suggest that the memory of the acute VOR motor learning is expressed mainly in FL while that of chronic involves other site as well. [Jpn J Physiol 54 Suppl:S34 (2004)]

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