Biomedical subjects

H Tatebayashi

Publications and source records attributed to H Tatebayashi.

At least 19 recordsLinked to original sources

PubMed1998

Alcohol modulation of single GABA(A) receptor-channel kinetics.

Alcohol modulation of single-channel kinetics of GABA(A) receptor currents was studied with rat dorsal root ganglion neurons using the excised outside-out patch clamp technique. GABA (1 microM) alone or GABA (1 microM) plus ethanol (30-300 mM) or n-Octanol (30-300 microM) were applied by pressure ejection to evoke single-channel currents. The main single-channel conductance was not changed by either ethanol or n-Octanol at 25 pS. Both alcohols exerted the same effects on the single-channel kinetics, although n-Octanol was more potent than ethanol. The frequency of openings, the mean open time, the percentage of open time, the frequency of bursts, and the mean burst duration were all increased, but the mean closed time was decreased. These changes in channel kinetics account for the increase in whole-cell current amplitude caused by ethanol and n-Octanol.

1-Octanol↗

PubMed1996

Interactions of tetramethrin, fenvalerate and DDT at the sodium channel in rat dorsal root ganglion neurons.

Type I and type II pyrethroids and dichlorodiphenyltrichloroethane (DDT) are known to modulate the sodium channel to cause the hyperexcitatory symptoms of poisoning in animals. However, since the degrees to which neuronal sodium channel parameters are altered differ, a question is raised as to whether these insecticides bind to the same site in the sodium channel. Competition patch-clamp experiments were performed using rat dorsal root ganglion neurons which are endowed with tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels. D-trans-Tetramethrin, S,S-fenvalerate and p,p'-DDT caused a slowly rising and slowly falling tail current to be developed in tetrodotoxin-sensitive sodium channels. In tetrodotoxin-resistant sodium channels, these insecticides, particularly tetramethrin and fenvalerate, generated a large and prolonged tail current upon repolarization. The effects of tetramethrin were reversible after washing with drug-free solution, whereas the effects of fenvalerate and DDT were irreversible. When fenvalerate application was followed by tetramethrin application, the characteristic changes in current by fenvalerate disappeared and the characteristic changes by tetramethrin appeared. After washout, the characteristic current pattern of fenvalerate reappeared. These results can be explained by assuming that the tetramethrin molecule displaces the fenvalerate molecule from the same binding site in the sodium channel protein, or that tetramethrin and fenvalerate bind to separate sodium channel sites which interact allosterically with each other. DDT interacted with fenvalerate and tetramethrin in the same manner.

Animals↗

PubMed1995

Sodium channels and GABAA receptor-channel complex as targets of environmental toxicants.

Voltage-activated sodium channels and GABAA receptor-chloride channel complex are among the most important target sites of various environmental neurotoxicants. Pyrethroids keep the sodium channels open for prolonged periods of time leading to hyperexcitation of the entire nervous system. In rat cerebellar Purkinje neurons and dorsal root ganglion neurons, only about 1% of sodium channel population needed to be modified by the pyrethroid tetramethrin to increase the depolarizing after-potential to the level of the threshold membrane potential for generation of repetitive after-discharges. This concept of toxicity amplification is applicable to other chemicals that go through a threshold phenomenon to exert their effects. The potency of pyrethroids on neuronal sodium channels increased with lowering the temperature with a Q10 value of 0.2. The selective pyrethroid toxicity between mammals and insects can be quantitatively explained on the basis of the differences in 5 factors, i.e. the intrinsic sodium channel sensitivity, the sodium channel modification due to temperature difference, the reversibility of sodium channel, the detoxication of pyrethroids, and body size. These 5 factors are multiplied to approximately 2000 which is in the same order of magnitude as that of the difference in LD50. Dieldrin had a dual action on the GABAA receptor-chloride channel complex of rat dorsal root ganglion neurons. The initial transient potentiation of GABA-induced currents after application of dieldrin was followed by a suppression. Dieldrin-induced potentiation of current was observed only when the gamma 2 subunit was present in embryonic kidney cells (HEK-293) transfected with GABA receptor subunits. Dieldrin-induced suppression was observed in the presence and absence of the gamma 2 subunit. The dieldrin suppression of GABA-induced currents is deemed directly responsible for hyperactive symptoms of poisoning in animals.

Animals↗

PubMed1994

Differential mechanism of action of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels.

Rat dorsal root ganglion neurons are endowed with tetrodotoxin-sensitive(TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels. The pyrethroid insecticides, which are known to keep sodium channels open for a prolonged period of time, cause differential effects on the two types of sodium channels. The whole-cell patch clamp experiments were performed with rat dorsal root ganglion neurons in primary culture. In TTX-S sodium channels, the slow sodium current during step depolarization was increased somewhat by tetramethrin, and a tail sodium current with a slowly rising and falling phase appeared upon repolarization. The tail current developed even after the sodium current during depolarization had subsided. In TTX-R sodium channels, the slow sodium current during step depolarization was increased markedly by tetramethrin, and upon repolarization a large instantaneous tail current was generated and decayed slowly. The steady-state sodium channel inactivation curve was shifted by tetramethrin in the hyperpolarizing direction in both TTX-S and TTX-R channels. The sodium conductance-voltage curve also was shifted by tetramethrin in the hyperpolarizing direction in both TTX-S and TTX-R channels, and the latter was affected more strongly than the former. At a concentration of 10 microM, the highest concentration tested, tetramethrin modified only 12% of the TTX-S sodium channels, whereas the modification was as high as 81% in the TTX-R. Even at 10 nM, 1.3% of TTX-R sodium channels were modified; this accounts for the high potency of tetramethrin as an insecticide.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Differential inhibition of a transient K+ current by chlorpromazine and 4-aminopyridine in neurones of the rat dorsal root ganglia.

1. K+ currents were recorded from neurones of the newborn rat cultured dorsal root ganglia, by a whole cell variation of the patch-clamp technique. 2. Chlorpromazine (CPZ), a neuroleptic, reversibly reduced the amplitude of the transient K+ current (referred to as 'IT' hereafter) with a dissociation constant (Kd) of 4.5 microM. The inhibition of the delayed rectifier K+ current (IDR) was much less potent (Kd, 120 microM). CPZ (100 microM) had no effect on the inward rectifier K+ current. 3. The blocking action of CPZ on IT was about seven times more potent than that of 4-aminopyridine (4-AP) which had a Kd of 31 microM. The inhibition of IT followed one-to-one binding stoichiometry with both drugs. 4. The decay time course of IT was not affected by CPZ, whereas 4-AP markedly accelerated the decay phase of IT. 5. The steady-state inactivation curve of IT was shifted in the negative direction (about 5 mV) by CPZ, whereas the curve was shifted in the positive direction (about 13 mV) by 4-AP. 6. The recovery from inactivation as measured by a conventional double pulse protocol was described by two exponential components in the control solution. CPZ markedly reduced the first component and slowed down the recovery from inactivation. In contrast, in the presence of 4-AP, the peak amplitude of IT was rather increased by a preceding IT possibly through voltage-dependent unbinding of 4-AP molecules. 7. These results indicate that CPZ has a preferential blocking action on IT and the mechanism underlying this block is markedly different from the mechanism underlying the blocking action of 4-AP.

4-Aminopyridine↗

PubMed1993

Kinetic analysis of two types of Na+ channels in rat dorsal root ganglia.

1. The gating properties of two types of Na+ channels were studied in neurones isolated from rat dorsal root ganglia using the whole cell variation of the patch electrode voltage-clamp technique. 2. Two types of Na+ currents (INa) were identified on the basis of their sensitivity to tetrodotoxin (TTX). One type was insensitive to TTX (up to 0.1 mM), while the other type was blocked by 1 nM of TTX. Whereas they were both insensitive to 50 microM Cd2+, a high concentration (2 mM) of Co2+ selectively inhibited the TTX-insensitive type. 3. The activation thresholds were about -60 and -40 mV for the TTX-sensitive and the TTX-insensitive INa, respectively. Activation of the TTX-sensitive INa developed with a sigmoidal time course which was described by m3 kinetics, whereas the activation of the TTX-insensitive INa was described by a single exponential function. A deactivation process, as measured by the tail current upon repolarization, followed an exponential decay in either type of INa. 4. The rate constant of activation indicated that under comparable membrane potential conditions, the TTX-insensitive channels open 4-5 times slower than the TTX-sensitive ones upon depolarization. Likewise, the rate constant of inactivation indicated that the TTX-insensitive channels inactivate 3-7 times more slowly than the TTX-sensitive ones upon repolarization. 5. The steady-state activation curve for the TTX-insensitive INa was shifted about 20 mV in the positive direction from that for the TTX-sensitive INa. 6. The steady-state inactivation curve for the TTX-insensitive INa as obtained with a 0.5 s prepulse was shifted about 26 mV in the positive direction from that for the TTX-sensitive INa, indicating a greater availability for the TTX-insensitive INa in depolarized membrane. However, on increasing the duration of prepulse, the inactivation curve for the TTX-insensitive INa, but not for the TTX-sensitive INa, shifted in the negative direction due to an extremely slow inactivation process in the TTX-insensitive INa. Consequently, an overlap between the activation and inactivation curves which causes a steady influx of Na+ (window current) became progressively reduce. 7. The time course of INa decay was best described by a single exponential process in either the TTX-sensitive or TTX-insensitive INa, whereas the development of inactivation and the recovery from inactivation, which were measured by a conventional double-pulse protocol, followed a second order process in either channel type.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1992

The mechanism of GABAB-mediated slowing of the activation phase of high voltage-activated Ca2+ channels in rat sensory neurons.

The mechanism underlying the slowed activation of the high voltage-activated Ca2+ current (HVA-ICa) in the presence of (-)-baclofen was studied in cultured neurons of rat dorsal root ganglia. The decay phase of the baclofen-sensitive component of HVA-ICa was described by a sum of two exponential functions. Although the inhibited portion in the amplitude of the baclofen-sensitive component of HVA-ICa was increased in a concentration-dependent manner, the two decay time constants remained unaffected regardless of the concentration of baclofen. Furthermore, the baclofen-sensitive component of HVA-ICa was largely inactivated by a depolarizing prepulse (-30 mV for 0.5 s). These results support the notion that the slowed activation of the HVA-ICa in the presence of baclofen is due to a preferential inhibition of the inactivating component of HVA-ICa rather than due to voltage-dependent unblocking of a single population of HVA-ICa.

Animals↗

PubMed1992

Use-dependent facilitation of L-like Ca2+ channels counteracts GABAB-mediated inhibition of N-like Ca2+ channels in rat sensory neurons.

Baclofen selectively blocked the inactivating N-like component of the high voltage-activated Ca2+ current (HVA-ICa) without affecting the sustained L-like component of the HVA-ICa in rat sensory neurons. The inhibition of the N-like component by baclofen was reversed by a large depolarizing prepulse to +50 mV as a result of facilitation of the L-like component. These results might indicate that a decrease in influx of Ca2+ through N-like Ca2+ channels due to the baclofen-induced block or due to the voltage-dependent inactivation induced by the depolarizing prepulse can be partially compensated by a rapid Ca2+ influx through extra L-like component facilitated by the depolarizing prepulse. Such a compensation of the Ca2+ influx by the use-dependent facilitation of L-like component may be a significant mechanism for adaptive regulation of Ca2+ channels in response to stimulation with a wide range of amplitude and frequency.

Animals↗

PubMed1992

Ontogenic development of the TTX-sensitive and TTX-insensitive Na+ channels in neurons of the rat dorsal root ganglia.

Developmental changes in the sensitivity of neurons to tetrodotoxin (TTX) were studied in relation to the cell size in rat dorsal root ganglia (DRG). Na+ currents were recorded from neurons of various stages of development. Two types of Na+ channels were identified on the basis of their sensitivity to TTX. One type was insensitive to a very high concentration (0.1 mM) of TTX, while the other type was blocked by a low concentration (1 nM) of TTX. These two types of Na+ channels were observed throughout the developmental stages examined from day 17 of gestation and adulthood. Thus, both types of Na+ channels are already established at the early stage of neuronal development and appear to be retained throughout the life-span of the DRG neuron. The concentration-response relationships for the block of TTX-sensitive Na+ current by TTX did not appreciably change during development. Although two types of Na+ channels had strikingly different kinetic properties, the kinetic properties of each channel type were basically similar throughout development. The TTX-sensitive Na+ channels were mainly concentrated in cells with large cell diameters throughout developmental stages examined. These large cells appear to correspond to the 'large-light' cells. On the contrary, the TTX-insensitive Na+ channels were found in smaller diameter cells which may correspond to the 'small-dark' cells. Thus, it is concluded that there are heterogeneous categories of neurons which have Na+ channels with different physiological and pharmacological properties. Since Na+ channels play a pivotal role in the action potential generation, these heterogeneity of DRG neurons appear to be instrumental in integrating the sensory signals.

Animals↗

PubMed1992

Slow inactivation of tetrodotoxin-insensitive Na+ channels in neurons of rat dorsal root ganglia.

Whole-cell patch-clamp experiments were performed with neurons cultured from rat dorsal root ganglia (DRG). Two types of Na+ currents were identified on the basis of sensitivity to tetrodotoxin. One type was blocked by 0.1 nM tetrodotoxin, while the other type was insensitive to 10 microM tetrodotoxin. The peak amplitude of the tetrodotoxin-insensitive Na+ current gradually decreased after depolarization of the membrane. The steady-state value of the peak amplitude was attained several minutes after the change of holding potential. Such a slow inactivation was not observed in tetrodotoxin-sensitive Na+ current. The slow inactivation of the tetrodotoxin-insensitive Na+ current was kinetically distinct from the ordinary short-time "steady-state" inactivation. The voltage dependence of the slow inactivation could be described by a sigmoidal function, and its time course had a double-exponential process. A decrease of external pH partially antagonized the slow inactivation, probably through an increased diffusion potential across the membrane. However, the slow inactivation was not due to change in surface negative charges, since a shift of the kinetic parameters along the voltage axis was not observed during the slow inactivation. Due to the slow inactivation, the inactivation curves for the tetrodotoxin-insensitive Na+ current were shifted in the negative direction as the prepulse duration was increased. Consequently, the window current activated at potentials close to the resting membrane potential was markedly reduced. Thus, the slow inactivation may be involved in the long-term regulation of the excitability of sensory neurons.

Animals↗

PubMed1992

Comparison of two types of Na+ currents with low-voltage-activated T-type Ca2+ current in newborn rat dorsal root ganglia.

Na+ currents and the low-voltage-activated T-type Ca2+ current (T-ICa) were recorded from neurons of rat dorsal root ganglia under similar ionic environments using the whole-cell patch-clamp technique. Two types of Na+ currents were identified on the basis of their sensitivity to tetrodotoxin (TTX) and channel kinetics. One type was blocked by 1 nM TTX and had a faster activation and inactivation time courses (F-INa), while the other type was insensitive to 100 microM TTX and had a much slower channel kinetics (S-INa). Activation thresholds were -60, -40 and -70 mV for F-INa, S-INa and T-ICa, respectively. Peak amplitudes were obtained in respective current/voltage curves at -30 mV (F-INa), 0 mV (S-INa) and -50 mV (T-ICa). The time to peak and the decay time constant measured at potential levels giving peak amplitudes were 0.5 and 1.5 ms for F-INa, 1.4 and 2.9 ms for S-INa and 8.1 and 17 ms for T-ICa, respectively. Cd2+ in a concentration of 50 microM totally blocked T-ICa, whereas it had no effect on either type of Na+ current. T-INa was found in 18 out of 25 cells which possessed F-INs, whereas it was found in only 2 cells among 15 which lacked F-INa. These three types of inward currents having different kinetic and pharmacological properties may mediate diverse functional roles in processing sensory signals.

Animals↗

PubMed1992

GABAB-mediated modulation of the voltage-gated Ca2+ channels.

1. The amino acid, gamma-aminobutyric acid (GABA), activates two different receptor types (Bowery et al., 1980; reviewed by Ogata, 1990a). 2. GABAA receptors are bicuculline-sensitive and are coupled to Cl- channels, while activation of bicuculline-insensitive GABAB receptors has been implicated in the modulation of Ca2+ (Dunlap and Fischbach, 1981) and K+ (Gahwiler and Brown, 1985; Inoue et al., 1985a,b; reviewed by Ogata, 1990b) channels. 3. Baclofen is a specific agonist for GABAB receptors (Bowery et al., 1980). In rat sensory neurones, baclofen suppresses the membrane Ca2+ current (ICa) by a mechanism involving a partussis toxin-sensitive G protein (Holz et al., 1986; Scott and Dolphin, 1986). 4. It has been shown that the inhibitory effect of baclofen is more potent on the early portion of ICa than on the later portion and consequently the rate of ICa activation is slowed (Deisz and Lux, 1985; Dolphin and Scott, 1986). 5. The mechanisms underlying these GABAB-mediated modulation of ICa is not fully understood. This article reviews the inhibitory action of baclofen on ICa in sensory neurones.

Animals↗

PubMed1992

Na+ current kinetics are not the determinants of the action potential duration in neurons of the rat ventral tegmental area.

Na+ currents were recorded from two morphological subpopulations of neurons acutely dissociated from the rat ventral tegmental area (VTA). About 45% of 56 VTA cells examined possessed only the ordinary type of Na+ current which was blocked by a low concentration (0.2 microM) of TTX. However, the remaining 55% had the Na+ current which contained a small fraction of the TTX-insensitive component, irrespective of morphological variations and action potential durations of VTA cells. The peak amplitude of the TTX-insensitive component was less than 10% of the peak amplitude of the total Na+ current. The activation and inactivation kinetics of the TTX-insensitive component were much the same as those of the overwhelming TTX-sensitive component of the Na+ current in VTA cells but differed from those of the TTX-insensitive Na+ current reported in peripheral sensory neurons. Thus, it was concluded that the well-known different action potential durations found for subpopulations of VTA cells are not due to multiplicity of Na+ channel kinetics.

Action Potentials↗

PubMed1992

Kinetic analysis of the GABAB-mediated inhibition of the high-threshold Ca2+ current in cultured rat sensory neurones.

1. The action of baclofen on the voltage-gated Ca2+ current (ICa) was studied, using cultured neurones of the newborn rat dorsal root ganglia (DRG). Two major categories of ICa were identified: a small transient current activated positive to -60 mV (low voltage-activated ICa) and a large and slowly inactivating current activated positive to -30 mV (high voltage-activated ICa). 2. Baclofen reversibly blocked the high voltage-activated ICa and slowed the activation phase of the current in a concentration-dependent manner (0.5-50 microM). The half-maximal effective concentration was about 1.5 microM as measured by a peak of ICa. On the contrary, a high concentration of baclofen (100 microM) had no detectable effect on the low voltage-activated ICa. 3. The baclofen-sensitive component of the high voltage-activated ICa was largely inactivated by a depolarized holding potential (Vh) of -40 mV, whereas the baclofen-resistant component was not affected by a change in Vh ranging from -110 to -30 mV. 4. The high voltage-activated ICa had two components of current decay: an inactivating component and a quasi-sustained component, with time constants about 420 and 1220 ms, respectively. The time constant of decay for the inactivating component was not affected by replacement of external Ca2+ with Ba2+, whereas that for the quasi-sustained component was markedly prolonged, suggesting that the decay of this component may be due to Ca(2+)-induced block rather than voltage-dependent inactivation. A high concentration of baclofen (50 microM) selectively blocked the inactivating component. 5. The decay phase of the baclofen-sensitive component of the high voltage-activated ICa was best fitted by a sum of two exponentials, with 29.2 and 481 ms for the fast and slow components, respectively. The time constants of the two components were not affected by an increase in the concentration of baclofen, whereas the amplitudes changed concentration-dependently. 6. The slowed activation of the high voltage-activated ICa by baclofen was partially reversed by a large depolarizing pre-pulse. However, such an acceleration of the current was similarly observed in the control solution. Furthermore, the actual current size increased by the pre-pulse was similar in both the control and baclofen-containing solutions. 7. These results suggest that baclofen selectively blocks the inactivating component of the high voltage-activated ICa which forms a rapid rising phase of this current, thus slowing the activation phase of the total high voltage-activated ICa.

Animals↗

PubMed1991

A simple and multi-purpose "concentration-clamp" method for rapid superfusion.

A simple method for the rapid exchange of the medium used for a variety of electrophysiological experiments including patch-clamp experiments on cultured cells is described. The advantages of this method over the conventional ones are that this solution exchange system has a very simple structure and thus is inexpensive and can be easily installed in already existing experimental setups. The method involves the use of a fine double-barrelled polyethylene needle placed near the cell or other isolated preparations under recording. Electromagnetic valves are used to switch the solutions in the polyethylene tubes which enable a rapid and localized application of test solutions as well as their rapid washout. The latency of solution exchange, as measured by a change in the holding current under voltage-clamp experiments was fairly rapid (about 120 ms). Neither was the recording interrupted nor the sealing resistance affected during repeated exposure to test solutions. This method is proving to be useful, particularly for electrophysiological and pharmacological studies on immovable cells such as those in culture.

Animals↗

PubMed1991

Primary culture of neurons derived from the adult mammalian brain.

A method is described for the isolation and culture of neurons in the adult mammalian brain. The cell could be maintained in primary culture for more than several weeks. Whereas the neurons freshly dissociated from the adult brain did not respond to any of the neurotransmitter substances applied, the neurons regained the ability to respond to a variety of neurotransmitters when cultured. The cultured neurons of the adult mammalian brain may be an excellent model for physiological as well as pharmacological investigations of the central nervous system.

Animals↗

PubMed1991

[Pharmacology and physiological function of gamma-aminobutyric acid B type receptor].

Baclofen, a beta-chlorophenyl derivative of gamma-aminobutyric acid (GABA), depresses neuronal excitability in various parts of the central nervous system. The site of action for this drug had once been considered to be distinct from GABA recognition sites. In addition to the classical GABA recognition site (GABAA site), a new class of GABA receptor (GABAB site) has been characterized. GABAB sites are mainly present on nerve terminals and, when activated, result in diminished transmitter release, probably through a reduction in Ca2+ influx. Baclofen was shown to be a selective agonist for this novel GABAB recognition. Baclofen also directly hyperpolarizes the membrane of mammalian brain neurons, in addition to its presynaptic action. This postsynaptic action of baclofen was shown to result from an increase in K+ conductance when studied in hippocampal pyramidal neurons through postsynaptic GABAB receptors. Thus, the inhibitory neurotransmitter GABA activates two receptor subtypes that can be distinguished by their physiological and pharmacological properties. GABAA receptors mediate rapid alterations in the distribution of Cl- across the membrane. GABAA receptors are linked directly to an ion channel, thus contributing to the prompt inhibition of cellular excitability. On the contrary, the GABAB receptor does not contain an integral ion channel and is thus responsible for slower responses through receptor-G-protein-effector complexes. G-protein may be directly coupled to K+ or Ca2+ channels. In addition, G-protein may modulate a variety of regulatory proteins or second messengers, thus contributing to the slower alteration of cellular excitability or to the modulation of neurotransmitter release.

Animals↗

PubMed1991

[Primary culture of mammalian brain neurons and its application to patch-clamp recording].

A method is described for the primary culture of neurons from adult mammalian brain. The primary culture consists of five processes: 1) preparation of brain slices, 2) microdissection of the discrete area, 3) enzymatic treatment of the tissue, 4) dissociation of cells by mechanical agitation of the tissue fragments, and 5) plating and feeding of dissociated cells. The cells could be maintained in culture for more than several weeks. Whereas neurons freshly dissociated from the adult brain do not respond to exogenously applied neurotransmitter substances, probably due to destruction of receptors by the enzymatic treatment, the neurons regained the ability to respond to a variety of neurotransmitters when they were cultured. Cultured neurons from adult mammalian brain are proving to be an excellent model for physiological as well as pharmacological investigations on the central nervous system.

Animals↗