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F Kukita

Publications and source records attributed to F Kukita.

At least 19 recordsLinked to original sources
PubMed2001

Recurrent subthreshold electrical activities of rat neocortical neurons progress during long-term culture.

The properties of neocortical neurons during long-term culture on monolayers of astrocytes were examined using whole-cell recording and immunocytochemical techniques. The soma size of neocortical neurons became larger and most neurites reached neighboring neurons within 2 weeks. Recurrent subthreshold electrical activities mediated by synaptic activation were observed at 10 days of culture and became more frequent as the neurons grew. Their frequency reached to about 1 Hz at 4 weeks. While the total number of neurons decreased during culture, the ratio of gammaaminobutyric acid (GABA) positive to total (MAP2 positive) neurons increased. These results suggest that neurons grown on astrocytes become mature during cultivation, and that recurrent subthreshold electrical activities may be related to the development of GABAergic inputs.

Animals↗
PubMed2000

Intracellular [Cl(-)] modulates synchronous electrical activity in rat neocortical neurons in culture by way of GABAergic inputs.

The influence of GABAergic neurons on spontaneous electrical activities of neocortical neurons in culture, which was estimated to be about 9.5% of the total neurons by immunohistochemistry, was examined using dual whole-cell recording. Synchronized depolarization or hyperpolarization was observed in recorded neurons with pipettes containing low [Cl(-)] solution, while synchronized bursting of action potentials (APs) was observed with pipettes containing high [Cl(-)] solution. Spontaneous currents (SCs) were synchronous in all pairs tested with either pipettes containing low or high [Cl(-)] solution and spontaneous outward currents (SOCs) observed at around -30 mV were sensitive to the GABA-A receptor antagonist, bicuculline. Their reversal potential (V(rev)) was linearly related to the logarithm of Cl(-) activity in the pipette (-56.9 mV/decade). The intracellular chloride concentration was estimated from the V(rev) of SCs with gramicidin perforated-patch recordings and was between 5.9 and 28.1 mM (mean: 13.0 mM). These results suggest that GABA depolarized some neurons and hyperpolarized others, depending on the E(Cl). Bicuculline decreased the frequency of periodic depolarized potentials and increased their amplitudes. However, perfusion with low [Cl(-)] bath solution did not decrease the frequency. Our data indicate that recurrent subthreshold electrical activities by GABAergic inputs along with glutamatergic inputs take part in deterring synchronized bursting and that intracellular [Cl(-)] can modulate this bursting.

Action Potentials↗
PubMed2000

Solvent effects on squid sodium channels are attributable to movements of a flexible protein structure in gating currents and to hydration in a pore.

1. Solvent effects on the time course of gating and sodium currents were analysed in squid sodium channels using four non-electrolytes of different size, glycerol, erythritol, glucose and sucrose, to separate effects of viscosity from those of osmolarity and to obtain viscosity and osmolarity parameters that were independent of molecular size. 2. The gating and sodium currents were reversibly slowed in a voltage-independent manner as the non-electrolyte concentration increased. 3. Solvent effects were analysed using a model in which the percentage change in time constant was expressed by an equation involving the viscosity parameter alpha and the osmolarity parameter delta: t/t0 = alpha (eta/eta 0) - 1 + 100 alpha-1)exp(delta delta pi), where eta/eta 0 is solution viscosity and delta pi is increase in osmolarity. Since the solution viscosity was found experimentally to be a function of the solution osmolarity, solvent effects are described by an equation with one independent variable eta/eta 0 or delta pi. 4. Voltage sensor movement, reflected in gating currents, was primarily sensitive to viscosity, as its decay time constant was a function of eta/eta 0, with only a minor sensitivity to osmolarity (delta was 2-3 water molecules). 5. For sodium currents, alpha was equal to that of gating currents but delta was 2-3 times greater, suggesting that the final channel opening was primarily sensitive to osmolarity (delta delta was 5 water molecules). The relative ineffectiveness of the largest non-electrolyte, sucrose, suggested that this osmolarity-sensitive step in channel opening occurred in the narrow pore region. 6. Sodium channel inactivation was primarily sensitive to osmolarity (delta delta was 8-12 water molecules). 7. The observed viscosity dependence of the sodium current activation and inactivation processes was attributable to the viscosity-dependent process accompanying the gating current. 8. This model explains why non-electrolytes slow sodium currents while electrolytes do not. 9. Viscosity effects on gating currents can be explained by a process in which non-electrolytes interact with the flexible hydrophilic parts of sodium channel proteins, but osmolarity effects on the final step need to be explained by a local interaction of several water molecules with fluctuating protein segments in the pore.

Animals↗
PubMed1999

Dual intracellular recording of neocortical neurons in a neuron-glia co-culture system.

Measurement of synchronized activities in neocortical networks is critical for understanding integrated brain function. We describe here a procedure for intracellular recording of activity in two functionally connected neurons using patch electrodes in neuron-glia co-cultures. Rat neocortical neurons survive on monolayers of astrocytes for far longer periods (3-4 weeks) than in their absence. This prolonged survival facilitates the detection of functional synapses which are readily identified in vitro by means of dual intracellular recording using two patch electrodes. The neuron-glia co-culture system appears to be useful not only for studying the formation of neural networks in vitro but also for determining the effects of physiologically active substances and chemicals on synapse formation.

Animals↗
PubMed1998

Functional synapses in synchronized bursting of neocortical neurons in culture.

Spontaneous electrical activities in pairs of neocortical neurons in culture were simultaneously recorded using a whole cell current clamp technique. Synchronous bursting activities were observed in all 59 pairs tested. In 52 pairs of neurons electrically stimulated, EPSPs were recorded in 20 pairs (39%), among which 3 pairs (6%) showed bidirectional coupling. The response latency observed was 4. 05+/-0.61 ms (mean+/-S.E.M.). The synaptic delay was estimated at 1. 5-1.9 ms, suggesting the response latency is derived from a polysynaptic connection. The burst latency which was defined as the time difference of the onset of bursting in each neuron was 5.87+/-0. 47 ms (mean+/-S.E.M.), and was weakly correlated with the spatial distance between the neurons (37.5-600 micro(m) apart) (Rs=0.362, tied P value=0.0065). No synchronized bursting was observed in bathing solution with a low Ca2+ concentration (0.4 mM) or in bathing solution containing 50 microM D-AP5 and 15 microM CNQX. No dye-coupling between bursting neurons was observed on injection of the small molecule dye Lucifer yellow or the neurotracer neurobiotin. Disrupting neural connections completely by cutting the cell layer, caused disappearance of synchronized bursting with each neuron bursting independently. In conclusion, these results are consistent with the hypothesis that synchronized bursting in cultured neocortical neurons is attributed to connections by way of several synapses rather than by way of gap junctions and/or diffusible factors.

2-Amino-5-phosphonovalerate↗
PubMed1997

Solvent-dependent rate-limiting steps in the conformational change of sodium channel gating in squid giant axon.

1. The time course of sodium currents (INa) in squid giant axon was analysed using viscous non-electrolyte solutions on both sides of the axolemma. It slowed reversibly as the non-electrolyte concentration increased. The activation, deactivation (closing) and inactivation processes were slowed in a similar manner. The gating current of the sodium channel was also slowed to the same extent as the activation time constant. 2. The voltage dependence observed in a time constant vs. voltage relationship and a chord conductance vs. voltage relationship (activation curve), did not change significantly. 3. The gating kinetics have a similar temperature dependence in non-electrolyte solutions, showing that the basic gating mechanism did not change in these solutions and only a slight increase in the activation free energy was one of the main causes of slowing. 4. Eight non-electrolytes, formamide, ethylene glycol, glycerol, erythritol, glucose, sorbitol, sucrose and polyethylene glycol (mean molecular weight 600) were used. The amount of slowing was correlated with the gram concentration (g l-1) of non-electrolytes, but not with molar concentration (M) and solution osmolarity (osmol l-1). 5. The percentage changes of the time constant were expressed as a function of the relative change in solution viscosity, eta/eta0. The proportionality constants alpha in the relationship alpha (eta/eta0), and gamma in the relationship 100 (eta/eta0)gamma, obtained using different non-electrolytes, were close to 100% and 1, respectively. The simplest model to explain the results assumes that a slowing of a global conformational change is a consequence of sequential viscosity-dependent movements of local structures (viscosity model). 6. Values of alpha and gamma deviated frequently from those in an ideal case, i.e. 100% for alpha and 1 for gamma, and they scattered, having a tendency to decrease as a function of molecular weight. 7. The slowing was also expressed as an exponential function of the solution osmolarity. A predicted solute-inaccessible volume Va ranged (in nm3 per molecule) between 0.09 and 1.45. The value of Va increased as a logarithmic function of the molecular weight of the non-electrolyte. 8. This solute-inaccessible volume should be distributed in all hydrophilic parts of the sodium channel protein, but is not located in the channel conducting pore itself. The slowing of gating could be explained by a model in which a rate-limiting step is a hydration process that occurs after local small structural changes have exposed new, unhydrated faces (transient hydrated-states model). 9. Considering the opposite dependencies of parameters alpha (or gamma) and beta on the molecular weight, sodium channel gating is likely to reflect a combination of these two models, which are coupled in microscopic segment movements. We emphasize with this combination of models that fluctuating hydrophilic structures play an important role in determining time constants in the gating process.

Animals↗
PubMed1995

Interaction stabilizing tertiary structure of bacteriorhodopsin studied by denaturation experiments.

The structural stability of bacteriorhodopsin was studied by denaturation experiments, using aliphatic alcohol as denaturants. The disappearance of a positive peak at 285 nm of the circular dichroism spectra, the change in the intrinsic fluorescence decay time, and the decrease of the regeneration activity bacteriorhodopsin indicated the denaturation of the tertiary structure of this protein at a methanol concentration of about 3 M. The circular dichroism band at 222 nm was unchanged by the denaturation. It was concluded that the alcohol-denatured state in water was similar to the molten globule state of soluble proteins, in which only the tertiary structure was destroyed. Solvent substitution from water to hexane did not cause denaturation of bacteriorhodopsin. However, further addition of alcohol destroyed the secondary as well as the tertiary structures. Comparing the alcohol effects of bacteriorhodopsin in water to that in hexane, the dominant interactions for the structure formation of this protein could be revealed: the hydrophobic interaction that arose from the structure of water is essential for the stability of membrane spanning helices, while the interaction which binds the helices is polar in nature.

Alcohols↗
PubMed1993

Kinetic analysis of the denaturation process by alcohols of sodium channels in squid giant axon.

1. The effects of several aliphatic alcohols on sodium currents were examined in the intracellularly perfused squid giant axon when the same concentration of alcohol was applied on both sides of the membrane. 2. An irreversible suppression of sodium currents, accompanied by anaesthesia at high alcohol concentration, was examined in detail using four aliphatic alcohols, that is, ethanol, 1-propanol, 1-butanol and 1-pentanol. 3. This irreversible effect seemed to be attributable to the sequential denaturation of sodium channels, because the kinetics, the current-voltage relation and the sodium channel activation-voltage curve did not change after the sodium current decreased. 4. The time course of the remaining sodium conductance was measured as a function of the sum of the alcohol application time by repeating the process of applying and completely washing out alcohol. The remaining sodium conductance decayed as a function of time in a single exponential manner. This decay time constant depended strongly on the concentration of alcohol and could be assumed to be the denaturation time constant of the sodium channel. 5. The denaturation time constant decreased as the alcohol concentration increased. This time constant is proportional to the Nth power of the alcohol concentration. The N values are 4.3, 4.5, 5.8 and 7.6 for ethanol, 1-propanol, 1-butanol and 1-pentanol, respectively. This implies that alcohol molecules bind to a restricted number of specific sites in the sodium channel protein to cause the denaturation. 6. The concentration of alcohol which caused the same amount of denaturation is related to the exponential function of the carbon number of the alcohol. Considering the partition coefficient of alcohol between lipid and aqueous solution, the concentration of alcohol in the membrane which denatured half of the sodium channels in 2 h can be calculated to be 0.5 M for all alcohols.

Action Potentials↗
PubMed1988

Removal of periaxonal potassium accumulation in a squid giant axon by outward osmotic water flow.

1. Periaxonal potassium accumulation does not occur in a squid giant axon when outward water flow is maintained by an osmotic gradient across the axolemma. Potassium concentrations in the periaxonal space were calculated from the potassium potentials (EK) for the tail K+ current. With outward water flow, the periaxonal K+ concentration was maintained at values less than or close to the K+ concentration in the bathing solution. 2. Outward osmotic water flow was produced by adding 1 M-urea to the isotonic external solution. This was sufficient to prevent K+ accumulation, but it had no effect if applied to both sides of the axolemma. 3. The thickness of the periaxonal space (theta s) and the permeability of the extracellular barrier (Ps) were estimated using a three-compartment model. Under isotonic conditions they were 25 nm and 3.7 micron/s, respectively. With outward water flow, either Ps or theta s or both must increase by a large factor, since K+ accumulation is prevented. 4. Instantaneous I-V relations with outward water flow showed outward rectification and no time-dependent changes in their shape. When external K+ concentration was increased, the curves became more linear.

Animals↗
PubMed1984

Pressure dependence of sodium gating currents in the squid giant axon.

Asymmetric displacement currents, Ig, were measured in squid axons at different hydrostatic pressures, P, up to 60 MPa. Potassium and sodium currents were abolished by intracellular Cs+ and TEA+, by extracellular Tetrodotoxin (TTX), and by Na+ substitution with Tris+. The time course of Ig became progressively slower with increasing pressure, and the amplitude decreased. With appropriate scaling in time and amplitude, Ig records at any given P could be made to superimpose very well with those obtained at atmospheric pressure. The same scaling factors yielded a good superposition of all records obtained for voltage steps to membrane potentials in the range -30 to +42 mV. The ratio between the amplitude and time factors was larger than unity and increased with P, indicating a progressive decrease (up to 35% at 60 MPa) of the total charge displaced, Q, with no significant change in its voltage dependence. The time-scaling factor increased exponentially with P, as expected if all the steps involved in the opening of a sodium channel, and producing a major charge redistribution, have the same activation volume, delta V not equal to g approximately 17 cm3/mol. This value is roughly one-half of that characterizing the pressure dependence of sodium current activation, suggesting that some late, rate-limiting step in the opening of sodium channels has a large activation volume without being accompanied by an easily detected charge movement. Part of the decrease of Q with pressure could be attributed to an increase in sodium inactivation. However, we cannot exclude the possibility that there is a reversible reduction in the number of fast activating sodium channels, similar to the phenomenon that has been reported to occur at low temperatures (Matteson and Armstrong 1982).

Animals↗
PubMed1983

Effects of an outward water flow on potassium currents in a squid giant axon.

The excitation of the squid giant axon was analyzed under an outward water flow through the membrane product by an osmotic gradient. The outward water flow made an undershoot of the action potential larger by about 25 mV without decreasing its peak largely. It also made EK more negative but not ENa. The effect of the outward water flow was specific for the potassium channel. The outward current increased and its decline during a long-lasting depolarization became less prominent under the outward water flow. At the same time, inward tail currents for the potassium channel decreased extraordinarily without a large change in the time course. The potassium conductance had a marked rectification in the direction of the water flow. The undershoot of the action potential under the outward water flow was very sensitive to potassium ions in the external solution. Eight mM KCl was effective to diminish the undershoot and to restore the change in EK by about 60% but gave no effect on the reduced tail current. The outward water flow effect can be explained not only by the change in a local concentration of potassium ions at the mouths of the potassium channel due to a sieving but also by the rectification in a hydrodynamic manner.

Action Potentials↗
PubMed1982

Properties of sodium and potassium channels of the squid giant axon far below 0 degrees C.

Squid giant axon could be excited in concentrated glycerol solutions containing normal concentrations of electrolytes, when osmolalities of solutions inside and outside the axon were matched. These glycerol solutions did not freeze at the temperature as low as -19 degrees C. The nerve excitation in these solutions were observed at this low temperature. The excitation process at this low temperature was slowed down and time constants of the excitation kinetics were several hundredfold larger than those in normal seawater at 10 degrees C, under which temperature the squid habituated. The temperature coefficients for the electrophysiological membrane parameters under this condition were larger than those in normal seawater above 0 degrees C. The Q10 value for the conduction velocity was 2.0 and that of the duration of the action potential was around 8.5. The time course of the membrane currents was also slowed with the Q10 value of around 5 and the magnitude decreased with the Q10 value of around 2 as the temperature was lowered. The Q10 values for the kinetics of the on process of the Na-channel were around 4.5 and were almost the same as those of the off process of the Na-channel in the wide range of the temperature below 0 degrees C. The Q10 value of the on process of K-channel was around 6.5 and was larger than those for Na-channel. The Q10 values increased gradually as the temperature was lowered.

Action Potentials↗
PubMed1981

Excitation of squid giant axons below 0 degree C.

The excitation of the squid giant axon that had been perfused intra- and extracellularly with solutions containing a high concentration of glycerol could be observed below 0 degree C. The action potential could be elicited at normal strengths of electrical stimuli. The time-course of the action potential was slowed, whereas the resting potential and the amplitude of the action potential changed only slightly. The membrane current under the voltage clamp at -6.3 degrees C was about 100-fold slower than that in normal sea water at 8.7 degrees C because of the large viscosity of glycerol solutions and the low temperature. The Q10 values of the magnitude and the time-course of the membrane current were 2.3 and 1/4.0, respectively.

Action Potentials↗
PubMed1979

Slowing of the time course of the excitation of squid giant axons in viscous solutions.

The time course of excitation of intracellularly perfused squid giant axons was slowed as the solution viscosity was raised by adding neutral molecules, i.e., glucose and glycerol. By twofold increase of the solution viscosity, the duration of action potential was prolonged to 2.7-fold and the maximum rate of rise decreased to one-half. At the same time, the membrane resistance at resting state increased by 60%. These effects were reversible. The time course of inward and outward currents was slowed also. When the solution viscosity increased to twofold, the time to peak inward current increased by 80%, and the amplitudes of peak inward and steady outward currents decreased by 60% and by 70%, respectively. These effects were not specific for the sodium or the potassium channel. Effects of solution viscosity occurred in both hypotonic and hypertonic solutions. Q10 values of temperature dependence of the time course of the action potential were equal in any viscous solutions. These effects in viscous solutions were explained by the change in solution viscosity but not by the change in solution osmolarities, ionic activities, or solution resistivity.

Action Potentials↗
PubMed1979

Excitation of squid giant axons in hypotonic and hypertonic solutions.

Excitability of intracellularly perfused squid giant axons was maintained in hypotonic solutions (down to 300 mOSM) and in hypertonic solutions (up to about 10 OSM), when osmolalities of internal and external solutions were adjusted to be equal with glycerol, glucose, or sucrose. Molar concentrations of ions were kept constant during one series of experiments. The resting potential and the amplitude of the action potential did not change in both hypotonic and hypertonic solutions. With reduction of osmolality, the duration of action potential decreased and the maximum rate of rise and conduction velocity increased. By raising osmolality, the duration was prolonged and the maximum rate of rise and the conduction velocity decreased. Effects of osmolality change were almost reversible. However, these effects were not directly related to the osmolality change but seemed to be related to the viscosity change of the solutions. When the osmolality of external solution was raised with NaCl (up to 2.6 M NaCl), the overshoot increased in porportion to the logarithm of the NaCl concentration. The slope of increase was about 50 mV/decade. However, the resting potential showed little change. With increase of the NaCl concentration, the duration of the action potential increased.

Animals↗