Slow Time Constants Research Articles

The osmotic response of the isolated tectorial membrane of the chick to isosmotic solutions: Effect of Na +, K +, and Ca 2+concentration

Changes in the size, shape, and structure of the isolated tectorial membrane of the chick were measured in response to isosmotic changes in the ionic composition of the perfusion solution. Substitution of artificial perilymph (AP) for artificial endolymph (AE) caused a small (~ 15%), slow (time constants τ ~ 12 min) shrinkage of the thickness of the tectorial membrane that was largely reversed on return to AE. Substitution of AP for AE alters not only the predominate cation (from K + to Na t) but also the Ca 2+ concentration (from < 7 μmol/1 to 2 mmol/1). Additional experiments were performed to separate effects of each of these changes. When a high-Na +, low-Ca 2+ solution was substituted for a high-K t, low-Ca 2+ solution (AE), the tectorial membrane swelled significantly, often to more than twice its original thickness (the largest swelling was 337%), with a slow time course (τ ~ 23 min). Addition of Ca 2+ to either high-K + or high-Na + solutions caused rapid shrinkage of the tectorial membrane (τ ~ 2–3 min). Addition of the Ca 2+ chelator EGTA caused rapid swelling (τ ~ 4 min). Large osmotic responses were only partially reversible and caused long-lasting changes. For example, long-duration solution changes that produced large, rapid osmotic responses early in an experiment tended to produce smaller and slower responses later in the experiment. In contrast, the small osmotic responses to short-duration solution changes were repcatable for tens of hours. Changes in ionic composition of the bath affected not only the thickness of the tectorial membrane but also its other dimensions. Responses were not generally isotropic; both the size and shape of the tectorial membrane generally changed. Consistent changes in microstructure accompanied the osmotic changes.

Two components of calcium currents in the soma of photoreceptors of Hermissenda.

1. The proposed mechanism of cellular plasticity underlying classical conditioning of Hermissenda involves Ca2+ influx through voltage-activated channels. This influx triggers several molecular cascades and leads to the phosphorylation of K+ channels in identified photoreceptors. We studied Ca2+ currents from isolated photoreceptors of Hermissenda with the whole cell patch-clamp technique. Two distinct Ca2+ currents were identified in isolated photoreceptors on the basis of differences in their voltage dependence, kinetics, and pharmacology. 2. One Ca2+ current was transient (ICa(t)), with a fast onset (approximately 5 ms), activated at -50 mV from a holding potential of -90 mV, and peaked at 0 mV. The second Ca2+ current, designated as sustained (ICa(s)), exhibited a delayed time-to-peak, activated at -30 mV, and reached maximum at 30 mV. 3. Steady-state activation curves for both currents were generated from normalized currents and fitted with the Boltzmann function; estimates of half-activation voltages for ICa(t) were -38.8 +/- 6.7 mV (mean +/- SD; n = 9) and 3.2 +/- 8.2 mV for ICa(s) (n = 11) with maximum slopes of 8.9 +/- 1.6 mV (n = 9) and 11.0 +/- 2.4 mV (n = 11). 4. The inactivation of ICa(s) was slow (time constants > 3 s) whereas ICa(t) inactivated rapidly (time constant of inactivation at various voltages; 75-600 ms). 5. Ni2+ (0.8 mM), Gd3+ (0.5 mM), and amiloride (10 microM) produced a reversible block of ICa(t) without affecting ICa(s). omega-Conotoxin GVIA (10 nM) irreversibly blocked ICa(s) whereas nitrendipine (20 microM) produced a reversible block. 6. ICa(t) may be responsible for steady-state membrane potential oscillations. ICa(s) may contribute to the maintenance of the amplitude of the plateau phase of the generator potential.

Effect of ivermectin on γ-aminobutyric acid-induced chloride currents in mouse hippocampal embryonic neurones

The effect of ivermectin on γ-aminobutyric acid (GABA)-induced Cl − currents was studied in embryonicse hippocampal cells in culture. When 0.1 μM ivermectin was applied to the perfusion medium, the responses to 2 μM GABA were enhanced to 273% within 60 s, and the GABA EC 50 was reduced from 8.2 to 3.2 μM. Half-maximal potentiation of GABA responses was found with 17.8 nM ivermectin. The potentiating effect of ivermectin diminished to 146% within 10 min but the GABA EC 50 did not change any further. At the same time, the maximal GABA-induced Cl − current decreased to 64%. Both the fast and slow desensitization time constants of GABA-activated membrane currents were shortened after ivermectin application. The final effect of ivermectin was irreversible. Modulation of the GABA responses by ivermectin did not interfere with the potentiation induced by diazepam and pentobarbital or with the sensitivity to blockade by bicuculline, picrotoxin and Zn 2+. These results support the view that ivermectin binds to a novel site on the GABA A receptor and allosterically enhances the affinity of the GABA binding site. The more slowly occurring conformational changes in the ivermectin-GABA A receptor complex apparently accelerate the desensitization of the GABA A receptor, reducing the amplitude of maximal GABA-induced currents.

Carrier transport effects and dynamics in multiple quantum well optical amplifiers

The gain recovery dynamics of multiple quantum well semiconductor optical amplifiers, following gain compression caused by ultrashort optical pulse excitation, have been studied for several devices of different structures. Fast, slow, and intermediate time constants are identified. The fast component (0.6 to 0.9 ps) corresponds to cooling of the dense, inverted electron-hole plasma. The slow component (150 to 300 ps) corresponds to replenishment of carriers from the external bias supply, with the dynamics dominated by spontaneous recombination (primarily Auger) of the electron-hole plasma. The intermediate time constant (2 to 14 ps) is caused by carrier capture by the quantum wells and is structure-dependent. For most of the devices, the capture process is dominated by diffusion-limited transport in the cladding/barrier region. The variation of carrier density and temperature also affects the refractive index profile of the devices and, hence, affects the waveguiding properties. Dynamical variation of the mode confinement factor is observed on the fast and slow timescales defined above.

Selective block of delayed rectifying potassium current in the rabbit sinoatrial node by a novel class III antiarrhythmic agent MS-551

Electrophysiological actions of MS-551, a novel class III antiarrhythmic agent, were studied using small preparations (0.2 × 0.2 × 0.1mm) of the rabbit sinoatrial (SA) node. MS-551 (0.1–3 µM) exerted a negative chronotropic action by prolonging the action potential duration and diastolic interval. Automaticity was completely suppressed in 5 of 6 preparations at 1–3 µM. Voltage clamp experiments using double microelectrode techniques revealed that MS-551 (0.1–10µM) blocked the delayed rectifying K+ current (IK) in a concentration-dependent manner, and the block was almost saturated at >1 µM, attaining 60% ± 10% at 10 µM (n = 5). The MS-551-sensitive IK tail (Kd = 0.4µM, Hill r = 1.4,n = 5) had fast and slow components of deactivation. MS-551 (1 µM) reduced the amplitudes of control IK fast and IK slow from 20 ± 4 and 11 ± 4 nA to 8 ± 3 and 5 ± 3 nA, respectively (P < 0.01,n = 4). Although the fast deactivation time constant at −60mV remained unaltered (127 ± 12 vs 113 ± 13ms), the slow deactivation time constant was prolonged from 1,117 ± 130 to 1,555 ± 407ms by 1µM MS-551 (P < 0.05). This agent shifted the steady-state activation curve for IK from −21 ± 2 to −26 ± 4mV and increased the slope factor from 8 ± 1 to 9 ± 1mV (P < 0.05,n = 4). The fully-activated IK exhibited prominent inward rectification and was reduced by MS-551. These results suggest that (1) MS-551 prolongs the action potential duration and diastolic interval, and exerts a negative chronotropic action by blocking IK, (2) MS-551 has a higher affinity for the activated than the resting state K+ channel, and (3) this agent may either preferentially block one type of IK, or stabilize a single population of IK in a subconductance state in the rabbit SA node.

Large amplitude variability of GABAergic IPSCs in melanotropes from Xenopus laevis: evidence that quantal size differs between synapses.

1. We made in situ whole-cell recordings from melanotropes in the intermediate lobe of the pituitary gland of Xenopus laevis. Melanotropes received spontaneous synaptic inputs that had a fast rise time and a much slower decay. These inputs were GABAergic inhibitory postsynaptic currents (IPSCs): they followed the reversal potential for chloride ions and they were blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline. 2. Because of the very low baseline noise it was possible to see discrete levels in the tails of IPSCs that corresponded to the opening of one or more synaptic GABAA receptor channels. "All-points" histograms of the IPSCs showed that the chord conductance of the channels in the tails of the IPSCs was 21.6 +/- 0.6 pS (mean +/- SE, n = 6). 3. The amplitudes of the spontaneous IPSCs were very variable, ranging from 3 to 390 pA at a holding potential of -80 mV. The average of the median amplitudes was -67.5 +/- 5.9 pA (n = 28). The amplitude distributions of the IPSCs were well described by the sum of two lognormal distributions with large SDs. The average of the means of the first lognormal distribution was 27.8 +/- 5.3 pA (n = 10); the average of the SDs was 24.7 +/- 8.1 pA. For the second lognormal distribution these values were 87.0 +/- 13.4 and 33.7 +/- 7.4 pA. An average of 41.8 +/- 6.9% of the IPSCs originated from the first lognormal distribution. 4. The large variability in the amplitudes of spontaneous IPSCs was not the result of presynaptic action potentials because it was not reduced by tetrodotoxin (TTX), Ca(2+)-free extracellular solution, or the combined application of TTX and Mn2+. 5. The time course of the IPSCs from the first and the second lognormal distributions were very similar: averages of the median 20- to 80% rise times were 585 +/- 64 and 488 +/- 28 microseconds, respectively (n = 8), whereas the decays were well described by the sum of two exponential functions, with fast time constants of 8.9 +/- 1.1 (n = 7) and 9.3 +/- 3.3 ms and slow time constants of 29.5 +/- 3.3 and 31.7 +/- 2.6 ms, respectively. 6. The decay of the IPSCs was voltage dependent; it was approximately 3 times slower at a holding potential of +40 mV than at -80 mV (n = 5).(ABSTRACT TRUNCATED AT 400 WORDS)

Modelling the Dynamic Ventilatory Response to Hypoxia in Normal Subjects

We have developed a mathematical model to describe the dynamic ventilatory response to hypoxia. The ventilatory response to both transient (two to three breaths nitrogen) and 3 min step change hypoxic stimuli were measured in ten normal subjects during moderate exercise (oxygen consumption 0·96 ± 0·08 1 min -1). The simplest model relating ventilation to ear oxygen saturation which adequately described the responses in all subjects consisted of two linear differential equations in parallel; both using the fall in oxygen saturation as input, and with the outputs summed to give the rise in ventilation. One equation had a fast time constant (< 3 sec), and the other a slow time constant. Non-linear terms included were (i) a "saturating" effect, similar to that described by the Michaelis-Menten equation, reducing the gain of the equation with the slow time constant as oxygen saturation falls, and (ii) "inhibition" or "potentiation" of the gain of the equation with a slow time constant as the output of the fast time constant equation increased. Repeated measurements in four subjects showed intra- and inter-subject variability for all parameters, with significant between-subject variability for the gain of the fast time constant equation. The final model structure is similar to that describing the peripheral chemoreceptor-mediated hypoxic ventilatory response in anaesthetized cats.

Properties of voltage-gated K+ currents expressed in Xenopus oocytes by mKv1.1, mKv1.2 and their heteromultimers as revealed by mutagenesis of the dendrotoxin-binding site in mKv1.1.

Two similar mouse Shaker-like K+ channel genes, mKv1.1 and mKv1.2, have been shown to form heteromultimers in vivo. The predicted amino acid sequence of each channel is nearly identical in mice, rats and humans, suggesting that each has been highly conserved evolutionarily. Here we report the biophysical and pharmacological properties of each channel when expressed alone or when coexpressed in Xenopus oocytes. The voltage sensitivities of activation were similar for both, but the voltages at which the K+ conductances were half-maximal (V1/2) were -37 mV and -27 mV for mKv1.1 and mKv1.2 respectively. Both displayed voltage-dependent, but incomplete, inactivation following a prepulse with mKv1.2 showing the greater degree of inactivation. For mKv1.1, the onset and recovery from inactivation could be described by single, slow time constants (2-4 s), whereas for mKv1.2 the onset and recovery from inactivation displayed a second, faster time constant (< 400 ms). Using a mutant mKv1.1 that is 100-fold less sensitive to dendrotoxin-I than mKv1.1, we demonstrate that this mutant mKv1.1 and wild-type mKv1.2 subunits can form heteromultimeric channels. With some exceptions, of unknown significance, the biophysical properties of the heteromultimeric channels formed by wild-type mKv1.1 and mKv1.2 subunits were intermediate between those of mKv1.1 and mKv1.2 homomultimers, but quantitatively more similar to the more abundant subunit.

Fast and slow activation of voltage-dependent ion channels in radish vacuoles

The molecular processes associated with voltage-dependent opening and closing (gating) of ion channels were investigated using a new preparation from plant cells, i.e., voltage and calcium-activated ion channels in radish root vacuoles. These channels display a main single channel conductance of approximately 90 pS and are characterized by long activation times lasting several hundreds of milliseconds. Here, we demonstrate that these channels have a second kinetically distinct activation mode which is characterized by even longer activation times. Different membrane potential protocols allowed to switch between the fast and the slow mode in a controlled and reversible manner. At transmembrane potentials of -100 mV, the ratio between the fast and slow activation time constant was around 1:5. Correspondingly, activation times lasting several seconds were observed in the slow mode. The molecular process controlling fast and slow activation may represent an effective modulator of voltage-dependent gating of ion channels in other plant and animal systems.

Stretch-activated channels in airway epithelial cells.

Two type of stretch-activated (SA) ion channels were identified in the basolateral membrane of isolated rabbit airway epithelial cells by patch-clamp techniques. Pressure activation and deactivation of one channel, which had a conductance of 29 pS, occurred after a delay of approximately 20-30 s. The open probability of this delayed stretch-activated (DSA) channel was increased from < 0.01 to 0.45 at 50 mmHg of suction. The reversal potential of the DSA channel, calculated from the pipette potential at which membrane currents reversed [-31.3 +/- 3.6 (SD) mV] and the resting membrane potential (-27.8 +/- 3.3 mV) was +3.5 +/- 3.3 mV. None of the equilibrium potentials of the ions used were similar to the calculated reversal potential of the DSA channel, suggesting that this channel is nonselective for cations. The DSA channel gating behavior was characterized by bursts of rapid transitions between open and closed states. The distribution of the open and closed times revealed that this gating behavior could be fitted with two open states and two closed states. Only the slow time constant of the closed state was decreased by suction. The second SA channel was selective for K+ and had a conductance of 65 pS but a long delay was not associated with the pressure sensitivity of this channel. The open probability of the K(+)-selective SA channel was increased from < 0.01 to 0.30 by 50 mmHg of suction. The K(+)-selective SA channel was distinct from the well-characterized basolateral K+ channel.

Voltage dependence and activation kinetics of pharmacologically defined components of the high-threshold calcium current in rat neocortical neurons.

1. As a first step toward identification of the functional significance of the spatial distribution of calcium channels we examined the high voltage-activated calcium current (HVA current) in acutely isolated pyramidal neurons from rat sensorimotor cortex using whole-cell voltage clamp. The goals of this study were (1) to determine whether the pharmacologically separable components of the HVA current differed in voltage dependence or activation kinetics and (2) to develop an empirical model that could predict the HVA current evoked by action potentials or other physiological responses. 2. Cells with short dendrites were chosen for study. Input resistance averaged 3.5 +/- 0.4 (SE) G omega. Specific membrane resistance averaged 51.9 +/- 6.8 K omega-cm2 on the basis of estimated membrane surface area. 3. Using 2 mM calcium in the extracellular solution, we evoked the HVA current by depolarizations positive to -45 mV from a holding potential of -60 mV, a potential where the low-threshold calcium current is fully inactivated. Maximum HVA current amplitude (484.9 +/- 42.3 pA) occurred near -15 mV. The evoked current was completely and reversibly blocked by 200 microM cadmium. 4. Tail current amplitude at a fixed potential increased as a sigmoidal function of prepulse potential. A plot of normalized tail current amplitude, taken as the fraction of HVA channels open at each prepulse potential, was best described by a Boltzmann function (maximum slope: e-fold per 11.3 mV; half activation: -24.6 mV) raised to the power of 2. This relation was not altered by extracellular application of 5 microM nifedipine or 10 microM omega-conotoxin, each of which reduced a separate component of the HVA current uniformly at all potentials. We conclude that the pharmacologically separable components of the HVA current do not differ significantly in voltage dependence. 5. The time course of current onset during a step depolarization was best described by second-order activation kinetics. Activation time constants ranged from a maximum of 1.2 ms at -40 mV to 0.3 ms at +25 mV. Neither activation nor tail current time constants were altered by extracellular application of 5 microM nifedipine or 10 microM omega-conotoxin. After application of 1 microM Bay K 8644 tail current decay was best described by a fast time constant similar to control values and a slow time constant. We conclude that the pharmacologically separable components of the HVA current in these neurons do not differ significantly in kinetics.(ABSTRACT TRUNCATED AT 400 WORDS)

Pre-steady-state charge translocation in NaK-ATPase from eel electric organ.

Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-->E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s-1.

Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel.

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.

Phosphorylation enhances inactivation of N-type calcium channel current in bullfrog sympathetic neurons.

We have investigated the effects of phosphatase and protein kinase inhibitors on calcium channel currents of bullfrog sympathetic neurons using the whole cell configuration of the patch clamp technique. Intracellular dialysis with the phosphatase inhibitors okadaic acid and calyculin A markedly enhanced the decline of inward current during a depolarizing voltage step. Tail current analysis demonstrated that this was genuine inactivation of calcium channel current, not activation of an outward current. The rapidly inactivating current is N-type calcium current (blocked by omega-conotoxin and resistant to nifedipine). Staurosporine, a nonselective protein kinase inhibitor, prevented the action of okadaic acid, suggesting that protein phosphorylation is involved. Under control conditions, the time course of inactivation could be described by the sum of two exponentials (tau = 150 ms and 1200 ms), plus a constant (apparently noninactivating) component, during depolarizations lasting 2 s. Okadaic acid induced a rapid inactivation process (tau = 15 ms) that was absent or negligible under control conditions, without obvious effect on the two slower time constants. As in control cells, inactivation in okadaic-acid-treated cells was strongest near -20 mV, with less inactivation at more positive voltages. However, inactivation did not depend on calcium influx. Modulation of calcium channel activity by phosphorylation may underly the spontaneous shift between inactivating and noninactivating modes recently observed for N-type calcium channels. Differences in basal phosphorylation levels could also explain why N-type calcium channels, originally described as rapidly and completely inactivating, inactivate slowly and incompletely in many neurons.

An Adaptive Observer-Based Fault Detection Scheme for Nonlinear Dynamic Systems

An adaptive observer-based fault detection scheme is presented. This scheme is applicable not. only for detecting abrupt faults in a nonlinear dynamic system but also for estimating unknown parameters which may represent faults with slow time constants, some kinds of nonlinearities or time-varying system parameters.

Solutions for transients in arbitrarily branching cables: I. Voltage recording with a somatic shunt

An analytical solution is derived for voltage transients in an arbitrarily branching passive cable neurone model with a soma and somatic shunt. The response to injected currents can be represented as an infinite series of exponentially decaying components with different time constants and amplitudes. The time constants of a given model, obtained from the roots of a recursive transcendental equation, are independent of the stimulating and recording positions. Each amplitude is the product of three factors dependent on the corresponding root: one constant over the cell, one varying with the input site, and one with the recording site. The amplitudes are not altered by interchanging these sites. The solution reveals explicitly some of the parameter dependencies of the responses. An efficient recursive root-finding algorithm is described. Certain regular geometries lead to "lost" roots; difficulties associated with these can be avoided by making small changes to the lengths of affected segments. Complicated cells, such as a CA1 pyramid, produce many closely spaced time constants in the range of interest. Models with large somatic shunts and dendrites of unequal electrotonic lengths can produce large amplitude waveform components with surprisingly slow time constants. This analytic solution should complement existing passive neurone modeling techniques.

Kinetic properties of NMDA receptor‐mediated synaptic currents in rat hippocampal pyramidal cells versus interneurones.

1. Whole-cell tight-seal recordings were obtained from visually identified pyramidal cells (PCs) and interneurones (INs) in the CA1 field of thin hippocampal slices from 13- to 23-day-old rats. The INs sampled were classified according to their location either in the molecular layer (M-INs) or in the oriens layer and alveus (OA-INs). PCs and INs differed in their mode of firing when depolarized by a prolonged current pulse. Whereas PCs fired a single action potential, most INs responded with non-accommodating high frequency spike firing. 2. In the presence of 1 microM tetrodotoxin (TTX), bath application of either 50 microM L-glutamate with 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 2.5 microM N-methyl-D-aspartate (NMDA), induced a similar conductance increase in PCs and INs that was completely blocked by 200 microM DL-2-amino-5-phosphonovaleric acid (APV). The NMDA receptor-mediated currents reversed around 4 mV and exhibited an area of negative slope conductance at potentials more negative than -20 to -30 mV in the presence of 1-2 mM Mg2+. 3. Dual-component excitatory postsynaptic currents (EPSCs) were evoked in PCs and INs by stimulating afferent fibres close to the neurone. The NMDA receptor-mediated component of the EPSCs (NMDA EPSC) was isolated by adding 10 microM CNQX to block non-NMDA receptors. The NMDA EPSCs in all cell types reversed around 1.5 mV and were abolished by 50 microM APV. 4. In saline containing 1 mM Mg2+, the peak current-voltage (I-V) relationship of NMDA EPSCs in PCs and INs showed an area of negative slope conductance at voltages more negative than -20 to -30 mV. In nominally Mg(2+)-free saline, the peak I-V relation was linear over a much wider voltage range in both cell types. 5. The 10-90% rise times of NMDA EPSCs at -60 mV ranged from 4.5 to 16 ms in PCs (mean 8.7 ms; n = 25) and in M-INs (mean 9.1 ms; n = 10). Their decay could be best fitted with the sum of two exponentials. The decay of NMDA EPSCs in PCs was significantly slower than that recorded in INs. The average fast (tau f) and slow (tau s) time constants of decay were, respectively, 66.5 and 353.9 ms in PCs, and 34.4 and 212.5 ms in M-INs.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple effects of spermine on N-methyl-D-aspartic acid receptor responses of rat cultured hippocampal neurones.

1. The modulation by polyamines of responses to N-methyl-D-aspartic acid (NMDA) was studied using a rapid perfusion system and whole-cell voltage-clamp recording from rat hippocampal neurons in dissociated culture. 2. Concentration jump responses to 100 microM NMDA in the presence of 10 microM glycine revealed potentiation by 3 mM spermine at a membrane potential of +60 mV, but depression at -120 mV; the degree of potentiation at +60 mV was variable from cell to cell while marked depression at -120 mV was observed in all cells. The depression of responses to NMDA by spermine was highly voltage dependent (z delta = 1.17) with an apparent equilibrium dissociation constant for block at 0 mV of 27 mM. 3. Analysis of spermine dose-potentiation curves for responses recorded at +60 mV in the presence of 10 microM glycine revealed a half-maximal effect at 125 microM. Under the same conditions, but at -60 mV, analysis of spermine-evoked depression was performed for cells with less than 5% potentiation at +60 mV, and revealed half-maximal inhibition at 344 microM. 4. Dose-response analysis for the glycine-sensitive activation of NMDA receptors at +60 mV revealed a 3.5-fold increase in apparent affinity for glycine in the presence of 1 mM spermine. This increase in affinity for glycine was accompanied by a 3.3-fold decrease in the rate of development of glycine-sensitive desensitization, and a 2.4-fold decrease in the rate of dissociation of glycine from NMDA receptors, while the rate constant for dissociation of NMDA was not reduced. 5. In the presence of non-saturating concentrations of glycine, spermine-induced potentiation at +60 mV developed with two exponential components: a slow glycine-sensitive component, the amplitude and time constant of which decreased with increasing glycine concentration (30 nM glycine, amplitude = 80.2 +/- 5.1%, tau = 780 +/- 79 ms; 3 microM glycine, amplitude = 22.6 +/- 7.1%, tau = 45 +/- 13 ms), and a faster component (tau < 20 ms at all concentrations of glycine), the amplitude of which varied from cell to cell, and which became larger with increase in concentration of glycine. When responses to the application of spermine were measured in the presence 10 microM L-alanine instead of 100 nM glycine, the slow component of potentiation was absent.(ABSTRACT TRUNCATED AT 400 WORDS)

Epileptogenesis in chronically injured cortex: in vitro studies.

1. Field potentials and intracellular activities were examined in neocortical slices obtained through areas of chronic cortical injury produced by cortical undercutting and transcortical lesions made in vivo 7-122 days before the terminal in vitro slice experiment. 2. Abnormal field potentials characterized by long- and variable-latency multiphasic events could be evoked by layer VI-white matter or subpial stimulation in 9 of 15 animals that had adequate partial cortical isolations. These "epileptiform" field potentials were recorded in layers II-V and propagated across the cortex. They appeared at threshold in an all-or-none fashion and, in most slices, could be blocked by increasing stimulus intensity. In one slice, spontaneous epileptiform events occurred that were similar to those evoked by extracellular stimulation. 3. Intracellular activities during the epileptiform field potentials consisted of polyphasic synaptic events that were predominantly depolarizing and that could last < or = 400-500 ms, synchronous with the field potential activities. A variety of observations suggested that the neuronal activities underlying epileptiform field potentials were relatively asynchronous and much less intense than those previously found in chemically induced epileptogenesis within the neocortex. 4. Inhibitory postsynaptic potentials (IPSPs) were not prominent in neurons when threshold stimuli evoked epileptiform events; however, suprathreshold stimuli could elicit biphasic IPSPs and block the long-latency polysynaptic activity and abnormal field potential in most slices. Depolarizing components of the polysynaptic activity had the appearance of excitatory postsynaptic potentials in terms of their responses to alterations in membrane potential. 5. Comparison of spike parameters in layer V neurons of epileptogenic slices with those in control layer V neurons showed no significant differences in spike height, threshold, duration, or rise time. Resting membrane potentials were also not significantly different. 6. There was a highly significant difference in input resistance (RN) between layer V neurons in control and injured slices; the mean value for neurons in lesioned cortex was 68.1 M omega, whereas that in control cells was 30.5 M omega. There was also a significant prolongation of the slow membrane time constant in neurons of injured cortex (19.4 ms) as opposed to that in control cells (12.2 ms), suggesting that a change in specific resistivity or capacitance contributed to the higher RNS. 7. The relationship between adapted spike frequency and applied current (f-I slope) was steeper in layer V neurons from injured cortical slices (44.3 Hz/nA) than in normal layer V cells (28.2 Hz/nA).(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of some metal‐ATP complexes on Na(+)‐Ca2+ exchange in internally dialysed squid axons.

1. Na(+)o-dependent Ca2+ efflux (forward Na(+)-Ca2+ exchange), and in some cases the Na(+)i-dependent Ca2+ influx (reverse Na(+)-Ca2+ exchange) were measured in internally dialysed squid axons under membrane potential control. 2. We tested the effect on the Na(+)-Ca2+ exchange of the MgATP analogue bidentate chromium adenosine-5'-triphosphate (CrATP), substrate of several kinases, and cobalt tetrammine ATP (Co(NH3)4ATP), a poor substrate of most kinases. 3. CrATP completely blocked the MgATP and MgATP-gamma-S (ATP-gamma-S) stimulation of the Na(+)o-dependent Ca2+ efflux (forward exchange) and the Na+i-dependent Ca2+ influx (reverse exchange). The analogue only blocked the nucleotide-dependent fraction of the Na(+)-Ca2+ exchange without modifying any kinetic parameters of the exchange reactions. 4. The effects of CrATP were fully reversible with a very slow time constant (t 1/2 about 30 min). 5. The MgATP stimulation of the Na(+)-Ca2+ exchange was completely saturated at 1 mM. Higher MgATP concentrations (up to 15 mM) had no additional effects. Pentalysine (internal or external), the protein kinase C inhibitor H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine) and several calmodulin inhibitors did not inhibit Na(+)-Ca2+ exchange either in the absence or presence of MgATP. 6. Our results do not agree with the idea of an aminophospholipid translocase being responsible for the ATP stimulation of the Na(+)-Ca2+ exchange in squid axons; they suggest that this is due to the action of a kinase system.