Electrical activity in embryonic heart cell aggregates

Summary Aggregates were formed from dissociated heart cells of 7-day chick embryos. When spontaneous action potentials were blocked with 10^–8 to 10^–7 g/ml tetrodotoxin (TTX) oscillatory pacemaker potentials were sometimes seen. The emergence of these pacemaker potentials was critically dependent on the external potassium concentration. In 1.3 mM potassium medium TTX suppression of action potential generation always led to a stable resting potential close to the threshold level (–55 to –50 mV). In 4.3 mM potassium TTX suppression was followed by a train of pacemaker potentials which usually gave way to a stable resting potential of about –70 mV. Raising the calcium concentration from 1.8 to 5 mM often induced long lasting (3 hrs) pacemaker oscillations of 20 to 30 mV peak to peak amplitude. These were abolished by raising the potassium concentration to 8.3 mM or upon the addition of 1.5 mM Mn²⁺. The responses of TTX-treated aggregates are discussed in terms of Noble and Tsien's pacemaker theory for Purkinje fibers. The results are well described by assuming the existence of ani _{k 2}-like potassium current in embryonic heart cells. The role of calcium is unclear but it may help provide the inward current against which the outward potassium current can function.     

Role of calcium influx and buffering in the kinetics of Ca(2+)-activated K+ current in rat vagal motoneurons.

1. Intracellular recordings were obtained from neurons of the dorsal motor nucleus of the vagus (DMV) in transverse slices of the rat medulla maintained in vitro. These neurons had a resting potential of -59.8 +/- 8.7 (SD) mV. Single action potentials elicited by brief depolarizing current pulses were followed by a prolonged afterhyperpolarization (AHP). Under voltage clamp, the current underlying the AHP was found to be a calcium-activated potassium current. 2. The outward current (GkCa,1) was voltage insensitive and was not blocked by tetraethylammonium (TEA) (10 mM). Unlike the slower time course calcium-activated potassium current recorded in some other neurons, GkCa,1 was blocked by apamin (25-100 nM), indicating that SK type calcium-activated potassium channels underlie this current. 3. GkCa,1 was maximal within 10 ms of the action potential and its decay was well described by a single exponential. After a single action potential the time constant of decay of GkCa,1 was 155 +/- 66 (+/- SD) ms. 4. Calcium influx was increased by adding TEA to the extracellular solution or by firing more than one action potential. As the calcium load was increased, both the peak amplitude and the time constant of decay of GkCa,1 increased. In cells impaled with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-filled electrodes, the time constant of decay of GkCa,1 after a single action current was 71 +/- 19 ms. 5. A simple diffusion-based model that incorporates two intrinsic calcium buffers is developed that accounts for many of the properties of GkCa,1. It is concluded that the decay of GkCa,1 reflects the time course of removal of calcium that has entered the cell during the action potential.     

Loose-patch clamp currents from the hypothalamo-neurohypophysial system of the rat

The loose-patch clamp technique was used to study voltage-activated currents from the surface of rat neurohypophysial and hypothalamic regions in situ. In the neurohypophysis, depolarizing pulses of 4–8 ms duration yielded tetrodotoxin (TTX)-sensitive sodium currents, a 4-AP-sensitive "A"-type potassium current, and a long-lasting outward TEA- and tetrandrine-sensitive Ca²⁺-activated potassium current. All of these currents were elicited during the application of the pulse. With high external calcium there were long-lasting inward currents blocked by Ni²⁺ and Cd²⁺, identifying them as voltage-gated calcium currents. Depolarizing pulses of 0.3–0.7 ms duration yielded fast biphasic responses, of 1–3 ms duration, composed of mostly sodium and "A"-type potassium currents. With high external calcium there were fast inward currents blocked by Ni²⁺ and Cd²⁺, indicating that these were voltage-gated calcium currents. These responses have the characteristics of action potential currents: they were elicited after the cessation of the applied pulse and the "A" component is eliminated together with the sodium component upon application of TTX. Similar responses to long and short pulses were obtained from the surface of the associated magnocellular somata in the supraoptic nucleus, and their projections. The explant currents are similar to those previously characterized using conventional methods from somata and terminals.     

Action potentials and membrane currents of isolated single smooth muscle cells of cat and rabbit colon

Membrane potentials, action potentials and macroscopic currents in enzymatically dispersed, single smooth muscle cells of the circular layer of cat and rabbit colon were investigated. The cells did not exhibit spontaneous depolarizations and repolarizations (slow waves) or spontaneous action potentials. Single action potentials of smooth muscle cells were evoked by depolarizing current pulses of 5 ms to 3 s duration. A repetitive action potential discharge and an increase in the duration of the action potential was observed in cells during long depolarizing current pulses by superfusion with tetraethylammonium (TEA) or 4-aminopyridine (4-AP). Tetrodotoxin (TTX) did not alter the configuration of the action potential. Voltage-clamp experiments revealed two major outward macroscopic currents: a quasi-instantaneous (time-independent) and a time-dependent outward current. Both currents were identified as potassium (K) currents due to their pharmacological sensitivity to K antagonists [TEA, 4-AP and cesium (Cs)] and due to the reversal potential of outward tail currents. Barium selectively blocked the time-independent current. A time-dependent outward K current in colon cells was observed which appeared to be dependent upon entry of calcium ions (Ca²⁺) through voltage-dependent Ca-channels, since it was blocked by cadmium and low concentrations of nifedipine. The majority of cells did not exhibit transient outward currents. Inward currents were exposed in some of the cells when the K currents were blocked by external TEA and by replacement of K by Cs and TEA in the recording pipette. Inward currents were presumably carried by Ca²⁺, since they were not altered by TTX, were sensitive to external Ca concentrations and were abolished by the Ca channel antagonist, nifedipine. Carbachol augmented the amplitude of the inward Ca current.     

The A-type potassium current: catechol-induced blockage in snail neurons

The effects of catechol (1-12.5 mM) on membrane properties, action potential and membrane ionic currents were investigated in identified snail neurons under current- and voltage-clamp conditions. Catechol hardly influenced the resting membrane potential, or the action potential amplitude and duration, but it increased the spike voltage threshold and slightly decreased the input resistance. Catechol specifically decreased the amplitude of the potassium A-currents in a dose-dependent way (Kd = 5 mM), without significant modulation of other potassium currents. The time constants of decay of A-current increased and the steady-state activation or inactivation curve shifted to more positive potentials in the catechol solutions. The blocking effect of catechol on A-currents followed a one-to-one binding stoichiometry (nH = 0.8).     

Voltage-dependent ion channels in CAD cells: A catecholaminergic neuronal line that exhibits inducible differentiation

Cell lines derived from tumors engineered in the CNS offer promise as models of specific neuronal cell types. CAD cells are an unusual subclone of a murine cell line derived from tyrosine hydroxylase (TH) driven tumorigenesis, which undergoes reversible morphological differentiation on serum deprivation. Using single-cell electrophysiology we have examined the properties of ion channels expressed in CAD cells. Despite relatively low resting potentials, CAD cells can be induced to fire robust action potentials when mildly artificially hyperpolarized. Correspondingly, voltage-dependent sodium and potassium currents were elicited under voltage clamp. Sodium currents are TTX sensitive and exhibit conventional activation and inactivation properties. The potassium currents reflected two pharmacologically distinguishable populations of delayed rectifier type channels while no transient A-type channels were observed. Using barium as a charge carrier, we observed an inactivating current that was completely blocked by nimodipine and thus associated with L-type calcium channels. On differentiation, three changes in functional channel expression occurred; a 4-fold decrease in sodium current density, a 1.5-fold increase in potassium current density, and the induction of a small noninactivating barium current component. The neuronal morphology, excitability properties, and changes in channel function with differentiation make CAD cells an attractive model for study of catecholaminergic neurons.     

Histamine decreases calcium-mediated potassium current in guinea pig hippocampal CA1 pyramidal cells

The action of histamine on CA1 pyramidal cells was studied in a hippocampal slice preparation. In the presence of tetrodotoxin (TTX) and tetraethylammonium (TEA), histamine had little effect on the calcium spikes. Using the single-electrode voltage-clamp technique, the actions of histamine on membrane currents were tested. In TTX, histamine (1 microM) decreased outward current only at potentials more depolarized than approximately -50 mV, where calcium-mediated potassium current is predominant. In the presence of manganese, histamine was without effect. Histamine (10 microM) did not affect the transient outward potassium current (A-current), the inward M-current resulting from small hyperpolarizing steps, or the inward Q-current elicited by larger hyperpolarizing steps. Blocking potassium currents with TEA or replacing calcium with barium revealed a slow inward current normally carried by calcium. With TTX present to block sodium currents, histamine (10 microM) did not reduce the inward current. The outward current reduced by a maximally effective concentration of histamine (10 microM) can be further decreased by manganese. The results support the conclusion that histamine selectively decreases the calcium-mediated potassium conductance in CA1 pyramidal cells of hippocampus. The possibility is raised that there is a component of calcium-mediated potassium current that is insensitive to histamine.     

Contribution of presynaptic calcium-activated potassium currents to transmitter release regulation in cultured Xenopus nerve-muscle synapses

Using Xenopus nerve-muscle co-cultures, we have examined the contribution of calcium-activated potassium (K(Ca)) channels to the regulation of transmitter release evoked by single action potentials. The presynaptic varicosities that form on muscle cells in these cultures were studied directly using patch-clamp recording techniques. In these developing synapses, blockade of K(Ca) channels with iberiotoxin or charybdotoxin decreased transmitter release by an average of 35%. This effect would be expected to be caused by changes in the late phases of action potential repolarization. We hypothesize that these changes are due to a reduction in the driving force for calcium that is normally enhanced by the local hyperpolarization at the active zone caused by potassium current through the K(Ca) channels that co-localize with calcium channels. In support of this hypothesis, we have shown that when action potential waveforms were used as voltage-clamp commands to elicit calcium current in varicosities, peak calcium current was reduced only when these waveforms were broadened beginning when action potential repolarization was 20% complete. In contrast to peak calcium current, total calcium influx was consistently increased following action potential broadening. A model, based on previously reported properties of ion channels, faithfully reproduced predicted effects on action potential repolarization and calcium currents. From these data, we suggest that the large-conductance K(Ca) channels expressed at presynaptic varicosities regulate transmitter release magnitude during single action potentials by altering the rate of action potential repolarization, and thus the magnitude of peak calcium current.     

Ionic mechanisms underlying the firing properties of rat neonatal motoneurons studied in vitro

Ionic mechanisms underlying the firing properties of spinal motoneurons of neonatal rats (postnatal days 3-10) have been investigated using a hemisected, in vitro spinal cord preparation. These results demonstrate the presence of a high-threshold voltage-dependent calcium response and partial sodium-dependent spikes. The calcium current is evident during the falling phase of the action potential and is the major component of the after-depolarizing potential. The subsequent increase in intracellular calcium concentration activates a calcium-dependent potassium conductance (gK-Ca), the major component of the after-hyperpolarizing potential. The gCa, by activating gK-Ca, is the primary determinant of firing rate in neonatal motoneurons. For, when gCa was blocked by Cd2+, the interspike interval decreased, the maximum firing rate and the slope of the firing frequency-injected current relation increased. The calcium current is particularly robust during the first few postnatal days; during this period, tetrodotoxin resistant action potentials can be elicited by direct stimulation under control conditions. In animals older than 5 days such calcium spikes could be elicited only after decreasing gK with intracellular Cs+ or extracellular tetraethylammonium. This was the case even when 1 mM of the bath CaCl2 was replaced with BaCl2. The rising phases of calcium spikes recorded from neurons in both age groups demonstrate several components suggesting the calcium spikes comprise several discrete events, which probably originate across the dendritic membrane. When gK was decreased by bath application of tetraethylammonium+ and Cs+, neonatal motoneurons generated prolonged Ca-dependent spikes lasting for up to 6 s. Repolarization of Ca spikes occurred in two stages, the first was rapid (-2.11 +/- 0.8 V/s, n = 6) but incomplete. The second, was slower (-0.01 +/- 0.003 V/s, n = 5) and returned the membrane potential to the resting level after about 1-2 s. It is suggested that accumulation of extracellular potassium may contribute to the slow phase of repolarization. Motoneurons from the younger age group (3-5 days old) demonstrate all-or-none partial spikes rising from the after-depolarization of directly elicited sodium-dependent action potentials. Similar partial spikes were elicited from neurons from older animals during intracellular Cs+ loading. The partial spikes had faster rates of rise than the tetrodotoxin-resistant spikes and were not seen after tetrodotoxin treatment, suggesting that they are sodium-dependent.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of voltage-dependent excitatory responses to alpha,beta-methylene ATP, ATP and non-adrenergic nerve stimulation by dihydropyridines in the guinea-pig vas deferens

Simultaneous recordings of mechanical and intracellular electrical activity were obtained from the guinea-pig vas deferens, where nerve stimulation, ATP and the stable nucleotide analogue alpha,beta-methylene ATP elicited excitatory responses. Excitatory junction potentials and action potentials were elicited by low-frequency (trains of pulses, generally less than or equal to 2 Hz) field stimulation. alpha,beta-Methylene ATP and ATP elicited only concentration-dependent depolarizations at low concentrations, while higher concentrations elicited a superimposed action potential discharge which was accompanied by mechanical contraction. The voltage threshold at which action potential discharge was initiated by these three stimuli was about -45 mV (resting membrane potential averaged -66 mV). Action potential discharges and contractile responses were antagonized by nifedipine and augmented by Bay K 8644 at concentrations (1 and 0.5 microM, respectively) which exhibited only small effects on either excitatory junction potential amplitudes or nucleotide-induced depolarizations. Bay K 8644 enhanced and nifedipine antagonized the repolarization (rectification) phase of action potential discharge elicited by nerve stimulation and drugs; after-hyperpolarizations were prominent in the presence of Bay K 8644 (0.1-5 microM). Excitatory junction potentials were antagonized after exposure to alpha,beta-methylene ATP. This antagonistic effect of alpha,beta-methylene ATP was also observed following depolarizations elicited in the absence and presence of nifedipine (1 microM). Noradrenaline was approximately 50-100 times less potent than alpha,beta-methylene ATP in eliciting action potential discharge and contraction. It was only when a high concentration of noradrenaline was used (about 60-100 microM) that the noradrenaline-induced depolarization attained the voltage threshold for action potential initiation. These results illustrate the similarity of the electrical components which underlie excitation by nerve stimulation and adenine nucleotides in the vas deferens, and demonstrate the ability of dihydropyridines to regulate voltage-dependent events associated with both the generation and inactivation of muscle action potentials. These are probably voltage-dependent calcium currents and calcium-activated potassium currents, respectively. Neither excitatory junction potentials nor the mechanism of desensitization of the ATP purinoceptor by alpha,beta-methylene ATP involve voltage-dependent calcium channels.     

A-type potassium currents dominate repolarisation of neonatal rat primary auditory neurones in situ.

Spiral ganglion neurones provide the afferent innervation to cochlear hair cells. Little is known of the molecular physiological processes associated with the differentiation of these neurones, which occurs up to and beyond hearing onset. We have identified novel A-type (inactivating) potassium currents in neonatal rat spiral ganglion neurones in situ, which have not previously been reported from the mammalian cochlea, presumably as a consequence of altered protein expression associated with other preparations. Under whole-cell voltage clamp, voltage steps activated both A-type and non-inactivating outward currents from around -55 mV. The amplitude of the A-type currents was dependent on the holding potential, with steady-state inactivation relieved at hyperpolarised potentials. At -60 mV (close to the resting potential in situ) the currents were approximately 30% enabled. The inactivation kinetics and the degree of inactivation varied between cells, suggesting heterogeneous expression of multiple inactivating currents. A-type currents provided around 60% of total conductance activated by depolarising voltage steps from the resting potential, and were very sensitive to bath-applied 4-aminopyridine (0.01-1 mM). Tetraethylammonium (0.1-30 mM) also blocked the majority of the A-type currents, and the non-inactivating outward current, but left residual fast inactivating A-type current. Under current clamp, neurones fired single tetrodotoxin-sensitive action potentials. 4-Aminopyridine relieved the A-type current mediated stabilisation of membrane potential, resulting in periodic small amplitude action potentials.This study provides the first electrophysiological evidence for A-type potassium currents in neonatal spiral ganglion neurones and shows that these currents play an integral role in primary auditory neurone firing.     

Pre- and post- junctional effects of 1-fluoro-2,4-dinitrobenzene at the frog neuromuscular junction

1. FDNB increased by 60-90% the depolarization of the end-plate produced by applied carbachol in frog sciatic nerve-sartorius muscle preparations.2. In partially curarized preparations, FDNB (0.4 mM) increased the amplitude of the end-plate potential by a factor of 1.8.3. The quantal content of end-plate potentials was increased by FDNB (2 mM) as determined by the method of failures.4. After approximately 25-35 min, neuromuscular transmission was blocked by 0.4 mM-FDNB, as evidenced by abolition of neurally elicited end-plate potentials. At this stage miniature end-plate potentials could still be recorded, which indicates that the neuromuscular block was presynaptic.5. FDNB (0.4 mM) increased miniature end-plate potential frequency several hundred-fold when the Ringer solution contained normal calcium concentration (1.8 mM) or 0.45 mM calcium and 5.4 mM magnesium.6. During the first 60 min of exposure to 0.4 mM-FDNB there was a slight drop (4-6 mV) in resting potentials of muscle fibres. During this period directly initiated action potentials showed a marked decrease in the rate of repolarization and a small decrease in the amplitude and rate of rise.7. Using the technique of point voltage clamping in tetrodotoxin-treated muscles, it has been found that FDNB almost completely abolished the active increase in g(K) during stepwise depolarization of the nonjunctional muscle fibre membrane from -90 to 0 mV. The passive outward leakage current appeared unaffected by FDNB.     

Studies of solitary semicircular canal hair cells in the adult pigeon. II. Voltage-dependent ionic conductances

1. The ionic conductances present in putative type II hair cells enzymatically dissociated from the anterior, posterior, and lateral semicircular canal cristae of the white king pigeon (Columba livia) vestibule were studied under whole cell voltage clamp. 2. Two classes of voltage-dependent potassium conductances were distinguishable on the basis of the time course of activation and inactivation and pharmacologic sensitivity. The rapid potassium conductance, IA, as inhibited by 6 mM 4-aminopyridine (4-AP), whereas the slow potassium conductance, IK, was inhibited by 50 mM tetraethylammonium (TEA). These conductances were not affected by extracellular calcium removal. IA was quite similar to the rapidly-inactivating A-current of molluscan soma, whereas IK was more like the delayed rectifier of molluscan soma. 3. The steady-state inactivation of IA occurred over a potential range from -100 to -40 mV. The threshold for activation of IA occurred between -60 and -50 mV. The slope conductance of the I-V curve over a range of -50 to -20 mV was 13.7 nS when the conditioning pulse was -100 mV, and we estimate it to be approximately 1-2 nS from the resting membrane potential of -56 mV. 4. The steady-state inactivation of IK was approximately 60% at -40 mV and was completely removed at -80 mV. The threshold for activation of IK was between -50 and -40 mV. The slope conductance of the I-V curve over a range of -50 to -20 mV was 10.5 nS when the conditioning pulse was -80 mV, and we estimate it to be approximately 6-7 nS from the resting potential of -56 mV. 5. At -56 mV (the average resting membrane potential of putative type II semicircular canal hair cells), approximately 10-14% of IA channels and approximately 57-70% of IK channels were not inactivated: thus IA and IK can contribute to the outward current during small depolarizations from rest. 6. A small calcium-dependent outward current, IK(Ca), could be elicited during step depolarizations from a holding potential of -40 mV. This calcium-dependent current was active over the range of -20 to +40 mV. 7. Inward currents could not be detected when the cells were exposed to normal physiological solutions. However, when the outward currents were blocked with internal cesium and the external solution contained 20 mM barium, sustained inward currents with rapid activation kinetics could be detected. The threshold for activation of the inward current occurred at -40 mV, and the I-V relationship peaked at -10 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat hippocampal neurons in culture: potassium conductances

Two-electrode voltage-clamp methodology was used to analyze voltage-dependent ionic conductances in 81 rat hippocampal neurons grown in culture for 4-6 wk. Pyramidal and multipolar cells with 15- to 20-micron-diameter cell bodies were impaled with two independent KCl electrodes. The cells had resting potentials of -30 to -60 mV and an average input resistance of about 30 M omega. A depolarizing command applied to a cell maintained in normal medium invariably evoked a fast (2-10 ms) inward current that saturated the current-passing capacity of the system. This was blocked in a reversible manner by application of tetrodotoxin (TTX) (0.1-1.0 microM) near the recorded cell. In the presence of TTX, a depolarizing command evoked a rapidly rising (3-5 ms), rapidly decaying (25 ms) transient outward current reminiscent of "IA" reported in molluscan neurons. This was followed by a more slowly activating (approximately 100 ms) outward current response of greater amplitude that decayed with a time constant of about 2-3 s. These properties resemble those associated with the K+ conductance, IK, underlying delayed rectification described in many excitable membranes. IK was blocked by extracellular application of tetraethylammonium (TEA) but was insensitive to 4-aminopyridine (4-AP) at concentrations that effectively eliminated IA. IA, in turn, was only marginally depressed by TEA. Unlike IK, IA was completely inactivated when the membrane was held at potentials positive to -50 mV. Inactivation was completely removed by conditioning hyperpolarization at -90 mV. A brief hyperpolarizing pulse (10 ms) was sufficient to remove 95% of the inactivation. IA activated on commands to potentials more positive than -50 mV. The inversion potential of the ionic conductance underlying IA and IK was in the range of the K+ equilibrium potential, EK, as measured by the inversion of tail currents; and this potential was shifted in a depolarizing direction by elevated [K+]0. Thus, both current species reflect activation of membrane conductance to K+ ions. Hyperpolarizing commands from resting potentials revealed a time- and voltage-dependent slowly developing inward current in the majority of cells studied. This membrane current was observed in cells exhibiting "anomalous rectification" and was therefore labeled IAR. It was activated at potentials negative to -70 mV with a time constant of 100-200 ms and was not inactivated. A return to resting potential revealed a tail current that disappeared at about EK. IAR was blocked by extracellular CS+ and was enhanced by elevating [K+]0. It thus appears to be carried by inward movement of K+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regenerative potentials in rat neostriatal neurons in an in vitro slice preparation

Summary 1. Regenerative potentials in rat neostriatal neurons were studied using the in vitro slice preparation. Some of the recorded neurons were intracellularly labeled with HRP. All had the morphological characteristics of the medium spiny neuron. 2. Application of TTX (10^–5 g/ml) to the superfusing medium abolished fast action potentials generated by intracellularly injected depolarizing current. Application of TEA prolonged the spike duration by decreasing its repolarizing rate without affecting rising phase. After suppression of K-conductance by TEA, depolarizing current elicited both fast and slow all or none action potentials. 3. Combined treatment with TTX and TEA revealed two types of depolarizing potentials, a slowly rising graded depolarizing potential and slow action potential. Substitution of Ca⁺⁺ with Mg⁺⁺ in the medium diminished the amplitude of these potentials. They were also blocked by application of Co⁺⁺ into the superfusion medium. The duration of slow action potentials were increased (1) with increase in the intensity of current pulse, (2) with decrease in the resting membrane potential, and (3) with increase in the concentration of TEA in the bathing medium. 4. In the normal Ringer solution, local stimulation elicited depolarizing postsynaptic responses (DPSPs). Large DPSPs evoked by strong local stimulation triggered one or two fast action potentials. In some neurons, large DPSPs could trigger both fast and slow action potentials. They were consistently triggered after application of TEA (1 mM) to the medium. 5. When a relatively high concentration of TEA (4 mM) was applied to the Ringer solution, locally evoked DPSPs could trigger only slow action potentials. In double stimulation experiments, a large reduction in the amplitude and the duration of test DPSPs was observed up to about 150 ms interstimulus interval.     

Effects of halothane on the transducer and potential activated currents of the crustacean stretch receptor

Halothane was applied to the stretch receptor neuron of the crayfish (Astacus astacus) and the effects on the transducer properties and the potential activated currents were studied with potential clamp technique using two microelectrodes. Exposure to halothane reduced the frequency of action potentials during stretch. This was shown to be due to effects both on the action potential generating currents and the transducer current. Halothane partially blocked the TTX sensitive fast inward current in a dose-dependent manner (Apparent KD = 3 mM). Halothane also reduced the outward current produced by a positive potential step. Both the fast and the slow component were affected, although the fast outward current seemed to be most sensitive. There was little or no change in the currents resulting from negative potential steps. The peak of the receptor potential and the receptor current were very little affected by halothane. The amplitude of the static phase of the receptor potential was reduced to a greater degree than the static phase of the receptor current (cells treated with TTX). A change in reversal potential of about--13 mV was observed for the peak and the static phase of the receptor current in four cells indicating an increased cord conductance for the transducer channel.     

Differential inhibition of glial K(+) currents by 4-AP

Spinal cord astrocytes express four biophysically and pharmacologically distinct voltage-activated potassium (K(+)) channel types. The K(+) channel blocker 4-aminopyridine (4-AP) exhibited differential and concentration-dependent block of all of these currents. Specifically, 100 microM 4-AP selectively inhibited a slowly inactivating outward current (K(SI)) that was insensitive to dendrototoxin (< or = 10 microM) and that activated at -50 mV. At 2 mM, 4-AP inhibited fast-inactivating, low-threshold (-70 mV) A-type currents (K(A)) and sustained, TEA-sensitive noninactivating delayed-rectifier-type currents (K(DR)). At an even higher concentration (8 mM), 4-AP additionally blocked inwardly rectifying, Cs(+)- and Ba(2+)-sensitive K(+) currents (K(IR)). Current injection into current-clamped astrocytes in culture or in acute spinal cord slices induced an overshooting voltage response reminiscent of slow neuronal action potentials. Increasing concentrations of 4-AP selectively modulated different phases in the repolarization of these glial spikes, suggesting that all four K(+) currents serve different roles in stabilization and repolarization of the astrocytic membrane potential. Our data suggest that 4-AP is an useful, dose-dependent inhibitor of all four astrocytic K(+) channels. We show that the slowly inactivating astrocytic K(+) currents, which had not been described as separate current entities in astrocytes, contribute to the resting K(+) conductance and may thus be involved in K(+) homeostatic functions of astrocytes. The high sensitivity of these currents to micromolar 4-AP suggests that application of 4-AP to inhibit neuronal A-currents or to induce epileptiform discharges in brain slices also may influence astrocytic K(+) buffering.     

An inward calcium current underlying regenerative calcium potentials in rat striatal neurons in vitro enhanced by Bay K 8644

The single electrode voltage clamp technique was used to characterize the currents underlying the calcium potentials in rat caudate neurons in vitro. In current clamp experiments, long depolarizing current pulses evoked repetitive firing of fast somatic action potentials. These were abolished by tetrodotoxin (1 microM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 microM) abolished the hyperpolarizing notch, enhanced the slow graded depolarizing response and induced the appearance of a slow all-or-nothing action potential. Both the slow graded response and the all-or-nothing action potential were abolished by cobalt (2 mM), suggesting the involvement of voltage-dependent calcium conductances. When the neurons were loaded intracellularly with caesium the action potential duration increased. Substitution of the extracellular calcium by barium (1-3 mM) or external addition of tetraethylammonium (5 mM) further prolonged spike duration and induced the appearance of long-lasting plateau potentials. These were insensitive to tetrodotoxin and were reversibly blocked by the calcium antagonists cobalt (2 mM), manganese (2 mM) or cadmium (500 microM). The calcium potentials were enhanced by the calcium 'agonist' BAY K 8644 (1-5 microM). In voltage clamp experiments when intracellular caesium was used to reduce outward currents and tetrodotoxin to block fast regenerative sodium currents, depolarizing voltage steps from a holding potential of -50, -40 mV activated an inward current. This current peaked in 50-80 ms and inactivated in two phases: an initial one at 150-200 ms followed by a second one after several hundred ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of noradrenaline on some potassium currents in CA1 neurones in rat hippocampal slices

Pyramidal (CA1) cells in rat hippocampal slices were voltage clamped using a single electrode voltage clamp. In the presence of tetrodotoxin (TTX), depolarizing pulses from holding potentials of −60 to −70 mV elicited a slow inward calcium (Ca²⁺) current and two outward potassium (K⁺) currents: an A current and a slower, Ca²⁺-dependent K⁺ current. Noradrenaline (NA) (20 μM) depressed the amplitude of the K⁺ currents without affecting the Ca²⁺ current. The effect of NA could be blocked with propranolol and was mimicked by isoprenaline, suggesting that NA depresses the K⁺ currents by binding to β-receptors.     

S100 calcium binding protein affects neuronal electrical discharge activity by modulation of potassium currents

S100 calcium binding protein has been associated with a variety of intra- and extracellular calcium-mediated functions, including learning and memory. We have previously localized S100-immunoreactive neurons correlated with spontaneous discharge activity in the central nervous system of the mollusc, Helix pomatia. In this study, we further investigated the effects of S100 (S100B and S100A1) on electrical discharge activity and membrane currents of Helix neurons, using current- and voltage-clamp techniques. Extracellular application of disulphide-linked S100B (S100B-s-s) in pico- to nanogram/ml concentrations was found to hyperpolarize the membrane resting potential, to inhibit spontaneous discharge activity of action potentials, to alter the stimulus response behaviour from tonic to phasic, to decrease the duration and increase the afterhyperpolarization of action potentials, and to reduce the cell input resistance. Measurement of membrane currents revealed that the total outward current was increased by S100B-s-s. Separation of outward currents showed that three types of potassium currents were altered: (i) an inward rectifying current, (ii) a calcium-activated potassium outward current, both increased by S100B-s-s, and (iii) a delayed, voltage-dependent potassium outward current which was decreased by the protein. The transient potassium outward and the calcium inward currents were not affected by S100B-s-s. Immunocytochemistry showed intracellular labelling of the cytoplasm after extracellular application of the protein, indicating internalization and suggesting an internal site of action. Injection of S100A1 mimicked the effects of S100B-s-s on discharge activity and action potentials. We conclude from our experiments that S100 calcium binding protein, by modulation of potassium currents, may play a role as a neuromodulator in nervous functions.