Muscarinic receptor activation induces a prolonged post-stimulus afterdepolarization with a conductance decrease in guinea-pig olfactory cortex neurones in vitro.

The persistent excitation of guinea-pig olfactory cortical neurones in vitro by the muscarinic agonist oxotremorine-M (OXO-M) was investigated. In OXO-M (10-20 microM), a slowly-decaying afterdepolarization (sADP) accompanied by sustained repetitive firing was induced following a long depolarizing stimulus. The corresponding slow inward current (IADP) revealed under voltage clamp behaved like a K(+)-mediated tail current, but was associated with a decreased membrane conductance. IADP was insensitive to tetrodotoxin (TTX), Ba2+, Cs+, or 4-aminopyridine (4-AP), but was blocked by 500 microM TEA or TBA (tetrabutylammonium). The OXO-M response and IADP were also reduced by Cd2+ or Ca(2+)-free solution, suggesting a dependence on Ca(2+)-entry. We propose that OXO-M induces a novel outward K+ current that can be slowly de-activated by Ca(2+)-entry during a depolarizing stimulus. Summation of IADP tail currents could contribute to the sustained muscarinic excitation of mammalian cortical neurones.     

Calcium-dependent chloride current induced by axotomy in rat sympathetic neurons.

1. Seven to ten days after sectioning their axons, rat sympathetic neurons were studied using intracellular recording techniques in an in vitro preparation of the superior cervical ganglion. 2. In 75% of axotomized cells, an after-depolarization (ADP) was observed following spike firing or depolarization with intracellular current pulses. Discontinuous single-electrode voltage-clamp techniques were employed to study the ADP. When the membrane potential was clamped at the resting level just after an action potential, a slow inward current was recorded in cells that showed an ADP. 3. In the presence of TTX and TEA, inward peaks and outward currents were recorded during depolarizing voltage jumps, followed by slowly decaying inward tail currents accompanied by large increases in membrane conductance. The inward peak and tail currents activated between -10 and -20 mV and reached maximum amplitudes around 0 mV. With depolarizing jumps to between +40 and +50 mV, net outward currents were recorded during the depolarizing jumps but inward tail currents were still activated. 4. In the presence of the Ca2+ channel blocker cadmium, or when Ca2+ was substituted by Mg2+, the ADP disappeared. In voltage-clamped cells, cadmium blocked the inward tail currents. The reversal potential for the inward tail current was approximately -15 mV. Substitution of the extracellular NaCl by sucrose or sodium isethionate increased the amplitude of the inward tail current, and displaced its equilibrium potential to more positive values. Changes in extracellular [K+] did not appreciably affect the inward tail current amplitude or equilibrium potential. Niflumic acid, a blocker of chloride channels activated by Ca2+, almost completely blocked the tail current. 5. No ADPs were observed in non-axotomized neurons, and when depolarizing pulses were applied while in voltage clamp no inward tail currents were evoked in these normal cells. 6. It is concluded that axotomy of sympathetic ganglion cells produces the appearance of a Ca(2+)-dependent chloride current responsible for the ADP observed following spike firing.     

Long-lasting reduction of excitability by a sodium-dependent potassium current in cat neocortical neurons

1. The function and ionic mechanism of a slow outward current were studied in large layer V neurons of cat sensorimotor cortex using an in vitro slice preparation and single microelectrode voltage clamp. 2. With Ca2+ influx blocked, a slow relaxation ("tail") of outward current followed either (1) repetitive firing evoked for 1 s or (2) a small 1-s depolarizing voltage clamp step that activated the persistent Na+ current of neocortical neurons, INaP. When a depolarization that activated INaP was maintained, an outward current gradually developed and increased in amplitude over a period of tens of seconds to several minutes. An outward tail current of similar duration followed repolarization. The slow outward current was abolished by TTX, indicating it depended on Na+ influx. 3. With Ca2+ influx blocked, the onset of the slow Na+-dependent outward current caused spike frequency adaptation during current-evoked repetitive firing. Following the firing, the decay of the Na+-dependent current caused a slow afterhyperpolarization (sAHP) and a long-lasting reduction of excitability. It also was responsible for habituation of the response to repeated identical current pulses. 4. The Na+-dependent tail current had properties expected of a K+ current. Membrane chord conductance increased during the tail, and tail amplitude was reduced or reversed by membrane potential hyperpolarization and raised extracellular K+ concentration [( K+]0). 5. The current tail was reduced reversibly by the K+ channel blockers TEA (5-10 mM), muscarine (5-20 microM), and norepinephrine (100 microM). These agents also resulted in a larger, more sustained inward current during the preceding step depolarization. Comparison of current time course before and after the application of blocking agents suggested that, in spite of its capability for slow buildup and decay, the onset of the Na+-dependent outward current occurs within 100 ms of an adequate step depolarization. 6. With Ca2+ influx blocked, extracellular application of dantrolene sodium (30 microM) had no clear effect on the current tail or the corresponding sAHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Rat hippocampal neurons in culture: Ca2+ and Ca2+-dependent K+ conductances

Rat hippocampal neurons grown in dissociated cell culture were studied in a medium containing 1 microM tetrodotoxin (TTX) and 25 mM tetraethylammonium (TEA), which eliminated the Na+ and K+ conductances normally activated by depolarizing current injections. In this medium depolarizing current pulses evoked depolarizing regenerative potentials and afterhyperpolarizations in most cells. Both of these events were blocked by close application of Co2+ or Cd2+. These events resemble Ca2+ spikes reported previously in hippocampal pyramidal cells. The membrane potential at which these Ca2+ spikes could be triggered and the rheobase current necessary were dependent on the potential at which the cell was conditioned: the more depolarized the holding potential, the more negative the absolute potential at which a spike could be triggered and the less rheobase current required. The duration of these Ca2+ spikes was also sensitive to the holding potential: the more depolarized the holding level, the longer the duration of the triggered spikes. The amplitude and duration of the Ca2+ spikes were enhanced in a reversible manner by 0.5-1.0 mM 4-aminopyridine (4-AP) delivered in the vicinity of the cell. Two-electrode voltage-clamp analysis of cells studied in TTX, TEA-containing medium revealed an inward current response that peaked in 25-50 ms during depolarizing commands. This response first became detectable during commands to -30 mV. It peaked in amplitude during commands to -10 mV and was enhanced in medium containing elevated [Ca2+]0. It was blocked by either 20 mM Mg2+, 0.2 mM Cd2+, 5 mM Co2+, or 5 mM Mn2+. These results have led us to identify this inward current response as ICa2+. 4-AP enhanced the magnitude and duration of ICa2+ independent of the drug's depressant effects on a transient K+ current also observed under these same experimental conditions. In many but not all cells the Ca2+ spike was followed by a long-lasting hyperpolarization associated with an increase in membrane conductance. This was blocked by Co2+. Under voltage clamp ICa2+ was followed by a slowly developing outward current response that was attenuated by Co2+ or Cd2+. These properties observed under current- and voltage-clamp recording conditions are superficially similar to those previously reported for Ca2+-dependent K+ conductance mechanisms (IC) recorded in these and other membranes. Long-lasting tail currents following activation of IC inverted in the membrane potential range for the K+ equilibrium potential found in these cells.     

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.     

Norepinephrine selectively reduces slow Ca2+- and Na+-mediated K+ currents in cat neocortical neurons

1. The effects of norepinephrine (NE) and related agonists and antagonists were examined on large neurons from layer V of cat sensorimotor cortex ("Betz cells") were examined in a brain slice preparation using intracellular recording, constant current stimulation and single microelectrode voltage clamp. 2. Application of NE (0.1-100 microM) usually caused a small depolarization from resting potential; hyperpolarizations were rare. Application of NE reversibly reduced rheobase and both the Ca2+- and Na+-dependent portions of the slow afterhyperpolarization (sAHP) that followed sustained firing evoked by constant current injection. The faster Ca2+-dependent medium afterhyperpolarization (mAHP), the fast afterhyperpolarization (fAHP), the action potential, and input resistance were unaffected. 3. The changes in excitability produced by NE application were most apparent during prolonged stimulation. The cells exhibited steady repetitive firing to currents that were formerly ineffective. The slow phase of spike frequency adaptation was reduced selectively and less habituation occurred during repeated long-lasting stimuli. The relation between firing rate and injected current became steeper if firing rate was averaged over several hundred milliseconds. 4. During voltage clamp in TTX, NE application selectively reduced the slow component of Ca2+-mediated K+ current. The faster Ca2+-mediated K+ current was unaffected, as were two voltage-dependent, transient K+ currents, the anomalous rectifier and leakage conductance measured at resting potential. Depolarizing voltage steps in the presence of Cd2+ revealed an apparent time- and voltage-dependent increase of the persistent Na+ current after NE application. The voltage-clamp results suggested ionic mechanisms for all effects seen during constant current stimulation except the depolarization from resting potential. The latter was insensitive to Cd2+ and TTX and occurred without a detectable change in membrane conductance. 5. NE application did not alter Ca2+ spikes evoked in the presence of TTX and 10 mM TEA. Inward Ca2+ currents examined during voltage clamp in TTX (with K+ currents reduced) became slightly larger after NE application. We conclude that NEs reduction of the slow Ca2+-mediated K+ current is not caused by reduction of Ca2+ influx. 6. Effects on membrane potential, rheobase, and the sAHP were mimicked by the beta-adrenergic agonist isoproterenol, but not by the alpha-adrenergic agonists clonidine or phenylephrine at higher concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characteristics of potassium and calcium currents of hepatic stellate cells (ito) in rat

Hepatic stellate cells (HSCs) are known to play a role in the pathogenesis of the increased intrahepatic vascular resistance found in chronic liver diseases. The aim of this study was to evaluate the K+ and Ca2+ currents in cultured HSCs from rat liver, through the patch-clamp technique. Most cells were positive for desmin immunostain after isolation and in alpha-smooth muscle actin immunostain after 10 - 14 days of culturing. Outward and inward rectifying K+ currents were confirmed. Two different types of K+ currents were distinguished: one with the inward rectifying current and the other without. The outward K+ currents consisted of at least four components: tetraethylammonium (TEA)-sensitive current, 4-aminopyridine (4-AP)-sensitive current, pimozide-sensitive current and three blocker-resistant current. The peaks of the outward K+ currents evoked by a depolarizing pulse were decreased to 32.0 +/- 3.0, 62.8 +/- 3.7 and 32.8 +/- 3.5% by 5 mM TEA, 2 mM 4-AP and 15 micro M pimozide, respectively. Moreover, the combined application of three blockers caused 86.6 +/- 4.8% suppression. The inward currents evoked hyperpolarizing pulses were inwardly rectifying and almost blocked by Ba2+. Elevation of external K+ increased the inward current amplitude and positively shifted its reversal potential. Voltage- dependent Ca2+ currents which were completely abolished by Cd2+ and nimodipine were detected in 14 day cultured HSCs. In this study, the cultured HSCs were found to express outward K+ currents composed of multiple pharmacological components, Ba2+-sensitive inward rectifying K+ current and L-type Ca2+ current.     

Tachykinins modulate multiple ionic conductances in voltage-clamped rat spinal dorsal horn neurons

1. The membrane actions of substance P (SP) and a related tachykinin, neurokinin A (NKA), have been investigated by means of a single-electrode, voltage-clamp technique in the immature rat dorsal horn neurons using an in vitro spinal cord slice preparation. 2. When the membrane potential was held at the resting level of between -75 and -55 mV, bath application of SP or NKA (10(-7) to 10(-5) M, for 1-3 min) induced an inward shift in the holding current lasting several minutes. The magnitude of this effect varied between 10 and 400 pA depending on the concentration of the peptides and the holding potential. 3. When a dorsal horn neuron was held at the resting level and subjected to 1-s depolarizing commands to membrane potentials between -60 and -35 mV, slow inward relaxations and inward tail currents, the latter on repolarization to the holding potential, were recorded. During the tachykinin-induced inward shift in the holding current, the inward relaxation and the tail current were augmented in a dose-related manner. 4. The SP-induced augmentation of the slow inward relaxation and the inward tail current is likely to be due to the enhancement of the activation of the Ca2+ current, because the effect was present, and even augmented in a zero-Ca2+, Ba2+-containing solution, it was reduced or completely abolished by zero-Ca2+, Co2+-, or Mg2+-containing solutions and is largely independent of the changes in external Na+, K+, or Cl- ions. Moreover, in the presence of the K+-channel blocker, tetraethylammonium (TEA), the effect is increased. 5. Depolarizing voltage commands to potentials positive to -35 mV evoked a large, outward K+ current response in the dorsal horn neurons, which was in part Ca2+-sensitive. The outward current response was augmented by SP. The SP effect persists, although being reduced in a zero-Ca2+, Ba2+- or Co2+-containing solutions. 6. In a zero-Ca2+ solution containing Co2+ and TEA, the augmentation of the Ca2+ current and the outward K+ current by SP was abolished. However, the SP-induced increase in a Ca2+-sensitive, voltage-insensitive conductance remained, although being reduced, and the response showed a reversal at about -28 mV. This current may be a result of a tachykinin-activated nonspecific increase in cationic permeability of the membrane of dorsal horn neurons, because the current is reduced by more than one-half when Na+ or Ca2+ is removed from the bathing medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Calcium-dependent potassium currents in neurons from cat sensorimotor cortex.

1. Ca(2+)-dependent K+ currents were studied in large pyramidal neurons (Betz cells) from layer V of cat sensorimotor cortex by use of an in vitro brain slice and single microelectrode voltage clamp. The Ca(2+)-dependent outward current was taken as the difference current obtained before and after blockade of Ca2+ influx. During step depolarizations in the presence of tetrodotoxin (TTX), this current exhibited a fast onset of variable amplitude and a prominent slowly developing component. 2. The Ca(2+)-dependent outward current first appeared when membrane potential was stepped positive to -40 mV. Downsteps from a holding potential of -40 mV revealed little or no time-, voltage-, or Ca(2+)-dependent current. When membrane potential was stepped positive to -40 mV, a prolonged Ca(2+)-dependent outward tail current followed repolarization. The decay of this tail current at -40 mV was best described by a single exponential function having a time constant of 275 +/- 75 (SD) ms. The tail current reversed at 96 +/- 5 mV in 3 mM extracellular K+ concentration ([K+]o) and at more positive potentials when [K+]o was raised, suggesting that it was carried predominantly by K+. 3. The Ca(2+)-dependent K+ current consisted of two pharmacologically separable components. The slowly developing current was insensitive to 1 mM tetraethylammonium (TEA), but a substantial portion was reduced by 100 nM apamin. Most of the remaining current was blocked by the addition of isoproterenol (20-50 microM) or muscarine (10-20 microM). 4. The time courses of the apamin- and transmitter-sensitive components were similar when activated by step depolarizations in voltage clamp, but they were quite different when activated by a train of action potentials. Applying the voltage clamp at the end of a train of 90 spikes (evoked at 100-200 Hz) resulted in an Ca(2+)-dependent K+ current with a prominent rapidly decaying portion (time constant approximately 50 ms at -64 mV) and a smaller slowly decaying portion (time constant approximately 500 ms at -64 mV). The rapidly decaying portion was blocked by apamin (50-200 nM), and the slowly decaying portion was blocked by isoproterenol (20-50 microM). 5. When recorded with microelectrodes containing 2 mM dimethyl-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA), which causes prolonged afterhyperpolarizations, the Ca(2+)-dependent K+ current evoked by step depolarizations had an extremely slow onset and decay. The current recorded after a train of evoked spikes had a similar slow decay.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-dependent potassium channels in activated rat microglia.

1. Voltage-dependent currents of untreated (proliferating) and lipopolysaccharide (LPS)-treated rat microglial cells in culture were recorded using the whole-cell patch-clamp technique. 2. Membrane potentials showed prominent peaks at -35 mV and -70 mV. Membrane potentials of LPS-treated cells alternated between the two values. This may be due to a negative slope region of the I-V relation resulting in two zero current potentials. 3. From a holding potential of -70 mV, hyperpolarizing steps evoked an inwardly rectifying current both in proliferating and in LPS-treated cells, while depolarizing steps below -50 mV evoked an outwardly rectifying current only in LPS-treated microglia. The currents were K+ selective, as indicated by their reversal potential of approximately 0 mV in symmetric K+ concentrations (150 mM both intra- and extracellularly) and the reversal potential of the outward tail currents of approximately -90 mV at a normal extracellular K+ concentration (4.5 mM). 4. The activation of the outward current could be fitted by Hodgkin-Huxley-type n4 kinetics. The time constant of activation depended on voltage. 5. The inactivation of the inward and outward currents could be fitted by a single exponential. The time constant of the inward current inactivation was dependent on voltage, whereas the time constant of the outward current inactivation was virtually independent of voltage, except near the threshold of activation. Recovery of the outward from inactivation was slow and could be fitted by two exponentials. Responses to depolarizing steps were stable at 0.125 Hz, but greatly decreased from the first to the second pulse at 1 Hz. 6. The inactivation of the inward, but not of the outward, current disappeared in a low Na(+)-containing medium (5 mM). The inward current was selectively inhibited by extracellular Cs+ and Ba2+. The outward current was selectively inhibited by Cd2+, 4-aminopyridine and charybdotoxin. Replacement of intracellular K+ by an equimolar concentration of Cs+, and the extracellular application of tetraethylammonium and quinine inhibited both currents. 7. An increase of extracellular Ca2+ from 2 to 20 mM resulted in outwardly rectifying K+ channels activating at more positive potentials. Omission of Ca2+ from the extracellular medium had the opposite effect. When the intracellular free Ca2+ was increased from 0.01 to 1 microM, the outward current amplitudes were depressed. The Ca2+ ionophore A23187 had a similar effect. 8. LPS-treated microglial cells possess inwardly and outwardly rectifying K+ channels. The physiological and pharmacological characteristics of these two channel populations are markedly different.(ABSTRACT TRUNCATED AT 400 WORDS)     

Patch-clamp study of the calcium-dependent chloride current in AtT-20 pituitary cells

1. Voltage-clamp recordings were made from cultured AtT-20 pituitary cells using the whole-cell patch-clamp technique. Cells were perfused internally with Cs+ to block K+ currents and bathed externally with either 1 microM tetrodotoxin or with tetraethylammonium (TEA) as a Na+ substitute to block voltage-activated Na+ currents. 2. Depolarizing voltage steps from a holding potential of -80 mV to potentials positive to -30 mV evoked two currents: a fast inward current that activated between -30 and +70 mV and a slowly activating current (designated "slow step current") that was inward between -30 and near 0 mV (the Cl- equilibrium potential) and outward positive to about 0 mV. Repolarization to -80 mV revealed a slowly decaying, inward tail current, whose magnitude with respect to step potential closely matched the current-voltage relationship of the voltage-activated Ca2+ current. 3. Activation of the fast inward current, slow step current, and tail current, was prevented by extracellular application of Cd2+ or removal of extracellular Ca2+. Replacement of extracellular Ca2+ with Ba2+ potentiated the fast inward current but blocked the slow step and tail currents. Intracellular perfusion with greater than 1 mM of the Ca2+ chelators ethyleneglycol-bis(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA) or [1,2-bis(2)aminophenoxy]ethane N,N,N',N'-tetraacetic acid (BAPTA) prevented activation of the slow step and tail currents, but not the fast inward current. 4. The reversal potential of the slow inward current was sensitive to changes in the Cl- equilibrium potential but not to substitution of TEA for Na+. The slow step current, but not the fast inward current, was partially blocked by the Cl- channel blocker, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid. 5. These data indicate that both the slow inward tail current and the slowly activating, reversible step current were a Ca2+-dependent Cl- current, similar to that described in other neuronal and nonneuronal cell types. The fast inward current was a voltage-activated Ca2+ current, described previously in these and other cells. 6. In the absence of intracellular EGTA, the tail current decayed with complex kinetics, its time course apparently dependent on the magnitude of the voltage-activated Ca2+ current. In the presence of 200 microM intracellular EGTA, the tail current decayed significantly faster and often decayed exponentially.     

Currents under voltage clamp of burst-forming neurons of the cardiac ganglion of the lobster (Homarus americanus)

Crustacean cardiac ganglion neuronal somata, although incapable of generating action potentials, produce regenerative, slow (greater than 200 ms) depolarizing potentials reaching -20 mV (from -50 mV) in response to depolarizing stimuli. These potentials initiate a burst of action potentials in the axon and are thus termed driver potentials. The somata of the anterior-most neurons (cells 1 or 2) were isolated by ligaturing for study of their membrane currents with a two-electrode voltage clamp. Inward current is attributed to Ca2+ by reason of dependence of driver potential amplitude on [Ca2+]0, independence of [Na+]0, resistance to tetrodotoxin, and inhibition by Cd (0.2 mM) and Mn (4 mM). Ca-mediated current (ICa) is present at -40 mV. It is optimally activated by a holding potential (Vh) of -50 to -60 mV and by clamps (command potential, Vc) to -10 mV. Time to peak (10-30 ms) and amplitude are strongly voltage dependent. Maximum tail-current amplitudes observed at -70 to -85 mV are ca. 100 nA. Inward tail peaks may not be resolved by our clamp (settling time, 2 ms). Tails relax with a time constant (tau) of approximately equal to 12 ms (at -70 to -85 mV). ICa exhibits inactivation in double pulse regimes. Recovery has a tau of approximately equal to 0.7 s. Tail current analyses indicate an exponential decline (tau approximately equal to 23 ms at -20 mV) toward a maintained amplitude of inward current tails. Analysis of outward currents indicates the presence of three conductance mechanisms having voltage dependences, time courses, and pharmacology similar to those of early outward current (IA), delayed outward current (IK), and outward current (IC) of molluscan neurons. Analysis of tail currents indicates a reversal potential for each of these near -75 mV, indicating that they are K currents. Early outward current, IA, shows a peak at 5 ms followed by rapid decline. Response to a second clamp given within 0.4 s is reduced; recovery is exponential, with a tau of approximately equal to 200 ms (at Vh = -50 mV). The amplitude of IA tested at 0 mV shows activation or deactivation by subthreshold shifts of Vh. The extent and rate of these changes shows voltage dependence (tau approximately equal to 100-500 ms for subthreshold prepulses). At the normal cell resting potential of -50 mV the amplitude of IA is 25% of that tested from -80 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia.

The net outward current in bursting pace-maker neurones of the snail (Helix pomatia) during sustained and repeated voltage clamp pulses was studied. The properties of currents remaining in cobalt-Ringer or after TEA injection were compared with those in untreated cells. 2. With sustained voltage clamp depolarizations the net outward current first increases to a maximum at 150 msec and then declines to 60% or less of its peak intensity. This depression, which is greater during repetition of short pulses (e.g. 100 msec pulses at 0-5 sec intervals), represents a true decrease in the outward flow of K (designated IK) and is not due to a decreased driving force resulting from extracellular K accumulation. The steady-state current-voltage (I-V) relationship for IK is N-shaped (Heyer & Lux, 1976). 3. A component of IK persists when Ca and Mg in the medium are replaced by Co (ICo-res). With voltage clamp depolarizations ICo-res increases rapidly to a maximum and then partially inactivates with voltage dependent time constants of hundredths or tenths of seconds. Repolarization removes the inactivation. Thus, repeated stimulation with short pulses does not increase the depression of ICo-res-ICo-res (e.g. measured during voltage steps from holding potentials of -50 to near 0 mV) is smaller in test pulses preceded by depolarization and larger in pulses preceded by hyperpolarization. The steady state I-V relationship is not N-shaped. ICo-res is blocked by intracellular injection of tetraethylammonium (TEA). 4. Repeated voltage clamp depolarization to near 0 mV with 100 msec pulses for neurones with large Ca currents in normal Ringer produces a long-term depression which is maximal with 300-400 msec repolarizations (to -50 mV) between pulses. This corresponds with stimulus parameters for the maximum Ca current (Heyer & Lux, 1976). Intracellular injection of Ca2+ (also Ba2+ and Co2+) likewise reduces the total net outward current and especially the delayed outward current under voltage clamp. 5. The component of IK which is removed by Co is identified as Ca dependent and designated IK(Ca). With single voltage clamp pulses IK(Ca) follows the approximate time course and voltage dependence of the slow inward Ca current (Iin slow; Heyer & Lux, 1976). Several lines of evidence suggest that Ca ions moving through the membrane activate IK(Ca). 6. Part of IK cannot be blocked by intracellular TEA injection. In different neurones the magnitude of the IK component resistant to TEA (ITEA-res) is approximately proportional to the relative magnitudes of Iin slow.ITEA-res does not inactivate with sustained depolarization and shows pronounced long-term depression with repetitive stimulation at intermediate intervals and an increased outward current at the onset of the second and subsequent pulses following short repolarizations. The steady-state I-V relationship is N-shaped. ITEA-res is abolished by extracellular Co. 7. A net inward current with low depolarizations can be measured after TEA injection...     

Characterization of an early afterhyperpolarization after a brief train of action potentials in rat hippocampal neurons in vitro

1. In rat hippocampal pyramidal cells in vitro, a brief train of action potentials elicited by direct depolarizing current pulses injected through an intracellular recording electrode is followed by a medium-duration afterhyperpolarization (mAHP) and a longer, slow AHP. We studied the mAHP with the use of current-clamp techniques in the presence of dibutyryl cyclic adenosine 3',5'-monophosphate (cAMP) to block the slow AHP and isolate the mAHP. 2. The mAHP evoked at hyperpolarized membrane potentials was complicated by a potential generated by the anomalous rectifier current, IQ. The mAHP is insensitive to chloride ions (Cl-), whereas it is sensitive to the extracellular potassium concentration ([K+]o). 3. At slightly depolarized levels, the mAHP is partially Ca2+ dependent, being enhanced by increased [Ca2+]o and BAY K 8644 and depressed by decreased [Ca2+]o, nifedipine, and Cd2+. The Ca2(+)-dependent component of the mAHP was also reduced by 100 microM tetraethylammonium (TEA) and charybdotoxin (CTX), suggesting it is mediated by the voltage- and Ca2(+)-dependent K+ current, IC. 4. Most of the Ca2(+)-independent mAHP was blocked by carbachol, implying that IM plays a major role. In a few cells, a small Ca2(+)- and carbachol-insensitive mAHP component was detectable, and this component was blocked by 10 mM TEA, suggesting it was mediated by the delayed rectifier current, IK. The K+ channel antagonist 4-aminopyridine (4-AP, 500 microM) did not reduce the mAHP. 5. We infer that the mAHP is a complex potential due either to IQ or to the combined effects of IM and IC. The contributions of each current depend on the recording conditions, with IC playing a role when the cells are activated from depolarized potentials and IM dominating at the usual resting potential. IQ is principally responsible for the mAHP recorded at hyperpolarized membrane potentials.     

Inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons in the ventrolateral medulla of neonatal rat are different in intrinsic electrophysiological properties

This study investigates the firing properties of the inspiratory-activated and inspiratory-inhibited airway vagal preganglionic neurons located in the external formation of the nucleus ambiguus. The results showed that inspiratory-activated and inspiratory-inhibited neurons are distributed with different density and site preference in this area. Inspiratory-inhibited neurons exhibit significantly more positive resting membrane potential, more negative voltage threshold and lower minimal current required to evoke an action potential under current clamp. The afterhyperpolarization in inspiratory-activated neurons was blocked by apamin, a blocker of the small-conductance Ca(2+)-activated K(+) channels; and that in inspiratory-inhibited neurons by charybdotoxin, a blocker of the large-conductance Ca(2+)-activated K(+) channels. Under voltage clamp, depolarizing voltage steps evoked tetrodotoxin-sensitive rapid inward sodium currents, 4-aminopyridine-sensitive outward potassium transients and lasting outward potassium currents. 4-Aminopyridine partially blocked the lasting outward potassium currents of inspiratory-activated neurons but was ineffective on those of inspiratory-inhibited neurons. These findings suggest that inspiratory-activated and inspiratory-inhibited neurons are differentially organized and express different types of voltage-gated ion channels.     

Properties of subthreshold response and action potential recorded in layer V neurons from cat sensorimotor cortex in vitro

Properties of the action potential and subthreshold response were studied in large layer V neurons in in vitro slices of cat sensorimotor cortex using intracellular recording and stimulation, application of agents that block active conductances, and a single-microelectrode voltage clamp (SEVC). A variety of measured parameters, including action-potential duration, afterpotentials, input resistance, rheobase, and membrane time constant, were similar to the same parameters reported for large neurons from this region of cortex in vivo. Action-potential amplitudes and resting potentials were greater in vitro. Most measured parameters were distributed unimodally, suggesting that these parameters are similar in all large layer V neurons irrespective of their axonal termination. The voltage response to subthreshold constant-current pulses exhibited both time and voltage dependence in the great majority of cells. Current pulses in either the hyperpolarizing or subthreshold depolarizing direction cause the membrane potential to attain an early peak and then decay (sag) to a steady level. On termination of the pulse, the membrane response transiently overshoots resting potential. Plots of current-voltage relations demonstrate inward rectification during polarization on either side of resting potential. Subthreshold inward rectification in the depolarizing direction is abolished by tetrodotoxin (TTX). The ionic currents responsible for subthreshold rectification and sag were examined using the SEVC. Steady inward rectification in the depolarizing direction is caused by a persistent, subthreshold sodium current (INaP) (54). Sag observed in response to a depolarizing current pulse is due to activation of a slow outward current, which superimposes on and partially counters the persistent sodium current. Both sag in response to hyperpolarizing current pulses and rectification in the hyperpolarizing direction are caused by a slow inward "sag current" that is activated by hyperpolarizing voltage steps. The sag current is unaltered by TTX, tetraethylammonium, (TEA), Co2+, Ba2+, or 4-aminopyridine. Fast-rising, short-duration action potentials can be elicited by an intracellular current pulse or by orthodromic or antidromic stimulation. Spikes are blocked by TTX. The form of the afterpotential following a directly evoked spike varies among cells with similar resting potentials. Biphasic afterhyperpolarizations (AHPs) with fast and slow components were most frequently seen. About 30% of the cells displayed a depolarizing afterpotential (DAP), which was often followed by an AHP. Other cells displayed a purely monophasic AHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Separation of two voltage-sensitive potassium currents, and demonstration of a tetrodotoxin-resistant calcium current in frog motoneurones.

1. Depolarization-induced voltage and conductance changes were studied in frog montoneurones in isolated, perfused spinal cord slices. Two types of afterhyperpolarization are observed following action potentials in normal Ringer, a fast afterhyperpolarization lastin 5-10 msec and a slow afterhyperpolarization lasting 60-200 msec. Both afterhyperpolarizations are mediated by an increased K+ conductance. 2. The slow afterhyperpolarization and conductance increase underlying it are selectively and reversibly inhibited by perfusion with solutions containing low [Ca2+] (less than or equal to 0-2 nM) or the Ca2+ antagonists Mn2+ (1mM) or Co2+ (5 mM), and are enhanced by perfusion with high [Ca2+]. 3. Addition of 2-5 mM tetraethylammonium ion (TEA+) to the perfusing solution prolongs the falling phase of the action potential and abolishes the fast afterhyperpolarization, but does not inhibit the slow afterhyperolarization. 4. When the voltage-dependent Na+ current is blocked by perfusion with TTX (10-5 M), intracellularly applied depolarizing current steps evoke fast and slow hyperpolarizations with kinetics and pharmacological sensitivities similar to those of the fast and slow afterhyperpolarizations, respectively. The fast hyperpolarization is maximally activated by brief, intense depolarizations, the slow hyperpolarization by prolonged, less intense depolarizations. 5. These pharmacological and kinetic data demonstrate that in frog motoneurones the repolarization-fast afterhyperpolarization sequence and the slow afterhyperpolarization are produced by different K+ conductance systems. The fast K+ conductance activates rapidly on depolarization, decays rapidly on repolarization, and is TEA+ sensitive, while the slow K+ conducatance activates and decays more slowly and is Ca2+-dependent. 6. Motoneurones perfused with TEA+ and TEA often show a slow, regenerative depolarizing response to applied depolarizing currents. These regenerative depolarizations are probably produced by an influx of Ca2+, because they persist in isotonic CaCl2 and are blocked by Mn2+ or low [Ca2+]. The Ca2+-dependence of the slow afterhyperpolarization and the increase in slow afterhyperpolarization magnitude observed following the slow Ca2+ potentials suggest that a depolarization-evoked Ca2+ influx activates the K+ conductance underlying the slow afterhyperpolarization. 7. Motoneurones in which the slow Ca2+ and K+ conductance systems have been enhanced by high [Ca2+] or blocked by Mn2+ show altered discharge patterns in response to intracellularly applied depolarizing current steps. Perfusion with twice normal [Ca2+] (4 mM) causes montoneurones to discharge more slowly at all current intensities, and reduces the slope of the 'steady-state' frequency-current relationship. Mn2+-perfused motoneurones exhibit fairly normal high-frequency discharge at the onset of the current step, but unlike normal motoneurones, do not discharge at frequencies below 60/sec...     

Ionic currents of cultured horizontal cells isolated from white perch retina

Horizontal cells from the retinas of white perch were isolated and maintained in cell culture for 3 days to 3 wk. Four morphologically distinct types of horizontal cells could be identified in culture and were labeled types H1, H2, H3, and H4. Whole-cell patch-clamp techniques were used to study the ionic currents present in the four cell types. In all cells, depolarizing commands above threshold elicited a fast-inward current followed by an outward current. The fast-inward current was abolished by tetrodotoxin (TTX) or 0 Na+ Ringer's, indicating the current was carried by Na+. In H1, H2, and H3 cells, the outward current, carried by K+, consisted of two components: a transient current (IA), blockable with 4-aminopyridine (4-AP), tetraethylammonium (TEA), or intracellular cesium and a sustained current that could be blocked with TEA. The H4 cell had only the sustained current. An inward rectifying K+ current (anomalous rectifier) was observed in the four cell types. The current was sensitive to the extracellular K+ concentration. Its activation showed two components: an instantaneous component and a slower component. The slow component becomes faster with greater hyperpolarizations. The four cell types possessed a small, sustained Ca2+ current that, under normal conditions, was masked by the inward Na+ current and outward K+ currents.     

Functional properties of a slowly inactivating potassium current in guinea pig dorsal lateral geniculate relay neurons.

1. The time- and voltage-dependent properties of a slowly inactivating and depolarization-activated potassium current and the functional consequences of its activation was investigated with current and single-electrode voltage-clamp techniques applied to guinea pig dorsal lateral geniculate neurons maintained as a slice in vitro. 2. In current clamp, application of a step depolarization to near firing threshold resulted in a slowly rising membrane potential that took up to 10 s to reach steady state and firing threshold. In voltage clamp, step depolarization of the membrane potential to values positive to approximately -65 mV resulted in the rapid activation followed by slow inactivation of an outward current. In both cases the sudden depolarization was associated with a large increase in membrane conductance, which gradually lessened in parallel with the slow depolarization in current clamp or with the decrease in outward current in voltage clamp. 3. The time course of inactivation of the outward current, which we refer to as IAs, was well fitted by a two-exponential function with time constants of 96 and 2,255 ms, suggesting the presence of a fast and slow phase of inactivation. The activation threshold for IAs was about -65 to -60 mV, whereas inactivation was incomplete even at -50 mV, suggesting the presence of a substantial "window" current. The time course of removal of inactivation of IAs at -85 to -100 mV was well fitted by a single exponential function with time constant of 91 ms. 4. IAs appears to be mediated by K+. Increasing [K+]o from 2.5 to 10 mM resulted in a reduction in amplitude of IAs, whereas changing from 10 to 2.5 mM [K+]o enhanced this current. Intracellular injection of Cs+ resulted in an abolition of IAs, whereas extracellular application of Ba2+ resulted in a large decrease in the apparent input conductance but relatively little reduction of IAs. 5. Both phases of inactivation of the transient outward current were completely blocked by low doses (100 microM) of 4-aminopyridine (4-AP), but not by extracellular application of Cs+, tetraethylammonium (TEA), tetrodotoxin (TTX), or after block of transmembrane Ca2+ currents. Local application of 4-AP to neurons depolarized to near firing threshold resulted in depolarization associated with a decrease in apparent input conductance, thereby confirming the presence of a window current.4+ this bias against depolarizing inputs.(ABSTRACT TRUNCATED AT 400 WORDS)     

The ionic mechanism of the slow outward current in Aplysia neurons

A slow outward current associated with spike frequency adaptation has been studied in the giant Aplysia neurons R2 and LP1. The current was observed during 60-s voltage clamp commands to potentials just below spike threshold. The slow outward current shows a marked voltage dependence at membrane potential less negative than -40 mV. The slow outward current is associated with increased membrane conductance. The K+ sensitivity of the slow outward current was studied by varying the extracellular K+ concentration and also by measuring potassium efflux with a K+-sensitive electrode. Both procedures indicated that the slow outward current was K+ dependent. Tail currents following the activation of the slow outward current were examined. They were shown to have a similar potassium sensitivity as the slow outward current and had a reversal potential near the potassium equilibrium potential for these cells. The sensitivity of the slow outward current to known blockers of K+ currents, tetraethylammonium and 4-aminopyridine, was tested. The sensitivity was much less than that reported for other K+ currents. The sensitivity of the slow outward current to changes of the extracellular concentrations of Na+ and Cl- ions, as well as electrogenic pump inhibitors, was tested. The results indicate that the slow outward current is much less sensitive to these changes than to the manipulations of the extracellular K+ ion concentration. We tested the sensitivity of this current to manipulations of intracellular and extracellular Ca2+ ion concentrations. We found that the current persisted at a slightly reduced level in the absence of extracellular calcium or in the presence of calcium blocking agents, cobalt and lanthanum. Intracellular injection of the calcium chelator EGTA at a concentration sufficient to block the Ca2+-dependent K+ current, seen after a brief (1.4-s) burst of action potentials, had minimal effects on the slow outward current. Procedures thought to increase intracellular Ca2+ were tested. We found that exposure of the cell to solutions containing elevated Ca2+ concentrations for prolonged periods increased the slow outward current. Also, treatment with drugs thought to elevate intracellular Ca2+ increased the slow outward current. In conclusion, the slow outward current results from an increased K+ conductance.(ABSTRACT TRUNCATED AT 400 WORDS)