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.     

K+-channel blockade in trigeminal root ganglion neurons: effects on membrane outward currents

1. The effects of the applications of three K+-channel blockers on the membrane outward currents of neurons were studied with single-electrode voltage-clamp techniques in in vitro slice preparations of the trigeminal root ganglion (TRG) of guinea pigs. The investigations are the first reported attempts to apply these techniques to TRG neurons. 2. During perfusion with tetrodotoxin (TTX; 1 microM), transient outward currents were elicited at the termination of hyperpolarizing voltage commands from holding potentials near -40 mV. The amplitudes of these currents were reduced in conditions of high extracellular [K+]. The activation of such currents was rapid (less than 5 ms), and inactivation was complete at potentials within the activation range. Perfusion with low-[Ca2+], Co2+-containing solutions only slightly and inconsistently reduced the transient outward currents. 3. During combined application of TTX (1 microM) and tetraethylammonium (TEA) (10 mM), fast-activating sustained currents (greater than 1 s) were evoked by depolarizing commands from holding potentials near -70 mV. These currents were blocked completely by the additional inclusion of 5 mM 4-aminopyridine (4-AP) in the perfusing solution. 4. Applications of TEA (0.1-10 mM) produced dose-dependent reductions in the amplitudes of the transient outward currents. Applications of Cs+ also greatly reduced the currents. However, administrations of 4-AP (50 microM-5 mM) diminished these currents only slightly, and high doses of muscarinic agonists had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma.

Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma. The cell body of the cockroach (Periplaneta americana) fast coxal depressor motoneuron (Df) displays a time-dependent change in excitability. Immediately after dissection, depolarization evokes plateau potentials, but after several hours all-or-none action potentials are evoked. Because K channel blockers have been shown to produce a similar transition in electrical properties, we have used current-clamp, voltage-clamp and action-potential-clamp recording to elucidate the contribution of different classes of K channel to the transition in electrical activity of the neuron. Apamin had no detectable effect on the neuron, but charybdotoxin (ChTX) caused a rapid transition from plateau potentials to spikes in the somatic response of Df to depolarization. In neurons that already produced spikes when depolarized, ChTX increased spike amplitude but did not increase their duration nor decrease the amplitude of their afterhyperpolarization. 4-Aminopyridine (4-AP) (which selectively blocks transient K currents) did not cause a transition from plateau potentials to spikes but did enhance oscillations superimposed on plateau potentials. When applied to neurons that already generated spikes when depolarized, 4-AP could augment spike amplitude, decrease the latency to the first spike, and prolong the afterhyperpolarization. Evidence suggests that the time-dependent transition in electrical properties of this motoneuron soma may result, at least in part, from a fall in calcium-dependent potassium current (IK,Ca), consequent on a gradual reduction in [Ca2+ ]i. Voltage-clamp experiments demonstrated directly that outward K currents in this neuron do fall with a time course that could be significant in the transition of electrical properties. Voltage-clamp experiments also confirmed the ineffectiveness of apamin and showed that ChTX blocked most of IK,Ca. Application of Cd2+ (0.5 mM), however, caused a small additional suppression in outward current. Calcium-insensitive outward currents could be divided into transient (4-AP-sensitive) and sustained components. The action-potential-clamp technique revealed that the ChTX-sensitive current underwent sufficient activation during the depolarizing phase of plateau potentials to enable it to shunt inward conductances. Although the ChTX-sensitive conductance apparently makes little contribution to spike repolarization, the ChTX-resistant IK,Ca does make a significant contribution to this phase of the action potential. The 4-AP-sensitive current began to develop during the rising phase of both action potentials and plateau potentials but had little effect on the electrical activity of the neuron, probably because of its relatively small amplitude.     

Membrane properties underlying patterns of GABA-dependent action potentials in developing mouse hypothalamic neurons

Spikes may play an important role in modulating a number of aspects of brain development. In early hypothalamic development, GABA can either evoke action potentials, or it can shunt other excitatory activity. In both slices and cultures of the mouse hypothalamus, we observed a heterogeneity of spike patterns and frequency in response to GABA. To examine the mechanisms underlying patterns and frequency of GABA-evoked spikes, we used conventional whole cell and gramicidin perforation recordings of neurons (n = 282) in slices and cultures of developing mouse hypothalamus. Recorded with gramicidin pipettes, GABA application evoked action potentials in hypothalamic neurons in brain slices of postnatal day 2-9 (P2-9) mice. With conventional patch pipettes (containing 29 mM Cl-), action potentials were also elicited by GABA from neurons of 2-13 days in vitro (2-13 DIV) embryonic hypothalamic cultures. Depolarizing responses to GABA could be generally classified into three types: depolarization with no spike, a single spike, or complex patterns of multiple spikes. In parallel experiments in slices, electrical stimulation of GABAergic mediobasal hypothalamic neurons in the presence of glutamate receptor antagonists [10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), 100 microM 2-amino-5-phosphonopentanoic acid (AP5)] resulted in the occurrence of spikes that were blocked by bicuculline (20 microM). Blocking ionotropic glutamate receptors with AP5 and CNQX did not block GABA-mediated multiple spikes. Similarly, when synaptic transmission was blocked with Cd(2+) (200 microM) and Ni(2+) (300 microM), GABA still induced multiple spikes, suggesting that the multiple spikes can be an intrinsic membrane property of GABA excitation and were not based on local interneurons. When the pipette [Cl-] was 29 or 45 mM, GABA evoked multiple spikes. In contrast, spikes were not detected with 2 or 10 mM intracellular [Cl-]. With gramicidin pipettes, we found that the mean reversal potential of GABA-evoked current (E(GABA)) was positive to the resting membrane potential, suggesting a high intracellular [Cl-] in developing mouse neurons. Varying the holding potential from -80 to 0 mV revealed an inverted U-shaped effect on spike probability. Blocking voltage-dependent Na+ channels with tetrodotoxin eliminated GABA-evoked spikes, but not the GABA-evoked depolarization. Removing Ca(2+) from the extracellular solution did not block spikes, indicating GABA-evoked Na+ -based spikes. Although E(GABA) was more positive within 2-5 days in culture, the probability of GABA-evoked spikes was greater in 6- to 9-day cells. Mechanistically, this appears to be due to a greater Na+ current found in the older cells during a period when the E(GABA) is still positive to the resting membrane potential. GABA evoked similar spike patterns in HEPES and bicarbonate buffers, suggesting that Cl-, not bicarbonate, was primarily responsible for generating multiple spikes. GABA evoked either single or multiple spikes; neurons with multiple spikes had a greater Na+ current, a lower conductance, a more negative spike threshold, and a greater difference between the peak of depolarization and the spike threshold. Taken together, the present results indicate that the patterns of multiple action potentials evoked by GABA are an inherent property of the developing hypothalamic neuron.     

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.     

Phasic stimuli evoke precisely timed spikes in intermittently discharging mitral cells

Mitral cells, the principal cells of the olfactory bulb, respond to sensory stimulation with precisely timed patterns of action potentials. By contrast, the same neurons generate intermittent spike clusters with variable timing in response to simple step depolarizations. We made whole cell recordings from mitral cells in rat olfactory bulb slices to examine the mechanisms by which normal sensory stimuli could generate precisely timed spike clusters. We found that individual mitral cells fired clusters of action potentials at 20-40 Hz, interspersed with periods of subthreshold membrane potential oscillations in response to depolarizing current steps. TTX (1 microM) blocked a sustained depolarizing current and fast subthreshold oscillations in mitral cells. Phasic stimuli that mimic trains of slow excitatory postsynaptic potentials (EPSPs) that occur during sniffing evoked precisely timed spike clusters in repeated trials. The amplitude of the first simulated EPSP in a train gated the generation of spikes on subsequent EPSPs. 4-aminopyridine (4-AP)-sensitive K(+) channels are critical to the generation of spike clusters and reproducible spike timing in response to phasic stimuli. Based on these results, we propose that spike clustering is a process that depends on the interaction between a 4-AP-sensitive K(+) current and a subthreshold TTX-sensitive Na(+) current; interactions between these currents may allow mitral cells to respond selectively to stimuli in the theta frequency range. These intrinsic properties of mitral cells may be important for precisely timing spikes evoked by phasic stimuli that occur in response to odor presentation in vivo.     

Highly 4-aminopyridine sensitive delayed rectifier current modulates the excitability of guinea pig cerebellar Purkinje cells

The effects of low concentrations of 4-aminopyridine (4-AP) on the membrane properties of guinea pig cerebellar Purkinje cells were investigated in slice preparation using intracellular recordings. It was found that 1–10 μM 4-AP did not affect the resting potential or the input resistance of the cells, but reduced markedly the duration of the slowly depolarizing potential (SDP), and thus the latency to the firing of Ca²⁺ spikes in response to intracellular current pulses. Intradendritic recordings in the presence of tetrodotoxin, Cd²⁺, and low [Ca²⁺]_o, which blocked all the regenerative responses, exhibited prominent membrane outward rectification in response to depolarizing current pulses. Under these conditions, the SDP was abolished and, in contrast, a slowly developing hyperpolarization was consistently observed. Application of 10 μM 4-AP reduced the outward membrane rectification in a reversible manner, but did not affect the transient hyperpolarization, which is usually attributed to the activation of potassium "A" current. These results demonstrate, for the first time, the presence of a highly 4-AP sensitive delayed rectifier in guinea pig cerebellar Purkinje cells, which prominently affects their excitability. The results also indicate that the slowly depolarizing potential of guinea pig Purkinje cells does not involve inactivation of transient potassium currents, which has been suggested previously as an underlying mechanism for this phenomenon in turtle Purkinje cells. Electronic Publication     

Consequences of 4-aminopyridine applications to trigeminal root ganglion neurons

1. The effects of 4-aminopyridine (4-AP) on the electrical properties of 30 trigeminal root ganglion (TRG) neurons were determined from the membrane voltage responses to step and sinusoidal current injections using intracellular microelectrode techniques in in vitro slice preparations (guinea pigs). 2. Comparisons of results from 4-AP applications (0.05-5 mM) with those from tetraethylammonium (TEA) applications (0.1-10 mM) revealed very different actions of these agents. Both agents produced an increase in input resistance and a decrease in threshold for spike generation. Applications of 4-AP increased subthreshold oscillations of the membrane potential and enhanced the repetitive spike firing evoked by intracellular injections of current pulses. However, TEA applications blocked the potential oscillations and did not exaggerate repetitive spike discharges. Spontaneous spike activity or bursts were observed in four neurons that received 4-AP applications. 3. Membrane properties were determined in 20 of the 30 neurons by fitting impedance data in the frequency domain with a four-parameter membrane model by the use of computer-intensive techniques. In the majority of neurons, the time-invariant and time-dependent membrane conductances decreased during 4-AP application. The time constant for the time-dependent conductance also decreased, suggesting that the closing of K+-channels was facilitated in the membrane. 4. Applications of 4-AP in a dose range of 50 microM-5 mM produced rapid (approximately tens of seconds) responses of the neurons, resulting in a dose-dependent increase of the impedance magnitude functions and in a leftward shift of the resonant "humps" to lower frequencies. This shift indicates that the TRG neuronal membrane is capable of producing large voltage responses to current inputs at low frequencies. Recovery from the effects of 4-AP was slow (usually greater than 30 min). 5. Applications of 4-AP at high doses (greater than or equal to 1 mM) and at various imposed membrane potentials in four neurons resulted in poorly reversible unspecific changes in certain membrane parameters (increased input capacitance and conductance) and an insensitivity of the input conductance to the imposed membrane potential. These effects could be interpreted as membrane breakdown. 6. The tendencies of TRG neurons to fire repetitively and in bursts of spikes during 4-AP application result from the increased oscillatory behavior of their membrane potentials and changes in membrane resonance induced by presumed blockade of K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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)     

Intrinsic theta-frequency membrane potential oscillations in hippocampal CA1 interneurons of stratum lacunosum-moleculare

The ionic conductances underlying membrane potential oscillations of hippocampal CA1 interneurons located near the border between stratum lacunosum-moleculare and stratum radiatum (LM) were investigated using whole cell current-clamp recordings in rat hippocampal slices. At 22 degrees C, when LM cells were depolarized near spike threshold by current injection, 91% of cells displayed 2-5 Hz oscillations in membrane potential, which caused rhythmic firing. At 32 degrees C, mean oscillation frequency increased to 7.1 Hz. Oscillations were voltage dependent and were eliminated by hyperpolarizing cells 6-10 mV below spike threshold. Blockade of ionotropic glutamate and GABA synaptic transmission did not affect oscillations, indicating that they were not synaptically driven. Oscillations were eliminated by tetrodotoxin, suggesting that Na+ currents generate the depolarizing phase of oscillations. Oscillations were not affected by blocking Ca2+ currents with Cd2+ or Ca2+-free ACSF or by blocking the hyperpolarization-activated current (Ih) with Cs+. Both Ba2+ and a low concentration of 4-aminopyridine (4-AP) reduced oscillations but TEA did not. Theta-frequency oscillations were much less common in interneurons located in stratum oriens. Intrinsic membrane potential oscillations in LM cells of the CA1 region thus involve an interplay between inward Na+ currents and outward K+ currents sensitive to Ba2+ and 4-AP. These oscillations may participate in rhythmic inhibition and synchronization of pyramidal neurons during theta activity in vivo.     

Membrane currents of cultured rat sympathetic neurons under voltage clamp

Sympathetic neurons, dissociated from neonatal rat superior cervical ganglia, were voltage clamped with two microelectrodes. Depolarization from resting potential activated a rapid transient inward current carried by sodium and a slow inward current blocked by cobalt. Depolarization from resting potential also activated up to three kinetically distinct outward currents, which were further studied by tail current analysis. Following long depolarizing steps, outward current decayed biphasically. The fast phase (delayed rectifier) decayed over 10-20 ms. The slow phase (calcium dependent) required as much as 1-2 s to decay to base line. A small component of the total outward current was a persistent current activated between -70 and -30 mV (M-current), which decayed over 200-300 ms. This current was studied in isolation following hyperpolarizing steps from potentials negative to the threshold for activation of the other delayed outward currents. Tetraethylammonium (TEA) blocked the fast tail current, partially inhibited the slow tail current, and reduced M-currents. Cobalt selectively decreased the slow tail current. Muscarine blocked M-current but not other outward currents. A transient outward current was activated by depolarization from only holding potentials negative to -60 mV. This current peaked in 10-20 ms and decayed over about 50 ms. A persistent ("anomalous") inward current was evoked by hyperpolarizing steps from only holding potentials negative to -50 to -60 mV. These seven membrane currents may be separately characterized on the basis of their voltage- and time-dependent properties. Further identification is aided by the use of channel-blocking chemicals, although the latter may lack specificity, especially when used to study potassium channels.     

Changes in membrane currents of hippocampal neurons evoked by brief anoxia

1. Effects of anoxia (2-4 min of 95% N2-5% CO2) on membrane currents of CA1 neurons were studied by single-electrode voltage clamp in hippocampal slices (from Sprague-Dawley rats) kept in an interface-type chamber at 33.5 degree. 2. When recording with KCl electrodes at a holding potential (VH) near-70 mV, anoxia evoked a slow outward current [0.18 +/- 0.06 (SE) nA], accompanied by a conductance increase ( + 46 +/- 20%, mean +/- SE). The difference current evoked by N2 had a reversal potential near-100 mV. It was much smaller in presence of 2-4 mM extracellular Cs, and any remaining outward current was abolished by 10 mM tetraethylammonium (TEA). Only inward currents were observed when recording with CsCl electrodes. 3. Inward relaxations evoked by large hyperpolarizing pulses from VH less than or equal to - 70 mV (Q-type) were not significantly depressed by anoxia (-1.5 +/- 6.0%). 4. Some voltage-dependent outward currents (evoked by 200-ms depolarizing pulses) were depressed during anoxia: 1) a fast-inactivating (A-like) current, obtained at VH less than or equal to -70 mV and suppressed by 200 microM 4-AP, was reduced by 25.6 +/- 7.3% (n = 5); 2) a slower, noninactivating (C-like) current, suppressed by TEA, was reduced by 52 +/- 7.2% (n = 16). Neither of these currents (1 or 2) was observed when recording with 2- to 3-M CsCl electrodes; and 3) small (M-like) inward relaxations, observed at VH approximately -40 mV 5. Net inward currents could be evoked after blockage of GK with 10 mM TEA when recording with KCl electrodes or by recording with CsCl electrodes. At VH less than or equal to -70 mV, large, transient, and incompletely controlled currents were evoked by depolarizing pulses; at VH less than or equal to -50 mV, smaller and more persistent currents were evoked by depolarizing pulses (L-like), and transient currents (T-like?) were seen immediately after hyperpolarizing pulses. 6.L-type currents (at VH less than or equal to -50 mV) were nearly abolished after 1-2 min anoxia (by approximately 90%). This was equally true of the currents evoked by constant pulses or peak currents in I-V plots. After reoxygenation, recovery was biphasic, with a quick early phase (to 50-80% in 2 min) and then a much slower one (to 60-90% by 10-15 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

Transient voltage and calcium-dependent outward currents in hippocampal CA3 pyramidal neurons

Membrane currents activated by step changes in membrane potential were studied in hippocampal pyramidal neurons of region CA3 using the single microelectrode voltage-clamp technique. The transient outward current activated by depolarizing steps appeared to be composed of two transient currents that could be distinguished by differences in voltage sensitivity, time course, and pharmacological sensitivity. The more slowly decaying current was activated by voltage steps positive to -60 mV and declined exponentially with a time constant between 200 and 400 ms. This current inactivated as the holding potential was made more positive over the range of -75 to -45 mV and was 50% inactivated near -60 mV. The more slowly decaying transient current was selectively blocked by 0.5 mM 4-aminopyridine (4-AP) but not by 5-10 mM tetraethylammonium (TEA) or 2-5 mM Mn2+. The second transient current had a much faster time course than the 4-AP-sensitive current, having a duration of 5-20 ms. This very fast transient current was observed during potential steps positive to -45 mV. The fast transient current was inactivated when the holding potential was made positive to -45 mV. The amplitude of the fast transient current was greatly reduced by the application of 4 mM Mn2+ or Ca2+-free artificial cerebrospinal fluid (CSF). The fast transient current appeared to be unaffected by 0.5 mM 4-AP but was greatly reduced by 10 mM TEA. These results suggest that the transient outward current observed during depolarizing steps is composed of at least two distinct transient currents. The more slowly decaying current resembles the A-current originally described in molluscan neurons (9, 32, 42) in voltage sensitivity, time course, and pharmacological sensitivity. The faster transient current resembles a fast, Ca2+-dependent transient current previously observed in bull-frog sympathetic neurons (5, 27).     

Primary afferent-evoked synaptic responses and slow potential generation in rat substantia gelatinosa neurons in vitro

1. Primary afferent fiber-evoked synaptic responses and the mechanisms of spike and slow potential generation have been examined in adult rat substantia gelatinosa (SG) neurons in an in vitro transverse spinal cord slice preparation in which an attached dorsal root is retained. Intracellular recordings were made from SG neurons identified by morphological and electrophysiological criteria. Afferent fiber-evoked fast excitatory postsynaptic potentials (fast EPSPs) and slow EPSPs have been analyzed. 2. SG neurons had mean resting membrane potentials of -67.1 +/- 0.5 mV (mean +/- SE), mean input resistance of 257 +/- 17.7 (SE) M omega, and a mean time constant of 21.3 +/- 1.9 ms and exhibited spontaneous EPSPs. 3. Single low-intensity stimuli applied to the dorsal root using a suction electrode produced, in 70% of SG neurons, short-latency, presumed monosynaptic fast EPSPs which had a half decay time of 10-30 ms and an amplitude of 8-28 mV. The conduction velocity of afferent fibers evoking fast EPSPs was 2-7 m/s, corresponding to that of thinly myelinated A-delta-fibers. Dorsal root stimulation at higher intensities evoked, in 10% of SG neurons, long-latency and apparently monosynaptic EPSPs which had a time course and amplitude similar to that evoked by low-intensity stimulation. The conduction velocity of fibers evoking long-latency EPSPs was 0.4-2 m/s, suggesting that they constitute predominantly C-fibers. A-delta- and C-fiber-mediated fast EPSPs were detected in 20% of SG neurons examined. 4. Low-intensity stimuli produced slow EPSPs in 20% of SG neurons. Slow EPSPs were 3-15 mV in amplitude and of up to 2 min in duration. A-delta-fibers appeared to be responsible for the generation of slow EPSPs. Slow EPSPs were associated with an increase in membrane resistance and were decreased in amplitude with membrane hyperpolarization. 5. Action potentials in SG neurons had a mean amplitude of 76.3 +/- 1.1 mV and a mean duration of 1.0 +/- 0.07 ms. Na+ ions represent the main charge carrier during the rising phase of the action potential and Ca2+ ions contribute to the shoulder on the falling phase. 6. In 20% of SG neurons, subthreshold depolarizing pulses were followed by long-lasting slow-inactivating depolarizing potentials which were able to initiate spikes. The slow depolarizing potentials were blocked by TTX and enhanced by application of TEA and Ba2+, suggesting that Na+ and K+ are involved in this slow-inactivating potential.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of granule cell excitability by a low-threshold calcium spike in turtle olfactory bulb

Granule cells excitability in the turtle olfactory bulb was analyzed using whole cell recordings in current- and voltage-clamp mode. Low-threshold spikes (LTSs) were evoked at potentials that are subthreshold for Na spikes in normal medium. The LTSs were evoked from rest, but hyperpolarization of the cell usually increased their amplitude so that they more easily boosted Na spike initiation. The LTS persisted in the presence of TTX but was antagonized by blockers of T-type calcium channels. The voltage dependence, kinetics, and inactivation properties of the LTS were characteristic of a low-threshold calcium spike. The threshold of the LTS was slightly above the resting potential but well below the Na spike threshold, and the LTS was often evoked in isolation in normal medium. Tetraethylammonium (TEA) and 4-aminopyridine (4-AP) had only minimal effects on the LTS but revealed the presence of a high-threshold Ca2+ spike (HTS), which was antagonized by Cd2+. The LTS displayed paired-pulse attenuation, with a timescale for recovery from inactivation of about 2 s at resting membrane potential. The LTS strongly boosted Na spike initiation; with repetitive stimulation, the long recovery of the LTS governed Na spike initiation. Thus the olfactory granule cells possess an LTS, with intrinsic kinetics that contribute to sub- and suprathreshold responses on a timescale of seconds. This adds a new mechanism to the early processing of olfactory input.     

Properties of persistent sodium conductance and calcium conductance of layer V neurons from cat sensorimotor cortex in vitro

Properties of the persistent sodium conductance and the calcium conductance of layer V neurons from cat sensorimotor cortex were examined in an in vitro slice preparation by use of a single microelectrode, somatic voltage clamp, current clamp, intra- and extracellular application of blocking agents, and extracellular ion substitution. The persistent sodium current (INaP) attained its steady level within 2-4 ms of a step change in voltage at every potential where it could be examined directly [to about 40 mV positive to resting potential (RP)]. Because of its fast onset INaP can be activated during a single excitatory postsynaptic potential (EPSP) and can influence the subsequent voltage time course and cell excitability. Application of a depolarizing holding potential greater than or equal to 20 mV positive to RP could inactivate spikes, thus allowing examination of INaP at voltages positive to spike threshold. At every potential where INaP was visible, it was mixed with a slow outward current. After depressing potassium currents with blocking agents, INaP could be observed during depolarizations to about 40 mV positive to RP where it is normally hidden by the larger outward currents. Indirect evidence suggests that INaP is present and large during prolonged depolarizations greater than 50 mV positive to RP. INaP was blocked by intracellular injection of the lidocaine derivative QX-314, as well as by extracellular tetrodotoxin (TTX). INaP was much more sensitive to QX-314 than was the height and rate of rise of the spike. This observation and the results in paragraph 3 above are best explained by separate INaP and spike sodium channels. After blockade of INaP and sodium spikes, Ca2+ spikes could be evoked only if potassium currents were first depressed. The Ca2+-dependent nature of the regenerative potentials was indicated by their disappearance when Co2+ or Mn2+ was substituted for Ca2+ in the perfusate and by the appearance of greatly enhanced potentials of similar form when Ba2+ was substituted for Ca2+. Ba2+ substitution greatly enhanced evoked and spontaneous synaptic potentials. Prolonged-plateau action potentials could be evoked in the presence of TTX and Ba2+. Ca2+ spike threshold was 30-40 mV positive to RP, which is significantly more positive than sodium spike threshold. Results of voltage clamp in the normal perfusate and in the presence of Ca2+-blockers or Ba2+ indicated that little or no Ca2+ conductance is activated in the voltage range 25 mV positive to RP where INaP is the dominant ionic current.(ABSTRACT TRUNCATED AT 400 WORDS)     

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)