On the ionic mechanism underlying adrenergic-cholinergic antagonism in ventricular muscle

In atrial muscle, acetylcholine (ACh) decreases the slow inward current (Isi) and increases the time-independent outward K+ current. However, in ventricular muscle, ACh produces a marked negative inotropic effect only in the presence of positive inotropic agents that elevate cyclic adenosine monophosphate (AMP). A two-microelectrode voltage-clamp method was used on cultured reaggregates of cells from 16--20-d-old embryonic chick ventricles to determine the effects of ACh on Isi and outward current during beta-adrenergic stimulation. Only double penetrations displaying low-resistance coupling were voltage-clamped. Cultured reaggregates are advantageous because their small size (50-- 250 microns) permits better control of membrane potential and adequate space clamp. Tetrodotoxin (10(-6) M) and a holding potential of --50 to --40 mV were used to eliminate the fast Na+ current. Depolarizing voltage steps above --40 mV caused a slow inward current to flow that was sensitive to changes in [Ca]o and was depressed by verapamil (10(- 6) M). Maximal Isi was obtained at --10 mV and the reversal potential was about +25 mV. Isoproterenol (10(-6) M) increased Isi at all clamp potentials. Subsequent addition of ACh (10(-6) M) rapidly reduced Isi to control values (before isoproterenol) without a significant effect on the net outward current measured at 300 ms. The effects of ACh were reversed by muscarinic blockade with atropine (5 X 10(-6) M). We conclude that the anti-adrenergic effects of ACh in ventricular muscle are mediated by a reduction in Ca2+ influx during excitation.     

Slow inward and outward currents of rat ventricular fibers under anoxia

Voltage and current clamp experiments were performed on rat ventricular strips under anoxia. 1. Under the influence of anoxia the membrane depolarized by 5 to 10 mV and the action potential amplitude decreased by 15 mV. The plateau disappeared and the duration of the action potential was shortened. 2. The slow inward current was reduced by 50 to 80% and its reversal potential became more negative by about 31 mV. The conductance of the slow inward channel decreased by 26%. 3. The net outward current was slightly depressed.     

Membrane Currents in Mammalian Ventricular Heart Muscle Fibers Using a Voltage-Clamp Technique

Bundles of sheep ventricular fibers were voltage-clamped utilizing a modified sucrose gap technique and intracellular voltage control. An action potential was fired off in the usual way, and the clamp circuit was switched on at preselected times during activity. Clamping the membrane back to its resting potential during the early part of an action potential resulted in a surge of inward current. The initial amplitude of this current surge decreased as the clamp was switched on progressively later during the action potential. Inward current decreasing as a function of time was also recorded if the membrane potential was clamped beyond the presumed K equilibrium potential (to -130 mv). Clamping the membrane to the inside positive range (+40 mv to +60 mv) at different times of an action potential resulted in a step of outward current which was not time-dependent. The results suggest that normal repolarization of sheep ventricle depends on a time-dependent decrease of inward current (Na, Ca) rather than on a time-dependent increase of outward current (K).     

An Assessment of the Double Sucrose-Gap Voltage Clamp Technique as Applied to Frog Atrial Muscle

The homogeneity of voltage clamp control in small bundles of frog atrial tissue under double sucrose-gap voltage clamp conditions was assessed by intracellular microelectrode potential measurements from cells in the test node region. The microelectrode potential measurements demonstrated that (1) good voltage control of the impaled cell existed in the absence of the excitatory inward currents (e.g., during small depolarizing clamp pulses of 10-15 mV), (2) voltage control of the impaled cell was lost during either the fast or slow excitatory inward currents, and (3) voltage control of the impaled cell was regained following the inward excitatory currents. Under nonvoltage clamp conditions the transgap recorded action potential had a magnitude and waveform similar to the intracellular microelectrode recorded action potentials from cells in the test node. Transgap impedance measured with a sine-wave voltage of 1,000 Hz was about 63% of that measured either by a sine-wave voltage of 10 Hz or by an action potential method used to determine the longitudinal resistance through the sucrose-gap region. The action potential data in conjunction with the impedance data indicate that the extracellular resistance (R_s) through the sucrose gap is very large with respect to the longitudinal intracellular resistance (R_i); the frequency dependence of the transgap impedance suggests that at least part of the intracellular resistance is paralleled by a capacitance. The severe loss of spatial voltage control during the excitatory inward current raises serious doubts concerning the use of the double sucrose-gap technique to voltage clamp frog atrial muscle.     

Omega-conotoxin blockade of calcium currents in cultured neonatal rat cardiomyocytes: different action on EGTA-modified calcium channels

Calcium currents from neonatal rat ventricular heart muscle cells grown in primary culture were examined using the "whole-cell" voltage clamp technique. An inward current characterized by large amplitude and slow inactivation decay was induced when the extracellular Ca2+ concentration was reduced by EGTA. This current was suppressed by extracellular Na+ removal, or by calcium antagonists, and increased by epinephrine and BAY K 8644. These findings suggest that this current is carried by sodium ions through Ca channels. Both Ca and Na currents through calcium channels were irreversibly blocked by omega-conotoxin. Complete blockade developed 10-15 minutes after the toxin introduction in the extracellular solution. Blockade of Na currents through calcium channels was characterized by a transient increase of current amplitude without any changes in its kinetics and voltage-dependent properties. Structural differences between calcium channels in rat and guinea-pig and frog cardiomyocytes were suggested.     

Electrical characteristics of frog atrial trabeculae in the double sucrose gap.

The electrical behavior of small single frog atrial trabeculae in the double sucrose gap has been investigated. The currents injected during voltage clamp experiments did not behave as predicted from the assumption of spatial uniformity of the voltage across a Hodgkin-Huxley membrane. Much of the difference is due to the geometrical complexities of this tissue. Nonetheless, two transient inward currents have been identified, the faster of which is blocked by tetrodotoxin (TTX). The magnitude of the slower transient varies markedly between preparations but always increases in a given preparation with increase of external calcium. The fast transient current traces, at small to intermediate depolarizations, are often marred by the presence of notches and secondary peaks due most probably to the loss of space clamp conditions. In many preparations these could be removed by reducing the current magnitude through application of a partially-blocking dose of TTX. Conversely, in the preparations whose fast transient was fully blocked by TTX, notches and secondary peaks in the slow transient could by induced through increasing calcium concentration and thereby the slow current magnitude. Previously used techniques for the measurement of the reversal potential of the fast inward transient have been shown to be invalid. In so far as they can be measured, the reversal potentials of the fast and slow inward transient are in the same neighborhood, i.e. around 120 mV from rest. The true values may be quite a bit apart. The total charge flow in the capacitive transient was measured for different sized nodes and preparations. From these data and estimates of plasma membrane area per unit trabecular volume, specific membrane capacitances of around 3 muF/cm2 were calculated for small bundles. The apparent ion current densities on this basis are approximately 1/10 of those measured in axons. The capacitive current occurring in small bundles decayed as the sum of at least three exponential functions of time. On the basis of these data and the anomalously large stable node widths, we suggest a coaxial core model of the preparation with the inner elements in series with an additional large extracellular resistance.     

Characterization of the inward-rectifying potassium current in cat ventricular myocytes

Whole-cell membrane currents were measured in isolated cat ventricular myocytes using a suction-electrode voltage-clamp technique. An inward-rectifying current was identified that exhibited a time-dependent activation. The peak current appeared to have a linear voltage dependence at membrane potentials negative to the reversal potential. Inward current was sensitive to K channel blockers. In addition, varying the extracellular K+ concentration caused changes in the reversal potential and slope conductance expected for a K+ current. The voltage dependence of the chord conductance exhibited a sigmoidal relationship, increasing at more negative membrane potentials. Increasing the extracellular K+ concentration increased the maximal level of conductance and caused a shift in the relationship that was directly proportional to the change in reversal potential. Activation of the current followed a monoexponential time course, and the time constant of activation exhibited a monoexponential dependence on membrane potential. Increasing the extracellular K+ concentration caused a shift of this relationship that was directly proportional to the change in reversal potential. Inactivation of inward current became evident at more negative potentials, resulting in a negative slope region of the steady state current-voltage relationship between -140 and -180 mV. Steady state inactivation exhibited a sigmoidal voltage dependence, and recovery from inactivation followed a monoexponential time course. Removing extracellular Na+ caused a decrease in the slope of the steady state current-voltage relationship at potentials negative to -140 mV, as well as a decrease of the conductance of inward current. It was concluded that this current was IK1, the inward-rectifying K+ current found in multicellular cardiac preparations. The K+ and voltage sensitivity of IK1 activation resembled that found for the inward-rectifying K+ currents in frog skeletal muscle and various egg cell preparations. Inactivation of IK1 in isolated ventricular myocytes was viewed as being the result of two processes: the first involves a voltage-dependent change in conductance; the second involves depletion of K+ from extracellular spaces. The voltage-dependent component of inactivation was associated with the presence of extracellular Na+.     

Fast Na+ channels in smooth muscle from pregnant rat uterus.

Smooth muscle cells normally do not possess fast Na+ channels, but inward current is carried through two types of Ca2+ channels: slow (L type) Ca2+ channels and fast (T type) Ca2+ channels. Whole-cell voltage clamp was done on single smooth muscle cells isolated from the longitudinal layer of the 18-day pregnant rat uterus. Depolarizing pulses, applied from a holding potential of -90 mV, evoked two types of inward current, fast and slow. The fast inward current decayed within 30 ms, depended on [Na]o, and was inhibited by tetrodotoxin (TTX) (K0.5 = 27 nM). The slow inward current decayed slowly, was dependent on [Ca]o (or Ba2+), and was inhibited by nifedipine. These results suggest that the fast inward current is a fast Na+ channel current and that the slow inward current is a Ca2+ slow channel current. A fast-inactivating Ca2+ channel current was not evident. We conclude that the ion channels that generate inward currents in pregnant rat uterine cells are TTX-sensitive fast Na+ channels and dihydropyridine-sensitive slow Ca2+ channels. The number of fast Na+ channels increased during gestation. The averaged current density increased from 0 on day 5, to 0.19 on day 9, to 0.56 on day 14, to 0.90 on day 18, and to 0.86 pA/pF on day 21. This almost linear increase occurs because of an increase in the fraction of cells that possess fast Na+ channels. The Ca2+ channel current density was also higher during the latter half of gestation. These results indicate that the fast Na+ channels and Ca2+ slow channels in myometrium become more numerous as term approaches, and we suggest that the fast Na+ current may be involved in spread of excitation. Isoproterenol (beta-agonist) did not affect either ICa(s) or INa(f), whereas Mg2+ (K0.5 = 12 mM) and nifedipine (K0.5 = 3.3 nM) depressed ICa(s). Oxytocin had no effect on INa(f) and actually depressed ICa(s) to a small extent. Therefore, the tocolytic action of beta-agonists cannot be explained by an inhibition of ICa(s), whereas that of Mg2+ can be so explained. The stimulating action of oxytocin on uterine contractions cannot be explained by a stimulation of ICa(s).     

Temperature effects on the Na and Ca currents in rat and hedgehog ventricular muscle

Cardiac transmembrane potentials and Na and Ca currents were recorded at different temperatures in rat and hedgehog ventricular muscle. At 35 degrees C in both species resting potential was about -80 mV and upstroke velocity (Vmax) of the action potential above 100 V/s. The shape of the action potential in hedgehog ventricular cells at 35 degrees C was similar to that in the rat showing a fast repolarization phase. When temperature was decreased, the membrane resting potential depolarized and action potential amplitude and Vmax declined. In rat ventricular cells at 10 degrees C, the resting potential was about -40 to -50 mV and Vmax was reduced to about 5 V/s. In hedgehog ventricular cells, however, the transmembrane potentials and Vmax were better maintained at low temperature. Phase 3 of the action potential was markedly prolonged below 20 degrees C in hedgehog but not in rat ventricular cells. When temperature was decreased to 10 degrees C the availability curve of the Na current shifted toward more negative potentials and ICa.peak declined in rat ventricular cells. In hedgehog cardiac preparations, the Na current was less influenced by the cooling and ICa.peak did not change very much at low temperatures. A transient inward current usually considered to induce cardiac arrhythmias could be recorded in rat ventricular cells below 20 degrees C but not in hedgehog preparations. These features of hedgehog cardiac membranes may contribute to the cold tolerance and the resistance to ventricular fibrillation during the hypothermia in mammalian hibernators.     

Effects of conditioning polarization on the membrane ionic currents in rat myometrium

Summary Membrane ionic currents were measured in pregnant rat uterine smooth muscle under voltage clamp conditions by utilizing the double sucrose gap method, and the effects of conditioning pre-pulses on these currents were investigated. With depolarizing pulses, the early inward current was followed by a late outward current. Cobalt (1mm) abolished the inward current and did not affect the late outward currentper se, but produced changes in the current pattern, suggesting that the inward current overlaps with the initial part of the late outward current. After correction for this overlap, the inward current reached its maximum at about +10 mV and its reversal potential was estimated to be +62 mV. Tetraethylammonium (TEA) suppressed the outward currents and increased the apparent inward current. The increase in the inward current by TEA thus could be due to a suppression of the outward current. The reversal potential for the outward current was estimated to be –87 mV. Conditioning depolarization and hyperpolarization both produced a decrease in the inward current. Complete depolarization block occurred at a membrane potential of –20 mV. Conditioning hyperpolarization experiments in the presence of cobalt and/or TEA revealed that the decrease in the inward current caused by conditioning hyperpolarization was a result of an increase in the outward current overlapping with the inward current. It appears that a part of the potassium channel population is inactivated at the resting membrane potential and that this inactivation is removed by hyperpolarization.     

Influence of intercellular clefts on potential and current distribution in a multifiber preparation.

A theoretical model is presented for current and voltage clamp of multifiber bundles in a double sucrose gap. Attention is focused on methodological errors introduced by the intercellular cleft resistance. The bundle is approximated by a continuous geometry. Voltage distribution, as a function of radial distance and time, is defined by a parabolic partial differential equation which is specified for different membrane characteristics. Assuming a linear membrane, analytical solutions are given for current step and voltage step conditions. The theoretical relations (based on Bessel functions) may be used to calculate membrane conductance and capacity from experimental clamp data. The case of a nonlinear membrane with standard Hodgkin-Huxley kinetics for excitatory Na current is treated assuming maximum Na conductances (gNa) of 120, 10, and 1 mmho/cm2. Numerical simulations are presented for potential and current distribution in a bundle of 60 microns diameter during depolarizing voltage steps. Adequate voltage control is restricted to the peripheral fibers of the bundle whereas the membrane potential of the inner fibers deviates from the command level during early inward current, tending to the Na equilibrium potential. In the peak current-voltage diagram the loss of voltage control is reflected by an increased steepness of the negative region and a decreased slope conductance of the positive region. With gNa = 120 mmho/cm2, the positive slope conductance is approximately 25% of the slope expected from ideal space clamping. With the lower values of gNa, the slope conductance ratio is in the order of 50%. Implications of the results for an experimental voltage clamp analysis of early inward current on multifiber preparations are discussed.     

Voltage Clamp of Cardiac Muscle in a Double Sucrose Gap: A Feasibility Study

A method of stabilizing the membrane potential of a small area of cardiac muscle membrane and the limitations of this method are described. Tiny bundles or strands, approximately 80 μm in diameter, of electrically interconnected fibers from the ventricles of rabbit hearts were used in a double sucrose gap. Current records associated with step changes in voltage were complicated by two capacitive surges of current of nodal and nonnodal origin and large “leakage” currents of nonnodal origin resulting mainly from the multifibered nature of the preparation and emphasized by the method. The transient, inward membrane currents in response to moderate depolarizing steps in command potential had the same duration as the upstroke of the action potential. In good runs, currents were smooth and free from notches. These initial currents behaved qualitatively like the initial sodium currents in squid axon and in other excitable membranes. A fraction of the initial sodium current persisted at least as long as 300 ms. The relationship between peak initial current and voltage was graded and linear in the positive direction. In the negative region the relationship was often very steep, indicating insufficient voltage control of all the membranes despite the squareness of the voltage record. Other indications of inadequacy of control could occur and thus even with this optimum preparation of cardiac muscle it was not feasible to analyze quantitatively either the initial or the prolonged sodium currents.     

Action potential-like responses due to the inward rectifying potassium channel

Summary This paper describes experiments carried out in the absence of sodium and calcium in the external solution. Frog atrial trabeculae were stimulated in current clamp with the double sucrose gap technique. The voltage responses looked like slow action potentials with a clear threshold. These responses were not suppressed in the presence of EGTA, in the presence of sodium or calcium channel blockers, or when sulfate ions replaced chloride. Guinea pig isolated ventricular myocytes were studied in whole cell clamp mode with a pathch pipette. Under current clamp, they displayed also voltage responses with a threshold. These responses were resistant to cadmium (5mm), and were suppressed by barium (0.5mm). A negative slope conductance is required to take into account these results. The membrane current in current clamp can be estimated by plotting the response in the phase plane. This analysis shows that on both types of preparations, the current responsible for the negative slope is not time dependent. This current is suppressed by barium. It can be concluded that it is the outward current flowing through the inward rectifying potassium channels. To confirm this hypothesis, data obtained in voltage clamp on the same preparations were introduced into a computer model to predict the response in current clamp. The results were in agreement with the experiments. Similar responses could be recorded and analyzed on skeletal muscle in isotonic potassium solution. These results show that the inward rectifier can induce by itself properties looking like excitability on different preparations. The physiological significance of this effect in normal conditions is discussed. The voltage responses described in this paper look similar to the slow action potentials on heart, which are sensitive to modifications of the calcium channels, but also of the potassium channels. Some implications in cardiac pharmacology are discussed.     

A fast-twitch oxidative-glycolytic muscle with a robust inward calcium current

This report describes the histochemical and physiological properties of a rat skeletal muscle with a robust activity-dependent slow inward Ca2+ current. The muscle, the flexor digitorum brevis (FDB), is a small plantar flexor from the hindfoot. It is a homogeneous muscle consisting of approximately 90% fast-twitch oxidative-glycolytic (type IIA) fibers. Stimulation of the FDB with repetitive stimulus trains (30 or 50 Hz for 330 ms, 1 train/s for 2-5 min) produced a slow increase in the base-line or resting tension of the muscle between trains. This progressive increase in resting tension appears to be due to the activation of voltage-dependent slow Ca2+ channels, since it could be eliminated (i) by stimulating the muscle in a medium containing 2 mM EGTA and without Ca2+, and (ii) by the addition of either Co2+ or verapamil. The presence of a slow current may be associated with an increase in K+ efflux as stimulation continues, and with a prolongation of relaxation time. We also propose that the slow Ca2+ current may contribute to the allosteric activation of phosphorylase kinase during muscle activity. The FDB provides an excellent preparation to investigate the regulation of muscle metabolism by intra- and extra-cellular Ca2+ during exercise.     

Steady-state and dynamic properties of cardiac sodium-calcium exchange. Sodium-dependent inactivation

Sodium-calcium exchange current was isolated in inside-out patches excised from guinea pig ventricular cells using the giant patch method. The outward exchange current decayed exponentially upon activation by cytoplasmic sodium (sodium-dependent inactivation). The kinetics and mechanism of the inactivation were studied. (a) The rate of inactivation and the peak current amplitude were both strongly temperature dependent (Q10 = 2.2). (b) An increase in cytoplasmic pH from 6.8 to 7.8 attenuated the current decay and shifted the apparent dissociation constant (Kd) of cytoplasmic calcium for secondary activation of the exchange current from 9.6 microM to < 0.3 microM. (c) The amplitude of exchange current decreased synchronously over the membrane potential range from -120 to 60 mV during the inactivation, indicating that voltage dependence of the exchanger did not change during the inactivation process. The voltage dependence of exchange current also did not change during secondary modulation by cytoplasmic calcium and activation by chymotrypsin. (d) In the presence of 150 mM extracellular sodium and 2 mM extracellular calcium, outward exchange current decayed similarly upon application of cytoplasmic sodium. Upon removal of cytoplasmic sodium in the presence of 2-5 microM cytoplasmic free calcium, the inward exchange current developed in two phases, a fast phase within the time course of solution changes, and a slow phase (tau approximately 4 s) indicative of recovery from sodium-dependent inactivation. (e) Under zero-trans conditions, the inward current was fully activated within solution switch times upon application of cytoplasmic calcium and did not decay. (f) The slow recovery phase of inward current upon removal of cytoplasmic sodium was also present under the zero-trans condition. (g) Sodium-dependent inactivation shows little or no dependence on membrane potential in guinea pig myocyte sarcolemma. (h) Sodium-dependent inactivation of outward current is attenuated in rate and extent as extracellular calcium is decreased. (i) Kinetics of the sodium-dependent inactivation and its dependence on major experimental variables are well described by a simple two-state inactivation model assuming one fully active and one fully inactive exchanger state, whereby the transition to the inactive state takes place from a fully sodium-loaded exchanger conformation with cytoplasmic orientation of binding sites (E1.3Ni).     

Investigations of the ionic mechanisms of bursting activity in Helix pomatia neurons

Steady-state current-voltage characteristics of the membrane and ionic currents arising during changes in membrane potential in bursting neurons ofHelix pomatia were studied by the voltage clamp method. The steady-state current-voltage characteristics of the membrane were shown to have a nonlinear region. Replacement of sodium ions by Tris-HC1 ions in the external solution completely abolishes this nonlinearity. Hyperpolarization of the membrane under voltage clamp conditions leads to the development of an outward current which reaches a maximum and then is inactivated. This current has a reversal potential in the region of the potassium equilibrium potential. Depolarization of the membrane to the threshold value for excitation of uncontrollable regions of the axon hillock causes the appearance of a slow inward current. After reaching a maximum, the inward current falls to zero. A model of generation of waves in a bursting neuron is suggested.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 10, No. 2, pp. 193–202, March–April, 1978.     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Inhibition of slow TTX-insensitive inward current by the anticonvulsant carbamazepine in an identified neuron of Lymnaea stagnalis

1. Pentylenetetrazol (PTZ), induces tonic depolarization and bursting activity in an identified neuron, B1, of Lymnaea stagnalis. This is due in part to activation of a slow, tetrodotoxin-insensitive inward sodium current.2. Carbamazepine (CBZ) reversed the effect of PTZ on both membrane potential and inward current, after a delay of up to 5 min. CBZ alone had no effect on voltage or current responses.3. These results suggest that CBZ blocks the slow sodium current, possibly via a decrease in intracellularly stored calcium ions.     

Current- and Voltage-Clamped Studies on Myxicola Giant Axons : Effect of tetrodotoxin

A new dissection procedure for preparing Myxicola giant axons for observation under voltage clamp is described. Preparation time is generally 40–45 min. 65–70% of the preparations attempted may be brought through the entire procedure, including insertion of the long internal electrode, and support an initial action potential amplitude of 100 mv or greater. Mean values for axon diameter, resting membrane potential, action potential amplitude, maximum peak inward transient current, and resting membrane resistance are 560 µ, —66.5 mv, 112 mv, 0.87 ma/cm² and 1.22 KΩ cm ² respectively. Cut branches do not seem to be a problem in this preparation. Behavior under voltage clamp is reasonably stable over several hours. Reductions in maximum inward transient current of 10% and in steady-state current of 5–10% are expected in the absence of any particular treatment. Tetrodotoxin blocks the action potential and both the inward and outward transient current, but has no effect on either the resting membrane potential or the steady-state current. This selective action of tetrodotoxin on the transient current is taken as an indication that this current component is probably carried by Na.