J. Phy.iol. (1975), 246, pp. 351-361 With 8 text-figures Printed in Great Britain

351

CALCIUM DEPENDENT ACTION POTENTIALS PRODUCED IN LEECH RETZIUS CELLS BY TETRAETHYLAMMONIUM CHLORIDE

By ANNA L. KLEINHAUS AND J. W. PRICHARD From the Department of Neurology, Yale University School of Medicine, New Haven, Connecticut 06510, U.S.A.

(Received 4 June 1974) SUMMARY

1. Retzius cells of leech segmental ganglia were exposed to tetraethylammonium chloride (TEA) presented both extracellularly, dissolved in the perfusing fluid, and intracellularly, by iontophoresis from a micro-

electrode. 2. Extracellular TEA, 10 and 25 mm, greatly prolonged the cells' action potentials, and the higher concentration increased their amplitude as well. At 10 mm the characteristic changes developed gradually over a period of about half an hour, while at 25 mM they appeared much more rapidly. However, at both concentrations the changes were reversible within minutes, even after long soaks in drug-containing solution. It is therefore probable that the drug acted at the outer surface of the membrane. 3. Intracellular TEA also prolonged the action potentials but there were several differences from the response produced by extracellular application. The changes developed gradually, and for a time, each firing of the cell was a complex event consisting of several early, brief depolarizations followed by a single much larger and more prolonged one. The large, late depolarization eventually obliterated the early ones; its gradual development suggested that it was produced only after TEA diffused to some extrasomatic portion of the cell. Intracellular TEA always caused progressive depolarization; this and the changes in the action potential were both irreversible, suggesting that the site of action was on the inner surface of the membrane. 4. Manipulations of external Na and Ca provided evidence that (a) in the absence of TEA, Retzius cell action potentials were exclusively Na-dependent, (b) that the early depolarizations in the complex action potentials produced by intracellular TEA were Na-dependent, while the later, large depolarization was Ca-dependent and (c) that the prolonged action potentials produced by extracellular TEA contained a large Ca-dependent component.

352 ANNA L. KLEINHAUS AND J. W. PRICHARD 5. We conclude that TEA, acting from either side of the membrane, caused a voltage-sensitive, slowly activated Ca current to become a major contributor to the inward current of the action potential, probably by blocking the outward K current which ordinarily counteracts it. However, we cannot rule out the possibility that TEA enabled a Ca current by some means independent of its presumed action on K conductance. 6. Data resembling ours in some respects have been obtained from studies of the action of TEA on frog dorsal root ganglion cells, frog neuromuscular junction, and squid stellate ganglion. No clear counterpart of our findings has been reported from experiments on squid and amphibian axons, molluscan neurones, or frog skeletal muscle fibres. INTRODUCTION

The tetraethylammonium ion (TEA) is known to prolong the duration of the action potential in many invertebrate and vertebrate neurones, and in some preparations this effect has been shown to result from selective blockade of the delayed outward K current responsible for repolarization (Hille, 1970 passim). As part of a series of studies on leech neuropharmacology (Prichard, 1972; Prichard & Kleinhaus, 1974), we undertook this work to determine whether the action of TEA on Retzius cells resembled its action in other preparations. In general, it did, though certain features of the response led us to consider the possibility that these neurones developed a Ca-dependent action potential in the presence of TEA. The experiments reported here were done principally to prove that this was the case. To avoid awkward and repetitious phrases, the expression 'TEAi' is used in parts of this paper to indicate intracellular application of TEA (drug iontophoresed into the cell soma from a micro-electrode); 'TEAo' is used to indicate extracellular application (drug dissolved in the fluid perfusing the experimental chamber). METHODS Segmental ganglia of Hirudo medicinali8 were dissected from the ventral nerve cord, pinned to small resin blocks and placed in a 1-0 ml. plastic chamber through which Ringer solution flowed continuously at a gravity-driven rate of 2-3 ml./min. A system of valves near the chamber allowed for rapid changes of solutions without interruption of flow. The Ringer solution contained (mm): NaCl 120; KCl 4 0; CaSO4 or CaC12 2-0; Tris-(hydroxymethyl) aminomethane 10; and sufficient 10 N-H2SO4 or HCl to bring the pH to 7*4. NaCl was replaced by choline chloride or sucrose in equivalent osmolar amounts. In solutions where Ca concentrations were raised, the additional Ca was added in the form of CaSO4 or CaCl2 and osmotically balanced by sucrose in the control solution. Tetraethylammonium chloride (Eastman, Rochester, N.Y.) was added to the Ringer solution in concentrations of 10

TEA AND Ca SPIKES

353

or 25 mM; the increased osmotic pressure and chloride content of the TEA solutions were balanced by addition of choline chloride to the control solution. Intracellular potentials were recorded from one channel of bevelled doublebarrelled micro-electrodes having single-channel d.c. resistances of 10-20 Mn when filled with 3M-KCl or 4 M-K acetate and tested in Ringer solution. The other channel was used for transmembrane current passage. In some experiments TEA was injected iontophoretically into the Retzius cell somata from micro-electrodes filled with 1-3 M-TEA. Injected current passed serially through a calibrated 109 Ql resistor, micro-electrode and cell membrane to system ground; its magnitude was determined by applying a measured voltage to the gigohm resistor. In the experiments of Fig. 4, the accuracy of this method of estimating iontophoretic current was checked by placing a 1 % 105 n resistor in series with the ground return and measuring the voltage drop across it with a Cary 401 electrometer. The two methods consistently gave current values which were within 5 % of each other. For details of electrode construction see Anderson, Kleinhaus, Manuelidis & Prichard (1974). Synaptic stimulation of Retzius cells was accomplished by shocking the interganglionic connective, snugly held in the polyethylene tip of a suction electrode. Bath and micro-electrode were connected through silver-silver chloride pellet electrodes to a negative capacitance electrometer amplifier (W-P Instruments M4). Current monitor and voltage signals were displayed on a storage oscilloscope and photographed with a Polaroid camera, and also recorded continuously on a Brush 220 pen writer. W-P Instruments Series 800 digital pulse generator and stimulus isolation units were used to generate current for transmembrane and nerve stimulation and iontophoretic injection of TEA. For measurement of rise times, potentials were led from the electrometer amplifier to a differentiating circuit with a time constant of 100 /tsec; the long time constant was imposed by a high frequency filter which was necessary to reduce the noise level for measurement of slow rise times. This circuit was calibrated by a saw-tooth wave of known rise time connected in series with system ground and displayed at the beginning of each oscilloscope sweep; it was linear over the range 1-50 V/sec. All experiments were performed at room temperature.

RESULTS

Effect of TEAo Exposure of a segmental ganglion to TEAo 10 mm characteristically modified the electrical properties of the Retzius cell. Fig. 1 illustrates the typical sequence of changes which occurred in each of forty-five cells with some variations in time course. Two to 15 min after introduction of TEAO, the cell hyperpolarized by 10 mV (Fig. 1 B, C) thereby increasing the threshold for stimulation. Several minutes later the membrane depolarized, causing a drop in threshold. Finally, after 30 min perfusion, the action potential developed a plateau during repolarization (F). Exposures up to 6 hr did not cause significant further change. These effects were reversible after short periods of washout (4-10 min) even in ganglia which had been soaked in TEAo 10mM for 6 hr. suggesting an extracellular site of action. A larger concentration of TEA0 (25 mM) initially produced hyperpolarization similar to the lower concentration, but a large action potential of

ANNA L. KLEINHAUS AND J. W. PRICHARD

354

about 400 msec duration appeared within 3 min (Fig. 2 A 2,B 2). During the plateau the voltage change caused by an injected current pulse declined in amplitude and the time constant of the membrane shortened, indicating increased membrane conductance to one or more ionic species (Fig. 2 A 3,

A

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Fig. 1. Effect of TEA 10 mm on action potential of leech Retzius cell. Data are photographic records of current monitor (upper trace of each pair) and intracellular potentials (lower traces). Current was injected through one side of a dual-channel micro-electrode and potential recorded from the other side. Micro-electrode filled with 3 M-KC1 for this and subsequent figures, except where otherwise noted. Numbers above right end of each voltage trace from B on are time in min after TEA 10 mm was introduced into bath. Note. (1) Immediate hyperpolarization and consequent elevation of threshold caused by TEA 10 mm (B, C). (2) Gradual prolongation of action potential duration and eventual failure of complete repolarization (D-G).

TEA AND Ca SPIKES A

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Fig. 2. Effect of TEA 25 mm on two cells (A 1-4, B 1-4). Calibration pulse at left of each voltage trace is 20 mV, 20 msec except in A 3 where it is 50 mV, 50 msec. Current pulses in A 3, B 1-3 are all 2 nA. Action potentials were elicited synaptically by shocks to interganglionic connective for all traces except B 1, for which a trans-membrane current pulse was used. Note. (1) Marked prolongation and increase in amplitude of action potentials 5 min after introduction of TEA 25 mm (A 2, B 2). (2) Increased membrane conductance indicated by decreased amplitude and shortened time constant of voltage changes caused by hyperpolarizing (A 3, B 3) and depolarizing (B 2) current pulses during plateau phase of prolonged action potentials. (3) Disappearance of prolonged action potentials within 3 min after removal of TEA (A 4, B 4).

ANNA L. KLEINHAUS AND J. W. PRICHARD 356 B 2, B 3). After potentials positive to the base line (e.g. A 2) were commonly observed with TEA0. Effect of TEA, The characteristic changes in the action potential produced by TEA1 in Retzius cells are illustrated in Fig. 3. A 1 is the synaptically evoked action potential recorded with an electrode containing 3 M-TEA before any current injection. A 2- 3 illustrate the progressive changes brought about by TEA1 culminating in an apparently unitary action potential some 200 msec in duration. Between A 1 and A 3 TEA was ejected from the micro-electrode by a steady 10 nA current passed for two 30 sec periods. Before B 1, the cell received an additional 10 nA for 40 sec. TEA1 always caused progressive depolarization and reduction in initial action potential amplitude. The relation Q = I t/F where Q is ion equivalents, F is Faraday's constant, I is current in amperes, and t is time in seconds, can be used to estimate the quantity of drug released from a micro-electrode by iontophoresis (Krnjevid, 1971). Assuming a cell diameter of 80 ,tm and a transport number of 0.5 for TEA, the above current should have produced an intrasomatic TEA concentration in the neighbourhood of 19 mm at B 1 of Fig. 3. B 1-C 3 represent the spontaneous transformation of the evoked action potential over a period of 15 min after the last iontophoretic current was passed. A striking and reproducible feature of the response to stimulation of TEA-injected cells was a late, large depolarization. The nerve shock triggered an excitatory post-synaptic potential, followed by a series of brief depolarizations with progressively impaired repolarizing phases, followed in turn by a second kind of depolarization, which when fully developed (C3) had an amplitude of 46 mV. This late depolarization often exceeded the amplitude of the pre-drug action potential, even though it arose from a depolarized membrane potential. The rate of rise of the late depolarization was much slower than the rates of rise of both the pre-drug action potential and the early depolarization (C3-D3). About 30 min after the late depolarization appeared the cells were considerably depolarized and were capable of generating responses only 20-30 mV in amplitude. The terminal membrane potentials of twenty-two such cells, determined by electrode withdrawal, ranged from -6 to -35 mV. It is noteworthy that a cell which reached the stage depicted in B 2 always developed the late, large depolarization within 5-20 min, without passage of any additional iontophoretic current, suggesting that the drug had to reach a region of the cell distant from the soma to exert its effect. No cell injected with an amount of TEA sufficient to alter its action potential showed any signs of recovery during periods of observation as long as 1 hr.

357 TEA AND Ca SPIKES The phenomena illustrated in Fig. 3 suggested to us that the early and late depolarizations might have different ionic mechanisms. We suspected that the early one might be the result of an increase in sodium conductance, whereas the later one, which appeared to have a different equilibrium potential, might be Ca-dependent. The remaining experiments were done to test this hypothesis. 1

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Fig. 3. Effect of intrasomatic iontophoresis of TEA and of reduced external Na on the resulting complex action potential. Calibration pulse at left of all traces is 20 mV, 20 msec. All action potentials were elicited synaptically by connective shocks of identical amplitude and duration. Micro-electrode filled with 3 M-TEA. TEA was ejected by a steady depolarizing 10 nA current passed for 30 see before A 2, 30 see before A 3 and 40 see before B 1. If the cell diameter were 80 /,m and the transport number for TEA 0 5, the putative intrasomatic concentrations of TEA produced by these currents would have been 6 mm at A 2, 12 mm at A 3 and 19 mm at B 1 (see text). Note. (1) Development of late depolarization, over the 4 min during which iontophoretic current was passed (A 2-B 1). (2) Spontaneous evolution ofthe complex response over the next 15 min (B 2, a 2) without passage of additional iontophoretic current. (3) Progressive decline in amplitude of first peak when Na lowered to 80 mm by replacement with choline (C 3-D 2, arrow) and prompt recovery when Na restored to 120 mm (D 3). There was no corresponding change in the late depolarization.

358

ANNA L. KLEINHAUS AND J. W. PRICHARD

Manipulation of external Na In Fig. 3, A 14J2, the ganglion was bathed in normal Ringer containing 120 mM-NaCl. C3 was recorded 2 min after the NaCl had been reduced to 80 mm while tonicity and other ions were kept constant. In this early stage the amplitude of the first peak fell by about 6 mV, whereas the second peak remained unchanged. Five minutes later (D 1), the first depolarization decreased by another 4 mV. After 10 min in the low Na solution, the first peak fell to about half the value it had in 120 mM-NaCl (D2, arrow). Three minutes after reintroduction of 120 mM-NaCl (D3) the amplitude of the first peak had increased by about 10 mV. During these manipulations the amplitude of the late depolarization did not follow the external Na concentration, though it did decline somewhat because of the progressive depolarization always caused by TEA1. Five cells were taken through the same steps and yielded similar results. However, these experiments were difficult because it was necessary to perform them against a background of progressive depolarization and during the limited period when the two peaks were both clearly identifiable.

Manipulation of external Ca The four traces in Fig. 4 illustrate the differences among TEA,-induced action potentials in external Ca concentrations of 1, 2, 5 and 10 mm. Four cells are represented; each was soaked in its respective Ca solution for at least 20 min before being impaled with a TEA-containing microelectrode. Each trace was taken 12-25 min after passage of a 5 nA iontophoretic current for 30 sec, and each represents a similar stage of response development. Electrode withdrawal shortly after the traces were made permitted accurate determination of the overshoot of the late depolarization, and these are plotted as a function of log Ca concentration in the graph. They define a slope (continuous line) quite close to that expected for an event caused by a change in conductance of a divalent cation (dashed line). The early depolarization is also larger in the higher Ca concentrations, as Retzius cell action potentials always are, probably because increased membrane resistance promotes their electrotonic spread into the soma from a site of active propagation elsewhere. However that may be, the progressively changing relationship between the early and late depolarizations suggests that the latter was more closely dependent on a Ca conductance change. The behaviour of the early event was frequently difficult to interpret in experiments of this sort, and exposure of the same cell to several Ca concentrations was often frustrated because equilibration of the Ca concentration in the bath with that of the extracellular spaces of the ganglion seemed to occur rather slowly in relation

TEA AND Ca SPIKES 359 to the development of the TEA,-induced changes in the action potential. For these reasons we performed the more definitive experiments to be described. ......................

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Fig. 4. Effect of various external Ca concentrations on TEAq-induced action potentials. For these experiments, all solutions were made with CaCl2. Each trace at the top is from a different Retzius cell, soaked at least 20 min in the Ca concentration which appears directly below it on the graph. Each trace represents a similar stage of response development after iontophoresis of TEA into the cell soma from the micro-electrode (see text). The dashed line on each trace is the potential obtained upon electrode withdrawal shortly after the trace was made, and all four calibration pulses are 20 mV, 20 msec. The four points on the graph denote the maximum overshoots of the late depolarizations, and the continuous line is the best least-squares fit to them. Note. (1) The slope of the experimental line is quite close to the slope expected for a divalent cation (dashed line). (2) The changing relationship between the peaks of the early and late depolarizations.

Na-free and Ca-free solutions The above findings prompted us to investigate whether a leech Retzius cell could be made to fire an action potential in the complete absence of external Na. When all Na is removed from the solution surrounding a leech ganglion, Retzius cells ordinarily lose their ability to generate action potentials (Kleinhaus & Prichard, 1974). Fig. 5 illustrates how these cells behaved in Na-free solution when impaled with an electrode containing 3 M-TEA. In normal Ringer the action potential had its usual appearance (A 1). External calcium was then increased from 2 to 10 mm, which caused

360 ANNA L. KLEINHAUS AND J. W. PRICHARD a slight increase in threshold but no additional change after equilibration (not illustrated; see Fig. 6 B). With the Ca concentration kept at 10 mm, all external Na was replaced by sucrose. Three minutes after removal of Na the cell was not excitable by current pulses up to 8 nA (A 2). Pulses of 1-8 nA were continuously delivered at a rate of 1 every 5 sec, thus ejecting small amounts of TEA into the cell. Ten minutes after the Na had been removed a large, prolonged action potential appeared, triggered by a 4 nA pulse (A 3). At this point the membrane potential had decreased by about 10 mV. If no iontophoretic current was passed such events did not appear unless the cell was impaled for a much longer time, presumably the time required for sufficient TEA to diffuse out of the electrode tip. Once present, these action potentials were elicitable for as long as an hour, though as the cell continued to depolarize by action of TEAi its threshold decreased. B 1 illustrates an action potential triggered by a 3 nA, 50 msec pulse 21 min after removal of Na. The relative refractory period of these action potentials was quite long; their amplitude and duration was directly related to interstimulus interval up to about 15 sec. B 2 shows four successively shorter action potentials elicited at 5 see intervals after a long stimulus-free period. When Na was reintroduced the action potential became considerably shorter (B 3). Cl and D 1 are potentials recorded from two other cells at the stage represented by B 1 (TEA1 no external Na). In both, the action potential disappeared almost completely within minutes when Ca was removed from the bath (C 2, D 2) and promptly reappeared when it was replaced (C3, D3). The typical response of a Retzius cell to TEA. 25 mm in Na-free solution is depicted in Fig. 6. A is the response to subthreshold and threshold depolarizing pulses in normal Ringer. B shows responses to the same currents several minutes after switching to a solution containing 10 mM-Ca, and C the effect of replacing NaCl with sucrose. In the absence of external Na, current pulses up to 10 nA and 150 msec did not trigger action potentials (C, D). The cell remained inexcitable when depolarized nearly 30 mV by a steady current (E), though some evidence of membrane response appeared with the stronger pulses. The small undershoots which follow the pulses in E were presumably due to K activation, which became more evident in the presence of the steady depolarization. The steady depolarizing current was then turned off, and 2 min after TEA 25 mm was added to the bath a typical prolonged action potential appeared in response to a 3 nA pulse (F), even though the membrane potential was more negative than in E. G shows that this action potential also had a long refractory period when stimulated every 5 sec after a long stimulus-free interval. Similar results were obtained in two cells when NaCl was replaced by choline chloride rather than sucrose but the high choline concentration

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Fig. 5. Internal TEA caused Ca-dependent action potentials in Na-free solution. Calibration pulses on each voltage trace 20 mV, 20 msec; pulse in A 1 also serves as 20 nA calibration for all current monitor (upper) traces. Current monitor trace inadvertently omitted from A 1. Micro-electrode filled with 3 M-TEA. External NaCl replaced by sucrose in all traces except A 1 and B 3. External Ca 2 mm in A 1, 10 mm in all other traces except 2 and D 2. Note. (1) Inexcitability of cell in Na-free solution; largest current pulse 8 nA (A 2). (2) Prolonged action potential elicited by 4 nA pulse after delivery of 1-8 nA depolarizing pulses every 5 see for 10 min (A 3). (3) Progressive shortening of duration of action potentials elicited every 5 see (B 2). (4) Shortening of action potential duration by re-introduction of NaCl in B 3. Compare with B 1; both action potentials elicited after a long stimulus-free interval. (5) Disappearance of action potentials in Ca-free solution in two other cells (C 1-3, D 1-3) previously taken through the same manipulations that led to B 1.

362 ANNA L. KLEINHAUS AND J. W. PRICHARD caused the cells to deteriorate significantly over the period required for the experiments. The Ca dependency of TEA,-induced potentials is demonstrated in Fig. 7. A 1 shows action potentials recorded in control Ringer. Removal of Na abolished them (A 2), but a typical prolonged event appeared when C

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Fig. 6. Failure of electrically induced depolarization and competence of TEAO 25 mm to produce prolonged action potentials in Na-free solution. Calibration pulse on voltage traces 20 mV, 20 msec. External NaCl replaced by sucrose in all traces except A and B. External Ca 2 mm in A, 10 mm in all other traces. Note. (1) Inexcitability of cell by current pulses up to 10 nA and 150 msec after removal of Na (C, D). (2) Continued inexcitability during 30 mY depolarization produced by steady current (E). (3) Production by TEA 25 mm of prolonged action potential with typical low threshold (F) and long refractory period (G).

TEAo 25 mm was introduced (A 3); B 1 illustrates the progressive disappearance of the action potential during removal of external Ca while the cell was stimulated at 15 sec intervals. In B2, 5 min after the change in solution, the cell became inexcitable. Replacement of Ca restored the response to a previously ineffective stimulus (B 3). Another example of the same phenomenon in another cell is shown in C 1-03. C 2 was obtained 5 min after removal of Ca and C3 10 min after its re-introduction.

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Fig. 7. External TEA caused Ca-dependent action potentials in Na-free solution. Calibration pulses on each voltage trace 20 mV, 20 msec; pulse in A 1 also serves as 20 nA calibration for current monitor (upper) traces. External Ca 2 mmi in A 1, 10 mm in all other traces except B 2 and (12. External NaCl replaced by sucrose in all traces except A 1. Note. (1) Action potential (A 3) produced by TEA 25 mim in previously inexcitable (A 2) cell. B 1 illustrates progressive decline of action potential elicited once every 15 sec during Ca washout. (2) Ca-dependency of the TEA-induced action potential (B 1-3). (3) Same phenomenon in another cell (0 1-3) previously brought to the stage of B 1.

The magnitude of the Ca current Using the maximum rate of voltage change (dV/dt) and membrane capacity (C), it is possible to estimate the maximum current (I) associated with a change in membrane potential from the relation I = C (dV/dt). Since C = tRO'IpIVp, where tRC is the membrane time constant and V. is the membrane potential change caused by the current pulse I , it is possible to express I in terms of measurable quantities by writing I = tRCIP(d V/dt)/ VP. I was calculated by this relationship from the data in Fig. 8 and had values of 11-5 and 6-8 nA for Ca-dependent action potentials in two Retzius cells from different leeches. The cell with the larger current (A) was exposed to 10 mm-Ca and the other one (B) to 2-0 mm-Ca. These are the maximum currents which flowed during the

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ANNA L. KLEINHAUS AND J. W. PRICHARD 364 depolarizing phases of the depicted events. The analysis provides no information about currents which flowed at other times or, by itself, about the ionic basis of I. a

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Fig. 8. Rate of rise measurements on TEAO-induced action potentials in two cells at external Ca concentrations of 10 mm (A) and 2 mm (B) and in absence of Na. Square calibration pulse in each voltage (lower) trace 20 mV, 10 msec and also serves as 20 nA calibration for current monitor (upper) traces. Rates of change of saw-tooth wave in voltage trace A 1 are 2*0 (positive-going) and 2-5 (negative-going) V/sec and serve to calibrate the differentiating circuit, the output of which constitutes the middle trace. In B 1 and 2 the positive and negative rates of change are 6*0 and 5.0 V/sec, respectively. Membrane time constant and resistance were determined from voltage responses to largest hyperpolarizing current pulses in A 2 and B 2, 5 nA in both cases. Note. Faster rise time of action potential in 10 mm (A) than in 2 mm (B) external Ca. Rise times correspond to whole-cell membrane currents of 11-5 and 6-8 nA (see text).

Insensitivity of normal and TEA-induced action potentials to tetrodotoxin Tetrodotoxin had no effect on either the Retzius cell action potential or the TEA-induced changes in it until the concentration was raised quite high. At 100 gm the drug first slowed the rise times and later reduced the amplitudes of both normal action potentials and the early and late components of the TEA response. These changes developed slowly over a

TEA AND Ca SPIKES 365 period of 10-20 min and were reversible. The tetrodotoxin used in these experiments was rapidly lethal to mice in a dose of 100 ptg/kg i.P., about 10 times the usual minimum lethal dose (Kao, 1966). The insensitivity of the Retzius cell was therefore not ascribable to lack of drug potency. DISCUSSION

TEA caused the action potential of the Retzius cell to develop a Cadependent component based on an inward Ca current which was large enough to be regenerative in the absence of Na. No such phenomenon could be demonstrated in the absence of the drug. These findings are compatible with any of three different relationships between TEA action and membrane Ca currents: (1) TEA might have blocked a large outward K current and thereby allowed an otherwise unaffected Ca current to dominate the behaviour of the excited membrane; (2) during excitation, prolonged depolarization or some other consequence of TEA-induced reduction of K current might have activated a Ca current which is ordinarily absent or does not flow in significant quantity; (3) TEA might have activated a Ca current by some means independent of changes in K current. We favour the first possibility because it is the most parsimonious and the most readily related to findings in other tissues. However, it should be noted that we have no direct evidence for TEA action on K conductance in the Retzius cell, and that a direct action of the drug on membrane Ca conductance in another tissue has been suggested (Beaulieu & Frank, 1967). The insensitivity of the normal, Na-dependent Retzius cell action potential to tetrodotoxin prevents any present conclusion about whether or not the Ca current which is responsible for the major portion of the TEA-induced action potential flows through the Na channel. In squid axon, a tetrodotoxin sensitive, Ca-dependent action potential can occur when Ca is the principal cation available to carry inward current and the inactivation mechanism of the Na channel is inhibited (Tasaki, 1968; Meves & Vogel, 1973). In our experiments the early (Na-dependent) and late (Ca-dependent) portions of the TEA-induced action potential both were affected by the same high concentration of tetrodotoxin. If the insensitivity of the Retzius cell to tetrotodoxin were due to failure of the drug to penetrate from the bath to the interior of the ganglion, a careful effort to follow up this preliminary finding would be justified. However, no such penetration barrier is known to exist, and Na-dependent, tetrodotoxin-resistant action potentials have been demonstrated in denervated muscle (Harris & Thesleff, 1971) and certain other tissues (Kao, 1966). Since the resistance of leech neurones to tetrodotoxin is itself a matter requiring further investigation, it would be premature to reach any

366 ANNA L. KLEINHAUS AND J. W. PRICHARD conclusion about the significance of the drug's effect, or lack of it, on the phenomena we have described. It is useful to compare our findings to the reported effects of TEA in other tissues, beginning with those which appear to have similar responses. Results similar in several respects to ours were obtained by Koketsu, Cerf & Nishi (1959), who observed Ca-dependent action potentials after both internal and external application of TEA to frog dorsal root ganglion cells. Internal application caused progressive depolarization, as in the Retzius cell. Prolonged action potentials were elicitable after complete replacement of Na with TEA, tetramethylammonium chloride (TMA), or choline, but not sucrose; however, with TMA and choline, the action potentials were briefer than with TEA and did not appear at all until the preparation had been soaked in Na-free solution for several hours. These authors found, as we did, that the duration of an action potential induced by TEA in Na-free solution was considerably shortened by re-introduction of Na. They interpreted this as evidence of competition between Na and TEA ions, but the additional possibilities that Na and Ca competed for the same channel or that the repolarization process was responsive to Cl ion were not eliminated by the conditions of their experiments or ours. Katz & Miledi (1969 a) described a Ca-dependent, tetrodotoxin-resistant local response after injection of TEA into presynaptic elements of the squid stellate ganglion. This response was an all-or-nothing action potential of long duration, sharply limited to the synaptic region of the presynaptic cell. Membrane resistance was decreased during the plateau, and repetitive stimulation at 2-5 see intervals caused the duration of the plateau to become progressively shorter. These features of the response are quite similar to our findings in the leech Retzius cell except that the insensitivity of the normal Retzius cell action potential to tetrodotoxin prevents, for the present, any conclusion about the significance of the failure of this drug to block the TEA-induced event (see above). The association of the Ca-dependent action potential with the synaptic portion of the membrane is especially interesting in view of the suggestion in our data that intracellular TEA did not exert its characteristic action until it diffused out of the synapse-free soma. These authors also described a spectacular augmentation of end-plate potentials produced by 5 mm external TEA in the frog neuromuscular junction (Katz & Miledi, 1969b). They presented indirect evidence that the phenomenon was based on a Ca-dependent action potential in presynaptic terminals similar to the one directly demonstrated in the squid ganglion. In several other studies on various tissues, there is little to indicate that TEA produced the sort of phenomena we have described. The most extensively studied preparations are squid axon (Armstrong & Binstock,

TEA AND Ga SPIKES 367 1965; Armstrong, 1966), toad axon (Koppenhofer & Vogel, 1969) and frog node of Ranvier (Hille, 1967; Armstrong & Hille, 1972). All three tissues were studied by voltage-clamp technique after both internal and external application of TEA. No inward currents corresponding to the Ca-dependent action potentials we have described were observed. The experiments were not designed to detect such currents, but it is unlikely that they would have gone unnoticed, had they been present. The action of TEA after external application was studied in unidentified molluscan neurones (Magura & Zamekhovsky, 1973), ligated somata of molluscan neurones (Connor & Stevens, 1971) and frog skeletal muscle fibres (Stanfield, 1970). In the first two studies, the use of TEA was a minor aspect of the work, and the data presented neither suggest nor preclude a TEA action like the one we have described. Stanfield, using a modified voltage-clamp technique, detected no TEA effects which were not attributable to blockade of outward K currents. Neher & Lux (1972) exposed unidentified snail neurones to internal and external TEA in the millimolar range and used a patch-clamp technique to measure currents flowing through a portion of soma membrane. They detected no currents with magnitude and time course corresponding to the Ca-dependent spike in the Retzius cell. In some of the above papers, the data are not sufficient to reach a conclusion about the role of Ca currents in the behaviour of the TEAtreated membrane. In the others, all based on voltage-clamp data, the possibility exists that a Ca current was present but was so small or so smoothly merged with other inward currents that it would not have been detected without specific efforts to reveal it. For these reasons we are not yet certain that the response of the leech Retzius cell to TEA is unequivocally different from the responses of all of the other excitable membranes mentioned above. Further experimentation is necessary to clarify the point. Although the range of methods and goals in these several studies is too broad to permit any firm generalization about the relation of TEA to Ca spikes, there is a suggestion that the latter are more likely to appear in preparations which include synapses than in those which do not. This brings to mind the fact that several kinds of vertebrate and invertebrate neurones can use divalent cations to fire action potentials in the absence of Na (Reuter, 1973), while it is less common, though not unknown (Greengard & Straub, 1959), for axons to do so. In at least two cases Cadependent soma spikes and Na-dependent axon spikes co-exist in the same neurone (Iwasaki & Sato, 1971; Wald, 1972). This is not to say that axonal membranes do not admit Ca ions during excitation. The squid axon certainly does (Baker, Hodgkin & Ridgway, 1971), but the Ca current is too

368 ANNA L. KLEINHAUS AND J. W. PRICHARD small to be regenerative in the absence of Na or when outward K current is blocked by TEA, unless the additional special conditions mentioned above are also created (Tasaki, 1968; Meves & Vogel, 1973). It may be that the function of transmitter release requires that membranes in or near synaptic regions pass larger Ca currents, and that the machinery for doing this favours the occurrence of both naturally occurring Ca spikes and those which appear in the presence of TEA. This work was supported by United States Public Health Service grants 1-RO1-NS-08851 and 5-PO1-NS-06208 and a grant from the Esther A. and Joseph Klingenstein Fund. Robert Morton provided indispensable technical assistance.

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