Light-evoked depolarizations in the retina of strombus: Role of sodium and potassium ions

Comp. Biochem. Physiol. Vol. 80A, No. 2, pp. 233 245, 1985 Printed in Great Britain

0300-9629/85 $3.00+0.00 ~ 1985 Pergamon Press Ltd

LIGHT-EVOKED DEPOLARIZATIONS IN THE RETINA OF STROMBUS: ROLE OF SODIUM A N D POTASSIUM IONS KEVIN S. CHINN and HOWARD L. G1LLARY Department of Physiology and Bekesy Laboratory of Neurobiology, University of Hawaii, Honolulu, HI 96822, USA

(Received 1 June 1984) Abstract--1. Light-evoked depolarizations (LED's) in retinal cells of Strombus luhuanus can exhibit an

early phase of depolarization (D E), a brief repolarizing phase (R E), and a later depolarizing phase (D r). 2. Lowering external Na ÷ by substitution with choline, tetramethylammonium or sucrose, reduced the amplitude of the entire LED, but D L was reduced more than D E. 3. Replacement of Na ÷ with Li ÷ reduced D E more than D r. 4. Lowering pH reduced D r more than D E, while raising it increased D r but not D E. 5. K + channel blocking agents, tetraethylammonium and 4-aminopyridine, increased R E. 6. During the LED, cell membrane conductance increased in two phases, corresponding to D E and D r . 7, The results suggest LED generation by two separable conductance increases to Na +, corresponding to D E and DL, and another to K ÷ during R E.

INTRODUCTION

A wide variety of invertebrate neurons depolarize in response to illumination. These include photoreceptors containing visual pigment as well as neurons coupled to them, such as the eccentric cell in the Limulus lateral eye (Hartline and Ratliff, 1972). Conductance changes to sodium and potassium ions can play important roles in generating light-evoked depolarizations (LEDs) (Brown et al., 1970; Hanani and Shaw, 1977; Alkon, 1979; M a a z et al., 1981; Fain and Lisman, 1981; O ' D a y et aL, 1982). In addition, in Limulus ventral photoreceptors, release of intracellular calcium seems important in shaping the L E D (Lisman and Brown, 1975; Brown et al., 1977; Fain and Lisman, 1981). Two types of retinal neurons in Strombus luhuanus, a marine gastropod, yield L E D s with waveforms which can exhibit two or more distinct peaks, or phases, of depolarization (Quandt and Gillary, 1979). F o r one o f these cell types (type II), it was shown (Quandt and Gillary, 1980) that the second phase was preferentially reduced by light adaptation, or elevated extracellular magnesium. Furthermore, in experiments involving the passage of intracellular current, the apparent reversal potential of the second phase was notably more negative than that of the first. Based on their results, they suggested that the two depolarizing phases arise from at least two separable voltage-insensitive conductance changes. The present studies were undertaken to examine the ionic bases for the different phases of the L E D of this type lI neuron. This paper focuses on the role of sodium and potassium ions in generating the response. The role of calcium ions will be considered in another paper. An abstract of some of the results has been published (Chinn and Gillary, 1980). MATERIALS AND METHODS

Except when indicated, the experimental procedures were carried out as described previously (Gillary, 1974; Quandt 233

and Gillary, 1979, 1980). In all experiments, the retina of a morphologically mature eye was isolated in artificial seawater (ASW), impaled with glass microelectrodes filled with 2 M potassium acetate (40-100 Mf~) and presented at 2 min intervals with 0.2 sec stimuli of white light (30 W/m 2) that had passed through an infrared absorption filter. During an experiment, the bathing solution in the preparation chamber (ca 0.2 ml) could be changed (in 8-12 sec) by means of a flow-through perfusion system. In experiments involving Na + substitution, the sodium of normal ASW (NaCI, 473 mM; KCI, 10 mM; CaCI2, 10 raM; MgCI2, 25 raM; MgSO4, 28 mM; HEPES, 5 mM; pH 7.7) was replaced by equimolar amounts of either choline, tetramethylammonium (TMA), lithium (Li*), or potassium (K+). In other studies, NaC1 was replaced by equimolar and equiosmolar amounts of sucrose. When tetraethylammonium chloride (TEA) of 25raM or less, or 4-aminopyridine (4-AP), were used, they were either added to the bathing medium or substituted for NaC1. In experiments in which TEA was 50 or 100 raM, the control ASW contained 100 mM choline, substituted for Na% and in the test solution, TEA replaced choline. This was to ensure that effects attributed to the high concentrations of TEA were not due to lowered Na * concentration. Other details regarding the perfusion media will be presented in the Results section. Unless otherwise indicated, a given bathing solution was changed only after the response attained a steady state, i.e, successive LEDs differed from each other by no more than 0.3mV, throughout the entire response. Except when specifically stated, experiments were performed at 23 + I°C on at least five cells. The methods have been described elsewhere in greater detail (Chinn, 1981). RESULTS

Resting potential The resting membrane potential in the dark (RP), typically - 81 _+ 3 mV in normal A S W for the several hundred cells impaled, was highly dependent on external potassium ion concentration (Ko). Raising K 0 above normal (10 mM), by substitution for Na ÷, caused an immediate depolarization. Within the range of 25-100 m M K0, this depolarization averaged

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Fig. 1. Waveform of LED. (A): Typical waveform. Resting Potential (RP) was - 8 0 mV. In this and subsequent figures, an arrow before the LED indicates the onset of a 0.2 sec flash of light, and an upward deflection indicates an increase in intracellular positivity, i.e. depolarization. The labelled phases of the LED are the early depolarization (D E), late depolarization (DL), early repolarization (R E), and late phase of repolarization (RL). At the vertical dashed line, the recorder speed was decreased. PIH indicates a post-illumination hyperpolarization, typically 1-2 mV in amplitude. (B) and (C): Three-peaked LEDs. Note that in (B) the LED exhibits two peaks of depolarization during the time normally occupied by D E, and in (C), a third peak of depolarization (at ca 1.5 sec) follows the typical D L phase. In this and subsequent figures the right-hand asterisk indicates RP, i.e. the dark membrane potential. 62mV per tenfold change (62mV/10×) in K0, a value close to that predicted by the Nernst equation at 23°C (59 mV/10 × ), if one assumes that the RP is entirely dependent on K ÷, and the intracellular K ÷ concentration (K~) is unaffected by changing K0. Between 10 and 2 5 m M K 0 , the decrease (ca 41mV/10× K0) was somewhat smaller than predicted. Omitting KCI entirely from the bathing medium caused the cells to hyperpolarize, to RPs of - 1 2 0 to - 1 4 0 m V . The above results cannot be explained by the variations in Na0; reducing Na0 by more than 300 mM had negligible effects on RP.

Photoresponse waveform The LEDs in response to brief light stimuli, often exhibit two distinct phases of depolarization, separated by a brief repolarizing phase (Fig. 1A). The relative amplitudes of these phases could vary under different conditions of stimulus frequency, intensity, and duration (Quandt and GiUary, 1980). These phases will be referred to as the early and late depolarizations (D E and DL) and the early and late repolarizations (RE and RL), as indicated in Fig. 1A. In addition to the LED, a small post-illumination hyperpolarization occurred. Although the responses examined in the present studies were all evoked by 0.2 sec flashes (that usually terminated during RE), responses of similar waveform were evoked by stimuli which ranged in duration from 35 msec to several seconds. A stimulus duration of 0.2 sec was chosen because for an interstimulus interval of 2 min, it consistently evoked LEDs with two distinct depolarizing phases (i.e. D E and DL), whereas briefer stimuli did not, and required longer interstimulus intervals to obtain LEDs with two such phases. The response amplitudes under the conditions of the present experiments were not saturated (i.e. maximal).

On rare occasions, cells having LEDs with more than two phases of depolarization were encountered. For example, the LED in Fig. IB exhibits two peaks during what would normally be the DE phase, and that in Fig. 1C exhibits an extra depolarizing phase after D L, The complexity of these responses suggests that certain processes underlying the LED may be obscured during the usual, two-peaked response. In the experiments to be described, the conditions of stimulation (including stimulus intensity, duration and frequency) were chosen to yield LEDs with fairly distinct DE and DL phases in normal ASW. Under such control conditions, the LED waveform evoked from different cells did show some variation (Figs 3B, 7A, 9A) of unknown origin; it could not be attributed to differences in the condition of the retinal preparation or electrode impalement procedures. However, for a given cell, the LED waveform was remarkably constant. After achieving a steady state, successive LEDs evoked by repetitive stimuli under constant conditons, showed variations in amplitude of no more than 0.3 mV, throughout the entire response. Such variations were considerably less than those attributed to the experimental manipulations of the bathing media described below. It is upon such reproducible, experimentally induced variations in LED waveform that the inferences of this paper are based.

Effects of reduced Nao Lowering Na0 by partial replacement with choline (Fig. 2A) or tetramethylammonium (TMA) (Fig. 3A), caused a reduction in amplitude of the entire response. This decrease was approximately a linear function of log Na 0; however, it was proportionately less for DE than for the rest of the response. Typical data are plotted in Figs 4, 5 and 6, the last two include values predicted by the Goldman-Hodgkin-

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Fig. 2. Effects of substituting choline for sodium. (A): Partial substitution of choline for Na +. In this and subsequent figures, the records are aligned so that the arrow below the lowest trace indicates the stimulus onset for those above it. In addition, unless indicated otherwise, responses in a given figure are from a single cell and presented in the sequence in which they were obtained experimentally. 1: Response in normal ASW, i.e. 473 m M Na ÷, 0 m M choline (RP, - 8 1 mV). 2: 210raM Na +, 263 m M choline (RP, - 7 9 mV). 3: Recovery in normal ASW (RP, - 8 0 mV). Note that decreasing Na0 caused a decrease in amplitude of the entire LED but that the potentials during RE and DL were reduced proportionately more than those during D E. (B): Total replacement of Na ÷ by choline and subsequent recovery in ASW, for a preparation different from that for (A). 1: Response in ASW (RP, - 79 mV). 2: Response in a different cell after 6 hr 40 rain in 0 mM Na ÷, 473 mM choline (RP, - 6 8 mV). (Four stimuli, delivered at 2 rain intervals preceded that for this record; before that, the preparation was dark adapted. An LED 2 mV in amplitude was evoked by the first stimulus to the dark adapted preparation.) 3: Same cell as for B2, 16 min after return to ASW (RP, - 72 mV). 4: Different cell, 23 rain after return to ASW (RP, - 81 mV). (C): Rebound in LED amplitude during recovery from total replacement of Na ÷ by choline. 1: After I hr in 0 m M Na ÷, 473 mM choline (RP, - 7 1 mV). Note small LED. 2: LED 2 rain after return to ASW (RP, - 7 9 mV). (The preceding stimulus was delivered when the preparation was in 0 mM Na +.) 3: Response to next stimulus, 4 rain after return to ASW (RP, - 81 mV). 4: "Steady state" response 12 rain after return to ASW (RP, - 8 0 mV). The vertical calibration marker is 5 mV for traces 1 and 4, and 10 mV for 2 and 3. Note the unusually large amplitude of the LED to the first stimulus following the return to normal ASW, which subsquently declined to normal. Such large LEDs exhibited only a single peak.

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A Fig. 3. Effects of substituting TMA for sodium. (A): Partial substitution of TMA for Na ÷. 1: Response in normal ASW, i.e. 4 7 3 m M N a ÷ 0 m M T M A . 2: 1 4 0 m M N a ÷, 3 3 3 m M T M A . 3: Final recovery in normal ASW. R P s for all records were - 8 5 + 1 inV. Note that the LED amplitude decreased as Na o was lowered. (B): Total replacement o f Na ÷ by TMA, for a preparation different from that for (A). 1: Response in ASW (RP, - 81 mV). 2: After 20 rain of perfusion with the zero Na + solution (RP, - 73 mV). After return to normal ASW, recovery was usually complete (not shown).

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Fig, 4. Effects on relative LED amplitude of partial substitution of sodium by choline (A) or T M A (B). The displacements from RP of three successive points on the LED (triangle, open circle, closed circle; see inset) are used as measures of response amplitude for DE, RE and D D. respectively, as indicated in the text and Fig. 1. (Insets in Figs 4-6 show typical LED waveform from Fig. IA.) For each of a number of cells, each value of LED amplitude corresponding to DE, R E and D L recorded in A S W with lowered Na + (315, 210 or 140 m M ) was expressed as a percentage of the corresponding value of the response in normal ASW (Na + = 473 mM); the averaged values ( + S E ) were then plotted as a function of N a + on a logarithmic scale. After each exposure to solutions with lowered N a +, the L E D was nllowed to recover in normal ASW. In all of these experiments the test sequence was randomized. Note that the values for DE, RE and D L all decreased in lowered Na ÷, but that the decrease was proportionately less for DE than for R E and DL. The number of cells tested (N) at 315, 210 and 140 m M Na +, respectively was 6, 5 and 6 in (A) (including those for Figs 2A and 5A), and 7, 8 and 9 in (B) (including those for Figs 3A and 5B).

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Fig. 5. Effects of sodium concentration on the absolute membrane potential of single cells during the LED. Values corresponding to D E, R e and D e are plotted as a function of log Na 0. The data in (A) are from a single cell (different from that in Fig. 2A) for which external N a + was partially replaced by choline; that in (B) is from another cell for which T M A replaced Na +. Sodium concentrations were 140, 210, 315 and 473 m M (normal). The dashed lines indicate values predicted by the G o l d m a n - H o d g k i n - K a t z ( G H K ) equation corresponding to D e (triangle), R E (open circle), and D e (closed circle) of responses in lowered Na0. The actual data for normal ASW and for N a 0 (140raM) indicates that in (A), the value at D E decreased by 7 mV per ten-fold decrease in N a 0 (7/10 x N a 0), and that at D L decreased by 11 mV/10 × Nao; in (B), the respective changes, in mV/10 x Na0, for D E and D L were 17 and 18. Note that for these cells, the values could be considerably less than the predicted values. (For average differences, see Fig. 6.) Resting potential was relatively unchanged by lowered Na0; for 473, 315, 210 and 140raM Na +, the respective values were - 80, - 80, - 79 and - 79 mV for (A), and - 84, - 85, - 86 and - 87 mV for (B).

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Fig. 7. Effects of total replacement of sodium by lithium. (A)-(C): LEDs from a single cell in response to continuous immersion in 0 m M Na +, 473 m M Li ÷. (A): Just before immersion. (B) and (C): After 8 and 10 min of immersion, respectively. (The respective R P s were - 8 6 , - 71 and - 71 mV.) Note the greater reduction in amplitude of D E than of D L. (D)-(E): LEDs from another cell after immersion of 8 and 10min respectively ( R P s , - 6 9 and - 6 6 m V ) . Note effects similar to those for (A)-(C). (The difference in D L amplitude between (D) and (E) is within the normal range of experimental variation.) (F)-(H): L E D recovery in another cell after return to normal A S W from 0 m M Na +, 473 m M Li +. (F): Response after 68 rain in 0 m M Na ÷ ( R P , - 62 mV). After 72 min in 0 m M Na ÷ ( R P , - 58 mV), the perfusate was changed to ASW. (G): 8 min after return to A S W ( R P , - 6 5 mV). (H): 16 min after return to A S W ( R P , - 89 mV). Upper vertical calibration marker applies to (F) and (G), and the lower one to (H). Note the unusually large L E D amplitude in (H), the m a x i m u m of which, in response to succeeding stimuli (not shown), remained larger by ca 50% than those of the LEDs before exposure to 0 m M Na +, 473 m M Li ÷, as in (A). C.B.P 80,2A

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238

KEVIN S. CHINN and HOWARD L. GILLARY

Katz (GHK) equation. The calculation of these values and their relation to the data is considered further in the Discussion. Similar results were also obtained when the sodium substitute was sucrose, although with sucrose, the quality of the intracellular recordings usually deteriorated within 4 to 10min. Total substitution of sodium by choline (Fig. 2B) or TMA (Fig. 3B) almost entirely abolished the response within 20min. However, LEDs of very small amplitude usually persisted for up to 6 hr. Short term treatment ( < 1 hr) caused the cells to depolarize by 4--10 mV within 15 min to a new steady membrane potential. Upon reimmersion in normal ASW, these effects were completely reversible within 2-12 rain. However, the sequence with which the LED amplitudes attained control values depended on how long before the next light flash the solution was changed. When the change occurred 15 sec before the next flash, LED recovery was gradual. However, when cells, immersed for 20-60rain in solutions with choline as the Na + substitute, were returned to normal ASW ca 2 min before the next flash, the response to that flash showed an amplitude considerably larger than the steady-state control value (Fig. 2C), which was progressively attained by successive LEDs, usually within 12rain. All TMA solutions were changed 15 sec before the next flash, after no longer than 30 min of immersion, so it is not known whether this effect can occur when TMA is substituted for N a ' . The above observations may be related to Na-mediated changes in intracellular calcium (see Discussion).

Effects q[ lithium, pH and tetrodotoxin Lithium can pass through certain types of Na t channels but not others (Obara and Grundfest, 1968). Total replacement of Na ~ by Li ~ caused a reduction in LED amplitude, although the latency to the peak of DE was unaffected. However, unlike the Na + substitutes, TMA or choline, Li + reduced DE proportionately more than DE (Fig. 7A-E), which remained substantial even 18rnin after immersion. As with choline or TMA, total substitution with Li + also caused RP to decrease progressively (4-10mV in 15 min). Upon return to normal ASW, the RP returned to normal and the LED attained an unusually large amplitude (Fig. 7F-H), However, in contrast to the choline experiments (Fig. 2C), the LED amplitude remained above the pre-immersion control value for the remainder of the experiment, which could be several hours, although some reduction over time did Occur.

Hydrogen ions are known to block certain types of Na t channels (Woodhull, 1973). Therefore, the effects of pH on the LED were examined in an attempt to distinguish between different membrane channels which might open during the LED. The results are illustrated in Fig. 8. The pH of the ASW perfusate was lowered from 7.7 (normal) to 5.5. After 2 min, D E was reduced and was completely abolished after about 20 rain, so that R E was no longer evident. After 30min DE, although reduced, was not abolished, In fact, DE was initially increased in amplitude within 2 min after the solution change. Immersion in ASW of pH 9.5 sometimes reduced D E, and always

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Fig. 8. Effects of pH. pH was changed from the normal of 7.7, to either 5.5 or 9.5. Note the reduction ofD Lafter 8 rain at pH 5.5. After 10min at pH 9.5, DE was slightly reduced, while D L was significantly increased (by 2.5 mV or 40}i;). (The final recovery at pH 7.7 was not complete when the experiment was terminated.) increased DE. As with the ASW of pH 5.5, these changes began within 2 min after shifting to pH 9.5. For exposures of 10 rain or less, the latency to the peak of D E was unaffected by pH (Fig. 8), although prolonged exposure to pH 5.5 (ca 30 rain) could cause it to increase by as much as 0.2sec. The above changes in LED waveform were independent of the test sequence, which was randomized in different experiments, and were usually reversible upon return to normal ASW although complete recovery could take as long as 30 rain. Resting potential and input resistance were unaffected by the changes in pH. Tetrodotoxin blocks voltage-dependent sodium channels in squid giant axons in nanomolar concentrations (Narahashi, 1974). In Strombus, introducing TTX into normal ASW in concentrations as high as 6 x 10-6 M had no effect on the LED or RP.

Effects of K + channel blockers To test whether an outward K + current occurred during the LED, the effects on the response of exposure to 4-aminopyridine (4-AP) and tetraethylammonium (TEA) were examined, since these agents block such currents in other preparations (see Discussion for references). One would expect

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Fig. 9. Effects of 5 mM 4-aminopyfidine. (A): Response in normal ASW. (B): In 5 mM 4-AP. (C): After recovery in ASW. Note the increase in response amplitude, particularly during RE. (D): Recording in dark after ca 8min in 5 mM 4-AP. (Lower trace follows and is continuous with upper.) Note small, rapid oscillations (ca 0.5 mV and 1 Hz) and slow fluctuations in potential (ca 0.1 Hz). RP mean value was -84 mV. Preparation was different from that for (A)-(C). blockage of an outward K + current to increase LED amplitude. Addition of 0.5 mM 4-AP (N = 3) to the bathing medium did not affect RP, but could significantly increase the amplitude of the LED, especially during R E and D L. Similar effects could be obtained with 2.5 mM TEA (N = 3). The effects on the LED, of exposure to either TEA or 4-AP (for the concentrations just described as well as for the higher ones below), were usually, but not always, fully reversible. For somewhat higher doses of TEA (5 and 13mM), or 4-AP (2.0, 2.5 and 5raM), response amplitudes usually increased (Fig. 9), although for some cells these were reduced, especially D E. In addition, 5 mM 4-AP usually caused fluctuations in R P (Fig. 9D). These were evoked after 1 to 10 rain in the high 4-AP and consisted of small oscillations (ca 0.5mV, 1 Hz) superimposed on somewhat larger fluctuations (ca 1 mV) of lower frequency (ca 0.1 Hz). These effects on the R P were almost always reversible within 1-10 min after return to normal ASW. The highest doses of TEA (50 and 100mM), always caused a reduction in the response (e.g. Fig. 10). These high doses of TEA also caused small fluctuations in R P (ca 1-2 mV, 0.2 Hz) superimposed on larger, slower fluctuations (ca l0 mV, 0.01 Hz; Fig. 10D). Lower doses of TEA (13 and 25mM) produced only the small fluctuations. Such effects were clearly distinguishable 2-10 min after exposure to TEA and were reversible within 2-10 min following return to normal ASW.

Fig. 10. Effects of 50 and 100mM TEA. (A): Response in control solution of ASW in which 100raM choline was substituted for 100 mM of Na ÷. (B): Response in ASW with 50 mM TEA and 50 mM choline substituted for 100 mM of Na ÷. (C): Response 62 rain after return to control solution used in (A). Recovery was slow. RP for (A)-(C) was -79, -80 and -78mY, respectively. (D): Recording in dark after ca 7 rain in 100 mM TEA. Note small oscillations (ca 1-2 mV and 0.2 Hz) and slower fluctuations in potential (ca 10mV and 0.1Hz). These effects were not seen when 100 mM choline replaced 100 mM of Na +. RP mean value was -80inV. The impaled cell was the same as that for

(A)-(C). Effects o f substituting K + for Na + The effects of varying the concentrations of Na + and K + simultaneously, by substituting K + for Na +, were examined to obtain additional data for comparison with hypothetical values predicted by the G H K equation. It was anticipated that these results might be more difficult to interpret than those from the previously described experiments, involving reduced Na0, because unlike those experiments, the variation in RP was considerable. It was also uncertain to what extent the Na + conductance mechanism might be expected to exclude K +. In general, substituting K + for Na + caused RP to become more positive, as expected, and decreased the LED amplitude (Fig. 11). However, in many cases the actual LED potentials were significantly more negative (by as much as 7 mV) than predicted. Other alterations in the ionic composition of the medium did not affect the responses. Replacing all the sulfate by chloride did not alter either the LED or RP. Constant current injection Previous experiments involving the passage of constant current (Quandt and Gillary, 1979, 1980) indicated that: (1) the V-I curves of the cells studied here were linear in the dark and during the LED; (2) the LED amplitude tended to decrease during the passage of depolarizing current and increase during hyperpolarizing current; (3) the apparent reversal potentials during RE and D L were more negative than that during DE. The results of similar studies reported

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Fig. I I. Effects on the LED of substituting K ~ for Na ~. Upper trace: response in normal ASW (473mMNa +, 10mMK+; R P = - 7 2 m V ) . Lower trace: response of same cell in 315mMNa ~, 168mM K + (RP = -22mV). Response in 0mM Na +, 483mM K + (not shown; RP = +2mV) was further reduced but did not reverse polarity. here, generally confirmed those reported previously. Figure 12A shows typical responses. However, three out of 23 cells exhibited atypical responses illustrated in Fig. 12B. For such cells, a depolarizing current caused an increase in LED amplitude, especially during RE, whereas a hyperpolarizing current decreased the entire LED, especially at RE, which could become more negative than RP. Similar results have been described previously (Quandt, 1976). In previous experiments the voltage displacements evoked by brief repetitive pulses (ca 100 msec, 3/sec) of hyperpolarizing current ( - 1 hA) decreased during the LED, suggesting that a conductance increase occurred during the response (Quandt, 1976; Quandt and Gillary, 1980). Such experiments were repeated using shorter pulses (ca 30 msec) of higher frequency (10-12/sec) in order to improve the temporal resolution for measuring such variations in conductance, associated with different phases of the LED. These pulses were longer than the cell charging time (i.e. the latency to 85% maximum potential) of 0.4 + 0.1 msec (Quandt and Gillary, 1979). Two general patterns were found. In most cells (N = 11) the voltage displacements evoked by both depolarizing and hyperpolarizing pulses indicated a conductance increase, with two maxima, which o c -

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curred shortly after the peaks of DE and DL (Fig. 13A,B). In some cells (N = 6), however, the voltage displacements increased during the LED, indicating an apparent conductance decrease with a maximum decrease during RE; this was seen with either depolarizing (Fig. 13C) or hyperpolarizing pulses. The potential implications of these results and their compatibility with the atypical effects of constant current on the LED will be considered further in the Discussion.

DISCUSSION

General role of Na + influx in generation of L E D

Light-evoked sodium ion influx is primarily responsible for the LEDs in the photoreceptors of invertebrates other than Strombus, such as Limulus (Millecchia and Mauro, 1969a,b; Brown and Mote, 1974), barnacle (Brown et al., 1970), honeybee drone (Fulpius and Baumann, 1969) and Hermissenda (Alkon, 1979). The evidence thus far, indicates that this is also true for the "type II" cells in Strombus studied here. It was previously shown that the apparent reversal potentials for all phases of the LED were more positive than RP. That for DE was approx.

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Fig. 13. Effects of passing repetitive current pulses during the LED. Brief repetitive pulses (ca 30 msec) of constant current were passed through an intracellular single-channel microelectrode across a bridge circuit which was balanced prior to photic stimulation. (A) and (B): Responses from a typical cell during the passage of 1.5 nA of hyperpolarizing (A) or depolarizing (B) pulses. The bridge imbalance during the LED indicates apparent increases in conductance, with maxima occurring shortly after the respective peaks of the DE and D L phases (indicated by large arrowheads below traces). (C): Less typical responses of a different cell to depolarizing current pulses (+0,5 nA). For this cell, the bridge imbalance indicates an apparent conductance decrease during RE (large arrow). + 2 0 m V while that for De was approx. - 4 0 m V (Quandt and Gillary, 1980). Combined with the finding that cell conductance appears to increase during the LED, these results suggest that the LED is due to the influx of cations or the efflux of anions (or both), the equilibrium potentials of which are significantly more positive than R P (ca - 8 0 mV). Experiments reported here, in which sodium was replaced by sucrose, choline or TMA, all indicate that reducing Na0 causes a significant reduction in LED amplitude, approximately as a linear function of log Nao. An attempt was made to compare these reductions in LED amplitude caused by decreased Na o with values predicted by a version of the G H K equation, i.e. V = [ R T / F ] In [(Na0 + rKo)/(Nai + rKi)], where r = PK/PNa. (For the equation and definition of other symbols, see Junge, 1976.) The LED was assumed to arise only from changes in r, the relative permeability of the cell membrane to Na + and K +. Since the membrane behaves in the dark almost as a perfect potassium electrode, between 25 and 100 mM K0, it was assumed that in this range, R P depends solely on [K+]. Using the Nernst equation (Junge, 1976) and the experimental value of R P (ca - 33 mV) obtained when Ko was raised to 100 mM by substitution for Na0, K~ was calculated to be 340 raM. (This reduction of R P during increased K0 is apparently not caused by the decreased Nao, since lowering Na0 by concentrations such as 100raM does not affect R P when choline or TMA, rather than K +, are the Na + substitute.) Na~ was assigned a value of 50 mM, which is similar to those reported for other invertebrates, including squid (axoplasm) and barnacle (photoreceptors) (Rothschild and Barnes, 1953; Definer, 1961; Brown, 1976). However, the predicted results were quite insensitive to the accuracy of this choice; using a range of values for r (15-90), greater than those typically calculated in the present studies

(25-60), variations in Na~ between 0 and 200mM altered the predicted results by less than a millivolt. Values for r were calculated using (1) the membrane potentials at three points during the LED corresponding to DE, RE and DL, (2) the known values for Na0 and K0 and (3) the values for Nai and K~ and the G H K equation given above. For example, for the LED in Fig. 1A, the membrane potentials of Dr, RE and DL, which were - 6 6 . 5 , - 6 9 . 5 and - 6 8 . 5 mV respectively, yield calculated values for r of 31, 38 and 36. Assuming that the above values for r, K~ and Na~ did not change significantly when Na0 was lowered, the membrane potential at DE, RE and De in solutions of lowered Na0 could be calculated from the values of r at corresponding points of the LED in normal ASW. As evident from the data obtained from experiments in which choline or T M A were substituted for Na +, the actual values in potential during the LED were always more positive than those predicted (Figs• 5 and 6). Consistent with this pattern is the observation that reducing Na + to zero did not entirely abolish the response. There are several possible reasons for this. For example, as in other invertebrate photoreceptors, the cell membrane may permit the ions substituted for Na + to pass through it to some degree. Another possibility is that some residual Na + remains trapped outside the impaled cells by glial cells. These possibilities have been proposed for Limulus (Millecchia and Mauro, 1969a,b; Brown and Mote, 1974), barnacle (Brown et al., 1970) and honeybee drone (Fulpius and Baumann, 1969). In Fig. 5A, the predicted membrane potentials during the LED in 140 mM Nao were more negative than the R P in normal ASW. However, they were not more negative than the predicted R P in 140 mM Na0. Usually, the actual R P s in 140mM Na0 were not significantly different from the R P s in normal ASW. A limited permeability of the cell to the Na + substi-

242

KEVIN S. CHINN and HOWARD L. GILLARY

tute, or a value of Na0 at the membrane greater than that in the bathing medium could help explain why the actual RPs were more positive than predicted. In other systems, lowering Na 0 can inhibit sodium-~alcium pumps and exchange mechanisms (Blaustein, 1974; Erulkar and Fine, 1979) causing an increase in Ca,, which is thought to inhibit the LED (Lisman and Brown, 1972, 1975). It is therefore possible that increased Ca~ affected by reducing Na0 contributed to the reduction m LED amplitude in Strombus. The increase in LED amplitude above the control value, upon the return of the preparation to normal ASW following immersion in ASW lacking Na + (Fig. 2C), may also be related to N a + - C a 2÷ exchange. In Limulus ventral photoreceptors, lowering internal free Ca -~+ increases the LED amplitude (Lisman and Brown, 1975) and exposure to light increases internal free Ca 2+ (Brown et al., 1977). As indicated above, immersion of Strombus retinal cells in ASW lacking Na + may lead to reduced N a + - C a 2+ exchange and increased Ca,. Upon return to normal ASW, if the activity of a reactivated N a + - C a 2+ exchange mechanism initially exceeds control levels, this could lower internal free Ca 2+ below control levels and lead to enlarged LEDs. One might expect the timing of such a change back to normal ASW to affect the LED amplitude. Enlarged LEDs were seen when the return to normal ASW occurred 2 rain before the next flash, but not when it was 15 sec before. The 2 min interval would have allowed more time for the re-activated N a + - C a 2+ pump to lower internal free Ca 2~ before the next LED, when internal Ca 2+ would rise and reduce the amplitude of subsequent LEDs. The absence of a R E-associated notch in the enlarged LEDs (e.g. Fig. 2C2) may be due to a Ca 2+ dependence of outward K + current, normally associated with RE. The results from experiments, in which K + was substituted for Na +, can be interpreted in a way that is compatible with the view already mentioned, regarding Na + entry during the LED, despite the discrepancies between the actual values in potential and those predicted on the basis of the G H K equation (applied as previously described, regarding the experiments in which Na0 but not Ko was varied). For all such cells, the values were more negative than those predicted. A possible explanation is that the deviant LED values reflect a greater K + efflux than predicted, perhaps mediated by values of K0 near the impaled cell below those of the perfusion medium. Such reduction of Ko might involve glial cells, which have been implicated in regulating K 0 in the drone retina (Cole and Tsacopoulas, 1979). Contribution o f K + efftux Conductance increases to K + during the LED have been implicated in the photoreceptors of other invertebrates such as the honeybee drone (Fulpius and Baumann, 1969) and Sepiola (Duncan and Pynset, 1979). Such currents occurring late in the response (which in Strombus would be during RE and DL) have also been shown in barnacle (Hanani and Shaw, 1977) and Hermissenda (Detwiler, 1976). In Strombus, low doses of the K +-channel blockers, TEA and 4-AP, both caused an increase in the amplitude of the LED, especially during RE, implying that an

outward K+-current occurs during this phase. It should be noted that in Strombus, TEA (50-100 raM) and 4-AP (5 mM) sometimes reduced the LED amplitude, especially at the highest concentrations used. This could have been due to a nonspecific blockade of Na + currents by these compounds, as suggested for Hermissenda photoreceptors (Alkon, 1979). At high concentrations, both TEA and 4-AP caused fluctuations in R P (e.g. Figs 9D and 10D). One possible explanation is that the fluctuations reflect the electrotonic spread of axonal depolarizations in the impaled cell's proximity (e.g. Sperelakis et al., 1967). Another is that they are synaptically mediated. In fact, small fluctuations in RP, attributed to synaptic input, have been reported for Hermissenda photoreceptors (Alkon, 1976; Heldman et al., 1979). Furthermore, structures which are apparently synaptic occur in the neuropile of Strombus (Gillary and Gillary, 1979), and these could conceivably mediate similar RP fluctuations in the type I1 cells. If an outward K + current occurs during the lightevoked response, one might initially expect hyperpolarizing responses in preparations immersed in Na+-deficient ASW. However, that this was not seen need not be surprising. Whereas the respective RPs of barnacle photoreceptors and Limulus ventral photoreceptors are about - 4 0 mV (Brown et al., 1970) and - 6 0 mV (Millecchia and Mauro, 1969a), the RP in Strombus is considerably more negative ( - 80 mV in normal ASW, and - 7 0 to - 7 5 m V following exposure to ASW deficient in Na +) and relatively close to the K + equilibrium potential, as measured in other invertebrate photoreceptors such as the barnacle ( - 8 0 m V ; Hanani and Shaw, 1977). It is also possible that, as for the drone retina (Coles and Tsacopoulas, 1979), the [K +] surrounding the cell can exceed the [K +] of several millimoles in the bathing medium, which could make the K + equilibrium potential more positive than predicted and closer to RP. In view of this, one might expect an increase in K + conductance during the light-evoked response to cause only a slight or negligible increase in negativity, which might easily be offset by the influx of the Na substitute or residual Na + trapped outside the cell. Furthermore, such trapped Na + might also enter the cell through the K + channels themselves, as in Limulus where they are slightly permeable to Na ~ (Leonard and Lisman, 1981). Origin o f separable D e and Dt~ phases Two different types of light activated Na ~ currents have been implicated in the origin of LEDs in a number of invertebrate photoreceptors. Wulff and Mueller (1973) suggested that in Limulus lateral eye, different components of the Na+-dependent LED may originate in different cellular regions. Wulff et al. (1977, 1979) have been able to unmask, in that eye and in Limulus ventral photoreceptors, two distinct light initiated, Na+-dependent currents. Benolken and Russell (1967) showed that in Limulus lateral eye, the transient (which in terms of its time course seems to correspond in Strombus to DE) was reversibly abolished by TTX while the plateau (which in Strombus is similar to DL) was unaffected. Maaz et al. (1981) found in Limulus ventral photoreceptors,

Na + and K + in Strombus retinal depolarizations two types of light activated currents, differing only in their kinetics of activation and inactivation, and not in their ion specificity. They suggested that one set was mainly responsible for an early phase of depolarization (like De in Strombus) and the other set for a later depolarizing phase. In addition, it has been suggested that the photoreceptor potential in the crayfish, Astacus, is composed of two overlapping components (Stieve and Clal3en-Linke, 1980). Furthermore, LEDs in Aplysia photoreceptors can exhibit two phases of depolarization (Jacklet and Rolerson, 1982), and it has been proposed that there may be two different processes for the fast and slow components of the E R G in Sepiola, both of which involve Na + influx (Clark and Duncan, 1978). Previous studies on Strombus mentioned earlier (Quandt and Gillary, 1979, 1980), indicate that D E and DL arise from different processes. One difference is that D E exhibits an apparent reversal potential that is more positive than D E. Part of this difference could be due to a greater efflux of K + during D L, than during DE, as suggested by the present studies involving TEA and 4-AP. The present data and those from experiments on the effects of divalent cations on the LED, to be described in a later paper, indicate that Na + and K ÷ currents account for at least 85~ of the overall LED amplitude. One can attempt to account for the LED waveforrn in terms of a single, inward Na ÷ current of long duration upon which is superimposed an outward K + current, maximal during RE and capable of dividing the Na+-mediated depolarization into two apparent phases. However, it is difficult to explain the differential effects on the relative amplitudes of D E and DL of various experimental conditions, such as replacing Na ÷ by choline, TMA or Li ÷, or altering the pH. To account for these effects, one would have to postulate temporal shifts in the two hypothetical ionic currents. However, the failure of the latency to the peak of D E to change, during Li ÷ substitution for Na ÷, or certain conditions of altered pH, argues against this. The present results seem more compatible with the view that during generation of the LED in Strombus, light increases the Na ÷ permeability of two types of cationic channels. This would help explain the earlier data (Quandt and Gillary, 1980; see Introduction), the differential reduction in D z and DL, seen when Na 0 was replaced by TMA, choline, or Li ÷, and the differential effects on the two phases of altered pH. For example, certain types of Na + channels can be blocked by H ÷ (Hille, 1968; Woodhull, 1973). Perhaps in Strombus type II cells, Na ÷ channels underlying DE are less sensitive to such a blockade than another type underlying D c. Furthermore, in a number of preparations the membrane permeability to Li + is similar to that of Na + (Hille, 1970, 1972; Brown and Mote, 1974). However, in the crayfish stretch receptor, Li + permeability is apparently much less than Na ÷ permeability in its soma, although not its axon (Obara and Grundfest, 1968). This suggests that two types of " N a + channels" can coexist in the same cell. Perhaps in Strombus, the greater reduction of DE than DL, seen when Li + replaced Na +, is due to a lower permeability to Li + by different types of Na ÷ channels underlying these phases (Figs 3 and 7).

243

Effects on the LED on intercellular interactions and electrotonic decrement It is unlikely that the general LED waveform is determined by synaptic input. Experiments to be described in a separate paper, in which the effects of the Ca 2+ channel blockers, Mn 2+ and Cd 2+, as well as lowering Ca0, were examined, indicate that Ca 2+ current is not a major contributor to the waveform of the LED. Such manipulations would also be expected to block synaptic activity, which is dependent on presynaptic Ca 2+ current (Katz and Miledi, 1967, 1969; Hagiwara and Byerly, 1981; Llinas et al., 1981; Augustine and Eckert, 1982). It is also unlikely that voltage-dependent conductances seen at intercellular junctions in other systems (Spray et al., 1981) contribute to the LED waveform in Strombus (Quandt and Gillary, 1979). It is not known if the type II cells are photoreceptors or cells coupled to them, such as the eccentric cells in the lateral eye of Limulus (Hartline and Ratliff, 1972). If the latter is true, one would expect such coupling to be electrotonic since, as considered above, synaptic input makes little if any contribution to the LED waveform. Furthermore, the failure of these cells to exhibit voltage-sensitive conductances (Quandt and Gillary, 1979) makes it unlikely that such coupling involves a significant contribution from voltage-sensitive junctions. Consequently, even if the type II cells are not photoreceptors, the LEDs recorded most probably reflect very closely the events in photoreceptors to which they are coupled, permitting one to make inferences about these events on the basis of these LEDs. If the LED in the type II cells reflects potentials of cells to which they are coupled, electrotonic decrement could have caused the amplitude of the recorded light-evoked potential to be less than that at the site of the light-induced current. Such decrement would increase the values of r calculated from the G H K equation. However, it would not alter the conclusions regarding the relative changes in membrane permeability and conductance to Na +, K ÷ and the Na ÷ substitutes described in this paper (Quandt and Gillary, 1980). If the LED is generated by light evoked conductance changes in the type II cell itself, significant electrotonic decrement would not be expected (Quandt and Gillary, 1979). Current passing studies As discussed by Quandt and GiUary (1980), previous observations on the voltage displacements in the type II cells of Strombus, evoked by the passage of intracellular current during the LED, are compatible with the hypothesis that the LED arises from two separable conductance increases (during DE and DE, respectively) which are light-dependent but not voltage-dependent, since V-I curves in the dark are linear and independent of time. The present studies tended to confirm those previous findings; in most cells the voltage displacements, evoked by pulses of injected current (Fig. 13A,B), decreased during the LED. However, in contrast to such typical cells, approximately one-third showed an increase in the voltage displacement during R E (suggesting a conductance decrease), when either depolarizing or

244

KEVIN S. CHINN and HOWARD L. GILLARY

hyperpolarizing pulses of c o n s t a n t current were used (Fig. 13C). These data are compatible with those seen in certain experiments (Fig. 12B), in which the amplitude of RE increased during the passage o f c o n s t a n t depolarizing current (rather t h a n current pulses) and decreased with c o n s t a n t hyperpolarizing current. The above observations seem puzzling a n d suggest that such atypical cells may represent a distinct s u b p o p u l a t i o n of neurons identified by other criteria as type It. Perhaps the atypical type lI cells, but not the typical ones, exhibit an actual light-induced conductance decrease, such as that seen in Hermissenda (Alkon et al., 1982). It is also conceivable that in the atypical cells, light activates a voltage-dependent inward current, such as the calcium current in H e r missenda which requires b o t h light and depolarization for activation (Alkon, 1979). In such a case, a change in voltage evoked by current injection, that might be interpreted as a simple c o n d u c t a n c e decrease, could actually be due to voltage-sensitive inward current. (For example, the voltage deflection observed during a depolarizing current pulse, would be increased by the presence of inward ionic current triggered by the depolarization; and that during a hyperpolarizing pulse could be increased by the turning off of such an inward ionic current.) However, the data for type II cells in Strombus (which would be expected to include b o t h typical and atypical subtypes) do not s u p p o r t the presence of such a voltagedependent conductance. A tentative picture compatible with the data now available is t h a t in most type II cells (i.e. the "typical" cells), the L E D is generated primarily by three separable light-evoked c o n d u c t a n c e changes. Two are conductance increases to N a ~; one occurs during DE and the other during D~. In addition, a c o n d u c t a n c e increase to K ~ occurs during Re. The overlap of these three time-varying c o n d u c t a n c e changes could serve to explain, in part, such things as the time course of the a p p a r e n t conductance increases and their relation to the L E D waveform, as well as the data from the pharmacological and ion substitution studies. M a n y details await further clarification. Acknowledgements---The authors are grateful to J. Bacigalupo, [. M. Cooke, D. K. Hartline. J. E. Lisman and F. N, Quandt for comments on the manuscript. This study was supported by a research grant (to HLG) from the U.S. National Institutes of Health (EY01531). REFERENCES

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