analysis of repolarizatio ofn presynaptic motor …mechanism of larval presynaptic terminals, we...

J. exp. Biol. 170, 93-111 (1992) 9 3Printed in Great Britain © The Company of Biologists Limited 1992

ANALYSIS OF REPOLARIZATION OF PRESYNAPTIC MOTORTERMINALS IN DROSOPHILA LARVAE USING POTASSIUM-

CHANNEL-BLOCKING DRUGS AND MUTATIONS

BY MICHEL GHO* AND BARRY GANETZKYt

Laboratory of Genetics, 445 Henry Mall, University of Wisconsin-Madison,Madison WI53706, USA

Accepted 3 June 1992

Summary

In Drosophila melanogaster muscles and neuronal cell bodies at least fourdifferent potassium currents have been identified whose activity shapes theelectrical properties of these cells. Potassium currents also control repolarizationof presynaptic terminals and, therefore, exert a major effect on transmitter releaseand synaptic plasticity. However, because of the small size of presynapticterminals in Drosophila, it has not been possible to analyze the potassium currentsthey express. As a first approach to characterizing the ionic currents present atpresynaptic motor terminals of Drosophila larvae, we recorded synaptic currentsat the neuromuscular junction. From the alterations in evoked synaptic currentscaused by various drugs and by mutations known to affect potassium currents inother tissues, we suggest that the repolarizing mechanism in presynaptic terminalsconsists of at least four distinct currents. One is affected by aminopyridines or Shmutations, a second component is affected by the slo mutation, a third is sensitiveto quinidine and one or more additional components are blocked by tetraethyl-ammonium. Depolarization depends on a presynaptic calcium current, whichdisplays only slight voltage-dependent inactivation. Because the mechanism ofrepolarization exerts a major effect on synaptic activity, this analysis provides aframework for further genetic and molecular dissection of the basic processesinvolved in the regulation of transmitter release.

Introduction

The capability of combining genetic, electrophysiological and molecular tech-niques has made Drosophila melanogaster one of the best experimental systemsfor studying ion channels and membrane excitability. This multidisciplinaryapproach has permitted the electrophysiological and pharmacological characteriz-

* Present address: Laboratoire de Neurobiologie Cellulaire et Moleculaire, CNRS, 91190Gif-sur-Yvette, France.

tTo whom reprint requests should be addressed.

Key words: transmitter release, membrane repolarization, potassium currents, neurogenetics,Drosophila melanogaster.

94 M. G H O AND B. GANETZKY

ation of particular ion currents, the identification of the genes that specify thesecurrents, and molecular analysis of the encoded proteins (for reviews see Wu andGanetzky, 1988; Papazian etal. 1988). These currents, which determine theelectrical properties of excitable cells, have been studied in vitro and in vivo inmuscle cells, photoreceptor cells and neuronal cell bodies (Salkoff, 1985; Gho andMallart, 1986; Sole and Aldrich, 1988; Hardie, 1991). However, little is knownabout the currents present in other functional regions of neurons in Drosophila.For example, at the presynaptic terminal, the mechanisms underlying depolariz-ation and repolanzation are of fundamental importance in regulating transmitterrelease but little is known about the potassium currents that contribute torepolarization. Furthermore, little is known about the mechanisms underlying thesynaptic plasticity observed at the presynaptic terminal in Drosophila larvae(Zhong and Wu, 1991a). It is known that modification of potassium activity, viaseveral different second messenger systems, has important consequences forsynaptic modulation and plasticity (Kandel and Schwartz, 1982; Alkon, 1984).Direct study of the presynaptic terminals in Drosophila has been difficult becauseof their small size. Here, as a first approach to investigating the repolarizationmechanism of larval presynaptic terminals, we investigate the effects of drugs andmutations known to block potassium currents on the time course of transmitterrelease at the neuromuscular junction.

At least four distinct potassium currents have been described in Drosophilamuscles and neurons (Salkoff, 1985; Wu and Haugland, 1985; Gho and Mallart,1986; Wei and Salkoff, 1986; Sole and Aldrich, 1988; Saito and Wu, 1990). Theseinclude two fast, transient currents, IA and ICF. IA is voltage-dependent andsimilar to the molluscan A current (Connors and Stevens, 1971; Neher, 1971),whereas ICF is activated by calcium and is similar to other calcium-dependentcurrents previously described in muscles and neurons (Mounier and Vassort, 1975;Yamamoto and Washio, 1981; MacDermott and Weight, 1982). In addition, thereare two slow, sustained potassium currents, IK and Ics- IK corresponds to theclassical non-inactivating, voltage-dependent delayed rectifier first described inthe squid axon (Hodgkin and Huxley, 1952). I c s is a calcium-dependent currentsimilar to that originally described in molluscan neurons (Meech and Standen,1975). These currents are selectively affected by several drugs and mutations indifferent genes. For instance, aminopyridines as well as Sh mutations block IA inmuscles and some neurons (Salkoff and Wyman, 1981; Salkoff, 1983; Sole etal.1987; Baker and Salkoff, 1990; Saito and Wu, 1991). In other neurons, IA ismediated by a distinct type of channel sensitive to aminopyridines but not affectedby Sh mutations (Sole et al. 1987; Baker and Salkoff, 1990). ICF is eliminated by slomutations (Elkins etal. 1986; Singh and Wu, 1989; Komatsu etal. 1990) andquinidine selectively affects IK (Singh and Wu, 1989; Hardie, 1991). In addition,calcium blockers or low calcium concentration in the external solution affect thecalcium-activated currents IcF and ICs-

Previous studies have inferred the presence of potassium currents in thepresynaptic motor nerve terminal by the phenotypic effects of mutations on the

Presynaptic currents in Drosophila 95

postsynaptic potential, referred to as an excitatory junctional potential (EJP). Forexample, the presence of presynaptic potassium channels encoded by the Sh locushas been indicated by the enlarged prolonged EJPs observed in Sh mutants,consistent with a failure of normal repolarization of the presynaptic terminal (Janet al. 1977). Similarly, a potassium current specified by the slo locus, a structuralgene for calcium-activated potassium channels (Atkinson et al. 1991), may also bepresent at motor terminals because abnormally prolonged postsynaptic potentialsare also seen in slo mutants (B. Ganetzky, unpublished results). However,because slo is important for proper repolarization of muscles (Elkins andGanetzky, 1988; Singh and Wu, 1990), it is not clear whether the effect of slo onthe postsynaptic potential is pre- or postsynaptic. Thus, alteration of the EJP by aparticular mutation or drug does not always provide sufficient evidence that thepotassium channels affected by these agents are found in the presynapticmembrane and steps must be taken to eliminate the contribution of postsynapticconductances to the duration or time course of the synaptic current. We havecircumvented this problem in these experiments by recording the excitatoryjunctional current (EJC) while keeping the muscle held at a constant membranepotential under voltage-clamp conditions.

At the neuromuscular junction, transmitter release is triggered by an influx ofcalcium ions into the presynaptic terminal (Katz and Miledi, 1965, 1969; Dodgeand Rahamimoff, 1967; Llinds and Nicholson, 1975) and is not initiated until therepolarizing phase at the end of the presynaptic action potential (Llinas et al. 1981,1982). Prior to this, the presynaptic terminal is depolarized to a value near thecalcium equilibrium potential and there is no net calcium entry. The delay beforerelease of neurotransmitter and the resultant synaptic current in the postsynapticcell are therefore primarily dependent upon the kinetics of repolarization underthe control of potassium channels (Katz and Miledi, 1967a,b; Benoit andMambrini, 1970; Datyner and Gage, 1980; Mallart et al. 1991). By measuring theEJC in larval muscle cells, we were able to obtain quantitative measurements ofthe delay before transmitter release and the onset of this response. Thesemeasurements provide information about the time before onset of the repolarizingphase of the presynaptic action potential, which in turn depends upon activation ofpotassium currents. Using this experimental paradigm, we examined the effects onevoked synaptic currents of several different potassium-channel-blocking mu-tations and drugs. Our data suggest that at least four potassium currents contributeto the repolarization of motor nerve terminals. These results provide a foundationfor further genetic and molecular analyses of the molecular mechanisms involvedin the regulation of transmitter release in Drosophila.

Materials and methods

Animals

Experiments were performed on body-wall muscle 6 (for nomenclature seeJohansen et al. 1989) in the second and third abdominal segments of D. melano-

96 M. GHO AND B. GANETZKY

gaster third-instar larvae. The larval preparation was similar to that previouslydescribed by Jan and Jan (1976). Previous measurements have shown that thelarval muscles are essentially isopotential (Wu and Haugland, 1985). Wild-typeflies were of the Canton-S strain. The mutations used were ShKSJ33, slo' and eag1,which were all raised under standard laboratory conditions.

Voltage-clamp recording

Synaptic currents were recorded using a two-electrode voltage-clamp. By use ofthe voltage-clamp to measure the synaptic currents, we avoided the problem ofnon-linear summation normally observed when the synaptic potential is used as ameasure of synaptic activity (Martin, 1955; Augustine etal. 1985). Muscle fiberswere impaled with microelectrodes of about 5-10 MQ resistance filled withBmoi r 1 KC1. The current electrode was positioned at the center of the fiber.Unless otherwise indicated, the holding potential was — 60 mV.

Synaptic current records were filtered at 3 kHz, digitized and recorded on aVCR system. Off-line analyses were performed using pClamp software (AxonInstruments). The principal parameters determined were (1) the onset slope,measured as the slope of the line approximating the onset phase of the synapticcurrent at its midpoint and (2) the synaptic delay, measured as the intersectionbetween the baseline and a line representing the onset slope of the synaptic currentdrawn as described above.

Only those cells that gave reproducible responses in at least five successive trialswere used for data collection.

Nerve stimulation

The segmental nerves innervating the muscles were severed near the ventral1ganglion. To obtain electrotonic depolarization of the terminal,

tetrodotoxin (TTX) was added to the bathing solution to block action potentialsand the nerve was stimulated close to the terminal. Unless otherwise stated,electrotonic depolarizing current pulses were 50 or 200 ms in duration and threetimes the threshold for evoking synaptic current (about 4-5 V). Under theseconditions, the synaptic currents elicited always corresponded to the maximalresponse. Synaptic responses could not be evoked by current pulses of similarstrength but inverted polarity.

Solutions and drugs

The standard saline was (in mmolT1): NaCl, 128; KC1, 2; MgCl2, 4; CaCl2, 5.4;Hepes, 5; sucrose, 353; pH7.1. Temperature was maintained at 6°C using a Peltierdevice (Cambion). The high concentration of sucrose in this saline prevented themuscle contractions that otherwise occurred at the external calcium concen-trations used in these experiments. Hypertonic solutions have previously beenused with insect muscle preparations for this purpose (Yamamoto and Washio,1981; Ashcroft and Stanfield, 1982; Gho and Mallart, 1986). This procedure


allowed us to maintain stable cell recordings for at least lOmin without anyobserved changes in response.

The following drugs were used to block various ion channels: 3,4-diaminopyri-dine (DAP, Sigma); quinidine hydrochloride monohydrate (Sigma), tetraethyl-ammonium chloride (TEA+, Sigma) and tetrodotoxin (TTX, Sigma).

ResultsExperimental paradigm

Previous studies of Drosophila have shown that prolonged synaptic responsesare evoked in the presence of agents affecting potassium currents. These changescould result from effects on (1) the action potentials arriving at the terminal, (2)the postsynaptic membrane or (3) the presynaptic terminal itself. We usedexperimental conditions that enabled us to discriminate effects on the presynapticterminal from the other possibilities. To eliminate the possibility that effects onsynaptic response resulted from abnormal axonal action potentials arriving at thesynaptic terminal, we examined synaptic responses when action potentials wereblocked by the addition of 1/zmolP1 TTX. Under these conditions, transmitterrelease was evoked by direct electrotonic depolarization of the presynatic terminalby application of a sufficiently large current pulse via the suction electrode (Katzand Miledi, 1969; Jan et al. 1977). Electrotonic depolarization of the terminal wasachieved using stimuli of long duration (50-200 ms). The electrotonic nature of thedepolarization under these conditions has been shown by the occurrence of gradedresponses that increase progressively to the maximal response as the stimulusintensity is gradually increased (Wu et al. 1978). Such graded responses are neverobserved under conditions of active nerve conduction.

To prevent any possible contribution of the postsynaptic cell to the evokedsynaptic responses, the muscle cell was held at a constant voltage. Thus, anyvoltage-dependent conductances in the muscle activated during the synapticresponse would be eliminated. In addition there is the possibility that a musclecurrent could be activated by the influx of some ion carried by the synaptic current.In this case, changes in the synaptic response could result from the blockage ofthese ion-activated postsynaptic currents by mutations or drugs. If this wereoccurring, the synaptic current should also change in the absence of blockers whenthe holding potential was equal to the equilibrium potential of the ion carried bythis putative outward current. However, we observed that the shape of thesynaptic current was invariant across the entire range of holding potentials from— 100 to — 30mV (not shown). This eliminates the possibility that activation ofnon-voltage-dependent conductances in the muscle contributes to the synapticresponses recorded.

Because the onset of transmitter release occurs during the repolarizing phase ofthe nerve terminal, measurements of synaptic latency can be used to compare theeffects of different drugs and mutations on the repolarization of the nerve terminalfollowing electrotonic stimulation. Because current pulses of long duration


(50-200 ms) were used to depolarize the terminal electrotonically, if the terminalwere to repolarize with a normal time course, the resting potential should berestored prior to the end of the current pulse and synaptic current should beinitiated before the end of the stimulus. However, if repolarizing currents werereduced, the onset of the synaptic current should be delayed.

In these experiments, the intensity of the stimulus pulse was always about threetimes higher than the threshold required to elicit a synaptic response. Thisstimulus strength evoked the maximal response; further increases in stimulusstrength had no additional effect on the synaptic current.

Effects of drugs and mutations known to affect potassium currents on theelectrotonically evoked synaptic response

The synaptic current evoked by a current pulse of long duration in wild-typemuscle began 9 ms after the stimulus had been applied, peaked after about 20 msand was followed by a slow decay. The rising phase of the synaptic current (onsetslope) was about lOnAms"1 (Fig. 1). Neither the slo mutation, which eliminatesICF in muscle and neuronal cell bodies (Elkins etal. 1986; Singh and Wu, 1989;Komatsu et al. 1990; Saito and Wu, 1991), nor the application to wild-type muscleof DAP, which selectively eliminates IA in muscles and neuronal cell bodies(Salkoff and Wyman, 1981; Sole and Aldrich, 1988; Saito and Wu, 1991), had anysignificant effect on the synaptic delay or onset slope compared with wild-typemuscle (Figs IA, 3). However, when DAP (20^moll~l) was applied to slo larvae,the synaptic delay increased to 17 ms and the slope of the rising phase of thesynaptic current (onset slope) was reduced to 3nAms~\ Similar results wereobtained using a Sh mutation, which eliminates IA, at least in muscles (Salkoff andWyman, 1981; Wu and Haugland, 1985), instead of DAP (Figs IB, 3). No effectson synaptic delay were observed after application of DAP to Sh mutants (notshown), suggesting that in these experiments DAP is acting on the same set ofchannels altered by Sh mutations.

An off-response, recognized as a second surge of synaptic current at the end ofthe pulse, was clearly observed when the slo mutation was combined with theapplication of DAP or the presence of the Sh mutation (Fig. 1). This off-responsewas even more pronounced when additional potassium channels were blocked (cf.Figs 2 and 5). The fact that significant changes in synaptic latency and onset slopeof the synaptic current were observed only after the combined action of DAP (orSh) and slo indicates that the repolarizing current of the motor nerve terminalcontains at least two components: one is blocked by DAP or Sh and the other isblocked by slo. Apparently, under the experimental conditions used, either one ofthese currents alone is sufficient to repolarize the terminal.

The existence at the presynaptic terminal of additional repolarizing componentsother than those affected by DAP or slo was tested by examining whetherapplication of quinidine and TEA"1" had any additional effect on synaptic delaybeyond those caused by application of DAP to slo (Fig. 2). When quinidine(O.lmmoll"1), which selectively blocks IK in muscle and photoreceptors cells


slo+DAP

WT

80 nA

20 ms

Fig. 1. Effects of diaminopyridine (DAP), Sh and slo on the synaptic delay and theslope of the onset phase of electrotonically evoked synaptic currents. Each tracerepresents the synaptic current evoked from a different cell by a prolonged (50 ms)electrotonic stimulus. The individual traces have been superimposed to allow directcomparison of the effects of blocking different potassium currents singly and incombination. (A) The synaptic currents obtained from 5/0 mutant larvae in thepresence and absence of DAP are compared. (B) The synaptic currents obtained fromslo mutant and Sh;slo double mutant are compared. Note that in both cases asignificant increase in the delay before transmitter release and a decrease in the onsetslope occur only when both agents are used together (s/o+DAP trace in A and Sh;slotrace in B). In this and following figures, the duration of the long-lasting presynapticstimulation is indicated by a solid bar. The bathing solution contained l^molP 1

tetrodotoxin (TTX). WT, wild-type muscle.

(Singh and Wu, 1989; Hardie, 1991), was applied together with DAP to slo, thedelay before transmitter release was further increased to 39 ms (Figs 2, 3A) andthe slope of the onset phase was reduced further to OinAms" 1 (Figs 2, 3B).These results indicate that quinidine blocks an additional repolarizing componentin the terminal that is different from those affected by DAP and slo.

In the experiments with quinidine, there was considerable variability between


, i/o+DAP+Quin+Tea+

40 nA

60 ms

Fig. 2. Effects of quinidine and TEA"1" on the synaptic delay and the slope of the onsetphase of electrotonically evoked synaptic currents. Synaptic currents evoked byprolonged (200 ms) electrotonic stimuli from three different cells are superimposed forcomparison. All traces were obtained from slo larvae in the presence of DAP. Note theprogressive increase in synaptic delay and decrease in onset slope as additionalpotassium currents are blocked by addition of quinidine (Quin, O.lmmoll"1) orquinidine plus TEA"1" (20mmoll~'). In the latter case, the synaptic response wasobserved only at the very end of the pulse. The bathing solution contained 1

TTX.

larvae and also between segments within a single larva in the synaptic latency andthe onset slope of the synaptic current. In some cells, the effect of quinidine onsynaptic currents resembled the more extreme results obtained only after thefurther addition of TEA+ (see below). This variability was not the result of a slowaction of quinidine because the synaptic delay and the onset slope remained fairlyconstant during long (approximately 20min) recordings from the same cell (notshown). Instead, the variability may indicate that some class of TEA+-sensitivepotassium channels is absent or nonfunctional in a few terminals; depending onthe presence or absence of this class of channels in a particular terminal, theaddition of TEA+ would or would not be required to block repolarization fully(see below).

When TEA+, a more general blocker of potassium currents, was appliedtogether with quinidine and DAP to 5/0 mutants, the synaptic current was neverinitiated until the end of the current pulse. Thus, the synaptic current was alwaysobserved as an off-response (Fig. 2); the synaptic delay was the same as theduration of the stimulus and the onset slope was zero. This additional effect ofTEA+ on the synaptic current suggests that at least one other repolarizingpotassium current not affected by quinidine, DAP or slo is present at the terminal.The fact that the synaptic current was always observed at the end of the stimuluspulse in the presence of a generalized potassium channel blocker, such as TEA+,suggests that the intrinsic repolarization mechanisms of these terminals were


Fig. 3. Quantitative analysis of the effects of different potassium channel blockers onsynaptic currents. Synaptic delay (A) and the slope of the onset phase (B) weremeasured for a number of cells in experiments such as those shown in Figs 1 and 2.Each bar represents the mean of data pooled from the number of cells indicated inparentheses above each bar. The standard deviation for each set of measurements isalso shown. The data are grouped from left to right according to the progressive blockof additional potassium currents. The mean values of synaptic delay and onset slope forthe cells in each experimental set were compared with those in every other set usingStudent's Mest. According to this analysis the data fell into four discrete groups.Within a group none of the values was significantly different from any other (P>0.05),whereas significant differences were found (P<0.01) for all possible between-groupcomparisons. The groups so defined were (1) wild type, DAP, Sh and slo; (2)ito+DAP and Sh;slo; (3) s/o+DAP, Quin; and (4) sto+DAP, Quin, TEA"1". Note thatas additional currents were blocked there was a stepwise increase in the synaptic delayand a corresponding decrease in the onset slope.

virtually eliminated under these conditions. Thus, it is unlikely that any other non-potassium current, such as a chloride current, participates significantly inrepolarization of the presynaptic motor terminal.

Because the eag mutation has previously been shown to reduce the sustainedpotassium currents in muscle (Wu etal. 1983; Zhong and Wu, 1991b), we testedwhether this mutation also had effects on the synaptic terminal. Synaptic delay was


not further increased by the combination of eag with slo plus DAP (not shown).Furthermore, synaptic delay was increased by the application of quinidine orTEA+ to DAP-treated eag;slo larvae (not shown), suggesting that eag did notsubstantially diminish the quinidine- or TEA+-sensitive currents of these ter-minals. Because the effect of eag on muscle is more extreme at 20°C than at 6°C(C.-F. Wu, personal communication), we repeated these experiments at a highertemperature (21 °C), but we still failed to observe an effect of eag on the synapticcurrent.

Calcium action potentials at the presynaptic terminal

As shown in Fig. 4A, when presynaptic terminals were stimulated by shortelectrotonic stimuli, varying the stimulus strength over a narrow range couldproduce substantial differences in the synaptic current. The relationship betweensynaptic currents and stimulus intensity is plotted in Fig. 4B for slo larvae treatedwith DAP. A sudden jump in the amplitude of the synaptic current was observedwhen the stimulus was increased above a certain threshold. A similar result wasobserved in wild-type larvae (not shown). Stimuli lower than this threshold couldalso evoke these explosive synaptic responses if the pulse duration was increased.Subthreshold graded release, sometimes followed by a full-blown response(Fig. 4A), was observed only in the Sh;slo double mutant or after application ofDAP to slo larvae, suggesting that, in the wild type, potassium currents preventthe presynaptic terminal from depolarizing gradually.

This explosive synaptic current is probably triggered by an all-or-nothing

B Stimulus strength (V)

2 3A

40 nA20 mV

--0.2

o

--1.0

Fig. 4. Explosive synaptic release evoked by subthreshold electrotonic stimuli.(A) Two successive traces evoked from the same cell by electrotonic stimuli ofidentical magnitude (1.5 V and lms) are superimposed. In one trace, the stimulusevoked a small subthreshold response; in the second trace this response was followedby an all-or-nothing synaptic response. (B) The amplitude of the synaptic currentplotted against the stimulus voltage. Note the sudden jump in amplitude of the synapticcurrent when the stimulus strength increases above 2.7 V. The traces were obtainedfrom slo larvae in the presence of DAP. The bathing solution contained l/«noll~'TTX. A and B are from different cells.


40 nA

^ 800 ms

Fig. 5. Absence of voltage-dependent inactivation of presynaptic Ca2

Synaptic currents evoked from the same cell by electrotonic stimuli of differentduration are shown superimposed. In one set of two traces the stimulus duration was200 ms. In the second set of two traces the stimulus duration was 3 s. In both cases thesynaptic currents were always observed as off-responses. Note, however, that there isvery little reduction in amplitude of the synaptic current even after the presynapticterminal has been depolarized electrotonically for 3 s. The traces were obtained from5/0 larvae in the presence of DAP, quinidine and TEA+. The bathing solutioncontained l^moll" ' TTX.

regenerative potential at the presynaptic terminal. Because TTX was present inthe bath, this presynaptic action potential was probably sustained by the activationof an inward calcium current. The observation that graded responses could beevoked by lower-strength stimulation indicates that the depolarization allows theinternal calcium concentration to increase sufficiently to trigger synaptic release,even before the threshold for triggering the presynaptic action potential isreached.

The voltage-dependent inactivation of the presynaptic inward current could beexamined when the normal repolarization mechanism of the terminal wascompletely blocked by application of DAP, quinidine and TEA+ to 5/0 larvae.Terminals were depolarized with strong electrotonic stimuli (three times abovethreshold) for 0.2-3 s. Under these conditions, the synaptic response was alwaysobserved at the end of the pulse as an off-response (15 of 15 muscle fibers studied).Superimposed traces of off-responses obtained with 200 ms and 3 s stimulus pulsesare shown in Fig. 5. In this particular example, the off-response after a 3 s stimuluswas not diminished in amplitude compared with the off-response after a 200 msstimulus. In all the other cases examined, the synaptic currents evoked by the 3 sstimulus were never reduced by more than 30 % compared with those elicited bythe 200 ms stimulus.

Discussion

The results presented here indicate that mutations and drugs, which have knowneffects on potassium currents in muscles and neuronal cell bodies of Drosophila,also affect repolarization of presynaptic motor terminals. The failure of theterminals to repolarize properly was observed as an increase in the delay and a


decrease in the onset slope of the evoked synaptic current recorded from themuscle.

We believe that under our experimental conditions the effects of the drugs andmutations on synaptic release result from an action on the terminal itself for thefollowing reasons. (1) Any possible contribution of voltage-dependent musclecurrents to the synaptic response recorded was eliminated by holding themembrane potential of the muscle constant. (2) The effects were observed evenwhen the muscle was clamped at different holding potentials, suggesting thatcontributions to the synaptic response from any non-voltage-dependent post-synaptic current are also unlikely. (3) The presynaptic terminal was depolarizeddirectly by electrotonic stimulation using suprathreshold stimulation in thepresence of 11X to block axonal conduction. Because, under these conditions, thesynaptic currents evoked represent the maximal response, the possibility thatchanges in the passive properties of the axonal membrane contribute to themodification of the synaptic responses is unlikely. (4) To our knowledge, therehave been no reports demonstrating any direct actions of these drugs andmutations on the mechanism of neurotransmitter release itself or on the propertiesof postsynaptic receptors (Augustine, 1990). Thus, the most likely explanation ofour results is a selective effect on potassium currents in the presynaptic terminal.

Among the useful parameters that could be reliably quantified in theseexperiments were the synaptic latency and the slope of the onset phase of thesynaptic current. Each of these varied in stepwise fashion when the drugs andmutations were added progressively (Fig. 3). The synaptic latency dependsdirectly upon the time course of repolarization of the presynaptic terminal (Katzand Miledi, 1967a,b; Benoit and Mambrini, 1970; Augustine, 1990; Mallart etal.1991). Thus, it is expected that the synaptic latency will increase progressively asdifferent components of the repolarizing current at the terminal are blocked oreliminated.

The decreasing slope of the onset phase of synaptic current observed as thevarious potassium currents were progressively blocked is more difficult tointerpret. One possibility is that the slope is correlated with the rate of the fallingphase of the presynaptic action potential. When the terminal is electrotonicallydepolarized by long-duration current injection, repolarization of the presynapticterminal will be slower when some of the repolarizing currents are blocked. Since,in our conditions, calcium channels show only little, if any, voltage inactivationduring this time (see below), the entry of calcium into the terminal probablyfollows the driving force determined by the membrane potential. Partial blockageof potassium currents at the terminal could thus slow the falling phase of the actionpotential, leading in turn to a low rate of calcium entry and slow release ofneurotransmitter.

In muscle fibers of Drosophila larvae, morphological studies have shown twoclasses of synaptic boutons (Johansen et al. 1989) and electrophysiological studieshave shown two components of the evoked EJP (Jan and Jan, 1976) suggestingthat the fibers are innervated by at least two different axons. Similar polyinnerva-


tion of larval muscle has been found in other dipterans (Hardie, 1976). Thus, onecould envisage that the various repolarizing currents described in the present studycould be differentially distributed among different terminals. Focal studies ofsynaptic terminals will be necessary to examine this possibility.

Because of the small size of the terminals in Drosophila, direct study of ioniccurrents responsible for repolarization of the presynaptic terminal has not yet beenpossible. However, our results provide some insight about the number andproperties of the potassium currents present at the terminal. Repolarization of thepresynaptic terminal and release of neurotransmitter are known to depend onpotassium currents (Katz and Miledi, 1967a,b; Benoit and Mambrini, 1970;Datyner and Gage, 1980; Augustine, 1990; Jackson et al. 1991; Mallart et al. 1991).Therefore, if the drugs or mutation used alter the time course of synaptic release,we can infer the presence in the presynaptic terminal of potassium currentsaffected by these agents. The observation that the synaptic response changed in astepwise fashion when the different drugs or mutations were introduced one at atime suggests that at least four distinct components contribute to the repolariz-ation of presynaptic motor terminals of Drosophila larvae. One component isaffected by Sh or DAP. The observation that DAP and Sh produce identicaleffects on synaptic release indicates that DAP is not affecting any class ofpotassium channels at the presynaptic terminal not affected by Sh and vice versa.A second component is affected by mutations of slo, which is a structural gene fora subunit of calcium-activated potassium channels present in muscle and neuronalcell bodies (Atkinson et al. 1991), and a third is affected by quinidine. Theadditional effects of TEA+ beyond those caused by these agents suggest thepresence of at least one other TEA+-sensitive component. An alternativepossibility is that the effects of TEA+ are caused by a more complete blockage ofthe same channels that were only partially blocked by DAP or quinidine.However, we think this possibility is less likely because in other Drosophilaneurons IA and IK are blocked completely by the concentrations of DAP andquinidine used here. Although direct recording from synaptic terminals will benecessary to match with certainty a particular current with each of thesecomponents and to characterize their properties in detail, it is worth noting thatthe genetic and pharmacological sensitivities of the repolarizing currents in theterminal parallel those of the potassium currents identified by voltage-clampanalysis of larval and adult muscle. Thus, the effects of Sh and DAP onrepolarization of the terminal suggest the presence in the terminal of a current thatmay resemble IA; the effects of slo suggest the presence of an inactivating calcium-dependent potassium current (ICF-like). Since quinidine selectively blocks IK inboth muscle and photoreceptor cells at the concentration used here (Singh andWu, 1989; Hardie, 1991), the effect of quinidine on synaptic release suggests thepresence of a delayed rectifier current (IK-like). We cannot rule out the possibilitythat quinidine has some additional effect on the presynaptic terminal that alterssynaptic release by a mechanism not involving potassium channels. Nonetheless,this possibility is unlikely to be a significant factor in our experiments because the

106 M. G H O AND B. GANETZKY

application of quinidine alone in the absence of other potassium channel blockersdid not have any observed effect on synaptic release (Wu and Ganetzky, 1988; M.Gho and B. Ganetzky, unpublished observations) and at the concentrations usedhere no effects of quinidine in Drosophila other than the selective blockage of IKhave been reported (Singh and Wu, 1989; Hardie, 1991). The additional action ofTEA+ indicates the presence of at least one other potassium current, perhaps thenon-inactivating calcium-dependent current (Ics-Hke).

Despite the possible similarity between potassium currents in muscles andpresynaptic terminals suggested above, it is still necessary to interpret our resultswith caution because our analysis is indirect. For example, drugs such as DAPcould have different pharmacological effects in muscles and motor terminals.Similarly, although mutations such as Sh and slo may affect potassium channels inboth synaptic terminals and muscle, the channels in each location could representalternative gene products encoded by the same gene with distinct functionalproperties. In addition, a differential distribution of some subunits encoded byother genes could lead to the assembly of potassium channels with differentproperties in the two regions. Thus, the biophysical properties of the potassiumchannels and their molecular composition may not be identical in muscle andpresynaptic terminals. In view of such considerations and the variety of potassiumchannel genes identified in Drosophila (Butler et al. 1989), it is somewhatsurprising that the genetic and pharmacological effects on potassium currents inmuscles and presynaptic terminals are as comparable as they appear from thisanalysis. Ultimately, more detailed biophysical and molecular studies of all thepotassium channel subunits present in muscle and presynaptic terminals will benecessary for a definitive comparison to be made.

In contrast to the effects of Sh and slo, which alter repolarization of both muscleand presynaptic terminals, eag did not appear to have any pronounced effect onrepolarization of the motor terminal even though muscle potassium currents arealtered in eag mutants (Wu et al. 1983; Zhong and Wu, 1991ft) and previousevidence suggests that eag affects the motor terminal (Ganetzky and Wu, 1982,1983). Furthermore, synaptic transmission in eag;Sh double mutants is greatlyenhanced beyond that of Sh mutants alone. The synergistic interaction betweenthese two mutations has been interpreted as the result of a combined deficit inrepolarization of the synaptic terminal affecting potassium currents other than IA

(Ganetzky and Wu, 1982). Consistent with this interpretation is the observation ofvery large prolonged EJPs, resembling those in eag;Sh double mutants, afterapplication of quinidine to Sh larvae or larvae treated with 4-aminopyridine (Wuet al. 1989). These data suggest that, as in muscles, a current that is affected eitherby eag or by quinidine is present in the presynaptic motor terminal. We are as yetunable to reconcile this conclusion with our failure to observe effects of eag onrepolarization of the presynaptic terminal in these experiments. However, all ofthe earlier EJP analysis was performed in a low-calcium external solution, wherethe efficacy of calcium-dependent potassium currents in repolarizing the presynap-tic terminal is minimal. The lack of effect of eag on synaptic release under our


conditions could be because a partial reduction in one or more potassium currentsis not sufficient to change the rate of repolarization of the synaptic terminal if someother potassium current similar to IQS remains fully functional. It will be necessaryto await procedures to block selectively any remaining potassium currents to testthis possibility.

In addition to the inferences about potassium currents, these experimentsenabled us to derive some new insights about the presynaptic inward current. Theexplosive synaptic responses observed are probably due to the activation of acalcium current at the presynaptic terminal. The surge of synaptic current at theend of the stimulus (off-response) is probably the result of a presynaptic calciumtail current that occurs at the end of the stimulus (Katz and Miledi, \961b). Whenall of the presynaptic potassium currents were blocked with DAP, quinidine,TEA+ and slo, the synaptic current was always observed as an off-response at theend of the electrotonically applied current pulse. Under these conditions, theelectrotonic stimulus is apparently sufficient to depolarize the presynaptic terminalto near the calcium equilibrium potential such that, during the pulse, there is nocalcium entry and transmitter release is prevented. However, at the end of thestimulus pulse, the calcium driving force again favors calcium entry and thesynaptic current is immediately restored (Katz and Miledi, 1971). This off-response was observed even when the terminal was fully depolarized for as long as3 s by stimulus pulses of long duration. This result suggests that voltage-dependentinactivation of the presynaptic calcium current during prolonged depolarization ofthe motor terminal is only slight, if it occurs at all. Barium currents recorded fromcultured embryonic neurons of Drosophila have indicated the presence in this cellpopulation of inactivating as well as non-inactivating calcium channels (Leung andByerly, 1991), although barium itself can exert pronounced effects on theinactivation of calcium channels. Calcium currents not displaying voltage-depen-dent inactivation have been described in other excitable cells (Katz and Miledi,1971; Keynes etal. 1973; Llinds etal. 1976) including insect muscle fibers (Ashcroftand Stanfield, 1982). We cannot rule out the possibility that the off-response resultsfrom calcium channels in larval motor terminals undergoing an obligatory transitionthrough an open state during the recovery process after inactivation, as has beendescribed for calcium channels in mouse cerebellar neurons (Slesinger and Lansman,1991) and IA channels (Demo and Yellen, 1991). Nevertheless, the observation thatthe probability of calcium channels reopening during the transition from inactivationto rest states increases with long periods of inactivation (Slesinger and Lansman,1991) argues against this explanation of our results, because we never observed anincrease (and in some cases even observed a decrease) in the off-response when thestimulus duration was increased from 10 ms to 3 s.

The presence of several potassium currents in motor terminals of Drosophila isreminiscent of observations on synaptic terminals in other organisms. Forexample, it has been shown that transient and sustained voltage-dependent orcalcium-dependent potassium currents are present in motor terminals of frog(Mallart, 1984), lizard (Benoit et al. 1989; Lindgren and Moore, 1989; Morita and


Barrett, 1990) and mouse (Tabti etal. 1989). The diversity of potassium currentshas been interpreted as a general feature of presynaptic terminals. This diversityattests to the existence of an elaborate mechanism for repolarization which, inturn, controls the amount and time course of transmitter release. In Drosophila,regulatory mechanisms that modulate the strength of one or more of thesecurrents could underlie the plasticity that has been observed at this synapse (Sternand Ganetzky, 1989; Zhong and Wu, 1991a). The results of this study provide thebasis for further elucidation of these regulatory mechanisms as well as for theanalysis of other mutations whose effects on synaptic transmission are not yetunderstood.

We are grateful to G. Robertson, K. A. Schlimgen, M. Stern and C.-F. Wu forhelpful comments on the manuscript. This research was supported by grants fromthe National Institutes of Health (NS 15390) and the Markey Charitable Trust andfellowships from the Klingenstein and McKnight Foundations. M.G. was alsopartially supported by the Fondation pour la Recherche M6dicale. This is papernumber 3189 from the Laboratory of Genetics.

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