215

J. Phytiol. (1976), 262, pp. 215-236 With 7 text-figures Printed in Great Britain

L-GLUTAMATE AS AN EXCITATORY TRANSMITTER AT THE DROSOPHILA LARVAL NEUROMUSCULAR JUNCTION

BY L. Y. JAN AD Y. N. JAN From the Division of Biology, California Institute of Technology Pasadena, California 91125, U.S.A. (Received 4 May 1976) SUMMARY

1. The possibility that L-glutamate is the excitatory transmitter at the Drosophila larval neuromuscular junction and the ionic basis of its action on the muscle membrane are examined. 2. Iontophoretically applied L-glutamate causes muscle depolarization (L-glutamate potential) if and only if the L-glutamate pipette is within a few ,tm of the nerve ending. D-glutamate, substance P, ACh and GABA are ineffective. 3. Bath-applied L-glutamate produces similar changes in the time course and amplitude of miniature excitatory junctional potential (m.e.j.p.), excitatory junctional potential (e.j.p.) and the L-glutamate potential. 4. Neuromuscular transmission and excitation-contraction coupling are operative in a haemolymph-like solution containing 1 mM L-glutamate. 5. The reversal potentials of the e.j.p. and the L-glutamate potential are identical to each other, changing similarly with changes in the ionic compositions of the external medium (twelve solutions). 6. The ionic dependence of the reversal potentials is predicted from an extended constant-field equation using a ratio of sodium :potassium permeabilities of PN&/PK = 1-3, and a ratio of magnesium: potassium permeabilities of PMgIPK = 4-7. 7. It is concluded that L-glutamate is, or is an agonist of, the excitatory transmitter at certain Drosophila larval neuromuscular junctions. INTRODUCTION

At many crustacean and insect neuromuscular junctions, L-glutamate is the most likely candidate for the excitatory transmitter (Robbins, 1959; Takeuchi & Takeuchi, 1964; Usherwood, 1972; Taraskevich, 1975), however, the evidence is considerably weaker than that for acetylcholine

L. Y. JAN AND Y. N. JAN (ACh) at vertebrate neuromuscular junctions (for review, see Usherwood & Cull-Candy, 1975). If L-glutamate is the excitatory transmitter at the neuromuscular junction, it must have the same effect upon the postsynaptic membrane as the true transmitter released from the presynaptic terminals. Because of technical difficulties, a detailed comparison of the properties of the excitatory junctional potentials (e.j.p.s) and the Lglutamate induced post-synaptic response (L-glutamate potential) has been lacking. Only recently has a close agreement between the reversal potentials of the e.j.p.s and the L-glutamate potentials been demonstrated in some cases (Anwyl & Usherwood, 1974a, b; Taraskevich, 1975; Onodera & Takeuchi, 1975; Dudel, 1974). In the present paper, we used a Drosophila nerve-muscle preparation (Jan & Jan, 1976) with several favourable features: the nerve terminals can be viewed with Nomarski optics, so that precise physiological localization of nerve terminals was possible. The muscles are not electrically excitable and can be depolarized readily, so that the reversal potential of the e.j.p.s and that of the L-glutamate potential can be compared directly. In the first part of this paper, it is shown that L-glutamate and the true transmitter have similar effects at the Drosophila larval neuromuscular junction. The reversal potentials for the e.j.p. and the L-glutamate potential were the same, varying similarly with changes of the ionic compositions of the external medium. The second part of the paper reports that, at this neuromuscular junction, L-glutamate increases the membrane permeability for sodium, potassium and magnesium ions, and that the reversal potentials for the e.j.p. and the L-glutamate potential in different solutions can be predicted with an extended constant-field 216

equation. METHODS Experiments were done on the ventral lateral longitudinal fibre first and second from the ventral mid line, in the 3rd to the 5th abdominal segments of late 3rd instar larvae and early pupae. This preparation, the standard saline solution, the recording arrangement and the procedure for nerve stimulation are described in the preceding paper of this issue (Jan & Jan, 1976). Standard deviations are given for all measurements. A 1 M solution of L-glutamate, pH 8.0, was prepared by adding sodium hydroxide to L-glutamic acid. The L-glutamate was injected iontophoretically by applying a negative pulse to the micropipette. Diffusion of glutamate from the micropipette was stopped by applying a braking current when necessary. D-glutamic acid, y-aminobutyric acid (GABA), serotonin and octopamine are from Calbiochem, La Jolla, Ca. L-Glutamate, L-aspartate, L-asparagine, L-glutamine, glycine, L-glutamate-y-methylester and L-glutamate-diethylester are from Sigma, St Louis, Mo. Substance P is a generous gift from Dr Roger Guilleman from the Salk Institute.

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 217 PART I. ACTION OF L-GLUTAMATE RESULTS

Localizaton of L-glutamate-sensitive areas on muscle surface When the preparation is viewed with Nomarski optics, a nerve branch can be seen to terminate on the muscle surface in a compact cluster of varicosities or boutons (Jan & Jan, 1976), similar in appearance to junctions in snake twitch muscle (Kuffler & Yoshikami, 1975). Electron microscopy shows synaptic structures in these regions (Jan & Jan, 1976). Upon nerve stimulation, synaptic current can be detected by extracellular recording only when the recording electrode is near a bouton. Moreover, in calcium-free solution, transmitter release could be elicited only if a calcium iontophoresis electrode was near the boutons (L. Y. Jan, Y. N. Jan and M. J. Dennis, in preparation). Thus the boutons are the sites of transmitter release. Using Nomarski optics, the distribution of L-glutamate sensitivity was mapped on the surface of the muscle fibre with an accuracy of a few jtm. The L-glutamate micropipette was moved along the entire muscle surface in 5-10 jtm steps. At each position, the tip of the micropipette was moved to within a few ,tm of the muscle surface and a 2 msec negative pulse of 50 nA was passed through the micropipette. A response was observed if and only if the electrode was within about 10 Itm of the bouton. When the micropipette was moved further than 10 ,m from the bouton, no response could be elicited even with strong (100 nA) and long lasting (10 msec) pulses. Thus, the only L-glutamate sensitive region appears to be in the immediate vicinity of the transmitter release sites (i.e. the boutons). With the micropipette tip next to the bouton, the chemosensitivities obtained by measuring the slope of the linear portion of the dose-response curves were typically 100 mV/nC, occasionally, values as high as 200 mV/nC were seen. For the ventral lateral longitudinal fibres first and second from the ventral mid line, the bouton clusters are consistently near the 'bridge' structure halfway along the fibres (see P1. 2, Jan & Jan, 1976). Therefore in these fibres the glutamate sensitive area can be located easily under a dissecting microscope. Most of the experiments described below were done in this fashion. The L-glutamate potential The amplitude and time course of the L-glutamate potential depended critically on the distance between the micropipette tip and the muscle surface. As shown in Fig. 1A, a perpendicular movement of 10 Fm away

L. Y. JAN AND Y. N. JAN from the muscle fibre caused the amplitude of the L-glutamate potential to decline to a third of the original value and the time to peak to lengthen 1P5-fold. In Fig. 1 B the position of the micropipette was fixed and the pulse intensity was varied. With increasing iontophoretic current, the amplitude of the L-glutamate potentials increased in a non-linear way, while the time course of the rising phase changed very little. Since the fibres are short and essentially isopotential, the time course is probably determined mainly 218

A

1'

02 juA

100 msec

B~~~~~~

i

MV

0.54uA|

Fig. 1. A, L-glutamate potential obtained with fixed pulse intensity and varying distance of iontophoretic electrode from muscle surface. Traces read successively from below correspond to decreasing electrode distance. B, L-glutamate potential obtained with fixed distance and varying pulse intensity. Traces read successively from below correspond to increasing

pulse intensity.

by the diffusion time of L-glutamate from the micropipette to the sensitive area on the muscle fibre and then into the bath. The concentration of L-glutamate near the receptor site can be approximated using the theory of del Castillo & Katz (1955), in which the following assumptions were made: (1) the iontophoresis pipette may be considered as an instan-

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 219 taneous point source from which a quantity of transmitter substance is suddenly released into a large volume; and (2) the receptors may be regarded as situated at a point at a distance from the source. Given these assumptions, the diffusion equation can be solved and the peak concentration of L-glutamate is [L-Glu] = Q exp (-1.5)/8(7TDT)1-5, (1) where Q is the amount of substance applied instantaneously, D is the diffusion constant, and T is the time to peak potential, which increases as the distance between the micropipette and the receptors is increased. In the case of Fig. 1B, the first trace from below shows that a current pulse carrying 0 94 x 10-9 C produced almost no depolarization. However, increasing the amount of charge passed through the pipette to 1P29 x 10-9 C produced a depolarization of 3.7 mV (see second trace from bottom in Fig. 1B). Therefore this amount of change was a close estimate of the threshold for eliciting a detectable L-glutamate response. If the transfer number of L-glutamate is assumed to be 0 5 (the resistance of L-glutamate pipette was about 20 MQ and no backing current was used) and the diffusion constant is taken as 7-62 x 10-6 cm2/sec (Takeuchi & Takeuchi, 1964), the peak concentration of L-glutamate is calculated by formula (1) to be 0 05 mM. This value is within the same order as the lowest L-glutamate concentrationinthe bath that affects the size and shape ofthe e.j .p.s (seebelow). The depolarization seen during L-glutamate iontophoresis was due to L-glutamate, not the electric current itself. In experiments described later which test for ACh and GABA sensitivity of the neuromuscular junction, double-barrelled electrodes were used, one barrel filled with L-glutamate, the other barrel with GABA or ACh. At locations where inward current from the glutamate pipette caused depolarization, no membrane potential changes were caused by inward and outward current through the nonglutamate barrel, or by outward current from the glutamate pipette. The action of L-glutamate seems to be post-synaptic for the following reasons: (1) release of transmitter from nerve terminals in this preparation is quantal, but L-glutamate iontophoresis causes a continuously graded response; (2) the L-glutamate potential in low-calcium solution (0 1 mM) or calcium-free solution is similar to that in normal saline, in spite of the fact that neurally evoked transmitter release is blocked under these conditions. Therefore it is unlikely that L-glutamate is stimulating presynaptic transmitter release. Desensitization If two pulses of iontophoretically applied L-glutamate are given within a short time, the L-glutamate response produced by the second pulse is

L. Y. JAN AND Y. N. JAN 220 smaller than the first. This 'desensitization' phenomenon has been observed in crayfish muscles (Takeuchi & Takeuchi, 1964; Dudel, 1975). Fig. 2A shows desensitization in Drosophila larval muscle. The ratio (R) of the amplitudes of test (2nd) to conditioning (1st) responses is about 0 5 when the interval between the two is 0*2 sec, and increases to 1-0 when the interval is longer than 2 sec (Fig. 2B). A

20 MV

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.~~~~~~~~~~~~~~~~~

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100 1

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-

601-

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0*5

Interaction between L-glutamate and e.j.p.s It was found by UJsherwood & Machili (1968) that L-glutamate is a potent excitatory substance in causing locust muscle contraction. When applied to their preparation in concentrations higher than 106 M, this substance evoked a single phasic contraction followed by a decline in

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 221 neurally evoked contractions. Maximal contractions were obtained at about 104 M. Above 5 x 104 M, the neurally evoked responses were completely abolished. Insect haemolymph usually contains high concentrations of amino acids, including L-glutamate at levels which might result in complete desensitization of the muscle to the L-glutamate. This could be a serious objection to L-glutamate being the excitatory neurotransmitter. In the [L-glU]

e.j.p.

m.e.j.p.

(mm) 0

0.1

05

I I

1.0

10

mV

4 mV 40 msec

0 4 sec

Fig. 3. E.j.p.s and m.e.j.p.s in solutions with different L-glutamate concentrations.

locust, Usherwood & Machili (1968) found that direct application of fresh haemolymph had no depolarizing effect unless exogenous L-glutamate was added to it. Their interpretation was that in locust haemolymph there was actually very little 'free' L-glutamate. Drosophila haemolymph contains a high concentration of L-glutamate

L. Y. JAN AND Y. N. JAN (0.9 mM) (Chen, Kubli & Hanimann, 1968), s0 if L-glutamate is the excitatory neurotransmitter in Drosophila, the same problem arises. The interaction between L-glutamate and e.j.p.s was therefore studied by adding L-glutamate to the bath in various concentrations, and recording both the spontaneous m.e.j.p.s and the evoked e.j.p.s. Since the electrical properties of the muscle fibre are sensitive to pH changes (Jan & Jan, 1976), solutions were carefully maintained at pH 7 0. Upon application of L-glutamate, the change of resting potential was absent, or small and transient. Equilibrating the preparation in solution containing L-glutamate did not significantly change the resting potential or the specific 222

A

5 mV

F

0-05

B

sec

2mVL 0.1

sec

Fig. 4. L-glutamate potential (A) before and (B) after addition of 1P0 mmL-glutamate to the bath. The location of the L-glutamate pipette and the strength of the iontophoretic current pulse were the same. After flushing the preparation with L-glutamate-free solution to remove the 1.0 mM Lglutamate in the bath, the L-glutamnate potential recovered, both in size and in time course.

membrane resistance, but did affect the e.j.p. and the m.e.j.p. Fig. 3 shows that, at 10- M L-glutamate, the e.j.p. and m.e.j.p. were essentially unchanged. At 5 x 104 M, the amplitude of the first peak of the e.j.p. decreased and the amplitude of the second peak increased relative to the first; the time course of both the e.j.p. and the m.e.j.p. became much slower, about 3 times that in absence of L-glutamate. At 1 mm, which is about the concentration in haemolymph, the e.j.p. amplitude was about 30-60 % of that in L-glutamate-free solution; the time course of both e.j.p. and m.e.j.p. lengthened six- to sevenfold. Neurally evoked muscle contraction observed under a dissecting microscope was still vigorous at 1 mM L-glutamate, probably even more so than in L-glutamate-free solution. This presumably was due to the great lengthening of the e.j.p.s time course.

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 223 The mechanism by which L-glutamate changes the size and shape of the e.j.p. is not understood yet. Nevertheless, it is true that, in Drosophila larvae, neuromuscular transmission and E-C coupling are operative even in the presence of 1 mM L-glutamate. Therefore, the fact that the haemolymph contains L-glutamate does not exclude L-glutamate as the excitatory neurotransmitter. The L-glutamate potential evoked by iontophoresis was also affected by the presence of L-glutamate in the bath. Fig. 4A shows the L-glutamate potential in normal saline. Fig. 4B shows it for the same fibre after 1 0 mML-glutamate was added to the bath, with the L-glutamate pipette at approximately the same position. The falling phase of the L-glutamate potential lengthened about threefold (note that the time scale is different in Fig. 4A and B). After the preparation was flushed with L-glutamatefree solution, both the time course and the amplitude of the L-glutamate potential recovered to those before the application of the L-glutamate. Pharmacological studies of the e.j.p. The results described so far are quite compatible with the idea that Lglutamate is the excitatory transmitter at the Drosophila larval neuromuscular junction. It remains to be determined whether some other putative transmitter substances can also affect the e.j.p. The experiments were done by adding to the bath solutions at pH 7 0 of the substance to be tested, and determining whether the neurally evoked e.j.p. was affected. Chemicals tested: (1) D-glutamate, L-glutamine, L-aspartate, L-asparagine, glycine. Lglutamine and L-aspartate at concentrations about 1 mm can cause contraction in locust (Usherwood & Machili, 1968). In Drosophila none of these five amino acids had any effect on the e.j.p. at concentrations up to 5 mM; (2) L-glutamate-y-methylester and L-glutamate-diethylester were found to be L-glutamate antagonists at a crayfish neuromuscular junction at concentrations of about 1 mm (Lowagie & Gerschenfeld, 1974). They had no effect on the e.j.p. in the Drosophila larva at concentrations up to 10 mM; (3) octopamine and serotonin had no effect on the e.j.p. at concentrations up to 10 mM; (4) GABA is the inhibitory transmitter at certain crustacean and insect neuromuscular junctions (Kravitz, Kuffler & Potter, 1963; Usherwood & Grundfest, 1965). Although ACh apparently is not an excitatory transmitter at those neuromuscular junctions, it may be the excitatory transmitter at certain crayfish neuromuscular junctions (Futamachi, 1972). The effects of ACh and GABA on the Drosophila larval neuromuscular

224 L. Y. JAN AND Y. N. JAN junction were tested. GABA (up to 10 mM) or ACh (up to 5 mM) in the bath had no effect on the e.j.p. In other experiments, doublebarrelled electrodes were used for iontophoresis, one barrel was filled with L-glutamate, the other with GABA or ACh. The entire exposed muscle surface was explored; at each position, current pulses of appropriate polarity were injected alternately from the two barrels. We failed to detect any GABA or ACh sensitivity; (5) Substance P was 1000-9000 times more potent than L-glutamate in causing depolarization of spinal motor neurones (Otsuka & Konishi, 1976). It had no effect on the e.j.p. at the Drosophila larval neuromuscular junction at concentrations up to 0 4 mM.

Reversal potential If L-glutamate is the excitatory transmitter, it must produce the same post-synaptic permeability changes as the neurally occurring transmitter. Therefore the reversal potential of the L-glutamate potential and the e.j .p. was determined in solutions of varied ionic composition. The Drosophila muscles used in this study are about 80 /um wide, 25 utm thick, and only 400 jm long, so that the entire fibre is effectively isopotential (Jan & Jan, 1976) and the membrane potential can be altered uniformly by injecting current through a micropipette. Therefore, polyinnervation is not a serious problem in determining the accurate values of reversal potentials. Contraction accompanying depolarization presents a problem in the late 3rd instar larvae usually used. Moreover, it was very difficult to depolarize the muscle membrane beyond 0 mV for more than a few hundred milliseconds. Both problems can be avoided by using young pupae instead of larvae. After the 3rd instar, the larva pupates. Within the first 2 hr of pupation, the musculature appears unchanged except that the muscle contraction is reduced to a low level, even when a large depolarizing current is being injected. Also the muscle membrane can be depolarized easily to beyond +40 mV by injecting current. The I-V characteristics from membrane potential of - 100 to - 10 mV (Fig. 5), the specific membrane resistance at resting potential [(4.4 + 0 3) x 103 Q cm2 (five fibres)], the specific membrane capacitance [(7.0 + 2.2) /tF/cm2 (five fibres)], and both the e.j.p. and the L-glutamate potential in these muscles are the same in the pupa as in the 3rd instar larva. Therefore, the larval fibres in early pupae were used to study the reversal potential. Fig. 6A shows a typical record. In the experiment, one intracellular micropipette was used to pass current, another to record membrane potential. The e.j.p. was evoked by stimulating the abdominal nerve with a suction electrode. The L-glutamate potential was produced by passing a

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 225 brief negative current pulse through the L-glutamate-filled pipette 09 see after nerve stimulation. The amplitudes of the e.j.p. and L-glutamate potential are plotted as functions of membrane potential in Fig. 6B. In this experiment, the reversal potential for the e.j.p. and the L-glutamate potential were - 1 and -3 mV, respectively. The average of five experiments in solution containing 0 5 mM calcium, 5-5 mm magnesium, 128 mM

20

15

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-80

mV -60

40 0 -20

0 0

5

10

15

Fig. 5. Current-voltage relation in two ventral lateral longitudinal fibres at the early pupal stage in solution A (Table 1, Jan & Jan, 1976).

sodium, 2 mm potassium, and 142 mm chloride, were -10 + 1-4 mV and - 02 + 1-3 mV (mean + S.D.), respectively. Therefore, the reversal potentials for the e.j.p. and the L-glutamate potential appear identical. Addition of 025 or 0*5 mM-L-glutamate to the bath prolonged both the e.j.p. and the L-glutamate potential, but did not alter their reversal potential. The reversal potentials of e.j.p. and L-glutamate potential in salines of different ionic compositions are shown in Table 1. In all cases, the reversal potentials of the e.j.p. and the L-glutamate potential are the same within experimental error.

226 L. Y. JAN AND Y. N. JAN Close examination of the e.j.p. evoked from a membrane potential near the reversal level reveals that it is actually diphasic (Fig. 7). There seems to be a faster component which has a reversal potential about 2-3 mV more negative than that of a slower component. For the L-glutamate potential, this biphasic wave form has been observed only twice in more A

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Fig. 6. A, the e.j.p. and the L-glutamate potential at different membrane potentials. B, same data plotted with membrane potential as abscissa and amplitude of the responses as ordinate.

than forty experiments. One explanation is that the L-glutamate potential has a much slower time course than the e.j.p., so that any diphasic wave form would easily be masked. In experiments where the L-glutamate potential appeared to be monophasic, its reversal potential corresponded to that of the fast component of the e.j.p.

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L-glutamate appears to be, or to be an agonist of, the excitatory transmitter of the Drosophila larval neuromuscular junctions studied; (1) the L-glutamate sensitive region coincides with the morphologically and electrophysiologically identified synaptic regions; (2) the reversal potential of the neurally evoked e.j.p. was the same as that of iontophoretically evoked L-glutamate potential in twelve solutions of different ionic compositions. This shows that L-glutamate produces the same post-synaptic permeability changes as does the naturally occurring transmitter; (3) among many transmitter candidates tested, only L-glutamate could mimic the action of the natural transmitter; (4) the L-glutamate potential and the e.j.p. were prolonged in a parallel fashion by bath application of L-glutamate. In locust, extra-junctional L-glutamate receptors cause an increase in chloride permeability and give rise to a slower L-glutamate potential in response to iontophoretically applied L-glutamate. The sensitivity to L-glutamate is less for extra-junctional receptors than for junctional receptors (Cull-Candy, 1976). One possible explanation for the lengthening of the time course of e.j.p. in Drosophila larvae upon bath application of L-glutamate may be that bath-applied L-glutamate plus transmitter released from the nerve terminal can elicit response from the extrajunctional receptors in the vicinity of the junction, while either one alone cannot. This seems unlikely for two reasons: (1) in contrast to the locust muscle, L-glutamate receptors on the Drosophila larval muscle are localized only at junctional regions; and, (2) with L-glutamate in the bath, the prolonged e.j.p. and the prolonged L-glutamate potential reverse at the same membrane potential as they do in the absence of bath glutamate. Thus the lengthening of the e.j.p. time course by bath-applied glutamate seems to be due to properties of the junction.

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 229 There is evidence suggesting that, in insect neuromuscular junction, the inactivation of L-glutamate is achieved by rapid uptake rather than enzymatic degradation (Usherwood & Machili, 1968; Faeder & Salpeter, 1970; Faeder, Mathews & Salpeter, 1974). A possible explanation of the lengthening of the time course by bath application of L-glutamate is saturation of the uptake system. The effect of bath glutamate on the e.j.p. amplitude might result from a combination of uptake saturation effect and receptor desensitization. One argument against L-glutamate as the excitatory transmitter in insects has been that there is a high concentration of L-glutamate in haemolymph, which could desensitize the L-glutamate receptors. Our results showed that glutamate iontophoresis still caused muscle depolarization and that neurally evoked muscle contraction was still operative in the presence of L-glutamate at the same concentration as that in haemolymph. PART II. IONIC BASIS OF THE E.J.P. AND THE L-GLIJTAMATE POTENTIAL RESULTS

At the frog neuromuscular junction (Takeuchi & Takeuchi, 1960) and certain other nicotinic cholinergic excitatory synapses (Ito, Kuriyamu & Tashiro, 1969), the dependence of reversal potential on external sodium and potassium concentrations does not fit the Goldman-Hodgkin-Katz equation assuming constant permeability for channels opened by ACh. However, it does fit an equivalent-circuit equation, assuming increases in sodium and potassium conductance, which remain constant over a wide range of [K+]o and [Na+]o. In many other systems, however, this equivalent-circuit equation fails to predict the reversal potential. It has been suggested that the Goldman-Hodgkin-Katz equation might be used for these systems, if one assumes also an increase in calcium permeability (Anwyl & Usherwood, 1975). In Drosophila, it turns out that, for both the e.j.p. and the L-glutamate potential, there are increases in the permeabilities for sodium, potassium and magnesium ions, and that an extended constant-field equation predicts reasonably well the reversal potentials in all sixteen solutions listed in Table 1. The data analysis leading to this conclusion follows. (a) The extended constant-field equation The basic assumptions in the constant-field membrane model (Goldman, 1943) are a uniform, homogeneous membrane, and a constant electric field within the membrane. (If the membrane is permeable only to univalent ions, and if total ionic strength is equal on both sides of the

L. Y. JAN AND Y. N. JAN membrane, the Goldman-Hodgkin-Katz equation (Hodgkin & Katz, 1949) can be derived without any assumptions (Finkelstein & Mauro, 1963).) Assuming a constant field and taking into consideration both monovalent and divalent ions, one obtains the extended constant-field equation (Piek, 1975): (2) E =b+(b2-4ac)C T F ~~2a in which P~~~~ a = [K+]i + p [Cl-]o + 4 M [Mg2+]i + 4& Ca[Ca2+]i + [NaNa+]i, 230

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PK

Na+, Cl-, Mg2+, and Ca2+, respectively.

(b) PC1 and Pca If calcium ions contribute to the e.j.p. or the L-glutamate potential, increasing [Ca2+]0 should make the reversal potential more positive, provided that the external concentrations of other ions are kept constant. The observed reversal potential was essentially the same in expts. 1, 2, 3 and 4 (Table 1), where [Ca2]0 was increased from 041 to 1.0 mM, [K+]o, [Na+]o, and [Cl-]o were kept constant, and [Mg2+]o was varied only slightly. Moreover, the reversal potential became more negative rather than more positive, when [Ca2+]O was further increased to 1-8 mm (and [Mg2+]o decreased from 5 5 to 4 0 mM), indicating that PCa could not have contributed significantly to the reversal potential in the range of [Ca2+]0 used. Therefore, 2Ca/PK was assumed to be zero. (It is possible that the contribution due to PCa becomes significant when a much higher [Ca2+]O is used. Further experiments using voltage-clamp techniques are required to test this possibility.) Similarly, if chloride ions contribute to the e.j.p. or the L-glutamate potential, increasing [Cl-]o should make the reversal potential more negative. This does not seem to be the case because (a) in expts. 8 and 10 of Table. 1, the major difference in ionic composition is the 24 mm potassium and 99 mm chloride in expt. 8 as compared to 0 mm potassium and 0 mm chloride in expt. 10. The observed reversal potential was more positive in expt. 8 (about -5 mV) than in expt. 10 (about - 11 mV), indicating that the contribution of chloride ions must be much less than the contribution of potassium ions. (b) In expts. 10 and 11 of Table 1,

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 231 [Mg2+10 was increased from 5-5 mm in expt. 10 to 19-4 mM in expt. 11, and [Cl-]o was increased from 0 in expt. 10 to 126 mm in expt. 11, while the other ionic concentrations were kept constant. The reversal potential in expt. 1 1 was about 10 mV more positive than that in expt. 10, indicating that the contribution of chloride ions was much less than the contribution of magnesium ions. (c) In expts. 7 and 9 of Table 1, [Mg2+]o and [Na+]0 were varied slightly (5.9 mm magnesium and 15 mm sodium in expt. 7 versus 5-5 mm magnesium and 0 mm sodium in experiment 9), [Cl-]o was lowered from 50 mm in expt. 7 to none in expt. 9, but the reversal potential was slightly more positive in expt. 7, again indicating that the contribution of chloride ions was not significant. Therefore PC1/PK was also assumed to be zero, and eqn. (3) was reduced to a = [K+]i + b

N

[Na+Ji + 4

=([K+] [K+]o) +

c=

Na

M

[Mg2]

([Na+]i-[Na+]o)(4)

[K+] p [Na+]o-P4 M [g2+]O,

(C) PK) PNa and PMg If potassium and/or sodium ions contribute to the current for the e.j.p. or the L-glutamate potential, increasing [K+]. and/or [Na+]o should make the reversal potential more positive. In expt. 9 (Table 1), there was no sodium but 85 mm potassium, while in expt. 10 there was no potassium but 85 mm sodium, the concentrations of calcium, magnesium and chloride ions being the same in both experiments. The reversal potential was essentially the same in expts. 9 and 10. Therefore, the contribution of potassium ions must be comparable to the contribution of sodium ions. In expts. 9 and 10, the sum of [K+]o and [Na+]Qo was 85 mm, in expt. 8 it was 117 mm, and in expt. 2 the sum was 130 mm, the concentration of calcium and magnesium being the same. The observed reversal potential was more negative (about - 11 mV) in expts. 9 and 10, as compared to those in expt. 8 (about - 5 mV) and in expt. 2 (about - 1 mV), indicating that potassium and sodium ions contribute significantly to the e.j.p. and the L-glutamate potential. Similarly, if magnesium ions contribute significantly, the reversal potential should become more positive if [Mg2+]o is increased. This is the case because (a) in expts. 2 and 6 (Table 1), [Mg2+]o was decreased from 5-5 to 0.5 mm and the reversal potential was decreased from -1 to - 8-3 mV. (b) In the series of expts. 13, 14, 15, 16 (Table 1), [Mg2+]o was increased from 0-5 to 21 mm and the reversal potential was increased from

L. Y. JAN AND Y. N. JAN 232 - 13 to about + 2 mV. (c) In expts. 10 and 11, [Mg2+]. was increased from 5.5 to 19*4 mm and the reversal potential was increased from - 1I1 to about o mV, and (d) in expt. 12, there was 0 mm potassium, 0 mm sodium, and

48*5 mm magnesium, and the reversal potential was about +12 mV, more positive than that in any of the other fifteen experiments listed in Table 1. This indicates that the contribution of magnesium was larger than that of potassium and sodium ions.

(d) Pa.PK and P1g/PK The unknown parameters in eqn. (4) are: PNa/PK, PMg/PK, [Mg2+]i, and For a given [Mg2+]i, one can estimate PMg/PE and [K+]i + (PNa/Pk)/[Na+]i from expts. 9 and 12 of Table 1 (in which cases [Na+]o = 0), and then P.a/PK from any one of the other fourteen experiments. The remaining thirteen experiments can be used to check the predictions of the extended constant-field equation, using the assumed value of [Mg2+],, and the estimated values for PNa/PK, PMg/PB, and [K+]1, (P:Na/PK) [Na+]i. There is no information concerning [Na+]i, [K+]i, and [Mg2+],, except that, in fitting the resting potential data to the GoldmanHodgkin-Katz equation, [K+]1, + 0*23 [Na+]i was found to be about 173 mM (Jan & Jan, 1976). Hence in! sample calculations, [Mg2+], was varied from 0 to 10 mM systematically and the other parameters were estimated for each [Mg2+]1. P.NaIPK was found to be about 1*3 and quite insensitive to the value of [Mg2+]1. [K+]l + (PNa/P) [Na+]1, however, decreased with increasing [Mg2+]1, and became less than 173 mm if [Mg2+]f was more than 9-25 mm. Therefore, concentrations of magnesium smaller than 9-25 mM were used. P-Fg/P& was always much greater than 1 (about 5 if [Mg2+]i = 0 or 9 mm, about 8 if [Mg+2], = 5, 7, or 8 mM). The fitting of data by the extended constant-field equation was reasonable regardless of the value chosen for [Mg2+]i, as long as [Mg2+], was less than 9*25 mm. The best fit was obtained at [Mg2+], = 9-25 mM, Pa./PK= 1-3, and PMg/PK = 4*7, and is given in the last column of Table 1. For other values of [Mg2+]j, the predictions were generally further off by 1-3 mV.

[K+]i + (PNa/Pk)[Na+]j.

DISCUSSION OF PART II

For a uniform membrane, permeable only to monovalent ions, the steady-state relation between the membrane permeabilities, the internal and external ionic concentrations, and the membrane potential is given by the Goldman-Hodgkin-Katz equation (Goldman, 1943; Hodgkin & Katz, 1949). This equation can be rewritten in a form which can be interpreted as an equivalent circuit model of the membrane (Finkelstein & Mauro, 1963). In this model, the membrane is considered as many elec-

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 233 trical elements in parallel, each consisting of the integral resistance of an ion and a battery with an e.m.f. equal to the Nernst potential of that ion. In general, the conductance of an ion is not constant, but depends on the permeability, the internal and external concentration of that ion, and on the membrane potential (Finkelstein & Mauro, 1963). Takeuchi & Takeuchi (1960) found that the reversal potential of the e.p.p. at the frog sartorius neuromuscular junction fits an equivalentcircuit model in which the two electric elements in parallel are the AChinduced sodium and potassium channels. The specific assumption they made is that the ratio of the conductance changes is constant and independent of external sodium and potassium concentrations. This might result either from the channels being open for too short a-period or from the number of channels being so small that the availability of channels rather than external ions becomes the limiting factor. It has been suggested that, in frog muscle, the increase of sodium and potassium conductance by ACh is constant because the sodium and potassium channels open for a very short period. Opening these channels for a longer period by substituting subecholine for ACh or by lowering the temperature makes the [K+]o-dependence of the reversal potential conform to the predictions of the Goldman-Hodgkin-Katz equation, where the permeabilities rather than the conductances are assumed to be constant (Bregestovski, Chailachjan, Dunin-Barkovski, Potapova & Veprintsev, 1972). Among the many chemical synapses studied (for review, see Ginsborg, 1973; Gerschenfeld, 1973; Anwyl &lUsherwood, 1975), some, like the frog neuromuscular junction, conform to the equivalent-circuit equation assuming constant conductances (Takeuchi & Takeuchi, 1960; Ito et al. 1969), others, like the inhibitory synapse in Aplysia neurones (Kehoe, 1972) are described by the Goldman-Hodgkin-Katz equation assuming constant permeabilities. In a third class of junctions, the reversal potentials fit both equations poorly, and contributions due to divalent cations have been implicated. In fact, calcium, in addition to sodium and potassium, seems to contribute to the e.j.p. in crayfish (Dudel, 1974) and both calcium and magnesium seem to contribute to the e.j.p. in mealworm larvae (Kusano & Grundfest, 1967). In frog, both calcium and magnesium reduce the sodium contribution to the end-plate current and may themselves contribute to the end-plate current (Takeuchi, 1963). At the Drosophila larval neuromuscular junction, permeability changes to sodium, potassium and magnesium ions contribute to the e.j.p. and the L-glutamate potential. The chloride contribution is negligible, as is the calcium contribution in the range of [Ca2+]o used (0. 1-1 8 mM). Whether, at high [Ca2+]0, calcium ions can contribute at all to the e.j.p. remains to be tested.

L. Y. JAN AND Y. N. JAN Since magnesium ions contribute significantly to the e.j.p. and the Lglutamate potential in Drosophila larvae, an extended constant-field equation (Piek, 1975) for both monovalent and divalent ions has to be used instead of the Goldman-Hodgkin-Katz equation. The changes of reversal potential in solutions of different ionic compositions are predicted by this extended constant-field equation assuming constant permeabilities. Thus, the kinetics of channel opening and other basic properties may be different from those of the well-studied frog neuromuscular junction. Further studies using voltage-clamp techniques or noise analysis may be fruitful in revealing such differences. 234

We thank Dr Seymour Benzer for advice, equipment, and fly stocks. We appreciate the helpful criticisms of this manuscript by Drs Seymour Benzer, Jacsue Kehoe, Philippe Ascher, William Quinn, Jr., Michael Dennis and William Harris. This work was supported in part by Grant GB-27228 from the National Science Foundation to Seymour Benzer, the Scottish Rite Schizophrenia Research Programme grant to Yuh Nung Jan and the National Institutes of Health Postdoctoral Fellowship (GM 05431-01) to Lily Yeh Jan. REFERENCES

AVwYL, R. & USEERWOOD, P. N. R. (1974a). Voltage clamp studies of glutamate synapse. Nature, Lond. 252, 591-593. ANWYL, R. & USUERWOOD, P. N. R. (1974b). Voltage clamp studies of the glutamate response at the insect neuromuscular junctions. J. Phyiol. 242, 86-87P. ANwYL, R. & USlMRWOOD, P. N. R. (1975). The ionic permeability changes caused by the excitatory transmitter at the insect neuromuscular junction. J. Physiol. 249, 24-25P. BREGESTOVSKI, P. D., CHAILACHJAN, L. M., Du1NIN.-B~movsKI, V. L., POTAPOVA, T. W. & VEPRINTSEV, B. N. (1972). Effect of temperature on the equilibrium end plate potential. Nature, Lond. 236, 453-454. CHLEN, P. S., KuBLi, E. &Z HBiAwN, F. (1968). Auftrennung der freien Ninhydrinpositiven stofte IS Phormia und Drosophila mittels zweidimensionaler hoch spannungselektrophorese. Remse 8ti88e Zool. 75, 509-523. CuLL-C"DY, S. G. (1976). Two types of extrajunctional L-glutamate receptors in locust muscle fibres. J. Physiol. 255, 449-464. DEL CASTILLO, J. & KATZ, B. (1955). On the localization of acetylcholine receptors. J. Physqiol. 128, 157-181. DUDEL, J. (1974). Nonlinear voltage dependence of excitatory synaptic current in

crayfish muscle. Pfilger8 Arch. ges. Phyaiol. 353, 227-241. DUDEL, J. (1975). Potentiation and desensitization after glutamate-induced postsynaptic currents at the crayfish neuromuscular junction. Pfluger8 Arch. ge8. Phyaiol. 356, 317-329. FAEDER, I. R., MATHEWS, J. A. & SATPETER, M. M. (1974). 3H-glutamate uptake at insect neuromuscular junctions: effect of chlorpromazine. Brain Res. 80,

53-70. FAEDER, I. R. & SALPETER, M. M. (1970). Glutamate uptake by a stimulated insect nerve muscle preparation. J. cell Biol. 46, 300-307.

L-GLUTAMATE AS TRANSMITTER IN DROSOPHILA 235 FINKELSTEIN, A. & MAURO, A. (1963). Equivalent circuits as related to ionic systems. Biophy8. J. 3, 215-237. FuTircRI, K. J. (1972). Acetylcholine: possible neuromuscular transmitter in crustacea. Science N.Y. 175, 1373-1375. GERSCHENFELD, H. M. (1973). Chemical transmission in invertebrate central nervous systems and neuromuscular junctions. Phy8iol. Rev. 53, 1-119. GINSBORG, B. L. (1973). Electrical changes in the membrane in junctional transmission. Biochim. biophy8. Acta 300, 209-317. GowIMN, D. E. (1943). Potential, impedance and rectification in membrane. J. gen. Phy&iol. 27, 37-60.

HODGKIN, A. L. & KATZ, B. (1949). The effect of sodium ions on the electrical activity of the giant axon of the squid. J. Phy8iol. 108, 37-77. ITO, Y., KuIRIYAMA, H. & TASHIRO, N. (1969). Effects of y-amino butyric acid and picrotoxin on the permeability of the longitudinal muscle of the earthworm to various anions. J. exp. Biol. 50, 107-118. J", L. Y. & J", Y. N. (1976). Properties of the larval neuromuscular junction in Dro8ophila melanogaster. J. Phyaiol. 262, 189-214. KEHOE, J. (1972). Ionic mechanisms of a two-component cholinergic inhibition in Aplyaia neurones. J. Phyaiol. 225, 85-114. KRAVITZ, E. A., KUFFLER, S. W. & POTTER, D. D. (1963). Gamma-aminobutyric acid and other blocking compounds in crustacea. III. Their relative concentrations in separated motor and inhibitory axons. J. Neurophy8iol. 26, 739751. KUFFLER, S. W. & YOSHIKAMT, D. (1975). The distribution of acetylcholine sensitivity at the post-synaptic membrane of vertebrate skeletal twitch muscles: iontophoretic mapping in the micron range. J. Phyaiol. 244, 703-730. KusANo, K. & GRUNDFEST, H. (1967). Ionic requirements for synaptic electrogenesis in neuromuscular transmission of mealworm larvae (Tenebrio molitor). J. gen. Phyaiol. 50, 1092. LOWAGIE, C. & GERSCHENFELD, H. M. (1974). Glutamate antagonists at a crayfish neuromuscular junction. Nature, Lond. 248, 533-535. ONODERA, K. & Trx~uck, A. (1975). Ionic mechanism of the excitatory synaptic membrane of the crayfish neuromuscular junction. J. Phyaiol. 252, 295-318. OTSUKA, M. & KoNisi, S. (1975). Substance P and excitatory transmitter of primary sensory neurons. Cold Spring Harb. Symp. quaint. Biol. 40, 135-143. PInK, T. (1975). Ionic and electrical properties. In Insect Muscle, ed. USumERWOOD, P. N. R., pp. 281-336. London, New York and San Francisco: Academic Press. ROBBINS, J. (1959). The excitation and inhibition of crustacean muscle by amino acids. J. Physiol. 148, 39-50. TAXuCmi, A. & TAx~ucr, N. (1960). On the permeability of end-plate membrane during the action of transmitter. J. Physiol. 154, 52-67. TA uCmi, N. (1963). Effects of calcium on the conductance change of the end-plate membrane during the action of transmitter. J. Physiol. 167, i41-155. TAxKucmn, A. & TAKEuCiI, N. (1964). The effect on crayfish muscle of iontophoretically applied glutamate. J. Physiol. 170, 296-317. TAR~sKEvIcn, P. S.; (1975). Dual effect of L-glutamate on excitatory post-junctional membranes of crayfish muscle. J. gen. Physiol. 65, 677-691. UsHERwOOD, P. N. R. (1972). Glutamate as an excitatory neuromuscular transmitter in insects. Neuro8ci. Res. Prog. Bull. 10-2, 136-143. Us;EmRwOOD, P. N. R. & CuLL-CAwDY, S. G. (1975). Pharmacology of somatic nerve. muscle synapses. In Insect Muscle, ed. UsUERwooD, P. N. R., pp. 207-280. London, New York and San Francisco: Academic Press.

236

L. Y. JAN AND Y. N. JAN

USHERWOOD, P. N. R. & GRUNDFEST, H. (1965). Peripheral inhibition in skeletal muscle of insects. J. Neurophysiol. 28, 497-518. USHERWOOD, P. N. R. & MACHILI, P. (1968). Pharmacological properties of excitatory neuromuscular synapses in the locust. J. exp. Biol. 49, 341-361.