Denver, Colorado 80262, U.S.A. (Received 11 January 1979) ionophoretically applied glutamate and aspartate were studied.

Transcription

1 J. Physiol. (1979), 293, pp With 9 text-figures Printed in Great Britain GLUTAMATE AND SYNAPTIC EXCITATION OF RETICULOSPINAL NEURONES OF LAMPREY BY GARY MATTHEWS AND WARREN 0. WICKELGREN From the Department of Phy8iology, Univer8ity of Colorado Medical School, Denver, Colorado 80262, U.S.A. (Received 11 January 1979) SUMMARY 1. Intracellular recordings were made from the cell bodies and axons of giant reticulospinal neurones (Muller cells) of the lamprey, and responses to bath- and ionophoretically applied glutamate and aspartate were studied. 2. Bath-applied glutamate and asparatate depolarized both cell bodies and axons, but there appeared to be an associated conductance increase only in the cell bodies. The depolarization of Muller axons by the bath-applied drugs probably resulted from the passive flow of current into them from spinal cells to which the axons are coupled electrically. 3. The reversal potentials for responses to ionophoretically applied glutamate and for excitatory post-synaptic potentials (e.p.s.p.s) evoked by stimulation of the contralateral vestibular nerve were directly determined in Muller cell bodies which had been damaged by penetration with low-resistance electrodes. The glutamate and e.p.s.p. reversal potentials were identical, the average difference in eight cells being 031 mv. The absolute value of the e.p.s.p.-glutamate reversal potential varied from - 16 to -35 mv in different cells, with the more negative values occurring in less damaged cells with higher resting potentials. 4. Injection of Cl into Muller cell bodies had no effect on the e.p.s.p.-glutamate reversal potential. Reduction of the extracellular Na concentration to -L normal produced a negative shift in the glutamate reversal potential. 5. It is proposed that the natural excitatory transmitter and glutamate produce identical conductance changes in Muller cells, involving an increase in Na and K conductance. INTRODUCTION The dicarboxylic amino acids glutamate and aspartate have potent excitatory effects on a variety of neurones in the vertebrate central nervous system, and glutamate in partuclar has been suggested to be an excitatory neurotransmitter (for reviews, see Curtis & Johnston, 1974; Krnjevid, 1974; Johnson, 1978). An obstacle in the study of glutamate as a neurotransmitter in the vertebrate C.N.S. has been the difficulty of comparing its post-synaptic effect with that of the natural excitatory transmitter. In addition to the difficulty of recording intracellularly from most vertebrate C.N.S. neurones, it has been impossible for a variety of reasons to measure accurately the reversal potentials for the excitatory post-synaptic potential (e.p.s.p.) /79/ $ The Physiological Society 14 PHY 293

2 418 G. MATTHEWS AND W. 0. WICKELGREN and the glutamate response. Further, although a recent published abstract reports a similarity in the reversal potentials for glutamate and the e.p.s.p. produced by parallel fiber activation in frog cerebellar Purkinje cells (Hackett, Hou & Cochran, 1978), in most instances where estimates of both e.p.s.p. and glutamate reversal potentials have been obtained on the same cell, they have been seriously discrepant (cat spinal motoneurones, Curtis, 1965; Zieglginsberger & Puil, 1973; frog spinal motoneurones, Shapolvalov, Shiriaev & Velumian, 1978). Such discrepancies could have resulted from (1) errors in estimating the reversal potentials by extrapolation from a limited range of membrane potential measurements, (2) a difference in the electrotonic distances from the position of the intracellular electrodes to the locations of the conductance changes for the glutamate response vs. the e.p.s.p., or (3) a difference in the nature of the conductance changes produced by glutamate and the excitatory transmitter. Only the last possibility would exclude glutamate as a candidate for the excitatory transmitter. On the other hand, if the reversal potentials for the e.p.s.p. and the response to glutamate could be accurately determined and shown to be identical, it would indicate that glutamate and the excitatory transmitter produce identical conductance changes just as has been shown for the crustacean neuromuscular junction where glutamate almost certainly is the excitatory transmitter (Onodera & Takeuchi, 1975). The advantages of the lamprey giant reticulospinal cells (Muller cells) for the investigation of putative neurotransmitters in the vertebrate central nervous system have been mentioned in the previous paper (Matthews & Wickelgren, 1979). Briefly, the cells are large, individually identifiable in many cases, and both the somata and axons permit stable intracellular penetration with several micropipettes. Further, the extracellular environment of the cells is under experimental control since the lamprey central nervous system survives well in artificial saline. Thus, Muller cells are well suited for a comparison of the post-synaptic effects of glutamate and the natural excitatory transmitter. In this paper we report that Muller axons, which do not receive an excitatory synaptic input, also apparently lack glutamate receptors. On the other hand, Muller cell bodies and dendrites, which do receive an excitatory synaptic input, are sensitive to glutamate, and the reversal potentials for the actions of the excitatory transmitter and glutamate are identical. The post-synaptic effect of both glutamate and the natural excitatory transmitter appears to be an increase in both sodium and potassium conductance. An abstract describing some of this work has appeared (Wickelgren & Matthews, 1978). METHODS Experiments were conducted on isolated brains and spinal cord segments from young adult sea lampreys (Petromyzon marinu8) or American brook lampreys (Lampetra lamottenii). The dissections, experimental conditions and apparatus were similar to those described in the previous paper (Matthews & Wicklegren, 1979). Briefly, brains or spinal cord segments were dissected free with the supporting cartilage or notocord and pinned to the bottom of a Plexiglass chamber which allowed change of bathing saline. Unless stated otherwise in Results, the composition of the saline was (mm): NaCl; 2 KCl; 4 CaCl.; 8 MgCl2; 4 glucose; 2 Tris; titrated to ph 7-4 with HCl. Temperature of the saline was held at 7.5 ± 0 5 'C by electrothermal plates beneath the chamber. Most experiments on Miller cell bodies were done on two of the three pairs in the walls of the

3 GLUTAMATE AND SYNAPTIC EXCITATION third ventricle (M2 and M.) and the single visible pair of cells in the aqueductal region between the 3rd and 4th ventricles (I,) (nomenclature is from Rovainen, 1967). However, some experiments on reversal potential were done on small, unidentified reticulospinal cells in the floor of the 4th ventricle because of their higher input resistances. The Muller axons studied were unidentified, except that the bursting Muller axon, '2' which appears different from the other MUller axons in that it receives a chemical synaptic input (Rovainen, 1967), was not studied. Usually two micropipettes, one to pass current and the other to record membrane potential, were placed in a Muller cell body or axon. The micropipettes were filled with 4 M-K acetate and normally had resistances of MC, except for experiments in which it was desired to measure directly the reversal potentials for the e.p.s.p.s and drug responses and then the current-passing electrode was broken to a resistance of MO. Electrodes for ionophoresis were filled with 2 M-Na glutamate (ph 8.7). In order to prevent blockage of the glutamate electrodes, ionophoretic currents were restricted to 0 3 /za or less. Ionophoresis current was provided by an isolated stimulator through a 100 Mfl resistor. Leakage of glutamate from the pipette was retarded by applying a positive bucking voltage. For bath application, Na glutamate or Na aspartate was substituted for NaCl on an equimolar bases, and ph adjusted to 7-4. Whenever a change in bathing saline was made during an experiment, at least 100 ml. of the new solution was washed through the chamber, which had a volume of 9 ml. Conventional electrophysiological recording and stimulating techniques were used. Excitatory synaptic potentials were evoked in Muller cells by electrical stimulation of the vestibular nerves via bipolar platinum wire electrodes placed against the exposed labyrinthine membranes. Fast electrophysiological events were displayed on an oscilloscope and photographed. For slower events a strip chart recorder was used. RESULTS Effects of bath-applied glutamate and aspartate Muller axons When added to the saline bathing a spinal cord segment, both glutamate and aspartate caused a reversible, dose-dependent depolarization of Muller axons. The depolarization occurred within sec after the beginning of the wash-in period and did not desensitize to any appreciable extent. The maximum amplitude of the depolarization in axons with -80 to -85 mv resting potentials was mv, and the depolarization persisted when synaptic transmission was blocked with saline containing 0-Ca and 10 or 20 mm-mg and when voltage sensitive conductances were blocked with tetrodotoxin (TTX; 0 5 jug/ml.) and 4-aminopyridine (4AP; 1 mm). The maximum amplitude of the depolarization was usually greater when voltagesensitive K channels were blocked by 4-AP. The depolarization was recorded all along the length of a Muller axon with no obvious local variations in amplitude. Typical dose-response curves for glutamate and aspartate in the same axon are shown in Fig. 1. In this experiment, the saline contained 0-Ca, 10 mm-mg, TTX 0-5 jug/ml. and 1 mm-4-ap. In all experiments the threshold dose of both glutamate and aspartate was between 0-1 and 0.5 mm, but while 1*0-5 0 mm-glutamate was sufficient to produce the maximum response, 10 mm-aspartate was required to reach the same level of depolarization. In saline without 4-AP the drug-induced depolarization was accompanied by a reduction in the input resistance of the axon, as measured by passing a brief current pulse through one intracellular electrode and measuring the resultant voltage change with a second electrode less than 250 /um away. Fig. 2A shows current-voltage (I-V) curves for an axon with and without 1 mm-glutamate in saline containing 0-Ca,

4 420 G. MATTHEWS AND W. 0. WICKELGREN 20 mm-mg, and TTX 0*5 /tg/ml. but no 4-AP. The glutamate depolarized the resting axon from -78 to -70 mv and reduced the resting input resistance, as can be seen from the difference in control and 1 mm-glutamate I-V curves near the resting potential. However, with larger negative current pulses, the slope of the I-V curve in 1 mm-glutamate increased and approached that of the control curve at comparable membrane potentials. This implies that the reduction in input resistance + 20 Axon E- +10 E / Drug concentration (mm) Fig. 1. Dose-response curves for glutamate (Q) and aspartate (0) on a Muller axon. Drug concentrations were presented in ascending order, followed by thorough washing with normal saline. Synaptic transmission was blocked by O-Ca2+, 10 mm-mg2+, and TTX 0-5,ug/ml. and 1 mm-4 aminopyridine were present throughout. during the glutamate response was largely due to increased K conductance resulting from the depolarization. This was confirmed, as shown in Fig. 2B, when the voltagedependent K conductance was blocked by the addition of 4-AP. In this case, 1 mmglutamate depolarized the axon from -85 to - 74 mv at rest, but there was no apparent accompanying resistance change. Identical results were obtained using aspartate instead of glutamate. Thus, we conclude that the decrease in input resistance during drug-induced depolarization was due largely, or entirely, to the rectifying properties of the axonal membrane. What then causes the depolarization of Mfuller axons by glutamate and aspartate? One possibility is that the depolarization represents the influx of Na during Nadependent uptake of glutamate (Bennett, Logan & Snyder, 1972; for relevant discussion concerning GABA uptake, see Blaustein & King, 1976). This is unlikely, however, since complete substitution of Li for Na, which blocks high-affinity glutamate uptake (Bennett et al. 1972), had no effect on the drug depolarization. Another possibility is that glutamate and aspartate produce a small increase in Na conductance which is undetectable experimentally but is nevertheless capable of producing a substantial depolarization. From experiments on the relation between the external K concentration and membrane potential (G. Matthews & W. O.

5 GLUTAMATE AND SYNAPTIC EXCITATION Wickelgren, unpublished observations), we estimate that the value of P>N/KP is 002 in resting Muller axons. Thus, from the Goldman equation, a 10 mv depolarization, like that often produced by 1 mm-glutamate, could result from a doubling of PNa with a change in PNa/PK to However, since Na conductance is such a small part of resting membrane conductance, even a doubling of PFa in Muller axons would 421 ()T Em (mv) -65. mm -GIu a,control / / -~~-85 /(na) (B) TTX Em (mv) AP -70 mm-glu control I(nA) -95 Fig. 2. Effect of 1 mm-glhtamate on the current-voltage properties of Mfiller axons. Current-voltage curves were obtained by passing current pulses through one microelectrode and measuring the resulting voltage change with another intracellular electrode. Steady-state voltage was measured. A, current-voltage curves with (@) and without (Q) 1 mm-glutamate in saline containing 0-Ca2+, 20 mm-mg2+, and TTX, 0*5,ug/ml. B, current-voltage curves from another Mfiller axon with (@) and without (0) 1 mm-glutamate in saline containing O-Ca2+, 20 mm-mg2+, TTX, 05,sg/ml. and 1 mm-4-ap. Note the absence of a conductance change accompanying the glutamateinduced depolarization. produce a change of 2 % or less in total membrane conductance, a change which would be difficult to detect experimentally. However, we do not think the drug depolarizations were caused by such a direct effect of the drugs on the axon membrane, because we were unable, despite extensive efforts, to produce detectable depolarizations in single Muller axons by ionophoretic application of glutamate directly to the axon membrane, even with strong drug currents which readily depolarized and produced action potential activity in spinal motoneurones and interneurones when applied to them. This evidence is not conclusive since the hypothetical drug receptors on the axon membrane might be of sufficiently low density that only bath-application would activate an appreciable number of them. The above possibility notwithstanding, a likely explanation is that the depolar- yor-90

6 422 G. MATTHEWS AND W. 0. WICKELGREN ization of Muller axons by glutamate and aspartate is caused by a passive flow of current into the axons through electrical junctions with spinal neurones which are depolarized by the drugs. There is both physiological and anatomical evidence that Muller axons make frequent electrochemical synapses with spinal motoneurones and interneurones (Rovainen, 1974; Ringham, 1975; Matthews & Wickelgren, 1978b) Em(mV) mv mm -Glu aocontrol - 70 i~ C. A> '0 + 1' Z v / '' ~~~~~/(na) -110 Fig. 3. Effect of 10 mm-glutamate on the current-voltage properties of a Muller cell (cell M.). The saline contained 4 mm-ca2+, 8 mm.mg2+, 2 mmmco2+, 1 mm-4-ap, and TTX, 0-5 jug/ml. The current-voltage curves with (@) and without (0) glutamate cross at a membrane potential of -37 mv (the glutamate reversal potential). During bath application of glutamate or aspartate, it is likely that the cells coupled to Muller axons are depolarized. Some of this depolarizing current would flow through the gap junctions into Muller axons, producing a depolarization without a drug induced conductance change in the axon membrane. This mechanism is the same one proposed to explain the slight hyperpolarization of Muller axons by GABA and glycine (Matthews & Wickelgren, 1979) and to account for the 'synaptic-like' coupling potentials seen in Muller axons during massed activity in the spinal cord (Matthews & Wickelgren, 1978a). Quantitative information about the frequency of electrical junctions in Muller axons and their electrical characteristics is necessary in order to validate this hypothesis. Muiller cell bodies The somata of Muller cells also were depolarized by bath-applied glutamate and aspartate, but here there was a clear conductance increase attributable to the drugs. An example is shown in Fig. 3, which shows I-V curves for a Muller cell body in saline (4 mm-ca, 8 mm-mg, 2 mm-co, TTX 0.5 jug/ml., and 1 mm-4-ap) with and without 10 mm-glutamate. The slope of the I-V curve was reduced by glutamate, and extrapolations of the two I-V curves crossed at a membrane potential of -37 mv. The point of crossing was calculated from the two best-fitting linear

7 GLUTAMATE AND SYNAPTIC EXCITATION 423 regression lines and gives an estimate of the reversal potential for the response to glutamate. This estimate agrees well with directly measured reversal potentials for the glutamate response (see below). Ionophoretic application of glutamate to Miller cells In contrast to its lack of effect on Muller axons, ionophoretically applied glutamate depolarized Muller cell bodies whether applied directly to the soma or the dendrites. The responses to glutamate pulses were rather slow, the fastest observed times-topeak being about 1 sec. The cell input resistance was reduced during the response ^q / %] ~~10 mv lmmrlr n, 10 na 4 sec 0-4 0*3 E * Glutamate pulse duration (sec) Fig. 4. Example of the response of a Muller cell to ionophoretically applied glutamate. The sample traces are the responses to 0-2 sec (left), 0 5 see (middle), and 2.0 sec (right) pulses of glutamate. The data below show the complete relation between the maximum conductance change and the duration of the glutamate pulse. The conductance change was calculated by subtracting the reciprocal of the input resistance before each pulse from the reciprocal of the input resistance at the peak of the response. Each point is the mean of at least four observations; bars represent 1 S.D. to glutamate, and the size of both the depolarization and the conductance change were greater with longer ionophoretic currents (Fig. 4). The maximum conductance change was determined by subtracting the reciprocal of the input resistance just before the response from that at the peak of the response. Note that the conductance change necessary to produce even the largest depolarization in Fig. 4 (25 mv positive to the -72 mv resting potential) was not large, particularly when compared to the usual conductance changes accompanying glycine or GABA responses in Muller cells (see Matthews & Wickelgren, 1979). This is another indication that the reversal potential of the glutamate response is relatively far from the resting potential.

8 424 G. MATTHEWS AND W. 0. WICKELGREN Reversal potentials of glutamate response and e.p.s.p. An estimate of the glutamate reversal potential (-37 mv) was obtained from the I-V curves in Fig. 3. This method of determination is indirect, depending upon extrapolation of the control and drug curves. Further, long-term exposure of the cell to glutamate in the bathing saline theoretically could cause changes in the > +12 E -~+10 c' I: +4._< Em (mv) Fig. 5. Estimation of the reversal potentials for the e.p.s.p. evoked by contralateral vestibular stimulation and the glutamate response of a Muller cell (cell B2). Responses were recorded via one intracellular electrode as membrane potential was varied by passing current through another intracellul" r electrode. The resting potential was - 73 mv (arrow on abscissa). Straight lines were fitted to the data by the least-squares method, and the reversal potentials were calculated from the fitted linear equations. For the e.p.s.p. data ( ) the calculated reversal potential was mv. For the glutamate data (@) the calculated reversal potential was mv. ionic concentrations in the cell, preventing an accurate determination of the normal reversal potential. Also to obtain the linear I- V curves which make the extrapolation possible, it was necessary to add TTX and 4-AP to the saline, making it impossible to evoke e.p.s.p.s in the cell. Ideally one would like to be able to depolarize the cell sufficiently with injected current to reverse the polarity of both the glutamate ionophoretic potential and the e.p.s.p., thereby obtaining direct estimates of both. However, in practice it is impossible to reverse the e.p.s.p. or glutamate response of a Muller cell with a normal - 70 to - 80 mv resting potential, because the input resistance, which is low even at rest (2-3 MQ), falls rapidly as voltage-sensitive Na and K channels open as the cell is depolarized and because the micropipettes have limited current-passing capacity. Thus, in undamaged Muller cells with normal resting potentials it is feasible to obtain data on the amplitudes of the glutamate response and the e.p.s.p. over only a limited range of membrane potentials around the resting potential and extrapolate the curves to the estimated reversal potentials. An example of an experiment of this type is shown in Fig. 5. The amplitudes of the glutamate response and the e.p.s.p. evoked by stimulation of the contralateral vestibular nerve were measured by one

9 GLUTAMATE AND SYNAPTIC EXCITATION 425 intracellular electrode as the membrane potential was varied by passing DC current through another electrode. Data were plotted only for that range of membrane potential over which the cell's input resistance was approximately constant. The straight lines were fitted to the data by the method of least-squares, and the reversal potentials were calculated from the linear equations. The extrapolated reversal potential for glutamate was -33 mv, which is similar to the estimate of -37 mv obtained from the data on bath-applied glutamate in Fig. 3. The extrapolated reversal potential for the e.p.s.p. was mv, which is reasonably similar to the glutamate reversal potential. The potential for error when reversal potentials are estimated by extrapolation is large. Because the reversal potential is tens of millivolts away from the range of measurement, slight changes in the slope of the best-fit line can make large changes in the estimated reversal potential. The data shown in Fig. 5 were selected for presentation because we were able to change the membrane potential over a fairly wide range due to the relatively high input resistance of the cell and because we obtained measurements on both e.p.s.p.s and glutamate responses. Normally we were not able to vary the membrane potentials of undamaged Muller cells as much as in Fig. 5 and frequently we obtained data on only the e.p.s.p. In seven cells with resting potentials greater than -67 mv, the extrapolated e.p.s.p. reversal potentials ranged from -28 mv (the cell used for Fig. 5) to mv ( '3, mean + S.D.). Such extreme variability in extrapolated e.p.s.p. reversal potentials has been reported previously (Shapovalov et al. 1978) and presumably reflects the large potential for error of this method (see Smith, Wuerker & Frank, 1967). In order to obtain direct measurements of the reversal potentials for the e.p.s.p. and the glutamate response, we intentionally damaged Muller cells by using a low resistance (15-20 MQ) micropipette for passing current in addition to the normal higher resistance recording micropipette. In pilot experiments, we found that about half the Muller cells penetrated with a low-resistance micropipette were irreparably damaged, i.e. the membrane potentials declined rapidly to about zero and no synaptic potentials could be recorded. However, the other half reached a new, fairly steady level of membrane potential after the damage, and in many cases the input resistance slowly returned to near normal. These cells had resting potentials which ranged from - 12 to -55 mv, but the majority had resting potentials greater than -40 mv. In these cells the action potential mechanism was partially or wholly inactivated, but both synaptic potentials and glutamate responses could be recorded and often reversed in sign during the passage of steady polarizing currents. An example of the reversal of a glutamate ionophoretic response is shown in Fig. 6. In this case the resting potential of the cell was - 13 mv and the glutamate response, which was slightly hyperpolarizing in the resting cell, was evoked while the membrane potential was set at various levels by injected currents. The amplitude of the glutamate response depended on membrane potential and was zero at a membrane potential of - 17 mv, the reversal potential. In this example, the glutamate response was contaminated by synaptic activity resulting from excitation by the glutamate of nearby cells or terminals presynaptic to the Muller cell. Such glutamate-elicited synaptic activity has been reported previously (Martin, Wickelgren & Beranek, 1970; Wickelgren, 1977) and could be eliminated, leaving only the post-synaptic

10 426 C. MATTHEWS AND W. 0. WICKELGREN response to the glutamate, by blocking transmitter release either by appropriate adjustment of the Ca and Mg concentrations of the saline or by adding TTX to the saline which eliminates action potentials in the presynaptic elements. Usually, synaptic potentials during the glutamate response were a nuisance to be eliminated, but in this instance the synaptic activity served a useful purpose, particularly since it seemed to consist mostly of e.p.s.p.s. As can be seen in Fig. 6, the amplitude of the +6 mvw- -6 mv -17 mv mv- -48 mv 10 mv Fig. 6. Direct measurement of the reversal potentials for the glutamate response in a MUller cell (cell M2) and for e.p.s.p.s evoked by the glutamate. The Muller cell was damaged intentionally (see text), and the resting potential was - 13 mv. Membrane potential was set at various levels by passing current through one of two intracellular electrodes. e.p.s.p.s superimposed on the glutamate post-synaptic response also depended on membrane potential and reversed in sign at about the same membrane potential as the glutamate response, providing a direct indication that the reversal potentials of the glutamate response and the e.p.s.p. are similar. A more controlled comparison of the e.p.s.p. and the glutmate response is shown in Fig. 7. In this example, the reversal potential of the e.p.s.p. evoked by stimulation of the contralateral vestibular nerve was compared with that of the glutamate response. The resting potential of this Muller cell was -50 mv. As shown in the sample records of Fig. 7A, the e.p.s.p. reversed at about -35 mv, and the glutamate response reversed at about -33 mv. The complete data are plotted in Fig. 7 B. Table 1 shows the results for the eight different Muller cells in which direct measurements of both the glutamate and the e.p.s.p. reversal potentials were obtained. The average difference between the reversal potential of the e.p.s.p. and the reversal potential of the glutamate response was mv (mean + S.D.). The consistent similarity of the glutamate and e.p.s.p. reversal potentials was remarkable in that the glutamate ionophoretic pipette was sometimes positioned directly over the cell body and sometimes #sm lateral to the cell body, presumably over one of the dendrites. This indicates that in all cases the locations of the conductance changes for the e.p.s.p. and the glutamate responses were electrically close to the Muller cell bodies, a fact which undoubtedly helps to account for the relative ease with which the responses could be reversed. 4 sec

11 GLUTAMATE AND SYNAPTIC EXCITATION 427 Cell TABLE 1. Eight Muller cells and their responses to glutamate and e.p.s.p. Em* (mv) Erev (e.p.s.p.)t (my) * Em, resting potential. t E.,, reversal potential. Erev (glut.) (mv) Difference (e.p.s.p.-glut.) (mv) * * ± 2-69 (mean + S.D.) A -4_ E.p.s.p. -3- Glutamate _ B mV 50 msec L 5mV 4 sec E w + '. E + U) 0. C: Em (mv) Fig. 7. Direct measurement of the reversal potentials for the e.p.s.p. evoked by vestibular stimulation and the glutamate response of an intentionally damaged Muller cell. A, sample records showing the reversal of the e.p.s.p. (left) and the glutamate response (right). The number to the left of each trace is the membrane potential in mv. B, plot of the complete results of the experiment for the e.p.s.p. (0) and the glutamate response (*). Both responses reverse at approximately the same membrane potential. The arrow indicates the resting potential (- 50 mv).

12 428 G. MATTHEWS AND W. 0. WICKELGREN The absolute value of the glutamate-e.p.s.p. reversal potential ranged in different cells from - 16 to -35 mv (see Table 1). This appeared to be due to variation in the amount of damage to the cells, as indicated by the fact that the more positive reversal potentials were found in cells with more positive resting potentials. Table 2 summarizes the data from all cells in which actual reversal of the glutamate response or the e.p.s.p. or both was obtained. The cells are separated into those with resting TABLE 2. Cells in which glutamate and/or e.p.s.p. reversal potentials were determined Cells with Em > -40 mv n Em (mean + S.D.) Erev (mean + S.D.) E.p.s.p. Glutamate response * Cells with Em <-40 mv Em Erev n (mean + S.D.) (mean + S.D.) E.p.s.p ±1+3 Glutamate response ± ± 2-7 potentials above and below -40 mv. The close correspondence between the reversal potentials for the e.p.s.p. and the glutamate response is again apparent, as is the tendency for cells with more positive resting potentials to have more positive reversal potentials. The correlation coefficient between the resting potential and reversal potential was (P < 0*001) for glutamate responses and (P < 0.001) for e.p.s.p.s (Pearson product-moment correlation coefficient). We conclude from these data that the reversal potential for the post-synaptic effect of glutamate and that of the natural excitatory transmitter are identical. The value of the e.p.s.p.-glutamate reversal potential in undamaged Muller cells is probably near -35 mv, since direct determinations from the least damaged cells and the better extrapolated values from healthy Muller cells (see Figs. 3, 5, and 7) were always close to this value. The identity of the glutamate and e.p.s.p. reversal potentials suggests strongly that glutamate and the natural excitatory transmitter produce identical post-synaptic conductance changes in Muller cells. Evidence concerning the ions involved in the conductance changes Na The e.p.s.p.-glutamate reversal potential was positive to the normal resting potential of Muller cells, and, thus, an increase in Na conductance appeared to be involved. To demonstrate this, experiments were conducted in which the Na concentration of the saline ([Na]o) was reduced to 1-L normal by replacing NaCl with choline-cl on an equimolar basis. This eliminated action potentials, and, thus, it was impossible to evoke e.p.s.p.s. In order to increase the input resistance of damaged Muller cells and make it easier to reverse glutamate ionophoretic responses, voltagesensitive Na and K conductances were blocked by adding TTX (0.5 jtg/ml.) and 4-AP (1 mm) to the bathing saline. Fig. 8 illustrates the negative shift of the glutamate reversal potential produced by the reduction in [Na]0. In this case the shift

13 GLUTAMATE AND SYNAPTIC EXCITATION 429 was from -22 mv in normal saline to -34 mv in 9LO normal Na. The resting potentials of cells in low Na also became more negative, and the shift in this example was from -39 to -51 mv. In Table 3 the compiled results on glutamate reversal potential in low-na saline are shown in comparison with those from all the cells in normal saline which had TABLE 3. Normal Na 1 10 normal Na Comparison of glutamate reversal potentials in differing Na concentrations No. of cells 4 5 Em (mean + S.D.) (my) ± 3-8 Erev (glut.) (mean ± S.D.) (my) ± E CA 0 0. a, CA Tu Em (mv) Fig. 8. Effect of reduction in extracellular sodium concentration on the reversal potential of tile glutamate response of a Miiller cell. Normal-Na saline (0) contained mm-nacl. Saline with normal-na (0) contained 10-5 mm-nacl and mmcholine-cl. TTX (0.5 #sg/ml.) and 4-AP (1 mm) were present throughout. resting potentials over -50 mv. Since all the cells in the low-na saline had resting potentials greater than -50 mv, the resting potentials of the two groups were comparable. As can be seen, the reversal potentials of the glutamate responses of cells exposed to A normal Na were significantly (P < 0-01, t test) more negative ( mv, mean + s.p'.) than those of the cells in normal Na ( mv, mean + S.D.). Had we included data from cells in normal saline with resting potentials lower than -50 mv with correspondingly more positive glutamate reversal potentials, the differences between the reversal potentials in normal and low-na would have been even greater. We conclude from these data that glutamate, and undoubtedly the natural excitatory transmitter as well, produces an increase in the Na conductance of the postsynaptic membrane. 0

14 430 G. MATTHEWS AND W. 0. WICKELGREN Na is clearly not the only ion involved in the glutamate response and the e.p.s.p., since the glutamate-e.p.s.p. reversal potential was tens of millivolts negative to the Na equilibrium potential. The value of the Na equilibrium potential in a damaged Muller cell body was assumed to be near, or positive to, the peak of the action potential, which is a conventional Na action potential (G. Matthews & W. 0. Wickelgren, unpublished observations). As mentioned previously, the action potential Control +28 Clinjectione +24 * >E + 5 _ on o + 20 _ 0~~~~~~~0 0 0~~~~~ c ok a +81_ _ *~~~~~~~ _-i Cu ~~~~~0 wm 0mV) Em E Oe a Em, (mv) Em (mv) Fig. 9. Lack of effect of injection of Cl into Muller cells on the e.p.s.p. (left) and glutamate response (right). Data for the e.p.s.p. and the glutamate response were obtained from two different cells with resting potentials of -35 and -55 mv, respectively. The e.p.s.p. data were gathered after 10 min of Cl injection (-10 na) and the glutamate data were gathered after 12 min of Cl injection (- 10 na). 0O control; 0, Cl- injection. mechanism was inactivated in a damaged cell, and in order to remove this inactivation the cell was hyperpolarized to -70 mv. During this hyperpolarization, the cell's spinal axon was stimulated to produce an action potential which then propagated antidromically into the cell body. We found that even in somata with resting potentials of -35 mv the peaks of the action potential reached + 10 to + 15 mv. In such cells the glutamate-e.p.s.p. reversal potential was always mv negative to this and, thus, the glutamate response and the e.p.s.p. must involve, in addition to Na, one or more ions whose equilibrium potentials) are negative to the reversal potential. Cl The Cl equilibrium potential in Mifller cells is near -80 mv (Matthews & Wickelgren, 1979), and, thus, chloride could be involved in the e.p.s.p. and glutamate response. To test for this, the intracellular Cl concentration in single Muller cells was raised by passing negative current through an intracellular KCl micropipette. The duration and strength of the Cl current were similar to those which produced substantial changes in the reversal potential of the i.p.s.p. in Muller cells (Matthews & Wickelgren, 1979). As illustrated in Fig. 9, injection of Cl had no effect on either the

15 GLUTAMATE AND SYNAPTIC EXCITATION e.p.s.p. or glutamate response of Muller cells. We conclude that Cl is not involved in either the e.p.s.p. or glutamate response. K Direct experiments on the effect of changing either intracellular or extracellular K on the e.p.s.p. or glutamate response were not done. However, since potassium is the only ion other than Cl with an equilibrium potential negative to the e.p.s.p.- glutamate reversal potential, we conclude that an increase in K conductance accompanies the increase in Na conductance produced by the natural excitatory transmitter and glutamate. DISCUSSION The directly determined reversal potentials for the glutamate response and the e.p.s.p. in lamprey Muller cells were identical, and this indicates that glutamate and the natural excitatory transmitter have identical post-synaptic effects. The value of the normal e.p.s.p.-glutamate reversal potential appears to be about -35 mv, since the directly determined reversal potentials from the least damaged cells and the best extrapolated values from undamaged cells were near this value. An increase in Na conductance during the glutamate response was demonstrated, and chloride was shown not to be involved in either the e.p.s.p. or the glutamate response. Since the e.p.s.p.-glutamate reversal potential was approximately midway between the sodium and potassium equilibrium potentials, there appears to be an increase in conductance to both Na and K, similar to the effect of acetycholine at the vertebrate neuromuscular junction (Takeuchi & Takeuchi, 1960). This result is in contrast to results at the crustacean neuromuscular junction, where glutamate is probably the neurotransmitter and the conductance increase is largely, or entirely, to sodium (Onodera & Takeuchi, 1975). The depolarization of Mflller axons produced by bath-applied glutamate and aspartate was apparently a coupling potential, since the axons showed no conductance changes when voltage-sensitive channels of the axonal membrane were blocked pharmacologically (see Results). Coupled with the lack of effect of iontophoretically applied glutamate, this indicates that there are few or no glutamate receptors in the axonal membranes. Except for the bursting axon I2 (Rovainen, 1967), which was not investigated in this work, Muller axons rarely receive morphologically identifiable synaptic inputs (Wickelgren, 1977), and there is no evidence of chemical synaptic potentials in Muller axons (Matthews & Wickelgren, 1978a). The soma and dendrites of Muller cells, on the other hand, are studded with synaptic endings (Wickelgren, 1977) and show clear conductance changes to glutamate and aspartate. Thus, in Muller cells the absence of glutamate receptors is correlated spatially with the absence of excitatory synaptic input, and glutamate receptors are present in areas rich in excitatory synaptic input. In summary, the present work shows that glutamate and the natural excitatory transmitter produce identical postsynaptic effects in Mflller cells and that glutamate appears to produce conductance changes only in regions of the cells where there is excitatory innervation. Thus, glutamate could be the excitatory transmitter. How- 431

16 432 G. MATTHEWS AND W. 0. WICKELGREN ever, we do not know whether other substances, e.g. aspartate, would mimic the actions of the natural excitatory transmitter as well as did glutamate. Positive identification of the excitatory transmitter would be aided by the development of specific blocking substances for the various putative excitatory transmitters and some means of determining that a suspected excitatory transmitter is released in appropriate amounts during excitatory synaptic activation. We thank Mr Harold Purves at the U.S. Fish and Wildlife Service, Marquette, Michigan; Dr Joe Hunn, U.S. Fish and Wildlife Service, Millersburg, Michigan; and Mr Cliff Creech, N.Y. State Department of Conservation, Cortland, New York, for supplying us with lampreys. This study was supported by N.I.H. grants and and Basic Research Support grant RR REFERENCES BENNETT, J. P., LOGAN, W. J. & SNYDER, S. H. (1972). Amino acid neurotransmitter candidates: sodium-dependent high-affinity uptake by unique synaptosomal fractions. Science, N.Y. 178, BLAusTEIN, M. P. & KING, A. C. (1976). Influence of membrane potential on the sodiumdependent uptake of gamma-aminobutyric acid by presynaptic nerve terminals: experimental observations and theoretical considerations. J. membrane Biol. 30, CURTIS, D. R. (1965). The actions of amino acids upon mammalian neurones. In Studies in Phypiology Presented to J. C7. Eccles, ed. CURTIS, D. R. & McINTYRE, A. K. Heidelberg: Springer-Verlag. CuRTIs, D. R. & JOHNSTON, G. A. R. (1974). Amino acid transmitter in the mammalian central nervous system. Ergebn. Physiol. 69, HACKETT, J. T., Hou, S. M. & CocHRAN, S. L. (1978). Glutamate and synaptic depolarization of cerebellar Purkinje cells. Soc. Neurosci. AbN. 4, 580. JOHNSON, J. L. (1978). The excitant amino acids glutamic and aspartic acid as transmitter candidates in the vertebrate central nervous system. Prog. Neurobiol. 10, KRNJEVI6, K. (1974). Chemical nature of synaptic transmission in vertebrates. Physiol. Rev. 54, MARTIN, A. R., WICKELGREN, W. 0. & BERkNEK, R. (1970). Effects of iontophoretically applied drugs on spinal interneurones of the lamprey. J. Physiol. 207, MATTHEWS, G. & WICKELGREN, W. 0. (1978a). Evoked depolarizing and hyperpolarizing potentials in reticulospinal axons of lamprey. J. Physiol. 279, MATTHEWS, G. & WICKELGREN, W. 0. (1978b). Sustained depolarizing potentials in reticulospinal axons during evoked seizure activity in lamprey spinal cord. J. Neurophysiol. 41, MATTHEWS, G. & WICKELGREN, W. 0. (1979). Glycine, GABA and synaptic inhibition of reticulospinal neurones of lamprey. J. Physiol. 293, ONODERA, K. & TAKEUCHI, A. (1975). Ionic mechanism of the excitatory synaptic membrane of the crayfish neuromuscular junction. J. Physiol. 252, RINGHAM, G. L. (1975). Localization and electrical characteristics of a giant synapse in the spinal cord of the lamprey. J. Physiol. 251, ROVAINEN, C. M. (1967). Physiological and anatomical studies on large neurons of the central nervous system of the sea lamprey (Petromyzon marines). I. Muller and Mauthner cells. J. Neurophysiol. 30, ROVAINEN, C. M. (1974). Synaptic interaction of reticulospinal neurons and nerve cells in the spinal cord of the sea lamprey. J. comp. Neurol. 154, SHApovALov, A. I., SHIRIAEV, B. I. & VELUMIAN, A. A. (1978). Mechanisms of post-synaptic excitation in amphibian motoneurones. J. Physiol. 279, SMITH, T. G., WUERKER, R. B. & FRANK, K. (1967). Membrane impedance changes during synaptic transmission in cat spinal motoneurons. J. Neurophysiol. 30, TAKEUCHI, A. & TAKEUCHI, N. (1960). On the permeability of the end-plate membrane during the action of transmitter. J. Physiol. 154,

17 GLUTAMATE AND SYNAPTIC EXCITATION 433 WICKELGREN, W. 0. (1977). Physiological and anatomical characteristics of reticulospinal neurones in lamprey. J. Phy8iol. 270, WICKELGREN, W. 0. & MATTHEWS, G. (1978). Glutamate and excitatory synaptic transmission onto giant reticulospinal neurones of the lamprey. Soc. Neuro8ci. Ab8. 4, 580. ZIEGLGANSBERGER, W. & PUIL, E. A. (1973). Actions of glutamic acid on spinal neurons. Expl Brain Re8. 17,