Brain Research, 160 (1979) 479-487 © Elsevier/North-Holland Biomedical Press

479

P H E N Y T O I N A N D T R A N S M I T T E R RELEASE AT T H E N E U R O M U S C U L A R J U N C T I O N OF T H E F R O G

YOEL YAARI, JONATHAN H. PINCUS and ZOHAR ARGOV Department of Physiology, Hebrew University-Hadassah Medical School, Jerusalem (Israel) and Department of Neurology, Yale University School of Medicine, New Haven, Conn. (U.S.A.)

(Accepted May llth, 1978)

SUMMARY The effects of phenytoin (diphenylhydantoin, DPH) on transmitter release were studied at the frog neuromuscular junction. It was found that in Ringer's solutions containing a normal concentration of Ca z+ ions, D P H (1-2 × 10-4 M) depresses neurally evoked transmitter release, whereas in Cag'+-deficient Ringer's solutions it produces an increase in evoked release. Spontaneous transmitter liberation is augmented by D P H under all the above conditions. An abrupt disappearance of the evoked response occasionally occured with stimulation at 0.5 Hz, but a normal response could be elicited by a second stimulus delivered shortly after the first. At 100-200 Hz, D P H regularly induced a partial block in synaptic transmission. In 8 m M MgC12, this phenomenon appeared at 50 Hz and developed into a total neuromuscular blockade.

INTRODUCTION The effects of phenytoin (diphenylhydantoin, DPH), a commonly used antiepileptic drug, have been studied extensively on many neural preparations (for review, see ref. 24). Among the mechanisms proposed to account for the anticonvulsant action of D P H are 'stabilization' of excitable membranes 5,1°, decrease in post-tetanic potentiation (PTP) 9,19 and augmentation of presynaptic 7 and postsynaptic inhibition s, 16. Recently we have reported that D P H depresses synaptic transmission at the frog neuromuscular synapse 25. Such an effect occurring at central excitatory synapses may directly contribute to the reduced spread of seizure discharge by DPH. Indeed, a DPH-induced reduction of excitatory postsynaptic potentials has now been found in central synapses of both invertebrate 3 and vertebrate ~1 preparations. These findings stimulated a further search for the mechanisms by which D P H exerts its depressant effect on the neuromuscular synapse. The focus of this report is on how D P H alters neurotransmitter liberation from the presynaptic nerve terminals.

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Intracellular recording was performed at room temperature from superficial muscle fibres of the isolated frog sartorius muscle with single 3 M KCl-filled micropipettes of 10-20 M f~ resistance. Solutions were suspended within an electrically screened area in which the preparation was mounted. The flow rate, driven by gravity, was adjusted to approximately 10 ml/min. This allowed rapid exchange as the capacity of the chamber in which the muscle was mounted was 5 ml. Normal Ringer's (NR) solution consisted of (in mM): NaC1, I 15; KC1, 2; CaC12, 1.8; NaHPO4, 0.25 (pH 7.3). In order to prevent muscle contraction during nerve stimulation either d-tubocurarine (Squibb and Sons) 2-5 mg/1 or MgCI2 7-9 m M were added to the N R solution. Low calcium Ringer's solution contained 0.3 or 0.4 m M CaCI2 and 1.0 m M MgCI2. When calcium-free Ringer's solution was used, 1 m M EGTA was added to lower calcium concentration below 10 8 M2O. In some experiments, neostigmine (2 rag/l) was added to the Ringer's solution. D P H powder (Parke-Davis) was dissolved in N a O H 0.1 M, and this solution was used to prepare the DPH-containing Ringer's solution. The pH TABLE I

Effect t~f DPH on quantal content at intermediate release levels Averages of 4 experiments performed in NR with MgCI2 7.0-9.0 mM. All changes were reversed by washing (QC > 10 < 20).

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Fig. 2. The DPH-induced increase in EPP amplitude observed in low-calcium Ringer's solution. Each point is the mean amplitude of 25 consecutive EPPs (bin = 25, A bin = 25). DPH concentration = 1 × 10-4M. was a d j u s t e d with HC10.1 M. The same a m o u n t o f N a O H a n d HC1 were a d d e d to the c o n t r o l solution. N o cloudiness was n o t e d in solutions o f D P H o f u p to 2 × 10 -4 M. A f t e r 5 h, the solutions with D P H were passed t h r o u g h a millipore Sweenex filter (0.45 # m H A ) , a n d the filtrate was assayed for D P H by gas c h r o m a t o g r a p h y . The concentrat i o n was u n c h a n g e d and, therefore, it was a s s u m e d t h a t D P H h a d r e m a i n e d in solution d u r i n g the course o f the experiment. T h e nerve was s t i m u l a t e d by single s u p r a m a x i m a l shocks at a rate o f 0.5 o r 0.4 Hz. I n some e x p e r i m e n t s p a i r e d shocks with c h a n g e a b l e intervals or short (250 msec) trains o f high-frequency stimuli (50-200 Hz) were also used. I n e x p e r i m e n t s where e n d - p l a t e p o t e n t i a l s (EPPs) a n d miniature EPPs ( M E P P s ) c o u l d be r e c o r d e d simultaneously, q u a n t a l c o n t e n t (QC) was 'directly' d e t e r m i n e d f r o m the r a t i o o f m e a n E P P a m p l i t u d e to m e a n M E P P amplitude. I n N R solution,

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482 only EPPs could be recorded and quantal content was estimated by the coefficient of variation method, after correcting for non-steady-state conditions 6. The 'moving bin' method TMwas used for accurately describing the time course of the measured variables. RESULTS Effects of DPH on evoked transmitter release In a partially curarized preparation perfused with NR, DPH depresses EPP amplitude in a dose-dependent manner 2s. In 6 experiments, after 30 min of exposure to D P H 1 x 10-4 M and 2 x 10-4 M, the mean EPP amplitude was reduced by 38.4 413.4 ~ (S.D.) and 50.0 4- 7.1 ~ (S.D.), respectively. This effect was readily reversible on washing, as exemplified in Fig. 1. Since MEPP amplitudes are depressed in curarized preparations, QC could not be determined directly under these conditions. Estimation of QC was reduced by DPH. However, the extent of this reduction cannot be determined accurately by this method, for quantal release in N R approximates a binomial rather than a Poisson distribution process lz. Therefore, similar experiments were performed in N R containing added Mg z+ to a concentration which just blocked nerve-induced muscle contraction (7-9 mM). Under these conditions EPPs and MEPPs can be recorded simultaneously, yet QC is relatively high. The results from 4 such experiments are summarized in Table I, and it can be seen that, similar to its effect on curarized preparations, 10-4 M D P H caused a 30.0 4- 12.9 70 (S.D.) reduction in EPP amplitude. The QC in the control period, determined directly from mean EPP and MEPP amplitudes, ranged from 10.0 to 25.9 and D P H 1 × 10-a M caused, after 30 min of exposure, a 16.2 4- 7.170 (S.D.) reduction, which accounts for 5470 of the EPP depression. When the preparations were bathed in low Ca 2+ Ringer's solution, the effect of

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Fig. 5. The failure of response to low-frequency (0.5 Hz) nerve stimulation induced by DPH. The second stimulus of a pair succeeds in evoking an EPP when applied at short intervals after the first. A: sample records of paired stimuli taken during the control period, after 30 min exposure to 1 × 10.4 M D P H , and 10 min after starting to wash. B: the success of a second stimulus in evoking an EPP as a function of the time interval between the two stimuli. Note that, at the higher concentration of DPH, the duration of the 'facilitatory' effect of the first stimulus is reduced.

DPH on EPP amplitude was variable, being either a slight increase (in 6 out of 8 experiments, one of which is shown in Fig. 2) or a slight decrease (in 2 experiments). The control QC values were relatively low under these conditions, ranging from 0.6 to 8.9. The effect of D P H on quantal content paralleled its effect on the EPP amplitude. In 2 out of 8 experiments, D P H 1 × 10 -4 M reduced QC as it had in NR, but, in each of the other 6 experiments, D P H raised QC above control values, the mean increase being 38.8 -+- 26.1 ~ (S.D.) (Fig. 3).

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Effect of DPH on spontaneous transmitter release In NR, D P H augments the frequency of MEPPs 25. Similarly, in low Ca 2+ media as well as in Ca2+-free Ringer's solution containing 1 m M EGTA, 1-2 x 10-4 M D P H caused an increase in M E P P frequency (Fig. 4). The amount of increase in MEPP frequency seen in the different media ranged, in 11 experiments from 17 to 604 ~ , and bore no apparent relationship to the external Ca 2+ concentration. The MEPP amplitudes were also consistently reduced in all 11 experiments. Membrane potentials averaged --82.8 mV during the control period, --77.9 mV at the end of D P H perfusion and --78.0 mV at the end of the wash.

Other presynaptic effects of DPH In 3 experiments conducted in NR, D P H caused an abrupt disappearance of the EPP produced by single nerve stimuli. Quite surprisingly, when paired shocks were delivered to the nerve at short intervals, the second stimulus sometimes elicited an EPP (Fig. 5A). The success of the second stimulus in giving rise to an EPP depended on the time interval separating the two shocks, the shock being greater when the two stimuli were closer to each other (Fig. 5B). It could also be seen that increasing the D P H concentration from 1 to 2 x l0 -4 M shortened the maximum time interval in which this 'facilitatory' effect could be observed (Fig. 5B). When the D P H was washed away, the nerve quickly (5-10 min) regained its ability to elicit an EPP in response to a single stimulus (Fig. 5A). When the nerve was activated by a short train of high-frequency (100-200 Hz) stimuli in NR, each nerve stimulus gave rise to an EPP. However, when 1-2 x 10 -4 M D P H was applied, it was found in 3 out of 4 additional experiments that only a portion of the nerve stimuli could produce a postsynaptic response. In another experiment conducted in N R containing 8 m M MgCl2, this phenomenon appeared at an even lower frequency of stimulation, and gradually developed into a total neuromuscular block (Fig. 6). This effect was completely reversible on washing. DISCUSSION The presynaptic actions of D P H on transmitter release are complex. In

485 situations in which quantal content is relatively high, DPH depresses the neurally evoked transmitter release. In situations in which quantal content is low, the predominant effect of DPH is to enhance evoked release. On the other hand, the spontaneous liberation of transmitter quanta is augmented by DPH under all these conditions. The DPH-induced reduction is quantal content found in NR may result from a decrease in Ca 2+ influx into the presynaptic terminal during the nerve action potential. DPH is known to interfere with calcium transport across excitable membraneslS,zL Moreover, it has been shown recently that DPH inhibits Ca 2+ flux produced by depolarizing concentrations of K + ions into isolated presynaptic endings22. Alternatively, it has been shown that DPH exerts a 'local anesthetic' effect at the squid giant axon: it decreases the amplitude of the action potential by blocking the inward sodium current 12. If DPH exerted a similar effect at the presynaptic terminal, it would also result in a diminished Ca z÷ entry, and hence a reduction in quantal content. Although depressing the neurally evoked transmitter release in NR, DPH was found to increase MEPP frequency in NR as well as in CaZ+-free solutions. This seemingly paradoxical effect has been described for several other antagonists of calcium transport1,2,17. It is commonly accepted that the release of transmitter quanta is strongly dependent on the intracellular concentration of Ca 2+ ions (Ca)in which is kept very low at rest by intracellular buffering mechanisms (e.g. mitochondria)z,4. An increase in (Ca)in will be reflected as an increase in MEPP frequency. Substances which interfere with intracellular calcium sequestration augment MEPP frequency. Since DPH is known to penetrate nerve fibres well 10, it is possible that it also antagonizes the transport of calcium at these intracellular sites, causing an increase in (Ca)in and hence an increase in MEPP discharge. Supporting this hypothesis is the finding that the facilitatory action of DPH on MEPP frequency does not require the presence of extracellular Ca 2+ ions. This excludes the possibility that the effect is mediated by an increased influx of Ca 2+ ions into the resting nerve terminal. A rise in resting (Ca)in should also cause an increase in quantal content, for the amount of Ca z+ ions entering the terminal during the action potential will sum with the increased internal concentration to produce a higher quantal liberation. However, since DPH is assumed also to block the depolarization-coupled Ca 2+ entry, thereby depressing release, the actual change in quantal content will depend on the relative contributions of these two opposing processes. It would be expected that when the amount of Ca 9'+ ions entering the terminal is small in comparison to (Ca)in, the dominant effect of DPH will be to increase quantal content. Indeed, this was the main observed effect of DPH on quantal content in low-calcium media. On the other hand, when Ca z+ ion influx is relatively large and becomes the main determinant of quantal content, DPH should depress evoked quantal release. As discussed above, this may account for the reduction in quantal content found in NR. The DPH-induced reduction in EPP amplitude developed slowly during exposure to the drug. However, in 3 experiments an abrupt disappearance of the EPPs occurred. This effect could not be postsynaptic in nature, for a normal EPP could be elicited by a second stimulus if it was applied to the nerve shortly after the first. It is

486 therefore p r o b a b l e t h a t D P H can b l o c k the nerve a c t i o n p o t e n t i a l somewhere a l o n g its route o f p r o p a g a t i o n . S u b t h r e s h o l d d e p o l a r i z a t i o n at the p o i n t o f b l o c k a g e could then enable a second action p o t e n t i a l to o v e r c o m e the block. The site o f b l o c k m a y be at b r a n c h i n g points o f the p r e s y n a p t i c terminals, for such sites are k n o w n to have a low safety factor for impulse propagationll,14, 23. The occurrence o f this p r o p o s e d b l o c k m a y be related to the 'local anesthetic' or 'stabilizing' a c t i o n o f DPHS,10, ~2. This m a y also underly the failure in synaptic t r a n s m i s s i o n f o u n d at high frequencies o f stimulation. I n conclusion, our results suggest t h a t in a n o r m a l ionic e n v i r o n m e n t D P H depresses t r a n s m i t t e r release by interfering with the electrosecretory process a n d with the p r o p a g a t i o n o f action potentials along the p r e s y n a p t i c fiber. It is conceivable t h a t b o t h o f these actions involve an interaction o f D P H with m e m b r a n o u s calcium binding sites. It m a y also be t h a t the a n t i c o n v u l s a n t effect o f D P H is exerted in a p r i m a r y action o f the d r u g on c a l c i u m - m e d i a t e d n e u r o n a l activities. ACKNOWLEDGEMENTS The research r e p o r t e d in this p a p e r was a c c o m p l i s h e d in p a r t while Dr. Pincus was a M a c y F a c u l t y Scholar for the a c a d e m i c year 1974-1975. This w o r k was s u p p o r t e d by U S - I s r a e l Binational F o u n d a t i o n G r a n t 731 a n d grants f r o m the C.G. Swebilius T r u s t F u n d a n d the Dreyfus M e d i c a l F o u n d a t i o n .

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487 14 Parnas, I., Grossman, Y. and Spira, M. E., Changes in conductance and membrane potential associated with differential channeling of information in a bifurcating axon, Israel J. reed. ScL, 8 (1975) 106. 15 Pincus, J. H. and Lee, S. H., Diphenylhydantoin and calcium: relation to norepinephrine release in rat brain slices, Arch. Neurol., 29 (1973) 239-244. 16 Raabe, W. and Ayala, G. F., Diphenylhydantoin increases cortical postsynaptic inhibition, Brain Research, 105 (1976) 597-601. 17 Rahamimoff, R. and Alnaes, E., Inhibitory action of ruthenium red on neuromuscular transmission, Proc. nat. Acad. Sci. (Wash.), 70 (1973) 3613-3616. 18 Rahamimoff, R. andYaari, Y.,Delayed release of transmitter at the frogneuromuscularjunction, J. Physiol. (Lond.), 228 (1973) 241-257. 19 Raines, A. and Standaert, F. G., Pre- and post-junctional effects of diphenylhydantoin at the soleus neuromuscular junction, J. Pharmacol. exp. Ther., 153 (1966) 361-366. 20 Rotshenker, S., Erulkar, S. D. and Rahamimoff, R., Reduction in the frequency of miniature endplate potentials by nerve stimulation in low calcium solutions, Brain Research, 101 (1976) 362-365. 21 Selzer, M. E., The action of phenytoin on a vertebrate central nervous system synapse, Ann. Neurol., 1 (1977) 503. 22 Sohn, R. S. and Ferrendelli, J. A., Anticonvulsant drug mechanisms, Arch. NeuroL, 33 (1976) 626-629. 23 Spira, M. E., Yarom, Y. and Parnas, I., Modulation of spike frequency by regions of special axonal geometry and by synaptic inputs, J. Neurophysiol., 39 (1976) 882-889. 24 Woodbury, D. M., In H. H. Jasper, A. A. Ward and A. Pope (Eds.), Basic Mechanisms of the Epilepsies, Little, Brown, Boston, 1969, 647 pp. 25 Yaari, Y., Pincus, J. H. and Argov, Z., Inhibition of synaptic transmission by diphenylhydantoin, Ann. NeuraL, 1 (1977) 334-338.