Reciprocal modulation of glutamate and GABA release may underlie the anticonvulsant effect of phenytoin

Phenytoin alters glutamate and GABA release

Pergamon PII: S0306-4522(99)00468-6

Neuroscience Vol. 95, No. 2, pp. 343–351, 2000 343 Copyright q 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/00 $20.00+0.00

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RECIPROCAL MODULATION OF GLUTAMATE AND GABA RELEASE MAY UNDERLIE THE ANTICONVULSANT EFFECT OF PHENYTOIN M. O. CUNNINGHAM, A. DHILLON, S. J. WOOD and R. S. G. JONES* Department of Physiology, University of Bristol, School of Medical Sciences, University Walk, Bristol, BS8 1TD, U.K.

Abstract—Although conventional wisdom suggests that the effectiveness of phenytoin as an anticonvulsant is due to blockade of Na 1-channels this is unlikely to be it’s sole mechanism of action. In the present paper we examined the effects of phenytoin on evoked and spontaneous transmission at excitatory (glutamate) and inhibitory (GABA) synapses, in the rat entorhinal cortex in vitro. Evoked excitatory postsynaptic potentials at glutamate synapses exhibited frequency-dependent enhancement, and phenytoin reduced this enhancement without altering responses evoked at low frequency. In whole-cell patch-clamp recordings the frequency of excitatory postsynaptic currents resulting from the spontaneous release of glutamate was reduced by phenytoin, with no change in amplitude, rise time or decay time. Similar effects were seen on miniature excitatory postsynaptic currents, recorded in the presence of tetrodotoxin. Evoked inhibitory postsynaptic potentials at GABA synapses displayed a frequency-dependent decrease in amplitude. Phenytoin caused a reduction in this decrement without affecting the responses evoked at low frequency. The frequency of spontaneous GABA-mediated inhibitory postsynaptic currents, recorded in whole-cell patch mode, was increased by phenytoin, and this was accompanied by the appearance of much larger amplitude events. The effect of phenytoin on the frequency of inhibitory postsynaptic currents persisted in the presence of tetrodotoxin, but the change in amplitude distribution largely disappeared. These results demonstrate for the first time that phenytoin can cause a simultaneous reduction in synaptic excitation and an increase in inhibition in cortical networks. The shift in balance in favour of inhibition could be a major factor in the anticonvulsant action of phenytoin. q 1999 IBRO. Published by Elsevier Science Ltd. Key words: phenytoin, glutamate release, GABA release, entorhinal cortex.

Phenytoin has been widely used as an anticonvulsant for over 60 years. Despite this, it’s molecular mechanism of action is still not fully understood. The ability of phenytoin to block voltage-gated Na 1-channels in a voltage and use-dependent manner 29,35,47 has often been implicated in it’s anticonvulsant action. This local anaesthetic-like action results in a reduction of high-frequency firing of action potentials in central neurons, and has been thought to account for the efficacy of the drug in partial and generalized tonic-clonic seizures. 32 However, given the multiplicity of the effects of phenytoin on ion channels and synaptic transmission, 38 this is unlikely to account entirely for it’s actions as an anticonvulsant. Early studies suggested that effects on short-term plasticity at excitatory synapses could be of significance. Esplin 10 showed that monosynaptic excitatory responses in spinal cord (glutamatergic) and autonomic ganglia (cholinergic) evoked at low frequency (0.5 Hz) were little affected by phenytoin, but the post-tetanic potentiation which followed a highfrequency train (up to 500 Hz) was clearly reduced. Similar effects were reported for frog neuromuscular junction 40 and at the Schaffer collateral and mossy fibre synapses in the rat hippocampus. 14,30

The aims of the present study were to investigate the effects of phenytoin on aspects of glutamate and GABA-mediated synaptic transmission in the entorhinal cortex (EC), an area which is highly susceptible to seizure generation (see Ref. 18). With respect to epileptogenesis in the EC we have been studying a form of short-term plasticity which is prominent during relatively low frequency (1–3 Hz) repetitive activation of glutamate synapses in the entorhinal cortex EC in vitro. 18–20 This phenomenon is characterized by a progressive increase in amplitude of excitatory postsynaptic potentials (EPSPs) over a period of 10–15 s following a switch from low-frequency (0.1–0.2 Hz) activation. Responses remain stable at this level for as long as the higher stimulation rate is maintained, but revert rapidly (within 5–10 s) to control levels on returning to low frequency. In the present study we have examined the effect of phenytoin on this short-term, frequency-dependent enhancement of glutamate-mediated EPSPs, and show that it can reduce the enhancement without substantially affecting low-frequency responses. Previous studies of post-tetanic potentiation and our present results on frequency-dependent enhancement are indicative of a presynaptic effect of phenytoin on excitatory terminals. Crowder and Bradford 6 have shown that phenytoin can reduce the release of endogenous glutamate evoked by veratrine from rat cortical slices. In addition, the drug also reduced glutamate release from the rat ventral hippocampus in vivo following an electroshock-induced tonic-clonic seizure. 39 To examine the effects of phenytoin on glutamate release in more detail we have used the whole-cell patchclamp technique to record spontaneous, glutamate-mediated excitatory postsynaptic currents (EPSCs). We have found that phenytoin can reduce the frequency of glutamate-mediated EPSCs in entorhinal cortex with no detectable change in their amplitude distribution. Experiments on miniature

*To whom correspondence should be addressed. Abbreviations: ACSF, artificial cerebrospinal fluid; AMPA, a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid; 2-AP5, d,l-2-amino-5phosphonovalerate; BMC, bicuculline methochloride; CNQX, 6-cyano7-nitroquinoxaline-2,3-dione; EC, entorhinal cortex; EGTA, ethyleneglycolbis(aminoethylether)tetra-acetate; EPSCs, excitatory postsynaptic currents; EPSPs, excitatory postsynaptic potentials; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulphonic acid; IPSCs, inhibitory postsynaptic currents; IPSPs, inhibitory postsynaptic potentials; K-S, Kolmogorov–Smirnov; mEPSCs, miniature excitatory postsynaptic currents; mIPSCs, miniature inhibitory postsynaptic currents; NBQX, 6-nitro-7-sulphamoylbenzo[f]quinoxalone-2,3-dione disodium; NMDA, N-methyl-d-aspartate; PKC, protein kinase C; TTX, tetrodotoxin. 343

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EPSCs (mEPSCs) have shown that this effect on glutamate release is likely to divorced from blockade of either Na 1- or Ca 21-channels by the drug. In the past, some consideration has also been given to the possibility that phenytoin may potentiate synaptic inhibition. Although the drug has been reported to enhance GABAmediated, Cl-dependent inhibitory postsynaptic potentials (IPSPs) in crayfish stretch receptor, 1 the majority of evidence suggests that such effects do not occur at mammalian GABA synapses. 15,25 However, GABAergic IPSPs at cortical synapses also show short-term frequency-dependent changes during repetitive activation. Both GABAA and GABAB receptor-mediated IPSPs exhibit a frequency-dependent decrease in amplitude when evoked at moderate (1–5 Hz) frequencies. 7,18,30,45,46 We have examined the effect of phenytoin on GABA responses and found that neither GABAA nor GABAB receptor-mediated IPSPs evoked at low frequency (0.2 Hz) in EC are altered by phenytoin, but that the frequency-dependent fading of both potentials is attenuated by the drug. The studies of Crowder and Bradford 6 on rat cortical slices demonstrated a reduction in veratrine-stimulated GABA release by phenytoin which was similar to, or even greater than, it’s effect on glutamate release. In contrast, however, Rowley et al 39 found that GABA release following electroshock-induced seizure was unaffected by phenytoin. We conducted whole-cell patch-clamp recordings to look at the effect of phenytoin on spontaneous and miniature GABAmediated inhibitory postsynaptic currents (IPSCs). We found that phenytoin increased the frequency of spontaneous IPSCs and provoked the appearance of much larger events. The frequency of miniature IPSCs was also increased but without a concomitant change in amplitude. In summary, our findings have shown that the combined effects of phenytoin at excitatory and inhibitory cortical synapses would result in a switch in bias towards the latter. It is possible that these synaptic actions of the drug could be responsible for it’s anticonvulsant action. EXPERIMENTAL PROCEDURES

Slice preparation Experiments were performed on slices containing EC and hippocampus prepared from male Wistar rats (n ˆ 47, 120–150 g). All experiments were performed in accordance with the U.K. Animals (Scientific Procedures) Act 1986, European Communities Council Directive 1986 (86/609/EEC) and the University of Bristol ethical review document. All efforts were made to minimise the number of animals utilized in these experiments and to eliminate any suffering. Animals were decapitated under anaesthesia induced with ketamine (120 mg/kg) and xylazine (8 mg/kg) administered by intramuscular injection. The brain was rapidly removed and submerged in chilled (4–58C) artificial cerebrospinal fluid (ACSF) during dissection. Slices (450 mm) were cut using a Campden Vibroslice. They were transferred directly to the recording chamber where they were maintained at the interface between a continuous stream (1.2 ml/min) of ACSF and warm, moist carbogen gas (95% O2/5% CO2). The ACSF had the following composition (in mM): NaCl (126); CaCl2 (2.0); MgSO4 (2.0); NaH2PO4 (1.25); NaHCO3 (24.0); d-glucose (10) and had a pH of 7.4. The temperature of the recording chamber was maintained at 33 ^ 0.38C. Slices were allowed to equilibrate for at least 1 h before recording began. Intracellular recording of evoked responses Electrodes used for intracellular recording were filled with potassium acetate (3 M) and had resistances of 70–130 MV. Recordings

were made from neurons in layer II or layer IV/V of the medial division of the EC. Synaptic responses were evoked by electrical stimulation delivered via bipolar platinum wire electrodes placed on the surface of the slice. EPSPs were evoked by stimulating either in the white matter, when recording from layer IV/V or in layer I of the lateral EC when recording in layer II. Monosynaptic IPSPs were evoked by stimulation close to the recording site with excitatory transmission blocked with appropriate antagonists. Recorded neurons had stable resting potentials of 70–80 mV and input resistances of 40–90 MV. When control responses evoked at 0.1–0.2 Hz had remained stable for at least 10 min, the stimulation frequency was increased to 2 Hz and maintained at this frequency for 35–40 s. Thereafter, the frequency was returned to 0.1–0.2 Hz with no intervening period of non-stimulation. Phenytoin was used at a concentration of 50 mM in all experiments. After switching to the drug containing solution, stimulation at low frequency was continued for 10–15 min before re-testing the effects of 2 Hz stimulation. In a few cells we tested longer periods of perfusion (up to 45 min) but there was no additional effect of the drug during these long perfusion periods. There was little evidence of recovery either, even with prolonged (up to 2 h) periods of washing. To compare evoked potentials at different frequencies we averaged 8–10 responses at steady state. Statistical comparisons of pooled data were made with a Student’s t-test (significance level set at P , 0.05).

Whole-cell patch-clamp recordings Recordings were made “blind” from neurons in layers II and IV/V of the medial EC. Neurons were voltage-clamped and held at 260 mV using an Axopatch 200B amplifier. Electrodes were pulled from borosilicate glass of outer diameter 1.2 mm, and for recording EPSCs were filled with a solution containing (in mM) Cs-methansulphonate (130), HEPES (5), EGTA (0.5), MgCl2 (1), NaCl (1), CaCl2 (0.34) and QX314 (5). To record IPSCs the electrode solution contained (in mM): CsCl (135), HEPES (10), EGTA (5) MgCl2 (2), QX-314 (5), CaCl2 (0.5). Both solutions were adjusted to pH 7.3 and had an osmolarity of 290 mOsmol). Spontaneous synaptic activity was filtered at 2 kHz, digitized at 48 kHz and recorded on a DAT recorder or directly to computer hard disk using Axoscope software (Axon Instruments). Data was analysed using the Strathclyde Electrophysiological or MiniAnal (Jaejin Inc.) software. Events were detected automatically using a threshold crossing criterion. Threshold varied from neuron to neuron but was always maintained at a constant level in a given neuron. When data were pooled, at least 150 events from each neuron were included in the population analysis. Amplitudes and inter-event intervals are given as mean ^ S.E.M. For statistical comparison of data we used the Kolmogorov–Smirnov (K-S) non-parametric test applied to cumulative probability distributions of pooled data (significance level set at P , 0.005). Access resistance was monitored at regular intervals throughout each study and neurons were discarded if it changed by more than 10%. As with the intracellular studies, phenytoin was used at a concentration of 50 mM and spontaneous EPSCs or IPSCs were sampled 10–15 min into the drug perfusion. Again, the effect of the drug was already maximal at this stage, with this concentration. A lower concentration (10 mM, n ˆ 3, data not shown) was tested and gave similar results, but required longer perfusion periods (30–40 min). However, in a small number of neurons recorded in submerged slices, phenytoin at 10 mM (n ˆ 3, data not shown) produced similar effects after only 5–10 min. Neither effects on EPSCs nor IPSCs showed much recovery on washing for periods of up to 90 min.

Materials Salts used in the preparation of ACSF were obtained from BDH and were Analar grade. All drugs were applied by bath perfusion at concentrations stated in the text. The drugs used were phenytoin, (5,5 diphenylhydantoin sodium salt, Sigma); d,l-2-amino-5-phosphonovalerate (2-AP5, Tocris); bicuculline methochloride (BMC, Tocris); tetrodotoxin (TTX, Tocris, Sigma or Alamone); 6-cyano-7-nitroquinoxaline2,3-dione disodium (CNQX, Tocris); 6-nitro-7-sulphamoylbenzo[f]quinoxalone-2,3-dione disodium (NBQX, Tocris); 3-N[1-(S)-(3,4-dichlorophenyl)ethyl]amino-2-(S)-hydroxypropyl-P-cyclohexylmethyl phosphonic acid (CGP55845A, gift from Novartis).

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Table 1. Summary data for the effects of phenytoin on frequency-dependent changes in synaptic potentials

AMPA NMDA GABAA GABAB

Control

Phenytoin (50 mM)

n

69.1 ^ 8.5% 36.0 ^ 8.5% 33.2 ^ 5.3% 64.9 ^ 5.5%

33.8 ^ 11.8%* 11.0 ^ 9.1%* 21.9 ^ 5.3%* 48.3 ^ 6.0%*

6 6 4 5

In the case of AMPA and NMDA the numbers represent the mean (^S.E.M.) percentage increase in amplitude of EPSPs evoked at 2 Hz compared to 0.2 Hz. For the GABA potentials the figures are the mean percentage decrease in amplitude of the IPSPs at 2 Hz compared to 0.2 Hz. *P , 0.05.

Fig. 1. Phenytoin reduces frequency-dependent enhancement of EPSPs in entorhinal cortex neurons. The upper line shows NMDA receptor-mediated EPSPs evoked in a layer V neuron following blockade of AMPA (CNQX), GABAA (BMC) and GABAB receptors (CGP55845). The trace on the left is the average of eight responses evoked at low frequency (0.2 Hz). The middle trace is the average of eight responses recorded 32 s after starting stimulation at 2 Hz, when the increase in amplitude of the EPSPs had reached a steady state. The responses at the two frequencies are superimposed on the right. Equivalent responses recorded 12–15 min after starting perfusion with phenytoin (lower line) show that the enhancement of the EPSPs at 2 Hz was abolished, with no effect on the low-frequency response. Scale bars ˆ 80 ms and 15 mV.

amplitude of the EPSP increased by 31% after 30 s stimulation at 2 Hz compared to the response at 0.2 Hz. Following the addition of phenytoin, the enhancement was abolished. The pooled data from six cells (Table 1) showed a significant (P , 0.05) reduction in frequency-dependent enhancement by phenytoin. However, the drug had no significant effect on the peak amplitude of the responses evoked at low frequency (control 7.9 ^ 0.9 mV vs 7.2 ^ 0.9 mV in phenytoin). The reduction of frequency-dependent enhancement was not confined to the slow EPSPs. Summary data for six cells in which we isolated fast, AMPA receptor-mediated EPSPs are also shown in Table 1. The peak amplitude of the fast EPSP at 2 Hz was increased by around 70% compared to 0.2 Hz, in the control situation. Addition of phenytoin reduced the degree of enhancement by half. Again the drug had no effect on the amplitude of the EPSP evoked at low frequency (14.5 ^ 2.3 mV vs 14.6 ^ 2.8 mV).

RESULTS

The current studies have all been conducted on neurons in the rat EC. We have previously suggested 21 that the deeper layers IV/V may be more susceptible to epileptogenesis than the superficial layer (II), and provided information as to why this may be so. 2,18,50 In the present study, we studied neurons in both deep and superficial layers but could detect no difference in the effects of phenytoin in the different layers. Phenytoin reduces frequency-dependent enhancement of glutamate excitatory postsynaptic potentials We used intracellular recording from neurons in layer II and in layers IV/V to study the effect of phenytoin on frequency-dependent changes in glutamate and GABA transmission. The results presented are for layer IV/V cells. Recordings were made in the medial EC and EPSPs were elicited by electrical stimulation of afferent pathways in the white matter. Fast EPSPs mediated by a-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid (AMPA) receptors were isolated in six cells by perfusion with 2-AP5 (50 mM), BMC, (5 mM) and CGP55845A (1 mM). Slow, N-methyl-daspartate (NMDA) receptor-mediated EPSPs were recorded by substituting CNQX (10 mM) for 2-AP5 in a further six cells. Increasing the frequency of stimulation from 0.2 to 2.0 Hz resulted in a frequency-dependent enhancement of both fast and slow EPSPs, which reached a plateau over a period of 15 to 30 s. Responses returned to control levels within 10 s of switching from 2 Hz back to 0.2 Hz. An example of this enhancement of NMDA receptormediated responses is shown in Fig. 1. In this cell, the peak

Phenytoin decreases the frequency of glutamate-mediated excitatory postsynapic currents The reduction of frequency-dependent enhancement of EPSPs could reflect either a pre- or postsynaptic action of phenytoin although the former would seem more likely (see Discussion). To look more closely at pre- vs postsynaptic mechanisms we studied the effect of phenytoin on spontaneous release of glutamate from synaptic terminals, using whole-cell patch-clamp recordings. Neurons in either layer II or IV/V, voltage clamped at 260 mV, displayed frequent inward currents of up to 120 pA in amplitude. Previous studies have shown that the vast majority of these events reflect spontaneous EPSCs mediated by the release of glutamate from synaptic terminals. 2 The released transmitter acts primarily via AMPA receptors, although pure NMDA receptor-mediated EPSCs can be recorded in these neurons. 2 However, these occur very infrequently (every 15–40 s), and in the current experiments we have not tried to analyse them separately. Phenytoin reduced the frequency of spontaneous EPSCs without affecting their amplitude; an example is shown in Fig. 2a. In pooled data from five layer II neurons (Fig. 2a, minimum of 150 EPSCs from each neuron) spontaneous EPSCs had a mean amplitude of 11.7 ^ 0.2 pA and an interevent interval of 0.52 ^ 0.02 s. During perfusion with phenytoin there was no change in mean amplitude (11.4 ^ 0.2 pA) but the inter-event interval was almost doubled (0.99 ^ 0.05 s), reflecting a substantial decrease in the frequency. Analysis of cumulative probability distributions (Fig. 2a) using the K-S test showed this change in frequency

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Fig. 2. Phenytoin decreases the frequency but not amplitude of spontaneous glutamate-mediated EPSCs in entorhinal cortex neurons. (a) The three traces on the top left are consecutive sweeps, showing inward currents mediated by glutamate release in a layer II neuron in control conditions (without the addition of any drugs). The traces on the right were recorded in the same neuron in the presence of phenytoin, and show a clear decrease in frequency of events. The graphs below show cumulative probability distributions of event amplitudes and intervals pooled from five layer II neurons (at least 150 events from each cell in each situation). Distributions in control and phenytoin are indicated by the letters c and p, respectively. There was a shift to the right of the interval distribution in the presence of phenytoin, reflecting a decrease in frequency. The amplitude distribution was unaltered. (b) Control traces on the left are from a layer II neuron in the presence of TTX to isolate mEPSCs. In the presence of phenytoin there was again a clear decrease in frequency. The cumulative probability distributions show a decrease in frequency with no change in amplitude (pooled data from five neurons). (c) mEPSCs recorded in the presence of the GABAA blocker, bicuculline. Once again, phenytoin decreased the frequency with no change in amplitude distribution. (d) Averaged mEPSCs (of 30 events) recorded in the presence of BMC from one neuron. The superimposed traces demonstrate a lack of change in either amplitude, rise or decay time in the presence of phenytoin. The scale bar between (a) and (c) represents 250 ms and 15 pA with respect to all traces in a–c. The scale bar in (d) represents 10 pA and 40 ms.

to be highly significant (P , 0.0001). Rise times (10–90%) and decay times (t50%) were unaltered in the presence of phenytoin (not shown). Similar results were obtained in layer V neurons (n ˆ 5). The mean amplitude and interval were 16.0 ^ 0.35 pA and 1.02 ^ 0.07 s in control and 14.47 ^ 0.41 pA and 3.4 ^ 0.29 s in the presence of phenytoin, respectively. K-S analysis of cumulative probabilities confirmed a significant (P , 0.0001) reduction in frequency with no change in amplitude. Spontaneous excitatory activity in the EC reflects glutamate release which results from action potentials invading synaptic terminals, and release which is action potential independent, i.e. mEPSCs. 2 One possibility which we considered was that phenytoin was reducing the overall frequency of EPSCs by blocking presynaptic Na 1-channels, and thus removing the activity-dependent events. We therefore repeated the experiments in a further five layer II neurons, this time in the presence of TTX (0.5 mM). Perfusion with phenytoin again resulted in a reduction in frequency of mEPSCs (control interval: 0.67 ^ 0.03 s vs phenytoin: 2.64 ^ 0.4 s), with no change in amplitude (12.4 ^ 0.1 vs 11.9 ^ 0.2 pA). An example of the effect of phenytoin on mEPSCs is shown in Fig. 2b, together with the cumulative probability analysis of the pooled data from five neurons. Finally, in the above experiments, we did not attempt to block spontaneous inhibition since with the holding potential and pipette solutions we employed, the inward currents we recorded were almost abolished by the addition of NBQX and 2-AP5 (not shown, see Ref. 2). However, since we found that

phenytoin could modify evoked and spontaneous GABA transmission (see below), to exclude any possible complications from this, and to be certain we were looking at isolated glutamate-mediated mEPSCs, in three further layer II neurons we added BMC (5 mM) in addition to TTX (1.0 mM). Again, phenytoin substantially reduced the frequency of mEPSCs (control interval 1.15 ^ 0.08 vs 2.66 ^ 0.23 s in phenytoin) with no change in amplitude (8.78 ^ 0.15 vs 8.81 ^ 0.19 pA) (Fig. 2e). K-S analysis confirmed the significance of the interval change (P , 0.00001). Figure 2c shows an individual example and the pooled data from three neurons. The traces in Fig. 2d are averaged mEPSCs recorded in BMC in the control situation, and following addition of phenytoin. Superimposition of the traces shows that amplitude and rise and decay times were little affected. Thus, phenytoin is clearly able to reduce excitation at glutamate synapses. If epilepsy arises as a result of excess excitation over inhibition in cortical networks, then this action of phenytoin alone could be important in tilting the balance towards inhibition. However, in view of previous studies which have indicated that phenytoin may have a potentiating effect on GABA-mediated inhibition 13,30 it seemed pertinent to study the effects of phenytoin on GABA transmission as well. Phenytoin reduces the frequency-dependent fading of GABA inhibitory postsynaptic potentials GABA-mediated inhibition in EC, 18 as at other cortical synapses undergoes a frequency-dependent decrement if

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Phenytoin increases the frequency of spontaneous, GABAmediated inhibitory postsynaptic currents

Fig. 3. Phenytoin reduces frequency-dependent decrement of IPSPs in entorhinal cortex neurons. The upper line shows GABAA receptor-mediated EPSPs evoked in a layer V neuron following blockade of AMPA (CNQX), NMDA (2-AP5) and GABAB receptors (CGP55845A). The trace on the left is the average of eight responses evoked at low frequency (0.2 Hz) and the middle trace is the average recorded 32 s after starting stimulation at 2 Hz. The responses are superimposed on the right. The lower line shows equivalent responses recorded 12–15 min after starting perfusion with phenytoin, and shows that the decrement in the IPSPs at 2 Hz was reduced with no effect on the low-frequency response. Scale bars are 20 ms and 15 mV.

stimulation rates much above 0.5 Hz are employed. 7,30,45,46 Figure 3 shows GABAA-mediated IPSPs isolated by perfusion with 2-AP5, CNQX and the GABAB antagonist, CGP55845A. Stimulation at 2 Hz resulted in a 67% reduction in peak amplitude of the IPSP compared to that evoked at 0.2 Hz. During subsequent perfusion with phenytoin, the reduction at 2 Hz was only 35%. Summary data for four neurons is given in Table 1 and shows that the effect of phenytoin on the decrement in IPSPs at 2 Hz was significant (P , 0.05). As with the EPSPs, phenytoin did not alter the amplitude of the low-frequency IPSPs. In the four neurons tested, IPSPs at 0.2 Hz had a peak amplitude of 6.8 ^ 0.4 mV in the control situation and 6.5 ^ 0.2 mV in phenytoin. In five further neurons we isolated GABAB-IPSPs by perfusion with 2-AP5, CNQX and BMC; the data is summarized in Table 1. The mean peak amplitude of such responses evoked at 0.2 Hz was 5.8 ^ 0.9 mV. This was unchanged in the presence of phenytoin (5.4 ^ 0.6 mV). However, GABABIPSPs, like their GABAA counterparts decreased in amplitude when evoked at 2 Hz and this decrease in control conditions amounted to a 65 ^ 5% reduction in peak amplitude. Also, like the GABAA responses, the frequency-dependent attenuation of GABAB-IPSPs was significantly (P , 0.05) less in the presence of phenytoin (49 ^ 6%). The ability of phenytoin to alter the frequency-dependent decrement of both GABAA and GABAB responses, together with a lack of effect on low-frequency responses could suggest an effect of the drug on presynaptic GABA release. To clarify whether phenytoin was having pre- and/or postsynaptic effects at GABA synapses we conducted whole-cell patchclamp recordings of IPSCs.

With symmetrical intra and extracellular Cl 2 concentrations, and in the presence of 2-AP5 (50 mM) and NBQX (5 mM) both layer IV/V and layer II neurons, clamped at 260 mV, displayed inward currents which could be blocked by BMC (5–10 mM), confirming that they were spontaneous IPSCs mediated via GABAA receptors. In five layer II neurons, addition of phenytoin resulted in a decrease in the inter-event interval (0.29 ^ 0.01 vs 0.13 ^ 0.004 s) and hence an increase in frequency. In addition, there was an increase in mean amplitude of the IPSCs (control vs phenytoin: 17.94 ^ 0.26 vs 20.73 ^ 0.38 pA). Both the interval and amplitude changes were significant (P , 0.0001). An example is shown in Fig. 4a, together with pooled data for the five neurons. Also shown are cumulative probability distributions of rise times and decay times of the IPSCs in these neurons. There was no change in the presence of phenytoin. Control IPSCs had mean rise time (10–90%) of 3.96 ^ 0.23 ms and a decay time (t50%) of 9.04 ^ 0.31 ms. The corresponding values in phenytoin were 3.67 ^ 0.21 ms and 8.24 ^ 0.32 ms. The traces in Fig. 4b are averaged IPSCs recorded in the presence and absence of phenytoin. Although the amplitude of the IPSC was greater in phenytoin, when the control response was scaled to the same amplitude and superimposed there was clearly very little difference in rise or decay. Similar effects of phenytoin on spontaneous IPSCs have also been observed in layer V neurons. Pooled data from five neurons gave a mean amplitude of 10.62 ^ 0.21 with an inter-event interval of 0.5 ^ 0.02 s. Phenytoin increased the amplitude to 19.02 ^ 0.01 pA whilst decreasing the interval to 0.26 ^ 0.01 s. As with layer II neurons neither rise nor decay times for spontaneous or miniature events were altered in phenytoin (data not shown). In five further layer II neurons we have looked at the effect of phenytoin on IPSCs in the absence of action potential driven release following the addition of TTX, i.e. miniature IPSCs (mIPSCs). mIPSCs in these five neurones had a mean amplitude and interval of 40.7 ^ 1.03 pA and 0.16 ^ 0.009 s, respectively. During perfusion with phenytoin these values changed to 42.16 ^ 0.96 pA and 0.10 ^ 0.004 s. K-S analysis revealed a significant (P , 0.0001) increase in frequency. However, in contrast to the situation where Na 1-channels were not blocked, the change in amplitude was not significant. Neither rise (3.2 ^ 0.15 vs 2.96 ^ 0.13 ms) nor decay (8.04 ^ 0.23 vs 8.02 ^ 0.22) times of mIPSCs were altered by phenytoin. DISCUSSION

Glutamate transmission A number of studies have suggested that low-frequency responses at glutamate synapses, or responses to exogenous activation of AMPA and NMDA receptors, may be reduced by phenytoin. 3,14,23,48 In agreement with the data of others 24–26,30,32 we found little effect of phenytoin on either AMPA or NMDA-mediated responses evoked at low frequency, indicating that phenytoin is unlikely to interact directly with the postsynaptic receptors in the EC. However, the frequencydependent enhancement of EPSPs was reduced by phenytoin. Since such enhancement of evoked responses probably reflects an increased release of glutamate, 36 it seems likely

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Fig. 4. Phenytoin increases the frequency and amplitude of spontaneous IPSCs. (a) The traces show that following the addition of phenytoin the frequency of events clearly increased, and many larger events were evident. The cumulative probability distribution (pooled data from five neurons) for event amplitudes is shifted to the right in phenytoin (p) as a consequence of the presence of larger events. At the same time the interval distribution is shifted left-wards reflecting the increased frequency. Neither rise times (10–90%) nor decay times (t50%) of events were altered by the drug. This is shown by the graphs in the bottom panel of (a) and the averaged IPSCs (70 events) in (b). The averaged IPSCs in the presence and absence of phenytoin, scaled to the same peak amplitude and superimposed show very little difference in kinetics. (c) mIPSCs recorded in the presence of TTX. Despite the high frequency of events seen in control conditions in this neuron, phenytoin still caused a substantial increase in frequency. The cumulative probability distributions of event intervals (pooled data from five neurons) was significantly shifted to the left by phenytoin reflecting this increase. However, the amplitude distribution was not significantly altered. Scale bars represent 50 ms and 40 pA (a and c) and 10 ms and 30 pA (b).

that phenytoin was acting presynaptically, particularly since the enhancement of both fast and slow EPSPs was equally affected. The effect of phenytoin could be due to a direct action of the drug on glutamate terminals (see below), but another possibility is that it could result from a use-dependent blockade of axonal Na 1-channels. It has been repeatedly shown that blockade of Na 1-channels by phenytoin is increased with increasing frequency of activation. 29,35,47 Conventional wisdom suggests that it is this very property which underlies the anticonvulsant action, by enabling the drug to ameliorate sustained, high-frequency firing of somatic action potentials. 32 However, use-dependent blockade of Na 1-channels is already apparent at the relatively low frequencies (1–3 Hz) of activation 29,35,47 that we have employed to elicit enhancement of glutamate responses. It is possible that when we stimulate at 1–3 Hz in the presence of phenytoin the evoked action potentials are partially and progressively blocked, and this would result in reduced transmitter release from the glutamate terminals. This could also explain why the low-frequency responses were unaltered. Selzter et al. 39 attributed a reduced enhancement by phenytoin of cholinergic transmission at the frog neuromuscular junction to frequencydependent block of axonal action potentials. Our studies of spontaneous glutamate release suggest an alternative or additional presynaptic action of phenytoin. It’s lack of effect on the amplitude, rise time or decay time of EPSCs supports our conclusion that the drug does not block postsynaptic glutamate receptors, but the reduction in frequency is a clear pointer to a presynaptic reduction of glutamate release. Phenytoin has been shown to inhibit the release of glutamate and aspartate from brain tissue, 6 but this effect was relatively specific for release induced by veratrine as opposed to high K 1-stimulation. Because of this it was suggested that release was inhibited as a result of a reduction

in voltage-activated Ca 21 entry, secondary to blockade of presynaptic Na 1-channels (although direct inhibition of voltage-gated Ca 21 channels by phenytoin has also been reported 43). It is possible that such an effect could account for the reduced frequency of spontaneous EPSCs by phenytoin, since about 15–20% of these events are driven by action potentials invading terminals. 2 However, the decrease in frequency of mEPSCs by phenytoin seen in the presence of TTX, where action potential dependent release of glutamate would not occur, means that glutamate release can be reduced by phenytoin independently of an effect on presynaptic Na 1channels. The reduction in release is also unlikely to result from blockade of voltage-gated Ca 21 channels, since these would be unlikely to open in the absence of action potentials. Also, we have shown that phenytoin still reduces release in the presence of TTX plus Cd 21 (Woodhall, Evans, Cunningham and Jones, unpublished observations). Rather, it seems more likely that phenytoin has it’s effect downstream of Ca 21 entry. There is no reason a priori to assume that the effects of phenytoin on spontaneous and evoked release occur via the same mechanism, but it is tempting to think that they might. One possible target for phenytoin in this respect could be via inhibition of protein phosphorylation. Many of the proteins involved in the complicated sequence of interactions leading to vesicle fusion and transmitter release (see Ref. 44) have been shown to undergo phosphorylation 11,13,16,37,41 and protein kinase activation can alter transmitter release. In particular, transmitter release at various synapses is increased by activation of protein kinase C (PKC), 4,28,51 an effect which can occur downstream of Ca 21 entry. DeLorenzo 8 showed that phenytoin could block protein phosphorylation in rat and human neocortex, and has speculated 9 that the anticonvulsant effect of the drug could depend on PKC inhibition. Thus, blockade

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of PKC by phenytoin could be involved in the reduction in glutamate release that we have observed. We have recently found that inhibition of PKC can reduce the degree of frequency-dependent enhancement at glutamate synapses (Heltovics and Jones, unpublished observations), and this may provide a mechanism to reduce release downstream of Ca 21 entry. We are currently investigating this possibility.

GABA synapses There has been some argument over whether phenytoin can potentiate synaptic inhibition mediated by GABA. GABAmediated, Cl- 2dependent IPSPs in crayfish stretch receptor 1 were enhanced by the drug, as were responses to exogenously applied GABA in a number of different preparations. 12,22,32 However, other studies have shown that potentiation does not occur at mammalian GABA synapses. 5,15,24 In agreement with the latter observations we found that GABA-mediated responses evoked at low frequency were unaffected by phenytoin, arguing against a postsynaptic effect at the GABA receptor. However, the decrement in these responses seen at higher frequencies was lessened in the presence of the drug. The basis of the frequency-dependent reduction in IPSPs is still not fully known. A number of factors have been implicated, including changes in ionic reversal potentials due to repetitive activation of postsynaptic receptors, desensitization of postsynaptic receptors and presynaptic autoreceptor-mediated inhibition of release. 7,31,45,46 Paradoxically, perhaps, one explanation for the effect of phenytoin could be that at the higher frequencies, the drug exerts a use-dependent block of action potentials in GABA neurons or their axons, resulting in a ‘reduced’ release of GABA. The lowered concentration of GABA in the synaptic cleft would then result in weakened activation of whatever mechanism was responsible for the decrement in GABA responses. However, it could also be that evoked GABA release is increased by phenytoin and the increased GABA present is enough to produce a bigger IPSP at high frequency despite the operation of whatever mechanism is responsible for the response decrement. The latter scenario is easier to reconcile with data from our patch-clamp studies, where we found a clear increase in frequency of spontaneous IPSCs in phenytoin. The increase in frequency, which applied also to mIPSCs, clearly indicates an increase in GABA release from presynaptic terminals. In addition, in the absence of TTX, we found a significant shift in the amplitude distribution of IPSCs, with much larger events detected in the presence of phenytoin. An increase in amplitude could reflect a postsynaptic potentiation at GABA receptors. As noted earlier, some previous studies suggested that phenytoin can enhance responses to GABA. 1,22,33 However, we feel that this is unlikely to be the case since we found no potentiation of the evoked IPSPs at low frequency, and neither rise nor decay times of spontaneous IPSCs were altered by phenytoin. Also, when Na 1-channels were blocked with TTX the change in amplitude was no longer apparent, so it seems likely that the large amplitude events seen in phenytoin resulted from enhanced action potential-dependent release from presynaptic terminals. One possible explanation for this effect is that phenytoin may be blocking K 1 channels in GABA axons and/or terminals. Southan and Robertson 42 have recently reported that blockade of a-dendrotoxin-sensitive voltage-gated K 1 channels (Kv) in

cerebellar basket cell terminals causes a pronounced increase in amplitude and frequency of GABA IPSCs in cerebellar Purkinje cells. We do not know whether phenytoin has an effect on a-dendrotoxin-sensitive Kv, although it has been shown to block other K 1 channels. 33 There is a high density of the dendrotoxin-sensitive Kv1.2 sub-unit in the medial EC, 49 so it is conceivable that phenytoin could increase IPSCs by blocking such channels in inhibitory terminals in the EC. In preliminary experiments (n ˆ 5, not shown) we have found that a-dendrotoxin has a similar effect on GABA release in EC to that seen in cerebellum. 42 We are now testing whether the toxin may occlude the increase caused by phenytoin. Of course, such an action is unlikely to explain the increased frequency of mIPSCs by phenytoin seen in TTX. With analogy to the decrease in mEPSCs it seems likely that the effect of phenytoin on mIPSCs may occur downstream of Ca 21 entry at some point in the complex release machinery. Since the quantal release of glutamate and GABA is affected in opposite directions the effect of phenytoin in excitatory terminals must differ from that in inhibitory terminals. This also raises a speculative but interesting possibility. It could suggest that the release mechanism may be different for the two transmitters, a possibility not considered previously. It is likely that epileptogenesis in cortical networks results from a shift in balance between inhibition and excitation, in favour of the latter. The present experiments show that phenytoin can redress this balance, shifting the bias back towards inhibition. That the effect on evoked responses is confined to higher frequencies may be of significance. Synchronized epileptiform activity would involve high frequency firing within cortical networks, so the modification of short-term plastic changes in inhibition and excitation could well be involved in limiting the spread of epileptiform activity. The balance between spontaneous release of GABA and glutamate is certainly of importance in determining overall levels of excitability in cortical networks. 27,34 That phenytoin could set a lower level, by simultaneously increasing GABA release and decreasing glutamate release, may also be of importance in maintaining a seizure-free condition. It is of considerable interest that Hirsch et al 17 have recently reported that GABAergic mIPSCs in CA1 neurons are reduced in frequency in two models of temporal lobe epilepsy. They suggest that this effect is a result of impairment of the vesicle release machinery in GABA synapses, leading to depletion of the reserve pool. They also suggest that this may be a new target for anticonvulsant drugs. Our studies with phenytoin, indicate that a widely used and long established drug may actually act, at least in part, at this “new” target.

CONCLUSION

The present results show that phenytoin effectively increases synaptic inhibition and at the same time decreases synaptic excitation. These reciprocal effects on both background and evoked inhibition and excitation are highly desirable actions required in an effective anticonvulsant.

Acknowledgements—We thank the Wellcome Trust, the MRC and the Taberner Trust for financial support, Dr John Dempster for the Strathclyde Software, and Novartis for the gift of CGP55845A.

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