Effects of seven clinically important antiepileptic drugs on inhibitory glycine receptor currents in hippocampal neurons

Epilepsy Research 58 (2004) 27–35

Effects of seven clinically important antiepileptic drugs on inhibitory glycine receptor currents in hippocampal neurons Kameel M. Karkar a,b,1 , Liu Lin Thio a,b,c,d , Kelvin A. Yamada a,b,c,d,e,∗ a

Department of Neurology, Washington University School of Medicine, 660 South Euclid Avenue, Box 8111, St. Louis, MO 63110, USA b Center for the Study of Nervous System Injury, Washington University School of Medicine, St. Louis, MO 63110, USA c Department of Pediatric Neurology, St. Louis Children’s Hospital, St. Louis, MO 63110, USA d Pediatric Epilepsy Center, St. Louis Children’s Hospital, St. Louis, MO 63110, USA e Department of Pediatrics, St. Louis Children’s Hospital, St. Louis, MO 63110, USA Received 28 July 2003; received in revised form 11 December 2003; accepted 17 December 2003

Abstract Although potentiation of the inhibitory glycine receptor (GlyR) may contribute to the mechanism of action of antiepileptic drugs (AEDs), the effects of AEDs on GlyRs have not been investigated in detail in forebrain neurons. We examined the effects of seven clinically important AEDs on GlyR-mediated currents using whole-cell patch clamp recordings from cultured embryonic mouse hippocampal neurons. At high therapeutic concentrations, topiramate (in 24% of neurons) and pentobarbital reversibly decreased glycine currents to 78 ± 6% and 81 ± 7% of control, respectively. At or below therapeutic concentrations, carbamazepine, felbamate, gabapentin, phenytoin, and valproate had no effect on glycine currents, while at supratherapeutic concentrations these agents produced modest reversible inhibition. We conclude that GlyR potentiation does not contribute to the antiepileptic action of the seven AEDs examined. © 2004 Elsevier B.V. All rights reserved. Keywords: Carbamazepine; Felbamate; Gabapentin; Pentobarbital; Phenytoin; Topiramate; Valproate

1. Introduction Inhibitory glycine receptors (GlyRs) along with ␥-aminobutyric acidA receptors (GABAA Rs) are the principal mediators of fast inhibitory synaptic transmission in the mammalian spinal cord and brainstem (Rajendra et al., 1997). Although GlyRs are tradition∗ Corresponding author. Tel.: +1-314-362-3585; fax: +1-314-362-9462. E-mail address: [email protected] (K.A. Yamada). 1 Present address: UCSF Epilepsy Center, Department of Neurology, University of California, San Francisco, CA, USA.

ally thought to be absent from the mammalian cortex, multiple experimental methods support their presence in the cortex (Krishtal et al., 1988; Raiteri et al., 1990; Naas et al., 1991; Engblom et al., 1996; McCool and Botting, 2000; Chattipakorn and McMahon, 2002) where they may be involved in tonic inhibition rather than fast inhibitory neurotransmission (Mori et al., 2002). Therefore, GlyR modulators may be useful agents for treating epilepsy. The following observations support the use of GlyR potentiators as antiepileptic drugs (AEDs). First, systemic administration of strychnine, a selective GlyR antagonist, causes seizures in both humans (Yamarick

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et al., 1992) and rodents (Roches et al., 1979). Second, focal cortical applications of strychnine cause seizures in rodents (Mutani et al., 1974). Third, ␣-ethyl-␣-methyl-␥-thiobutyrolactone (␣EMTBL), an anticonvulsant in rodents (Ferrendelli et al., 1989), potentiates recombinant GlyRs composed of ␣1 homomers and ␣1 ␤ heteromers (Steinbach et al., 2000). Fourth, GlyR activation suppresses neuronal hyperexcitability and seizure-like events in the rat entorhinal cortex and hippocampus (Chattipakorn and McMahon, 2003; Kirchner et al., 2003). Theoretically, GlyR potentiation may contribute to the mechanism of action of AEDs, but the few studies that have examined how AEDs modulate GlyRs have focused on GlyRs outside the forebrain. For example, gabapentin had no effect on spinal cord GlyRs despite protecting mice from strychnine-induced convulsions (Rock et al., 1993). Interestingly, although levetiracetam does not directly modulate GlyRs, it potently reverses the inhibition of GlyRs and GABAA Rs by the negative allosteric modulators zinc and ␤-carboline in embryonic spinal cord neurons (Rigo et al., 2002). In the same study, carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, and valproate weakly inhibited GlyRs in mouse embryonic spinal cord neurons. Finally, pentobarbital potentiates expressed GlyRs having the same subunit composition found in the spinal cord (␣1 ␤ heteromers) (Pistis et al., 1997), it inhibits GlyRs in septal neurons (Kumamoto and Murata, 1996), and it has no effect on native GlyRs in spinal neurons (Ransom and Barker, 1976). While these studies indicate that AEDs modulate GlyRs, they do not address the importance of these effects to the treatment of epilepsy since forebrain GlyRs have a different subunit composition from brainstem and spinal GlyRs (Malosio et al., 1991) and may have different responses to modulators. GlyRs in spinal neurons switch postnatally from ␣2 homomers to ␣1 ␤ heteromers (Rajendra et al., 1997). In contrast, GlyRs in our cultured embryonic hippocampal neurons are predominantly ␣2 ␤ heteromers (Thio et al., 2003), and we have shown that these GlyRs are biphasically modulated by tropisetron (Thio et al., 2003) and by zinc (Thio and Yamada, 2003). Here we explore whether GlyR potentiation contributes to the mechanism of action of seven clinically important AEDs thought to have differing mechanisms of action: carbamazepine, felbamate, gabapentin, pen-

tobarbital, phenytoin, topiramate, and valproate. We found that none potentiated GlyR-mediated whole-cell currents in cultured embryonic mouse hippocampal neurons. A preliminary version of some of this work has been reported in abstract form (Karkar et al., 2001). 2. Methods 2.1. Embryonic mouse hippocampal cultures Embryonic mouse hippocampal neurons were prepared as described previously (Thio et al., 2003). Briefly, timed pregnant (E16) Swiss Webster mice were anesthetized with halothane and sacrificed by cervical dislocation. Embryos were removed by cesarean section and were decapitated. After the brains were removed, the hippocampi were dissected and sliced in Leibovitz’s L-15 media containing 0.4 mg/ml bovine serum albumin (BSA) before enzymatic digestion at 37 ◦ C for 20 min with 1 mg/ml of papain. The slices were then gently triturated with a glass pipette in plating media containing 1× minimum essential medium (MEM) with Earle’s salts, 10% Nu-Serum, 5 mg/ml glucose, 2.2 mg/ml NaHCO3 , 20 units/ml penicillin, and 20 ␮g/ml streptomycin. The cell suspension was then centrifuged for 5 min at 1000 rpm in the presence of 0.25 mg/ml BSA and 0.25 mg/ml trypsin inhibitor. The pellet was resuspended in plating media and then plated on a monolayer of passaged cortical astrocytes at a density of 2.5 × 105 cells/ml. 5-Fluoro-2 -deoxyuridine and uridine were added to a final concentration of 15 and 35 ␮g/ml, respectively, at 48 h to inhibit glial proliferation. Recordings were made from neurons cultured for 5–20 days. We could not distinguish the different hippocampal neuronal cell types in our culture preparation. Animal care and experimentation conformed to PHS Guide for Care and Use of Laboratory Animals, AVMA Panel on Euthanasia Guidelines, and were approved by the Washington University School of Medicine Animal Studies Committee. 2.2. Electrophysiology Whole-cell currents were recorded from neurons voltage-clamped at −65 mV using an Axopatch 200A amplifier (Axon Instruments, Inc., Union City, CA).

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Neurons were bathed in a solution containing (mM): 140 NaCl, 5 KCl, 1 MgCl2 , 1.5 CaCl2 , 10 HEPES, 10 d-glucose, and 0.0005 tetrodotoxin at a pH of 7.36–7.40. Recording pipettes had resistances of 2–4-M and were filled with a solution containing (mM): 140 CsCl, 4 NaCl, 0.5 CaCl2 , 5 EGTA, and 10 HEPES at a pH of 7.36–7.40. In some experiments, the 140 mM CsCl in the pipette solution was replaced by 140 mM Cs methanesulfonate (CsCH3 SO3 ) or 70 mM CsCl + 70 mM CsCH3 SO3 . All holding potentials were corrected for junction potentials, which were determined empirically (Neher, 1992). Series resistance compensation was set at 90–95%. Recordings were low pass filtered at 5 kHz. All experiments were performed at room temperature. Stock solutions of water-soluble AEDs were made with the extracellular solution. Stocks of water insoluble AEDs were made with dimethyl sulfoxide (DMSO) and diluted to the desired concentration with extracellular solution. The maximum DMSO concentration was 0.2%, and it was present in equivalent concentrations in all drug and control solutions when used. In the presence of 0.2% DMSO, 50 ␮M glycine-induced peak currents were 92 ± 2% (n = 4) of control peak currents obtained in the absence of DMSO. Drugs were applied using a multibarrel, gravity driven, flow tube system. Agonist applications were separated by 30 s to allow for recovery from desensitization. Only cells with stable control responses were used in experiments. Two-second applications of glycine alone and glycine plus the AED of interest were interleaved to control for rundown, which was usually less than 10%. The AED was pre-applied for 30 s prior to each glycine/AED co-application. The neuron being studied was continuously perfused with either the extracellular solution or a test solution at 2 ml/min during the drug application. Simultaneously, the bath was constantly perfused with extracellular solution at 0.5 ml/min.

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where R(Gly) is the response to a given concentration of the glycine, Glymax is the response to a saturating concentration of glycine, EC50 is the concentration producing a half-maximal response, and n is the Hill coefficient (as determined from a Hill plot). The Levenberg–Marquardt algorithm was used to fit the dose–response data. Peak currents in the presence of an AED were expressed as a percentage of the preceding control current. Statistical comparisons between groups were made using ANOVA followed by Tukey’s post hoc comparison of means. Data are presented as mean ± S.E.M. Statistical analysis was performed using Origin (Microcal Software, Inc., Northampton, MA). 2.4. Materials MEM with Earle’s salts were obtained from Gibco BRL (Grand Island, NY), Nu-Serum was obtained from Collaborative Biomedical Products (Bedford, MA) and topiramate was kindly provided by R.W. Johnson Pharmaceuticals. All other chemicals were obtained from Sigma (St. Louis, MO).

3. Results 3.1. Glycine induced a dose-dependent, strychnine sensitive chloride current Glycine elicited concentration-dependent, strychnine-inhibited, chloride currents with peak current amplitudes evoked by 50 ␮M glycine ranging from ∼100 to 10,000 pA (Fig. 1). In this embryonic hippocampal neuron culture preparation, we previously showed that glycine currents had an EC50 of 57±8 ␮M and a Hill coefficient of 1.2 ± 0.2 (Thio et al., 2003), comparable to another study in primary neuronal cultures (Rajendra et al., 1997), and indicative of GlyR activation in these neurons.

2.3. Data analysis Currents were digitized at 10 kHz and analyzed using pCLAMP (Axon Instruments, Inc., Union City, CA). Glycine dose–response data were fit to the logistic equation: Glymax × [Gly]n R(Gly) = , ECn50 + [Gly]n

3.2. Selection of AED concentrations We tested the effects of therapeutic concentrations of seven AEDs on glycine currents mediated by the GlyR. We defined therapeutic concentrations as serum, cerebrospinal fluid (CSF) or brain tissue concentrations in patients with controlled epilepsy (Table 1).

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K.M. Karkar et al. / Epilepsy Research 58 (2004) 27–35 Table 1 Therapeutic concentrations of seven AEDs

Fig. 1. Glycine evokes a strychnine sensitive chloride current. (A) Strychnine reversibly inhibited glycine currents. Currents evoked by 50 ␮M glycine (Control), 50 ␮M glycine and 5 nM strychnine (+5 nM Strychnine) after pre-applying 5 nM strychnine for 45 s, and 50 ␮M glycine alone after washout of strychnine (Recovery) in a neuron voltage-clamped at −65 mV. Bars above traces in this and all other figures indicate the duration of glycine application. The vertical spikes in current traces in this and all other figures are an artifact of the drug perfusion system. (B) Glycine activates a chloride conductance. (B1) Currents elicited by 50 ␮M glycine in a neuron voltage-clamped at potentials between −125 and +10 mV in 15 mV increments using the CsCH3 SO3 internal solution. (B2) Plot of the peak current vs. holding potential using the data in B1. (B3) Semilogarithmic plot of the reversal potential for 50 ␮M glycine currents vs. pipette chloride concentration. Circles indicate the mean reversal potential determined from data generated as shown in B1and B2 (n = 4–7). S.E.M. was smaller than the symbol for all points. The line illustrates a linear fit with a slope of 49 ± 5 mV.

Brain tissue concentrations are the most relevant measures but are only available for some of the agents. For others, serum/brain ratios are available from patient case studies (gabapentin—Ojemann et al., 1988; valproate—Shen et al., 1992) or animal experiments (pentobarbital—Hatanaka et al., 1988). 3.3. Effect of seven AEDs on cultured embryonic hippocampal neurons First, we examined whether any of the AEDs elicited a current on its own. Of the seven AEDs tested, only pentobarbital induced a current at the concentrations used in this study. At 100 ␮M, it directly

Antiepileptic drug

Concentrations1

Carbamazepine

4–13 ␮M (CSF)a 4–60 ␮M (brain)a

Felbamate

100–309 ␮M (brain)b

Gabapentin

12–117 ␮M (serum)c 0.8 (brain/serum ratio)d

Pentobarbital

80–177 ␮M (serum)e 1.5 (brain/serum ratio)f

Phenytoin

2–14 ␮M (CSF)g 20–80 ␮M (brain)g

Topiramate

1.5–100 ␮M (serum)h

Valproate

40–700 ␮M (serum)i 0.54 (brain/serum ratio)j

1 Free serum, CSF, or brain concentrations in patients with controlled epilepsy. a Johannessen et al. (1976). b Adusumalli et al. (1994). c Vollmer et al. (1986). d Ojemann et al. (1988). e Lowenstein et al. (1988). f Hatanaka et al. (1988). g Vajda et al. (1974). h Easterling et al. (1988). i Loscher (1993). j Shen et al. (1992).

activated a GABAA R-mediated current, as reported previously (Twyman et al., 1989). Pentobarbital selectively activated GABAA Rs and not GlyRs because 1 ␮M bicuculline decreased 100 ␮M pentobarbital currents to 12 ± 4% of control (n = 5), while 100 nM strychnine had no significant effect (data not shown). 3.4. Effects of seven AEDs on GlyR-mediated currents We examined the effects of seven AEDs on GlyR-mediated currents evoked by 50 ␮M glycine. We chose a concentration near the EC50 to allow both potentiation and inhibition to be detected. Of the seven AEDs tested, only topiramate and pentobarbital modulated GlyR-mediated currents at clinically relevant concentrations (Table 1, Fig. 2). In 24% of neurons (5/21), 100 ␮M topiramate reduced 50 ␮M glycine currents to 78 ± 6% of control (Fig. 2A1 and A2, left panel). In the remainder of the neurons (16/21),

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Fig. 2. High therapeutic concentrations of pentobarbital and topiramate reversibly inhibit GlyRs in hippocampal neurons. (A1 and A2) Topiramate slightly inhibited glycine currents in a subset of neurons. (A1) Currents evoked by 50 ␮M glycine alone (left trace), 50 ␮M glycine and 100 ␮M topiramate (middle trace), and 50 ␮M glycine alone after washout of topiramate (right trace) in a single neuron. (A2) Dose–response for the effect of topiramate on 50 ␮M glycine currents. Bars show the peak amplitude of 50 ␮M glycine currents in the presence of varying concentrations of topiramate expressed as a percentage of the control 50 ␮M glycine current. In 5/21 cells, 100 ␮M topiramate significantly reduced 50 ␮M glycine currents to 78 ± 6% of control (left panel). In these cells, lower concentrations of topiramate had no significant effect. In 16/21 cells, topiramate had no significant effect on 50 ␮M glycine currents at any concentration tested (right panel). (B1 and B2) Pentobarbital slightly inhibited glycine currents. (B1) Currents evoked by 50 ␮M glycine alone (left trace), 50 ␮M glycine and 100 ␮M pentobarbital (middle trace), and 50 ␮M glycine alone after washout of pentobarbital (right trace) in a single neuron. (B2) Dose–response for the effect of pentobarbital on 50 ␮M glycine currents. Pentobarbital at 100 ␮M significantly modulated 50 ␮M glycine currents, reducing them to 81 ± 6% of control (n = 8). Error bars show S.E.M. Asterisks indicate significant effects by ANOVA followed by Tukey’s post hoc comparison of means (∗ P < 0.05).

100 ␮M topiramate had no effect on 50 ␮M glycine currents (Fig. 2A2, right panel). At 100 ␮M, pentobarbital slightly and reversibly reduced 50 ␮M glycine currents to 81 ± 7% of control (n = 8) (Fig. 2B1 and B2). Lower concentrations of pentobarbital and topiramate had no significant effects (Fig. 2A2 and B2). Carbamazepine, felbamate, gabapentin, phenytoin, and valproate, at or below therapeutic concentrations (Table 1), had no significant effect on 50 ␮M glycine

currents (Fig. 3A1 and B). At concentrations above the therapeutic range, all five slightly and reversibly inhibited 50 ␮M glycine currents (Fig. 3A2 and B).

4. Discussion We found that therapeutic concentrations of seven clinically used AEDs did not enhance glycine currents

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Fig. 3. Supratherapeutic concentrations of five other AEDs inhibit glycine currents. (A) Supratherapeutic but not therapeutic concentrations of phenytoin inhibit glycine currents. Currents evoked by 50 ␮M glycine alone (left traces), 50 ␮M glycine and 50 ␮M (A1) or 500 ␮M (A2) phenytoin (middle traces), and 50 ␮M glycine alone after washout of phenytoin (right traces). Traces in A1 and A2 come from a single neuron. (B) Dose–response for the effect of carbamazepine (upper left panel, n = 6), felbamate (upper middle panel, n = 6), gabapentin (upper right panel, n = 8), phenytoin (lower left panel, n = 7), and valproate (lower middle panel, n = 9). Bars show the peak amplitude of 50 ␮M glycine currents in the presence of varying concentrations of the AEDs tested expressed as a percentage of the control 50 ␮M glycine current. Error bars show S.E.M. Asterisks indicate significant effects by ANOVA followed by Tukey’s post hoc comparison of means (∗ P < 0.05; ∗∗ P < 0.001).

mediated by GlyRs in cultured embryonic mouse hippocampal neurons. The seven included AEDs thought to act by a variety of different mechanisms including GABAA R potentiation, sodium channel inhibition, calcium channel inhibition, and N-methyl-d-aspartate receptor inhibition. In a separate study, we found that benzodiazepines, a well-known class of GABAA R potentiators, also do not enhance GlyRs (Thio et al., 2003). Our results indicate that GlyR potentiation does not contribute to the antiepileptic action of the agents we examined. In general, our results are similar to prior studies examining the effects of some AEDs on GlyRs using a single AED concentration in spinal neurons. For example, gabapentin did not alter the response

of spinal neurons to iontophoretically-applied glycine (Rock et al., 1993). When tested at a single concentration in the therapeutic range, carbamazepine, clonazepam, ethosuximide, phenobarbital, phenytoin, and valproate inhibited glycine currents in cultured mouse spinal cord neurons by <25% (Rigo et al., 2002). Although we observed a similar degree of inhibition only at supratherapeutic concentrations of the AEDs we used, this difference in the results may be secondary to a difference in the subunit composition between the two preparations. We expected that GABAA R modulators might potentiate GlyRs because the two receptors belong to the same superfamily and have significant homology. High therapeutic concentrations of two GABAA R

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potentiators, pentobarbital (Twyman et al., 1989) and topiramate (White et al., 1997, 2000), slightly inhibited rather than potentiated GlyR-mediated currents. The small degree of glycine current inhibition we observed with 100 ␮M pentobarbital, a concentration used in the treatment of refractory status epilepticus (Lowenstein et al., 1988), is similar to that observed in rat septal cholingeric neurons (Kumamoto and Murata, 1996). Although topiramate inhibited glycine in only a subset of neurons, it also potentiated GABAA R currents in only a subset of neurons (White et al., 2000). This limited inhibition may suggest variability in subunit composition of hippocampal GlyRs. The inhibition observed with pentobarbital and topiramate may contribute to the antiepileptic effect of these AEDs by blocking disinhibition mediated by GlyRs (Zheng and Johnson, 2001). Alternatively, GlyR inhibition may have an antiepileptic effect when GlyR activation is excitatory. GlyR activation is excitatory in immature neurons because of relatively high intracellular chloride concentrations (Ito and Cherubini, 1991). Like GABAA Rs, GlyR activation may be excitatory in mature neurons under some normal and pathological conditions including epilepsy (reviewed in Cohen et al., 2002; Stein and Nicoll, 2003). GlyR inhibition by supratherapeutic concentrations of the other five AEDs tested is of uncertain significance. However, this inhibition of an inhibitory receptor may contribute to the phenomenon of “paradoxical intoxication” whereby high doses of AEDs exacerbate seizures. Indeed, one of the postulated mechanisms of this observation is the superimposition of AED actions not usually operating at lower doses (Perucca et al., 1998; Genton, 2000). We designed this study to determine if AEDs directly modulate GlyRs in cultured embryonic mouse hippocampal neurons. The results do not exclude the possibility that these AEDs directly modulate GlyRs having a different subunit composition. For example, the anticonvulsant ␣EMTBL potentiates glycine currents mediated by ␣1 homomers and ␣1 ␤ heteromers, but it reduces glycine currents mediated by ␣3 homomers (Steinbach et al., 2000). Since ␣3 subunits are expressed in the postnatal but not embryonic hippocampus (Malosio et al., 1991; Thio et al., 2003), the AEDs we tested may directly modulate GlyRs in the adult hippocampus.

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In summary, we have demonstrated that seven commonly used AEDs do not directly potentiate hippocampal GlyRs. This finding is consistent with GlyRs not generally being exploited as a target in the treatment of epilepsy or other neurological diseases (Laube et al., 2002). The development of AEDs that selectively enhance GlyRs may provide drugs with a novel mechanism of action for treating epilepsy (Chattipakorn and McMahon, 2003; Kirchner et al., 2003). Such drugs may be of particular benefit to the 20–30% of patients with medically refractory epilepsy.

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