Elsevier

Neuropharmacology

Volume 42, Issue 1, January 2002, Pages 107-116
Neuropharmacology

Effect of phenytoin on sodium and calcium currents in hippocampal CA1 neurons of phenytoin-resistant kindled rats

https://doi.org/10.1016/S0028-3908(01)00148-4Get rights and content

Abstract

About 20–30% of patients with epilepsy continue to have seizures despite carefully monitored treatment with antiepileptic drugs. The mechanisms explaining why some patients' respond and others prove resistant to antiepileptic drugs are poorly understood. It has been proposed that pharmacoresistance is related to reduced sensitivity of sodium channels in hippocampal neurons to antiepileptic drugs such as carbamazepine or phenytoin. In line with this proposal, a reduced effect of carbamazepine on sodium currents in hippocampal CA1 neurons was found in the rat kindling model of temporal lobe epilepsy (TLE), i.e. a form of epilepsy with the poorest prognosis of all epilepsy types in adult patients. To address directly the possibility that neuronal sodium currents in the hippocampus play a crucial role in the pharmacoresistance of TLE, we selected amygdala-kindled rats with respect to their in vivo anticonvulsant response to phenytoin into responders and nonresponders and then compared phenytoin's effect on voltage-activated sodium currents in CA1 neurons. Furthermore, in view of the potential role of calcium current modulation in the anticonvulsant action of phenytoin, the effect of phenytoin on high-voltage-activated calcium currents was studied in CA1 neurons. Electrode-implanted but not kindled rats were used as sham controls for comparison with the kindled rats. In all experiments, the interval between last kindled seizure and ion channel measurements was at least 5 weeks. In kindled rats with in vivo resistance to the anticonvulsant effect of phenytoin (phenytoin nonresponders), in vitro modulation of sodium and calcium currents by phenytoin in hippocampal CA1 neurons did not significantly differ from respective data obtained in phenytoin responders, i.e. phenytoin resistance was not associated with a changed modulation of the sodium or calcium currents by this drug. Compared to sham controls, phenytoin's inhibitory effect on sodium currents was significantly reduced by kindling without difference between the responder and nonresponder subgroups. Further studies in phenytoin-resistant kindled rats may help to elucidate the mechanisms that can explain therapy resistance.

Introduction

A substantial number of epileptic patients continue to have seizures in spite of adequate treatment with antiepileptic drugs (Johannessen et al., 1995). In such patients with pharmacoresistant epilepsy, brain surgery may be an alternative treatment. The largest group of surgical candidates comprises patients with complex partial seizures of temporal lobe origin (Theodore, 1992). Surgical resection of epileptogenic tissue, particularly the hippocampus, often results in pharmacological seizure control after surgery (Theodore, 1992), indicating that the hippocampus plays a crucial role in the pharmacoresistance of temporal lobe epilepsy (TLE) in these patients. The mechanisms underlying pharmacoresistance most likely involve the functional and morphologic changes developing in regions such as the hippocampus in the course of the disease (Heinemann et al., 1994). Drugs of primary choice for treatment of TLE such as phenytoin or carbamazepine are thought to act via modulation of voltage-activated sodium and calcium channels (DeLorenzo, 1995, Macdonald, 1999). We have previously shown that the properties of these channels change in the hippocampus of patients with therapy-refractory TLE (Beck et al., 1997, Beck et al., 1998, Reckziegel et al., 1998), which could explain the loss of therapeutic efficacy of major antiepileptic drugs. Indeed, Wadman and colleagues found an impaired modulation of sodium current inactivation by carbamazepine in hippocampal neurons from patients with pharmacoresistant TLE that was associated with hippocampal sclerosis (Vreugdenhil et al., 1998b). A similar reduction in carbamazepine's effect on sodium channels was also determined in hippocampal CA1 neurons of kindled rats, i.e. a widely used model of TLE (Vreugdenhil and Wadman, 1999). However, the reduction in carbamazepine's effect was only transient in kindled rats (Vreugdenhil and Wadman, 1999). Furthermore, carbamazepine is a very potent and efficacious anticonvulsant in kindled rats (Albertson et al., 1984; Löscher et al., 1986, Hönack and Löscher, 1989) so that the transiently reduced carbamazepine effect on sodium channels described in the hippocampus of kindled rats by Vreugdenhil and Wadman (1999) is not associated with any resistance to the drug's anticonvulsant effect in vivo.

However, like individual epileptic patients, individual kindled rats differ in their response to anticonvulsant drugs, allowing selection of pharmacoresistant rats from large populations of kindled animals (Löscher, 1997). In such pharmacoresistant rats (nonresponders), which can be identified by repeated testing with phenytoin or its prodrug fosphenytoin, all major antiepileptic drugs, including phenytoin, carbamazepine, phenobarbital, valproate, vigabatrin, lamotrigine and others, are either ineffective or less effective against kindled seizures than in pharmacosensitive rats (responders) from the same kindled rat population (Löscher and Rundfeldt, 1991, Löscher et al., 1993, Ebert et al., 2000, Reissmüller et al., 2000). Kindled responders and nonresponders are thus ideally suited to investigate the mechanisms underlying pharmacoresistance in TLE (Löscher, 1997).

In the present study, we evaluated the modulation of voltage-dependent sodium and calcium currents by phenytoin in acutely isolated rat CA1 neurons in subgroups of kindled rats which were preselected by repeated in vivo testing with phenytoin into phenytoin responders and nonresponders. Furthermore, the properties of these ion channels were electrophysiologically characterized and compared between the subgroups.

Section snippets

Animals

Female Wistar outbred rats (Harlan-Winkelmann, Borchen, F.R.G.), weighing 200–230 g were used. The animals were purchased from the breeder at an age of 10 weeks. Following arrival in the animal colony, the rats were kept under controlled environmental conditions (ambient temperature 24–25°C, humidity 50–60%, 12/12 h light/dark cycle, light on at 6:00 a.m.) for at least 1 week before being used in the experiments. Standard laboratory chow (Altromin 1324 standard diet) and tap water were allowed ad

Sodium currents

Voltage-dependent sodium currents were elicited in whole cell voltage clamp in isolated CA1 pyramidal neurons with depolarizing command pulses starting from a hyperpolarizing prepulse of −120 mV (500 ms; Fig. 1B) or in some cases directly from the holding potential of −80 mV. Representative current families of sham (implanted but not kindled) animals and of phenytoin nonresponders as well as responders are shown in Fig. 1A. The kinetics of inactivation was found to be the same within the three

Discussion

The present study could not demonstrate differences in the phenytoin sensitivity of sodium and calcium currents of hippocampal CA1 neurons in kindled rats which are resistant to the anticonvulsant effect of phenytoin (phenytoin nonresponders). This finding was unexpected because a subsensitivity of voltage-activated sodium or calcium channels to phenytoin would have been a likely explanation for the pharmacoresistance to phenytoin's anticonvulsant effect found in nonresponders in vivo.

Phenytoin

Acknowledgments

We thank M. Weissing and E. Boening for technical assistance. The study was supported by a grant (Lo 274/9-1) from the Deutsche Forschungsgemeinschaft (Bonn, Germany).

References (39)

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