Elsevier

Neurobiology of Disease

Volume 31, Issue 2, August 2008, Pages 169-187
Neurobiology of Disease

Antiepileptic drug resistant rats differ from drug responsive rats in GABAA receptor subunit expression in a model of temporal lobe epilepsy

https://doi.org/10.1016/j.nbd.2008.01.005Get rights and content

Abstract

Epidemiological data indicate that 20–40% of the patients with epilepsy are refractory to treatment with antiepileptic drugs (AEDs). The mechanisms underlying pharmacoresistance in epilepsy are unclear, but several plausible hypotheses have emerged, including loss of AED target sensitivity in the epileptic brain, decreased AED concentrations at brain targets because of localized overexpression of drug efflux transporters in epileptogenic brain tissue, and network alterations in response to brain damage associated with epilepsy. Rat models of epilepsy in which part of the animals are resistant to treatment with AEDs offer a means to investigate the mechanisms underlying AED resistance. In the present study, AED-responsive and AED-resistant rats were selected from a model in which spontaneous recurrent seizures develop after a status epilepticus induced by electrical stimulation of the basolateral amygdala. For selection into responders and nonresponders, epileptic rats were treated over two weeks by phenobarbital. Subsequent histological examination showed neurodegeneration of the CA1, CA3 and dentate hilus in only one of eight responders but five of six nonresponders (P = 0.0256). Based on previous studies in AED-resistant rats of this model, we hypothesized that changes in the structure and function of inhibitory GABAA receptors may contribute to drug resistance. We therefore analyzed the distribution and expression of several GABAA receptor subunits (α1, α2, α3, α4, α5, β2/3, and γ2) immunohistochemically with specific antibodies in the hippocampal formation of responders, nonresponders and nonepileptic controls. In nonresponders, decreased subunit staining was observed in CA1, CA2, CA3, and dentate gyrus, whereas much less widespread alterations were determined in responders. Furthermore, upregulation of the α4-subunit was observed in the CA1 of nonresponders. Our data suggest that alterations in GABAA receptor subtypes may be involved in resistance to AEDs.

Introduction

Epilepsy is one of the most common serious brain disorders, and is associated with an increased risk of comorbidities and mortality (Chang and Lowenstein, 2003). Despite the availability of various newly developed antiepileptic drugs (AEDs), pharmacoresistance remains a major challenge in epilepsy management (Schmidt and Löscher, 2005). Unravelling the mechanisms underlying AED resistance has been the focus of intense efforts to develop new rationally designed therapies for as yet refractory epilepsies. Based on experimental and clinical studies, two major neurobiological theories have been put forward: (a) the multidrug transporter hypothesis, which suggests that increased brain expression of drug efflux transporters such as P-glycoprotein (P-gp) decreases AED levels at their brain targets, and (b) the target hypothesis, which suggests that AEDs are not effective because of target alterations in epileptogenic brain tissue (Löscher and Potschka, 2005, Remy and Beck, 2006). Most AEDs exert their effects either by modulation of voltage-dependent ion channels or by enhancing the inhibitory action of GABA (Rogawski and Löscher, 2004). There is some evidence that resistance to Na+ channel modulators such as carbamazepine may be due to target alterations in epileptogenic tissue of patients with refractory epilepsy (Remy and Beck, 2006). However, as yet no such clinical evidence is available for other AED targets such as GABAA receptors.

Animal models of epilepsy provide a means to investigate fundamental mechanisms underlying the development of epilepsy and AED resistance in epilepsy (Löscher, 2006). Combined molecular and functional studies in rat models of temporal lobe epilepsy (TLE) indicate a transcriptionally mediated switch in the alpha subunit composition of GABAA receptors in the hippocampal formation, characterized by a decrease of α1 subunits and an increase of α4 subunits in epileptic animals compared to controls (Brooks-Kayal et al., 1998, Coulter, 2000). This switch in the alpha subunit composition is associated with a reduced in vitro sensitivity to AEDs acting on the benzodiazepine site of the GABAA receptor (Coulter, 2001). However, none of these studies evaluated whether these changes in the GABAA receptor subunit expression in the hippocampus of epileptic rats were associated with altered in vivo responses of the animals to AED treatment, so that the relationship, if any, of the altered subunit composition for in vivo AED resistance is not known.

In a recent study in a rat model of TLE, we categorized epileptic rats into AED responders and nonresponders by prolonged treatment with phenobarbital (PB) and used GABAA receptor autoradiography to analyze the regional distribution of diazepam-sensitive and -insensitive binding sites on GABAA receptors (Volk et al., 2006). A marked enhancement of diazepam-insensitive binding was observed in the dentate granule cells of nonresponders compared to both responders and controls, whereas this was not observed in the hilus or CA1, suggesting a selective upregulation of diazepam-insensitive GABAA receptors in the dentate gyrus granule cells of nonresponders (Volk et al., 2006). These data thus suggested that the target hypothesis of drug resistant epilepsy may include GABAA receptors.

For further evaluation of this hypothesis, we repeated the selection of PB-responders and nonresponders and used GABAA receptor subunit immunohistochemistry to determine whether responders and nonresponders differ in their alpha subunit expression. Highly selective antibodies for the α1, α2, α3, α4 and α5 subunits were used for this purpose (Fritschy et al., 1992, Fritschy et al., 1999, Fritschy and Mohler, 1995). Furthermore, the β2/3 and γ2 subunits of the GABAA receptor were included in the study. In addition to characterizing the alpha subunit expression in PB-responders and nonresponders, we compared the morphological changes in the hippocampal formation in the two groups, because neuron loss affects the expression of GABAA receptor subunits. In a recent study, hippocampal damage was only determined in PB-nonresponders, whereas the hippocampus of PB-responders did not differ from non-epileptic controls (Volk et al., 2006). Because the group size in this recent study was relatively small, we evaluated in the present study whether the difference in brain damage between responders and nonresponders can be reproduced in larger groups.

Section snippets

Animals

As in our previous experiments in rats with spontaneous recurrent seizures (SRS) developing after status epilepticus (SE) induced by prolonged electrical stimulation of the basolateral amygdala (BLA) (Brandt et al., 2004, Volk and Löscher, 2005, Volk et al., 2006), adult female Sprague–Dawley rats (Harlan-Winkelmann, Borchen, Germany) were used for this study. All animal experiments were carried out in accordance with the European Communities Council Directive of 24. November 1986 (86/609/EEC)

Selection of PB-responders and nonresponders

With the dosing protocol used for selection of responders and nonresponders, PB induced marked sedation and ataxia, indicating that maximum tolerated doses were used. Analysis of plasma drug concentrations showed that drug concentrations within or above the therapeutic range (10–40 μg/ml) known from patients with epilepsy were maintained in all rats throughout the period of treatment.

In the 15 rats used for testing PB's anticonvulsant efficacy, six rats showed complete control of seizures and

PB-resistant rats differ from responsive rats in GABAA receptor subunit expression in the hippocampal formation

This is the first study demonstrating that AED-resistant rats differ from drug responsive rats in GABAA receptor subunit expression in a model of TLE. Overall, seven GABAA receptor subunits were analyzed in four hippocampal subregions in both hemispheres (plus α5-staining in the CA2), resulting in a total number of 58 sets of data in four groups of rats (Fig. 12, Fig. 13, Fig. 14, Fig. 15). PB-nonresponders significantly differed from controls in 44 of these 58 sets of data, compared to 20/58

Conclusions

As shown by our studies, epileptic rats that are resistant to treatment with PB exhibit complex morphological and neurochemical brain alterations compared to PB-responsive rats, including hippocampal damage, overexpression of P-gp in the hippocampus and piriform cortex, enhanced diazepam-insensitive GABAA receptor binding in the dentate gyrus, and widespread alterations in GABAA receptor subunits in the hippocampal formation (Volk and Löscher, 2005, Volk et al., 2006; present study). We have

Acknowledgments

We thank Dr. Richard W. Olsen (Department of Molecular and Medical Pharmacology, Geffen School of Medicine, University of California, Los Angeles) and Dr. Maharaj K. Ticku (Department of Pharmacology, The University of Texas Health Science Center, San Antonio Texas) for the discussion on PB's effects on GABAA receptors. Furthermore, we thank Ms. Nicole Ernst for technical assistance during the video/EEG-monitoring of seizures, Ms. Christina Fuest and Ms. Melanie Langer for help in the

References (56)

  • NishimuraT. et al.

    Altered expression of GABA(A) and GABA(B) receptor subunit mRNAs in the hippocampus after kindling and electrically induced status epilepticus

    Neuroscience

    (2005)
  • RacineR.J.

    Modification of seizure activity by electrical stimulation: II. Motor seizure

    Electroencephalogr. Clin. Neurophysiol.

    (1972)
  • RudolphU. et al.

    GABA-based therapeutic approaches: GABAA receptor subtype functions

    Curr. Opin. Pharmacol.

    (2006)
  • SchwarzerC. et al.

    GABA(A) receptor subunits in the rat hippocampus II: altered distribution in kainic acid-induced temporal lobe epilepsy

    Neuroscience

    (1997)
  • SomogyiP. et al.

    A note on the use of picric acid–paraformaldehyde–glutaraldehyde fixative for correlated light and electron microscopic immunocytochemistry

    Neuroscience

    (1982)
  • SperkG. et al.

    GABA(A) receptor subunits in the rat hippocampus I: immunocytochemical distribution of 13 subunits

    Neuroscience

    (1997)
  • SperkG. et al.

    Expression of GABA(A) receptor subunits in the hippocampus of the rat after kainic acid-induced seizures

    Epilepsy Res.

    (1998)
  • TsunashimaK. et al.

    GABA(A) receptor subunits in the rat hippocampus III: altered messenger RNA expression in kainic acid-induced epilepsy

    Neuroscience

    (1997)
  • VolkH.A. et al.

    Increased expression of the multidrug transporter P-glycoprotein in limbic brain regions after amygdala-kindled seizures in rats

    Epilepsy Res.

    (2004)
  • VolkH.A. et al.

    Antiepileptic drug resistant rats differ from drug responsive rats in hippocampal neurodegeneration and GABAA-receptor ligand-binding in a model of temporal lobe epilepsy

    Neurobiol. Dis.

    (2006)
  • WongE.H. et al.

    gamma-Aminobutyric acid activation of 36Cl-flux in rat hippocampal slices and its potentiation by barbiturates

    Brain Res.

    (1984)
  • BaulacM.

    Phenobarbital and other barbiturates: clinical efficacy and use in epilepsy

  • BethmannK. et al.

    Resistance to phenobarbital extends to phenytoin in a rat model of temporal lobe epilepsy

    Epilepsia

    (2007)
  • BrandtC. et al.

    Striking differences in individual anticonvulsant response to phenobarbital in rats with spontaneous seizures after status epilepticus

    Epilepsia

    (2004)
  • Brooks-KayalA.R. et al.

    Selective changes in single cell GABAA receptor subunit expression and function in temporal lobe epilepsy

    Nat. Med.

    (1998)
  • BureauM.H. et al.

    GABAA receptor subtypes: ligand binding heterogeneity demonstrated by photoaffinity labeling and autoradiography

    J. Neurochem.

    (1993)
  • ChangB.S. et al.

    Epilepsy

    N. Engl. J. Med.

    (2003)
  • CoulterD.A.

    Mossy fiber zinc and temporal lobe epilepsy: pathological association with altered “epileptic” gamma-aminobutyric acid A receptors in dentate granule cells

    Epilepsia

    (2000)
  • Cited by (0)

    View full text