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