Journal of Neuroscience Methods 200 (2011) 164–172 Contents lists available at ScienceDirect Journal of Neuroscience Methods j o ur nal homep age: www.elsevier.com/locate/jneumeth Functional test of multidrug transporter activity in hippocampal–neocortical brain slices from epileptic patients Richard Kovács ∗ , Claudia Raue, Siegrun Gabriel, Uwe Heinemann Institute for Neurophysiology, Charité-Universitätsmedizin Berlin, 13347 Berlin, Germany a r t i c l e i n f o Article history: Received 21 April 2011 Received in revised form 28 June 2011 Accepted 29 June 2011 Keywords: P-glycoprotein Multidrug transporters Multidrug resistance associated protein Pharmacoresistance Temporal lobe epilepsy Brain slice Calcein a b s t r a c t About 70% of the patients suffering from temporal lobe epilepsy (TLE) are resistant to currently avail- able antiepileptic drugs (AEDs). For them one therapeutic option to achieve seizure control is to undergo epilepsy surgery. Expression of multidrug transporters is upregulated in resected tissue specimens from TLE patients, as well as in animal models of chronic epilepsy, which might lead to altered tissue availability of AEDs and therefore contribute to drug refractoriness. Here we describe a functional test of multidrug transporter activity in brain slices from TLE patients based on intracellular accumulation of the fluores- cent multidrug transporter substrate calcein and compare functional data to the expression pattern of multidrug transporters. The rate of cytosolic calcein fluorescence increase was altered by inhibitors of multidrug transport such as probenecid (400 M) and verapamil (40 M) in a subset of slices, indicating the presence of functional multidrug transport proteins in human epileptic tissue. Interestingly, there were differences between the expression pattern of multidrug transporters and their ability to remove calcein-AM. Consequently, in vitro studies on multidrug transporters should always include functional tests of their activity as expression alone is not necessarily conclusive. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Multidrug resistance – a major hindrance to cancer chemother- apy – relies on the ability of tumor cells to expel multiple cytotoxic compounds. ATP-dependent plasma membrane trans- porters belonging to the ABC (ATP-binding casette) family such as the multidrug resistance (MDR1) gene product, P-glycoprotein (Pgp) and the multidrug resistance associated proteins (MRP1 to 5) are involved in transport of xenobiotics. These transporters are normally present on transport epithelia, and their expression is upregulated not only in cancer but also in epilepsy (Löscher and Potschka, 2005). Most epileptic patients respond well to available antiepilep- tic drugs (AEDs) but about 30% of all epileptic patients and 70% of patients suffering from temporal lobe epilepsy (TLE) have or develop poor pharmacological control of seizures (Semah et al., 1998). Epileptic patients not responding to two or more AEDs might undergo surgical intervention, where the epileptogenic area is removed. After resection, a considerable portion of the patients become seizure free or achieve a good seizure control indicating that drug refractoriness was related to the resected tissue (Engel, ∗ Corresponding author at: Institute for Neurophysiology and NeuroCure Research Center, Charité-Universitätsmedizin Berlin, Oudenarder Strasse 16, 13347 Berlin, Germany. Tel.: +49 30 450 528 357; fax: +49 30 450 528 962. E-mail address: richard.kovacs@charite.de (R. Kovács). 1987; Clusmann et al., 2002). Increased MDR1 mRNA levels and Pgp immunoreactivity was found at the brain capillary endothe- lial cells in resected tissue samples from epileptic patients (Tishler et al., 1995; Aronica et al., 2003; Calatozzolo et al., 2005; Sisodiya et al., 2006). In addition to Pgp, the expression of several MRPs was increased in tissue samples from epilepsy patients (Aronica et al., 2003; Sisodiya et al., 2002) as well as in animal models of epilepsy (Volk and Löscher, 2005). Some of these transporters were expressed ectopically on glial cells and neurons, i.e. in cell types where they are not present normally. One theory of drug refrac- toriness in epilepsy implies that overexpression of Pgp and MRPs at the blood brain barrier might decrease the tissue concentration of AEDs (Lee et al., 2001; Löscher and Potschka, 2002, 2005). In a previous study we found that epileptic activity induced in brain slices from patients suffering from pharmacoresistant epilepsy did not respond to AEDs in vitro (Jandová et al., 2006). As blood brain barrier is abrogated in this preparation we turned our attention to the role of ectopically expressed multidrug transporters. Conclusions on Pgp and MRP function are often based on the determination of expression pattern at protein or mRNA level. However, discrepancies between expression of Pgp as detected by monoclonal antibodies and transport capability of xenobiotics were described in cancer cell lines (Bailly et al., 1995; Xie et al., 1995). This fact necessitates the development of functional tests for multidrug transporter activity. Such tests are frequently used to prove the presence of multidrug transporters on cancer cell lines (Homolya et al., 1996) and they take advantage of the ability of mul- 0165-0270/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2011.06.032