European Journal of Neuroscience, Vol. 10, pp. 231–238, 1998 © European Neuroscience Association Functional GABA A receptors on human glioma cells Charalampos Labrakakis, 1 Stephan Patt, 1,2 Jana Hartmann 1 and Helmut Kettenmann 1 1 Department of Cellular Neurosciences, Max Delbru ¨ck Center for Molecular Medicine (MDC), Robert-Ro ¨ssle-Str. 10, D-13122 Berlin, Germany 2 Institute of Pathology, Friedrich Schiller University Jena, D-07740 Jena, Germany. Keywords: astrocytoma, GABA A receptor, glioblastoma, human, oligodendroglioma, patch clamp Abstract Glioma cells in acute slices and in primary culture, and glioma-derived human cell lines were screened for the presence of functional GABA A receptors. Currents were measured in whole-cell voltage clamp in response to γ- aminobutyric acid (GABA). While cells from the most malignant glioma, the glioblastoma multiforme, did not respond to GABA, an inward current (under our experimental conditions with high Cl – concentration in the pipette) was induced in gliomas of lower grades, namely in 71% of oligodendroglioma cells and in 62% of the astrocytoma cells. Glioma cell lines did not express functional GABA A receptors, irrespective of the malignancy of the tumour they originate from. The currents elicited by application of GABA were due to activation of GABA A receptors; the specific agonist muscimol mimicked the response, the antagonists bicuculline and picrotoxin blocked the GABA-activated current and the benzodiazepine receptor agonist flunitrazepam augmented the GABA-induced current and the benzodiazepine inverse agonist DMCM decreased the GABA current. Cells were heterogeneous with respect to the direction of the current flow as tested in gramicidin perforated patches: in some cells GABA hyperpolarized the membrane, while in the majority it triggered a depolarization. Moreover, GABA triggered an increase in [Ca 2+ ] i in the majority of the tumour cells due to the activation of Ca 2+ channels. Our results suggest a link between the expression of GABA receptors and the growth of glioma cells as the disappearance of functional GABA A receptors parallels unlimited growth typical for malignant tumours and immortal cell lines. Introduction Increasing malignancy of gliomas correlates with a decrease in the density of γ-aminobutyric acid (GABA) binding sites (Jussofie et al., 1994), suggesting an association of the proliferative activity and the expression of GABA receptors. Evidence that GABA and GABA A receptors play an active role by inhibiting proliferation was obtained for non-malignant but proliferating cells, the cortical progenitor neurones (LoTurco et al., 1995). In postmitotic neurones GABA affects the devel- opment and differentiation (Wolff et al., 1978; Meier et al., 1987; Spo- erri, 1988; Cherubini et al., 1991; Ben-Ari et al., 1994). GABA can regulate the extent of neurite outgrowth (Spoerri, 1988) and can influ- ence neuronal survival (Mount et al., 1993). At least some, if not all, of these actions are mediated through the GABA A receptor (Michler, 1990; Barbin et al., 1993). The GABAergic system is indeed present prior to synaptogenesis. GABAergic fibres have been found in prenatal nervous tissue, i.e. in the spinal cord at E13 (Lauder et al., 1986) and in the neocortex of E14 rats (Van Eden et al., 1989). While GABA hyperpolarizes the membrane of mature neurones, it acts depolarizing in glial cells and immature neurones. GABA A is an ionotropic receptor permeable for Cl – ions and HCO 3 – (Kaila, 1994). Depending mainly on the intracellular Cl – concentration GABA is able to hyperpolarize or depolarize the cell. GABA influences only in those neurones the proliferation and development in which it also induces depolarization. The link could be mediated by Ca 2+ : the depolarization Correspondence: Helmut Kettenmann, as above. E-mail: hketten@mdc-berlin.de Received 9 May 1997, revised 21 July 1997, accepted 28 July 1997 can activate voltage gated Ca 2+ channels and thus lead to an elevation of the intracellular Ca 2+ concentration (Connor et al., 1987; Yuste & Katz, 1991; Reichling et al., 1994). Similar physiological responses were also described for glial cells. Glial precursor cells as well as oligodendrocytes and astrocytes express GABA A receptors. Activation of the glial receptors always leads to a depolarization and for glial precursor cells and astrocytes the activation of Ca 2+ channels was reported (Kirchhoff & Kettenmann, 1992; Fraser et al., 1995; von Blankenfeld et al., 1995). Few studies have shown high-affinity binding sites for GABA and benzodiazepines in human glioma cells and in the rat glioma C6 cell line (Guidotti et al., 1979; Frattola et al., 1985; Jussofie et al., 1994). So far, all these studies relied on binding assays while the presence of functional GABA receptors on these malignant brain cells has not yet been established. We therefore set out to test for the presence of functional GABA A receptors on glioma cells. Fresh brain slices and in comparison primary cultures from the same tumour material were used. Methods Tumour material Ten oligodendrogliomas of the WHO (World Health Organization) grade II (Kleihues et al., 1993) (T5/93, T7/93, T13/93, T1/94, T3/94,