~ J Pergamon Neuroscience Vol. 83, No. 4, pp. 1161 1173, 1998 Copyright © 1998IBRO. Publishedby ElsevierScienceLtd Printed in Great Britain. All rights reserved PII: S0306-4522(97)00456-9 03064522/98 $19.00+0.00 EXPRESSION OF VOLTAGE-ACTIVATED CHLORIDE CURRENTS IN ACUTE SLICES OF HUMAN GLIOMAS N. ULLRICH,*§ A. BORDEY,* G. Y. GILLESPIEt and H. SONTHEIMER*:~ *Department of Neurobiology and ?Brain Tumor Research Laboratories, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A. Abstract--Using whole-cell patch-clamp recordings, we identified a novel voltage-activated chloride current that was selectively expressed in glioma cells from 23 patient biopsies. Chloride currents were identified in 64% of glioma cells studied in acute slices of nine patient biopsies. These derived from gliomas of various pathological grades. In addition, 98% of cells acutely isolated or in short-term culture from 23 patients diagnosed with gliomas showed chloride current expression. These currents, which we termed glioma chloride currents activated at potentials >45 mV, showed pronounced outward rectification, and were sensitive to bath application of the presumed C1- channel specific peptide chlorotoxin (~ 600 nM) derived from Leiurus scorpion venom. Interestingly, tow grade tumours (e.g., pilocytic astrocytomas), containing more differentiated, astrocyte-like cells showed expression of glioma chloride currents in concert with voltage-activated sodium and potassium currents also seen in normal astrocytes. By contrast, high grade tumours (e.g., glioblastoma multiforme) expressed almost exclusively chloride currents, suggesting a gradual loss of Na + currents and gain of CI- currents with increasing pathological tumour grade. To expand on the observation that these chloride currents are glioma-specific, we introduced experimental tumours in scid mice by intracranial injection of D54MG glioma cells and subsequently recorded from tumour cells and adjacent normal glial cells in acute slices. We consistently observed expression of chlorotoxin-sensitive chloride channels in implanted glioma cells, but without evidence for expression of chloride channels in surrounding "normal" host glial cells, suggesting that these chloride channels are probably a glioma-specific feature. Finding of this novel glioma specific C1- channel in gliomas in situ and it's selective binding of chlorotoxin may provide a way to identify or target glioma cells in the future. © 1998 IBRO. Published by Elsevier Science Ltd. Key words: ion channels, chloride, slice, chlorotoxin, glioblastoma, astrocytoma. Most neoplasms arise in tissues that have a high cell turnover during normal organ development. Neurons are postmitotic cells, and as a consequence have little capacity for the neoplastic changes that lead to tumour formation. Glial cells, by contrast, are able to re-enter the mitotic cycle in response to injury 12~24'31'33 or inflammation, s'26 and are able to proliferate even in the adult brain. 19 Consequently, the vast majority of tumours originating in the CNS are of glial origin. 2°'3° These glial-derived tumours are collectively called gliomas and include astro- :~To whom correspondence should be addressed. §Present address: Yale University School of Medicine, New Haven, CT 05611, U.S.A. Abbreviations: ACSF, artificial cerebrospinal fluid; DIDS, diisothiocyanatoostilbene-2,2'-disulfonic acid; DMEM, Dulbecco's modified Eagle's medium; DNDS, 4,4'- dinitrostilbene-2,2'-disulfonic acid; EDTA, ethylene- diaminetetra-acetate; EGTA, ethyleneglycolbis(amino- ethylether)tetra-acetate; FBS, fetal bovine serum; GCC, glioma chloride currents; GFAP, glial fibrillary acidic protein; HEPES, N-2-hydroxyethylpiperazine-N'-2- ethanesulfonic acid; LY, Lucifer Yellow; NGS, normal goat serum; PBS, phosphate-buffered saline; scid, severe combined immune deficiency; WHO, World Health Organization. cytomas, glioblastoma multiforme, ependymomas, oligodendrogliomas, and mixed gliomas. Gliomas present an enormous therapeutic challenge, and thus most gliomas are incurable despite radical surgery, high-dose radiotherapy and chemotherapy. 22,3° In two recent studies, 38'39 we investigated the elec- trophysiological and pharmacological properties of several glioma cell lines and described the consistent expression of a novel chloride current with distinct biophysical and pharmacological features. Chloride currents were exclusively observed in glial-derived tumour cells but not in tumours of non-glial origin and were termed glioma chloride currents (GCC). To establish that these chloride currents are indeed a glioma-specific feature, and are also present in glio- mas in vivo, we now studied glioma cells in acute slices of biopsy tissue from nine patients and in acutely isolated glioma cells from fresh surgical speci- mens from 14 patients and observed expression of GCC in all gliomas studied. Gliomas can be sub- classified into histopathological tumor grades I-IV [as defined by the World Health Organization (WHO) 17] by their degree of differentiation or malig- nancy on the basis of morphological and histo- pathological criteria such as nuclear atypia, cellular 1161