CHANGES IN GABA A AND GABA B RECEPTOR BINDING FOLLOWING CORTICAL PHOTOTHROMBOSIS: A QUANTITATIVE RECEPTOR AUTORADIOGRAPHIC STUDY M. QUE,*† O. W. WITTE,‡ T. NEUMANN-HAEFELIN,‡ K. SCHIENE,‡ M. SCHROETER‡ and K. ZILLES*§ *C. & O. Vogt Institute of Brain Research, ‡Department of Neurology, University of Du ¨sseldorf, P.O. Box 101007, D-40225 Du ¨sseldorf, Germany §Institute of Medicine, Research Center Ju ¨lich, D-52425 Ju ¨lich, Germany Abstract —Experimental cortical photothrombosis leads to pronounced alterations in the binding density of [ 3 H]muscimol and [ 3 H]baclofen to GABA A and GABA B receptors, both in the lesioned and the structurally intact cortex. The binding density of [ 3 H]muscimol to GABA A receptors was markedly increased in the “core” of the lesion during the first week, reaching a maximum on the third day post-lesion. Simultaneously, it dropped in the exofocal primary somatosensory cortex. Reductions in the binding density of [ 3 H]muscimol were also found in remote cortical areas of the contralateral hemisphere and lasted for several weeks. In contrast to the down-regulation of apparent binding density of [ 3 H]muscimol, a long-lasting up-regulation of that of [ 3 H]baclofen to GABA B receptors was measured in the exofocal primary somatosensory cortex and in remote cortical areas of both hemispheres. The greatest increase in the binding density of [ 3 H]baclofen was seen on the seventh day in the surroundings of the lesion. Our findings indicate that widespread alterations in the concentrations of GABA A and GABA B receptors are induced in remote cortical areas by a focal ischaemic lesion. Since GABA A receptor affinity is regulated by nitric oxide, we suggest that the observed down-regulation of GABA A receptors may be correlated with a lesion-induced increase in nitric oxide, whereas the up-regulation of GABA B receptors might be caused by other mechanisms, e.g., compensatory processes. In the centre of the lesion, however, a GABA A receptor-mediated mechanism, which limits the spread of lesion-induced hyperexcitability, is thought to be involved.  1999 IBRO. Published by Elsevier Science Ltd. Key words: cortical lesion, photothrombosis, GABA A and GABA B receptors, muscimol and baclofen binding. It is well known that normal brain functions are constantly being modulated by GABA receptor-mediated inhibi- tion, 4,23,36 and that epileptic activity may be induced by block- ing or impairment of GABAergic inhibition. 13,28,35 Functional studies have confirmed that a local and transient loss of GABA receptor-mediated inhibition may also affect physio- logical plasticity and memory. 9,17,24 Recently, we reported that GABA A receptor-mediated paired-pulse inhibition was reduced in areas surrounding a lesion, as well as in areas contralateral to it. 5,7,10,36 This reduction was associated with a down-regulation of GABA A receptor binding. 29,32 Since two subtypes of receptors—GABA A (bicuculline-sensitive) and GABA B (bicuculline-insensitive) receptors—differ pharma- cologically and functionally, 4,33 it is necessary to separately analyse the effect of a cortical lesion on their binding densi- ties. The present study examines the temporal development of alterations in the binding densities of [ 3 H]muscimol and [ 3 H]baclofen to GABA A and GABA B receptors after the induction of a cortical lesion. Survival times extending from 4 h to 30 days were chosen. EXPERIMENTAL PROCEDURES Animals Cortical photothrombosis was induced in adult SPF strain Wistar rats (two months old, body weight 250–300 g, n  30). Adult male Wistar rats (280–310 g) were anaesthetized with 1.5–2% halothane in a mixture of O 2 /N 2 or with enflurane in a mixture of O 2 /N 2 O. A catheter was inserted in the femoral vein. Focal lesions were induced in the parietal cortex. The skin of the skull was opened and an optic fibre bundle mounted on a cold light source (Schott KL 1500, Germany) was positioned exactly 4 mm posterior to bregma and 4 mm lateral to the midline. The illumination lasted 20 min; during the first minutes, Rose Bengal (Aldrich Chemie, Steinheim, Germany; 1.3 mg/100 g body weight) was injected into the femoral vein. Laboratory details are provided in Schiene et al. 32 After different survival times post-lesion (PL; 4 h, three, seven, 14 and 30 days), the brains were removed and frozen in isopentane at - 50 to - 70°C. Guided by the visibly damaged area on the surface of the brain, frontal sections (10 mm) were cut in a cryostat at - 18 to - 20°C and mounted on glass slides for receptor binding. Each 10th section was mounted for histo- logical assessment using a modified silver staining technique. 22 Sham-operated age-matched animals were used as controls (n  5). All experimental protocols were approved by government authori- ties and complied with the European Communities Council directive. Binding assays [ 3 H]Muscimol was used as ligand for demonstrating the GABA A receptor. 31 The GABA B receptor was labelled with [ 3 H]baclofen. For removal of endogenous substances such as GABA, not associated with receptors, the sections were pre-incubated with the respective buffers (see below) for 15 min (3 × 5 min) at 4°C. For labelling of GABA A receptors, sections were incubated in 3 nM [ 3 H]muscimol (12.05 Ci/ mmol; DuPont, Homburg, Germany) in 50 mM Tris–citrate buffer (pH 7.0) at 4°C for 45 min. Incubation was terminated with three rinses in cold buffer (3 s each). For demonstrating GABA B receptors, sections were incubated in 15 nM [ 3 H]baclofen (46.9 Ci/mmol; DuPont) in 50 mM Tris–HCl buffer (pH 7.2, containing 2.5 mM CaCl 2 ) at 22°C for 60 min. The binding of [ 3 H]baclofen to GABA B receptors was terminated in cold buffer (3 × 5 min). Non-specific binding was deter- mined with 10 -5 M unlabelled GABA (Merck, Darmstadt, Germany; for GABA A ) or 10 -4 M unlabelled baclofen (RBJ/Biotrend Chemicals, Cologne, Germany; for GABA B ). The tritium-labelled sections were Alteration of GABA A and GABA B receptors by cortical photothrombosis 1233 1233 Neuroscience Vol. 93, No. 4, pp. 1233–1240, 1999 Copyright  1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306-4522/99 $20.00+0.00 PII: S0306-4522(99)00197-9 Pergamon †To whom correspondence should be addressed. Tel.: + 49-211-81-12798; fax: + 49-211-81-12336. E-mail address: que@hirn.uni-duesseldorf.de (M. Que) Abbreviations: Fr1, primary motor cortex; Par1, primary somatosensory cortex; PL, post-lesion.