ELSEVIER Brain Research 647 (1994) 167-171 BRAIN RESEARCH Short Communication The benzodiazepine receptor antagonist flumazenil increases acetylcholine release in rat hippocampus Assunta Imperato a,,, Laura Dazzi b, M. Carmen Obinu a, Gian Luigi Gessa a, Giovanni Biggio b a Department of Neuroscience 'Bernard B. Brodie', University of Cagliari, Via Porcell 4, 09123 Cagliari, Italy b Department of Experimental Biology 'Bernardo Loddo" University of Cagliari, Via Palabanda 12, 09124 Cagliari, Italy (Accepted 10 March 1994) Abstract The benzodiazepine receptor antagonist flumazenil (2.5-20 mg/kg i.p.) increased acetylcholine (ACh) release by up to 85% in the hippocampus of freely moving rats. In contrast, the benzodiazepine receptor full agonist diazepam (2.5-10 mg/kg i.p.) decreased ACh release up to a maximum of 45% in the same brain area. Injection of flumazenil (10 pmol) or diazepam (10 pmol) into the medial septum increased (95%) or reduced (50%), respectively, ACh release in the hippocampus. The maximum effect produced by those drugs was of the same magnitude as that observed after systemic injection. The changes in hippocampal cholinergic function elicited by activation and blockade of benzodiazepine receptors in the medial septum may thus play a crucial role in the alterations of the cognitive processes elicited by benzodiazepine receptor ligands. Key words: Benzodiazepine; Flumazenil; Acetylcholine release; Hippocampus; Microdialysis Benzodiazepine (BDZ) receptor agonists, the most clinically effective anxiolytic drugs, decrease vigilance and impair cognitive processes [22,39,42,43] and habit- uation to novel environmental stimuli [11,15]. In con- trast, BDZ receptor antagonists, which block or reverse the BDZ-induced impairment in learning and memory [22,42], enhance vigilance and cognitive processes when administered alone [24,25,40] particularly if the task requires a rapid analysis of the environment [41]. The BDZ receptor antagonist flumazenil (Ro 15-1788) also protects against the amnesic action of scopolamine [26] suggesting that this drug may affect the cholinergic system. The BDZs diazepam and midazolam reduce the output of acetylcholine (ACh) in the hippocampus of freely moving rats indicating that y-aminobutyric acid (GABA) mediated transmission exerts a tonic modula- tion on ACh release in the hippocampus [18]. We have now investigated the effect of flumazenil on cholinergic transmission in the hippocampus, a brain area consid- ered to play an important role in the regulation of * Corresponding author. Fax: (39) (70) 65-7237. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0006-8993(94)00299-R learning and memory and in determining the state of attention and arousal [4]. Male Sprague-Dawley rats were anesthetized with chloral hydrate (0.4 g/kg i.p.), and dialysis tubes (AN 69-HF, with a wet tube outer diameter of 320 ~m; Hospal-Dasco, Bologna, Italy) were implanted at the level of the dorsal hippocampus (A 3430, V - 3.5, from the skull, according to the K6nig and Klippel atlas). Surgery was performed according to the transversal microdialysis technique recently revised in order to induce less tissue damage and reduce the glial reaction around the dialysis tube [17]. Experiments started 24 h after implantation of the dialysis tube as previously described [17]. The Ringer solution contained 3 mM KC1, 125 mM NaC1, 1.3 mM CaCI 2, 1.0 mM MgC12, 23 mM NaHCO3, 1.5 mM potassium phosphate buffer (pH 7.3), and 0.1 /zM neostigmine. The extracellular concentration of ACh was determined by high-performance liquid chro- matography with electrochemical detection, as de- scribed by Damsma and Westerink [8]. Tetramethylam- monium chloride (1 mM) was added to the mobile phase (0.2 M potassium phosphate buffer). The detec- tion limit for ACh was 0.02 pmol per injection.