Brain Research Bulletin. Vol. 32, pp. 385-389, 1993 0361-9230/93 $6.00 + .OO Printed in the USA. All rights reserved. Copyright 0 1993 Pergamon Press Ltd. Elevation of Hippocampal Extracellular Acetylcholine Levels by Methoctramine MICHAEL J. STILLMAN,‘,2.4 BARBARA SHUKITT-HALE,‘%2 ROBERTO M. KONG, AHARON LEVY3 AND HARRIS R. LIEBERMAN Military Performance and Neuroscience Division, United States Army Research Institute of Environmental Medicine, Natick, MA 01760-5007 Received 2 1 January 1993; Accepted 7 May 1993 STILLMAN, M. J., B. SHUKITT-HALE, R. M. KONG, A. LEVY AND H. R. LIEBERMAN. Elevation of hippocampal extracellular acetylcholine levels by methoctramine. BRAIN RES BULL 32(4) 385-389, 1993.-Previous studies suggest that m2 muscarinic receptors serve as presynaptic autoreceptors. Blocking these receptors by selective antagonists may, therefore, lead to increased acetylcholine (ACh) release. This study assessed changes in extracellular ACh levels, via in vivo microdialysis, following administration of the m2 antagonist methoctramine. Drug or placebo (Ringer’s solution) was perfused via a microdialysis probe into the CA 1 hippocampal region of unrestrained, awake male Fischer rats. HPLC-EC was used for online analysis of the dialysates. Methoctramine significantly enhanced ACh release in a dose-dependent fashion as compared to placebo for the doses employed (0.25-16 PM). The present in vivo data corroborate studies that show increased ACh levels in vitro following application of m2 antagonists. Acetylcholine Microdialysis Methoctramine Muscarinic receptors mz Receptor Autoreceptor A number of cholinergic receptor subtypes have been identified in various central nervous system (CNS) regions. Hippocampal muscarinic receptors belong to two main subtypes, currently designated m, and mz, which are distinguished by their binding affinity to pirenzepine (6). It appears that the majority of hip- pocampal mz receptors are localized presynaptically and may regulate ACh release by functioning as inhibitory autoreceptors. Presumably, synaptic ACh binds to presynaptic mz receptors, thereby inducing a decrease in ACh release (3,11,17). An m2 antagonist, by blocking these receptors, might suppress this in- hibitory activity, leading to enhanced hippocampal choline@ transmission. It appears that elevated hippocampal cholinergic function may improve performance on certain cognitive tasks. Recently, increased levels of hippocampal ACh accompanied by improved memory were demonstrated in our laboratory through the ad- ministration of the calcium channel blocker nimodipine (10). Peripheral administration of the m2 antagonist AF-DX 116 has been found to improve performance on a memory task in rats (14). It is also possible that elevated hippocampal cholinergic transmission following m2 antagonist administration may alle- viate deficits of Alzheimer’s Disease (AD) patients. Fewer m2, but not ml, receptors are present in the cerebral cortices of AD ’ Geo-Centers, Inc., Newton Centre, MA 02 159. * Department of Psychology, Boston University, Boston, MA 022 15. >- - ^_. patients, as well as in rats with experimental cortical choline& denervation ( 1 l), possibly suggesting down-regulation due to reduced ACh release. This study was conducted to assess in vivo changes in extra- cellular ACh following central administration of the m2 antag- onist methoctramine, a polymethylene tetraamine (2,12). The highly polar structure of methoctramine prevents it from readily crossing the blood-brain barrier. Therefore, it was delivered di- rectly into the hippocampus via the microdialysis probe. A sig- nificant increase in extracellular ACh after direct application of methoctramine would support the involvement of m2 receptors in the regulation of hippocampal cholinergic neurotransmission. METHOD Animals Twenty male Fischer 344 rats (Charles River Labs, Kingston, NY), 4-7 months of age, weighing between 250 and 360 g, were tested from one to five times each in this study (mean = 2.00, SEM = 0.2 1). The rats were individually housed in hanging wire mesh cages and maintained on a 12L: 12D cycle (lights on at 0600 h). J Current address: Department ot Pharmacology, Israel Institute for Biological Research, Ness Ziona, 70450 Israel. 4 Requests for reprints should be addressed to Michael J. Stillman, Military Performance and Neuroscience Division, United States Army Research Institute of Environmental Medicine, Kansas St, Natick, MA 0 1760-5007. 385