Behavioral Neuroscience 1998, Vol. 112, No. 2, 352-359 Copyright 1998 by the American Psychological Association, Inc. 0735-7044/98/$3.00 Endogenous Acetylcholine in the Dorsal Hippocampus Reduces Anxiety Through Actions on Nicotinic and Muscarinic i Receptors Sandra E. File, Luis E. Gonzalez, and Nick Andrews United Medical and Dental Schools Dorsal hippocampal cholinergic modulation of behavior in different tests of anxiety was investigated by direct injection of the muscarinic M! and M2 receptor antagonists, pirenzepine and gallamine, and the nicotinic receptor antagonist mecamylamine. In the social interaction test, the anxiogenic effect of pirenzepine (30-100 ng) provided evidence for a tonic choljnergic anxiolytic action mediated by postsynaptic M, receptors. The anxiogenic action of mecamylamine (30 and 100 ng) was most likely mediated by its action of presynaptic nicotinic receptors to reduce acetylcholine release. Gallamine (10-1,000 ng) was without effect, suggesting that M 2 receptors in this brain region do not play a significant role in this behavioral test. On Trial 1 in the elevated plus-maze, the receptor antagonists were without any effect, but in those with a previous 5-min experience of the plus-maze pirenzepine and mecamylamine had anxiogenic effects in the dose range of 30-300 ng; gallamine (100 and 300 ng) was without significant effect. Most research into the pharmacological mechanisms controlling anxiety has concentrated on the GABA A - benzodiazepine and 5-HT systems and evidence is accumu- lating as to the roles of these transmitter systems in limbic areas such as the hippocampus and amygdala. Stimulation of postsynaptic 5-HT 1A receptors with the agonist, 8-OH- DPAT, has anxiogenic effects following direct administra- tion to-the dorsal hippocampus (Andrews, Hogg, Gonzalez, & File, 1994) and basolateral nucleus of the amygdala (Gonzalez, Andrews, & File 1996; Hodges, Green, & Glenn, 1987). These are also areas in which direct administration of benzodiazepines has anxiolytic effects (Gonzalez et al., 1996; Hodges et al., 1987; Plaznik, Jessa, Bidzinski, & Nazar, 1994). There is growing evidence that the cholinergic system may also play a modulating role in anxiety, and anxiogenic effects have been reported after hippocampal administration of the muscarinic antagonist, scopolamine (Hess & Blozovski, 1987). Scopolamine has also been reported to have anxiogenic effects after systemic adminis- tration in mice (Rodgers & Cole, 1995; Smythe, Murphy, Bhatnagar, Timothy, & Costall, 1996) and normal volunteers (Curran, Shifano, & Lader, 1991). The novel muscarinic M r receptor agonist, xanomeline, has been reported to decrease fearfulness and agitation in geriatrics (Bodick & Offen, 1995). Nicotinic agonists have also been found to have anxiolytic effects (Brioni, O'Neill, Kim, & Decker, 1993; Sandra E. File, Luis E. Gonzalez, and Nick Andrews, United Medical and Dental Schools of Guy's and St. Thomas' Hospitals, Division of Pharmacology, Guy's Hospital, London, United Kingdom. These experiments were supported by a grant from the special trustees of Guy's Hospital. We are grateful to Peter Mabbutt for expert technical help. Correspondence concerning this article should be addressed to Sandra E. File, Psychopharmacology Research Unit, UMDS Divi- sion of Pharmacology, Guy's Hospital, London SE1 9RT, United Kingdom. Electronic mail may be sent to s.file@umds.ac.uk. Cao, Burkholder, Wilkins, & Collins, 1993; Decker et al., 1994; O'Neill & Brioni, 1994; Vale & Balfour, 1988), and anxiogenic effects have been found on withdrawal from chronic nicotine treatment (Costall et al., 1993). This accumulating evidence suggests that endogenous acetylcho- line has an anxiolytic role. The purpose of the present study was to explore hippocam- pal cholinergic modulation in different tests of anxiety by direct injection of nicotinic, M[ and M 2 receptor antagonists. Nicotinic and M! receptors are prevalent in the hippocampus (Rossner, Schliebs, Perez-Palo, Wiley, & Bigl, 1995) and there is evidence for low densities of presynaptic M 2 receptors (Stillman, Shukitt-Hale, Galli, Levy, & Lieber- man, 1996). The antagonists selected were mecamylamine, pirenzepine, and gallamine, respectively, all of which dis- solved in artificial cerebrospinal fluid (aCSF) at neutral pH, thus minimizing any nonspecific damage due to the injec- tions. Mecamylamine acts as an allosteric antagonist at brain nicotinic receptors (Takayama, Majewska, & London, 1989); pirenzepine is a muscarinic antagonist with a 37.2 fold selectivity for Mj over M 2 receptors and 11.2 fold selectivity for M, over M 3 receptors (Doods, Mathy, Davidesko, van Charldorp, & van Zwieten, 1987); gallamine is a muscarinic antagonist with highest affinity for M 2 receptors, but also with affinity for M, and M 3 receptors (Michel, Delmondo, Lopez, & Whiting, 1990). Centrally injected receptor antago- nists are particularly useful in establishing the endogenous tone of a neurotransmitter system (if there is no tonic activity, i.e., transmitter release acting at a particular recep- tor, then the appropriate antagonist will be silent). There is growing evidence that different animal tests are measuring different types or states of anxiety, and principal component analysis has shown that measures derived from different animal tests reflect different underlying factors (File, 1992). There is also evidence of quite distinctive roles for GABA and 5-HT in the control of behavior in the social interaction and elevated plus-maze tests (File & Gonzalez, 1996; 352 This document is copyrighted by the American Psychological Association or one of its allied publishers. This article is intended solely for the personal use of the individual user and is not to be disseminated broadly.