Introduction Muscarinic ACh receptors (MAChR), members of the seven-transmembrane protein receptors family whose response is mediated by G-proteins, 1 are wide- spread throughout the body, and are involved in many fundamental physiological processes. In the central nervous system (CNS) cholinergic transmis- sion is mainly mediated by MAChR and has been suggested that it is involved in learning and memory processing. Five subtypes of MAChR receptors are expressed in the mammalian brain (m1–m5) and their coding genes have been cloned. 2–4 There are differ- ences in the concentration of the receptor subtypes in various brain regions and more than one subtype is often expressed in the same cell. It has been diffi- cult to study their localization, quantification and function due to the lack of selective ligands that exclusively act on one receptor subtype. As the best example, pirenzepine, a relative selective antagonist for m1 MAChR, 5 also binds with rather high affinity to the m4 subtype and, although with lower affinity, binds to all the remaining subtypes. 6 Adem et al. 7 isolated two protein fractions from the venom of the green mamba snake (Dendroaspis angusticeps) that partially inhibited the binding of the radiolabelled muscarinic antagonist quinuclidinyl benzilate ([ 3 H]QNB) to rat cerebral cortex and were named MT1 and MT2 (MT, muscarinic toxins). We have also isolated these two proteins from the same venom, and showed that they inhibited half the binding of N-methyl-scopolamine ([ 3 H]NMS) to cerebral cortex membranes but only inhibited about 30% of this binding to brain stem membranes. 8 MT1 and MT2 almost completely inhibited the specific binding of [ 3 H]pirenzepine, the relative selective antagonist for the M 1 muscarinic receptor. 8 Karlsson and colleagues continued isolating different proteins with muscarinic actions, with close related mol. wt (around 7 kDa). 9 Among them, MT3 was also purified by the group of Potter and was named m4 toxin. 10 MT3 was selective for the m4 receptor, with 40-fold lower affinity for the m1 subtype. MT1 was shown to have almost the same affinity for both m1 and m4 subtypes, while MT2 showed lower affinity but higher selectivity for m1 receptor than Learning and Memory 1111 2 3 4 5 6 7 8 9 10111 1 2 3 4 5 6 7 8 9 20111 1 2 3 4 5 6 7 8 9 30111 1 2 3 4 5 6 7 8 9 40111 1 2 3 4 5 6 7 8 9 50111 1 2 3 4 5 6111p © Rapid Science Ltd Vol 9 No 7 11 May 1998 1407 THE selectivity of the muscarinic toxin MT3 from green mamba snake venom was corroborated by inhibition of the binding of [ 3 H]NMS, a classical muscarinic radioli- gand, to native and cloned muscarinic receptors, showing 214-fold higher affinity for m4 than for m1 subtype, without significant binding to the others. The highest concentrations of MT3 sites (putative m4 recep- tors) in the rat brain were found in striatum and olfac- tory tubercle, intermediate concentration in dentate gyrus and CA1, and lower but still conspicuous levels in CA3 and frontal cortex. MT3 caused retrograde amnesia of an inhibitory avoidance task, when injected into the dorsal hippocampus of rats after training, suggesting a positive role of these MT3 sensitive sites, which are prob- ably m4 muscarinic receptors, in memory consolidation of this task. NeuroReport 9: 1407–1411 © 1998 Rapid Science Ltd. Key words: Memory; Muscarinic m4 receptors; Muscarinic receptors; Muscarinic toxin Muscarinic toxin selective for m4 receptors impairs memory in the rat Diana Jerusalinsky, CA Edgar Kornisiuk, Paula Alfaro, Jorge Quillfeldt, 1 Mariana Alonso, Emiliano Rial Verde, Carlos Cerveñansky 2 and Alan Harvey 3 Institute of Cell Biology and Neuroscience ‘Prof. Eduardo De Robertis’, Faculty of Medicine, University of Buenos Aires, 2155 Paraguay Street, 3rd Floor, 1121 Buenos Aires, Argentina; 1 Department of Biophysics, Federal University of Rio Grande Do Sul (Campus), Porto Alegre, RGS, Brazil; 2 Department of Biological Investigations ‘Clemente Estable’, Montevideo, Uruguay; 3 Department of Physiology and Pharmacology, University of Strathclyde, Glasgow, UK CA Corresponding Author Website publication 3 April 1998 NeuroReport 9, 1261–1265 (1998)