EE ———_ Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3 Reprinted from Archives internationales de Pharmacodynamie et de Thérapie - Vol. 209 - No. 2 - fune 1974 Studies with Agents which Influence Acetylcholine Metabolism in Mouse Brain J. K. Saztens, J. Pp. Stuke, J. ScitUMAN AND M. P. ALLEN Research Department, Pharmaceuticals Division, CIBA-GEIGY Corporation, Summit, New Jersey, 07901—-U.S.A. a Abstract—By using an intravenous single pulsc injection of choline-C™ into the tail vein of mice and measuring the endogenous levels of choline and acctylcholine, the effects of agents which influence acetyl- choline metabolism in mouse brain were studied. The results were consistent with a feedback activation of cholinergic neurons with scopola- mine, choline-0-acetyltransferase inhibition with naphthylvinylpyridine, inhibition of acetylcholine release by morphine and cholinesterase inhibition with physostigmine. ‘The interactions of these agents with the cholinergic system further suggests certain characteristics of cholinergic neurons, ¢.g. that choline-0-acetyltransferase activity is accelerated by a decrease in intracellular acetylcholine levels and retarded by an increase in intracellular acetylcholine levels, that the choline transport mechanism is functioning at maximum capacity under normal physiol- ogical conditions and that the synaptic levels of endogenous choline play a role in tracer choline capture. ee Introduction In 1969 Schuberth, e¢ ai. (1) introduced a technique for measuring the rate of synthesis of acetylcholine (ACh) in whole mouse brain. In their procedure the rate of radiactive ACh formation in the brain was measured. following an intra- venous pulse injection of tritium-labelled choline (Ch). After determination of the isotope dilution of the Ch in the brain, the ACh rate of synthesis, in vivo, was calculated. We have now applied this technique involving four agents purported to affect central cholinergic neurons in different ways. The agents are the muscarinic cholinergic receptor blocking agent, scopolamine; the choline-0- acetyltransferase inhibitor, naphthylvinylpyridine (2, 3); the cholinesterase inhibitor, physostigmine; and an agent reported. to block the release of ACh, morphine (4, 5, 6). The results presented below are consistent with the proposed 250 Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3
rs Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3 ACETYLCHOLINE METABOLISM 251 mechanisms of the various agents and help to further define certain character- istics of central cholinergic neurons. Methods Administration of Choline-Methyl-C™ (Ch-C'*). Male CFS (Carworth Farms) mice, 18-20 g were used. Each mouse received 5 pe (0.66 moles) of Ch-C¥4 chloride (New England Nuclear, specific activity = 7.6 pc{ molec) intravenously in a tail vein. The tracer was dissolved in distilled water and administered in a volume of 0.1 ml with a gas-tight Hamilton syringe through a 30 gauge necdle. Preparation of Brain Samples. 'The mice were sacrificed at different times after injection by rapid immersion of the whole animal into a Dewar flask containing liquid N, for 3.5 seconds. Individual mouse brains were removed as rapidly as possible, dissected on a thermoelectric cold plate maintained at 1~2° C, and frozen in liquid N, . Where whole brain analysis was performed, the brain was placed directly in liquid N,. Timing was critical with this method. If the immersion lasted for more than 4 seconds, the surface of the cortex began to freeze and stick to the skull making removal of the tissue difficult. The frozen brains or brain parts were weighed on a Mettler electronic balance and then pulverized in a Thermovac (R) tissue crusher cooled with dry ice and liquid nitrogen. The remainder of the procedure was as described for rats by Saelens et al. (7). Determination of ACh and Ch. ACh and Ch were determined by the enzymatic method of Feigenson and Saclens (8) using the modifications recommended by Saelens et al. (9). Statistical Analysis. The disposition of Ch-C™, and the levels of Ch and ACh were determined in different groups of mice. In order to obtain the appropriate parameters, the product of two independent variables were analyzed by the method of Goodman (10). At least 5-10 mice were uscd. in all measurements. Agents. Test agents were dissolved in distilled water, and administered subcutaneously, or intraperitoneally, in a volume of 0.1 ml/10 g of body weight or intravenously in a volume of 0.05 ml/10 g of body weight. Mice received food and water ad libitum up until the time of sacrifice. The agents studied were scopolamine hydrobromide (Merck), naphthylvinyl- pyridine (Aldrich), physostigmine sulfate (Merck) and morphine sulfate (Merck). Results and Discussion Control Studies: The Schuberth model for estimating the rate of synthesis of ACh is essentially a single closed compartment model. To be valid, the level of Approved For Release 2000/08/07 : CIA-RDP96-00787R000500200004-3