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THE existence of high concentrations of N-acetylaspartic acid in the central nervous systems of mammals and birds has been known for a number of years (TALLEN, MOORE and STEIN, 1956) but its purpose there remains an enigma. It was felt that means for a rapid quantitative assay of this compound not only in extracts from whole brain but in histologically distinct layers of brain would be a valuable step towards elucidation of its function. E X P E R I M E N T A L General methods. The assay procedure is an enzymic one in which N-acetylaspartic acid is deacetylated to aspartic acid with acylase 11. When large amounts of material are available the aspartic acid is assayed spectrophotometrically with NADH, glutamate-oxalacetate transaminase, and malate dehydrogenase in a manner similar to that described by PFLEIDERER, GRUBER and WEILAND (1955) and JACOBSON (1959). To measure low levels of N-acetylaspartic acid, the sensitivity is increased by substituting fluorimetry for spectrophotometry. In this case, because NADH levels have to be kept low and the acylase I1 prepared by the present procedure contains considerable NADH oxidase activity, deacylation and transamination are carried out before NADH is added. O n heating to loo", the acylase I1 and transaminase (and NADH oxidase) are destroyed and the oxalacetate that has been formed is at the same time converted to pyruvate. This is subsequently measured with lactate dehydrogenase and NADH. For measuring still smaller quantities of aspartic acid and N-acetylaspartic acid in microgram sections of brain the cycling procedure described by LOWRY, PASSONNEAU, SCHULZ and ROCK (1961) is used to determine the NADI-I formed in the final step.Materials. N-acetylaspartic acid was made from L-aspartic acid and acetic anhydride according to the method of BARKER (1953). (This substrate is now available from Sigma Chemical Company, St. Louis, Mo.) L-aspartic acid was obtained from Sigma Chemical Company. Heart lactate dehydrogenase was obtained from Worthington Biochemical Corporation, and skeletal muscle lactate dehydrogenase and malate dehydrogenase from Boehringer and Sons through the California Corporation for Biochemical Research. The glutamate-oxalacetate transaminase used was a preparation made by Dr. JANET V. PASSONNEAU. This enzyme is now available from California Corporation for Biochemical Research and Sigma Chemical Company, and the quantities used are given in terms of the Boehringer preparation.Prepurarion aftissues. For study of changes in aspartic acid and N-acetylaspartic acid with anoxia, mice were frozen whole in Freon 12 kept at its freezing point in liquid nitrogen, or their heads were frozen at intervals after decapitation. The brain, minus the cerebellum, was dissected out in a cold room at -20", powdered in a mortar cooled in liquid nitrogen, weighed and placed on 0.3 ml of frozen 3M-HC10,. Subsequent dilution and buffering were made as described by LOWRY, PASSONNEAU, HASSELBERGER and SCHULTZ (1964). The final extract represented about 005 g of brain/ml.

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Some drugs with depressant properties on the peripheral nervous system have been examined for depressant effects on the reticular activating system of the cat. Large doses of nicotine, or of the anti-nicotinic agents dihydro-perythroidine and mecamylamine, failed to depress the reticular activating system; non-quaternary drugs with anti-muscarinic properties, such as atropine and hyoscine, depressed it readily. Hyoscine was the most potent depressant tested and its effects could be antagonized by physostigmine. In contradistinction, depression of the reticular activating system with pentobarbitone was not antagonized by physostigmine. Lignocaine was a weak depressant of the reticular activating system, and the possibility that this might be due to a central anti-muscarinic action is discussed. Adrenergic blocking drugs, such as dihydroergotamine, phenoxybenzamine or choline 2: 6-xylyl ether, did not appear to depress the reticular activating system: the significance of this is discussed. It was concluded that the hypothetical cholinergic transmitter, acting somewhere within the reticular activating system, displayed actions analogous to the muscarinic, and not to the nicotinic, actions of acetylcholine.The discovery by Moruzzi and Magoun (1949) that electrical stimulation of the brain stem reticular formation is followed by behavioural and electroencephalographic signs of arousal in animals was an outstandingly important contribution to neurological science. The recognition of such a reticular activating system has provided neurophysiologists, and in particular Magoun and his associates, with a means of disentangling some of the complex reactions of the brain to the influx of sensory information; it has stimulated neuroanatomists in their search for recognizable functional pathways inter-connecting cortex and midbrain; and it has given neuropharmacologists a valuable tool for investigating the effects of drugs upon the elusive central synapse.The demonstration that the reticular formation is particularly susceptible to the depressant effects of hypnotic and anaesthetic agents (French, Verzeano, and Magoun, 1953a;Arduini and Arduini, 1954) has been followed by several useful analyses of the effects of various drugs on the reticular arousal system (for example, Domino,