HPLC/EC Detection and Quantification of Acetylcholine in Dialysates

Acetylcholine (ACh) is a neurotransmitter widely distributed in the central nervous system (CNS) and peripheral nervous system (PNS). Its role as a neurotransmitter was first elucidated by Dale, who noted that ACh mimicked the effects of parasympathetic nerve stimulation and by Otto Loewi, who demonstrated the vagal release of a substance that slowed heart rate (1 –3 ). More recently ACh in the CNS has been implicated in sensorimotor arousal, attention, sleep regulation, and learning and memory (4 –8 ). Its distribution in the CNS includes the entire cortical mantle and hippocampus innervated by cholinergic neurons of the basal forebrain and the interpeduncular nucleus innervated by the medial habenula. The striatum, nucleus accumbens, and olfactory tubercle each contain cholinergic interneurons (9 ). The extraction of ACh from these areas in intact preparations historically has been largely via push-pull cannulae and now via microdialysis (10 ,11 ). Once extracted from the brain, however, the separation (by high-performance liquid chromatography [HPLC]) and quantification (by electrochemical detection) of ACh presents an unusually difficult problem. Such a difficulty arises first from the extremely rapid hydrolysis of ACh in vivo by ACh esterase and second from the resistance of the ACh molecule to electrochemical oxidation. The first of these problems can be overcome by in vivo application of an esterase inhibitor such as neostigmine, although the use of too high a concentration can dramatically disturb the physiological regulation of the cholinergic system and introduce artifactual experimental results (12 –14 ).