Presynaptic cholinergic degradation has been linked to cholinotoxic disorders such as Alzheimer's dementia. Direct quantitation of the changes in concentrations of neurochemical marker compounds upon selective cholinotoxic events is a challenging task. Here, the applicants will develop especially sensitive methodology to directly measure the changes in theses neurochemical markers in response to selective cholinotoxic events. They have prepared 6-hydroxycatecholine, a cholinomimetic analogue of 6-hydroxydopamine, and a number of related cholinotoxic agents in a series for the purpose of defining conditions for the introduction of selective cholinergic presynaptic lesions in the brain. Selective cholinotoxic events will be investigated in vitro with synaptosomal preparations to establish the selectivity and relative neurotoxicity of each neurotoxic agent. Subsequent in vivo studies will be conducted by microdialysis sampling in cannulated rat brain to monitor the near real-time changes which occur in a living system. Conditions for the application of selective neurotoxins will be sought to maximize the changes in levels of choline and acetylcholine, while at the same time minimize the changes in catecholamine and tryptophan neurotransmitters relative to controls. Analytical methods based on capillary electrophoresis with ultramicroelectrochemcial detection will be utilized to selectively and sensitively monitor changes of trace levels of neurotransmitters and their metabolites. Capillary electrophoresis techniques will permit efficient separation of uL to nL samples of species based on their electrophoretic mobility, while electrochemical detection with specifically designed ultramicroelectrode devices will allow for additional selectively and sensitive detection a the trace level. This strategy will allow sequential and time-resolved monitoring of altered metabolite an toxin uptake-efflux when coupled with microdialysis of tissues surrounding specific brain regions challenged with chemical toxins. The methodology will define the temporal and concentration-dependent acute and chronic selective intoxication of cholinergic presynaptic sites by 6-hydroxycatecholine and related quaternary ammonium catechol-based redox-affinity reagents. Thus, new methodologies to follow the chemical events in cholinotoxic states may be developed for future studies of animal models relevant to Alzheimer's dementia and for the study of underlying cholinergic contributions to mechanisms that govern consciousness and cognition.
