New Methods for Evaluating Cholinergic Toxicity

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DESCRIPTION (provided by applicant): Cholinergic degradation has been linked to cholinergic disorders such as Alzheimer's dementia. Direct quantitation of the changes in concentrations of neurochemical marker compounds associated with neurocompromising events is a challenging task. Here, we will develop especially sensitive methodology to directly measure the changes in these neurochemical markers in response to selective cholinergic degradation. We have prepared 6-hydroxycatecholine, a cholinomimetic analogue of 6-hydroxydopamine, and a number of related catechol-based chemical 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 catechol-based chemical agent. Subsequent in vivo studies will be conducted by microdialysis sampling in cannulated rat brain to monitor the near real-time changes that occur in a living system. Conditions for the application of selective neurocompromising agents 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 microelectrochemical detection have been developed to selectively and sensitively monitor changes of trace levels of neurotransmitters and their metabolites. Capillary electrophoresis techniques permit efficient separation of mu l to nL samples of species based on their electrophoretic mobility, while electrochemical detection with specially designed microelectrode devices allow for additional selectivity and sensitive detection at the trace level. This strategy will allow sequential and time-resolved monitoring of the catechol-based chemical agents and their metabolites uptake efflux when coupled with microdialysis of tissues surrounding specific brain regions compromised with the chemical agents. The methodology will define the temporal and concentration-dependent acute and chronic selective compromise 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.