New Methods for Evaluating Cholinergic Toxicity

描述（由申请人提供）：胆碱能降解与胆碱能疾病如阿尔茨海默氏痴呆症有关。直接量化与神经损害事件相关的神经化学标记化合物浓度的变化是一项具有挑战性的任务。在这里，我们将开发特别敏感的方法来直接测量这些神经化学标记物在选择性胆碱能降解反应中的变化。我们已经制备了6-羟基儿茶酚，一种6-羟基多巴胺的胆碱模拟物，以及一系列相关的基于儿茶酚的化学制剂，目的是确定在大脑中引入选择性胆碱能突触前病变的条件。选择性胆碱毒性事件将在体外用突触体制剂进行研究，以确定每种儿茶酚基化学制剂的选择性和相对神经毒性。随后的体内研究将通过微透析取样在插管的大鼠大脑中进行，以监测生命系统中发生的近乎实时的变化。将寻求应用选择性神经损害剂的条件，使胆碱和乙酰胆碱水平的变化最大化，同时使儿茶酚胺和色氨酸神经递质相对于对照组的变化最小化。毛细管电泳微电化学检测是一种选择性、灵敏地监测神经递质及其代谢物微量含量变化的分析方法。毛细管电泳技术可以根据物种的电泳迁移率有效地分离μ l到nL样品，而电化学检测采用特殊设计的微电极装置，可以在痕量水平上进行额外的选择性和敏感检测。该策略将允许对儿茶酚类化学制剂及其代谢物摄取外排进行连续和时间分辨监测，同时对受化学制剂损害的特定脑区域周围的组织进行微透析。该方法将定义6-羟基儿茶酚和相关的季铵盐儿茶酚氧化还原亲和试剂对胆碱能突触前位点的时间和浓度依赖性急性和慢性选择性损害。因此，可以开发新的方法来跟踪胆碱中毒状态下的化学事件，用于与阿尔茨海默氏痴呆症相关的动物模型的未来研究，以及研究潜在的胆碱能对控制意识和认知机制的贡献。

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.