NEW METHODS FOR EVALUATING CHOLINERGIC TOXICITY

描述(摘自申请人摘要)突触前胆碱能降解 与胆碱毒性疾病有关，如阿尔茨海默氏症。 直接定量测定神经化学标志物的浓度变化 针对选择性胆碱毒性事件的化合物是一项具有挑战性的任务。这里, 申请者将制定特别敏感的方法来直接 检测这些神经化学标记物对选择性免疫反应的变化 胆碱中毒事件。他们已经制备了6-羟基儿茶酚，一种 6-羟基多巴胺的类胆碱类似物，以及一些相关的 用于确定条件的一系列胆碱毒性药物 在大脑中引入选择性胆碱能突触前病变。 选择性胆碱毒性事件将在体外进行研究 建立突触小体准备的选择性和相对 每种神经毒剂的神经毒性。后续的体内研究将是 通过对空腹大鼠脑内进行微透析采样来监测 在一个有生命的系统中发生的接近实时的变化。适用的条件 将寻求应用选择性神经毒素来最大限度地改变 胆碱和乙酰胆碱的水平，同时将 儿茶酚胺和色氨酸类神经递质的变化 控制。基于毛细管电泳法的分析方法 将利用超微电化学检测来选择性地和 灵敏地监测神经递质及其微量水平的变化 代谢物。毛细管电泳法将使高效 基于电泳法的物种UL到NL样品的分离 机动性，同时电化学检测具有专门设计的 超微电极设备将允许额外的选择性和 灵敏的检测是一种示踪水平。这一策略将允许连续 和时间分辨监测改变的代谢物毒素摄取-外排 当结合特定脑组织周围组织的微透析时 面临化学毒素挑战的地区。该方法将定义 时间和浓度依赖的急性和慢性选择性 6-羟基儿茶酚和6-羟基儿茶酚对突触前胆碱能部位的毒性作用 相关的邻苯二酚基季铵盐类氧化还原亲和试剂。因此， 追踪胆碱中毒状态下的化学事件的新方法可能 将用于未来与阿尔茨海默氏症相关的动物模型的研究 并用于研究潜在的胆碱能对痴呆的贡献 支配意识和认知的机制。

DESCRIPTION (from applicant's abstract) 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.