Methods for Measuring Sub-second Neurotransmitter Interactions

描述(申请人提供)：谷氨酸和其他神经递质/神经调节剂亚秒控制多巴胺释放的特殊机制尚不为人所知，主要是因为解决这些重要问题所需的分析方法尚未充分发展。尽管碳纤维微电极上的快速扫描循环伏安法提供了足够的时间分辨率来测量多巴胺等电活性生物胺的释放和摄取，但它在表征这种亚秒级神经递质/神经调节剂相互作用方面的应用还很少。这一缺陷是一个严重的障碍，因为大脑中影响外部身体反应和认知事件的信号事件发生在这个亚秒级时间段内。因此，这项拟议研究的中心目标是开发和应用工具，以便对活组织和活体中的这些神经递质相互作用进行定量研究。对于时间相容的方法，快速扫描循环伏安法和对羟基苯基笼状谷氨酸的光活化(PHP-Glu)将被用作模型笼状系统，用于测量多巴胺释放对毫秒级时间尺度外源谷氨酸暴露的响应。这项研究的总体目标将通过成功完成两个具体目标来实现：(1)建立和验证同时定量测量多巴胺释放和光激活谷氨酸的电化学方法；(2)优化光活化探针/碳纤维微电极的构造和使用，以测量笼状化合物光激活对体内多巴胺释放事件的影响。这种方法是创新的，因为它是第一批同时将这两种高时间分辨率技术应用于活组织和动物的方法之一。这一拟议方法的开发和应用具有重要意义，因为它将使实验室研究人员能够测量响应毫秒时间刻度谷氨酸应用的亚秒时间刻度多巴胺释放。此外，这项工作应该会产生广泛的影响，因为这些技术的应用可以扩大到包括检测其他电活性神经递质和神经调节剂，如5-羟色胺、过氧化氢(H_2O_2)和一氧化氮(NO)，以及其他生物活性分子的光激活，如GABA和专门设计的合成激动剂/拮抗剂。重要的是，这项研究直接关系到NIH的使命，即寻求关于生命系统的性质和行为的基本知识，并减少疾病和残疾的负担，适用于但不限于：(1)基础神经生物学研究；(2) 与多巴胺相关的运动障碍的研究[例如亨廷顿病(HD)、帕金森病(PD)、多发性抽动症(TS)]；(3)成瘾和抑郁的研究；以及(4)中枢神经系统活性药理药物作用机制的研究。

DESCRIPTION (provided by applicant): The particular mechanisms underlying the sub-second control of dopamine release by glutamate and other neurotransmitters/neuromodulators are not well-known largely because the analytical methodology required to address these important questions has not been sufficiently developed. Although one technique, fast-scan cyclic voltammetry at carbon-fiber microelectrodes, offers sufficient temporal resolution to measure the release and uptake of electroactive biogenic amines such as dopamine, its use in characterizing such sub-second neurotransmitter/neuromodulator interactions is lacking. This deficiency represents a serious roadblock because signaling events in the brain that influence outward physical responses and cognitive events occur within this sub-second time regime. Therefore, the central aim of the proposed research is to develop and apply tools that will allow for the quantitative study of these neurotransmitter interactions in living tissues and in vivo. To temporally-compatible methods, fast-scan cyclic voltammetry and the photoactivation of the p- hydroxyphenyl caged form of glutamate (pHP-Glu), used here as a model caged system, will be applied to measure the sub-second response of dopamine release in response to millisecond timescale exposures to exogenous glutamate. The overall objective of this proposed research will be accomplished by successfully completing two specific aims: (1) Develop and validate an electrochemical method to quantitatively measure dopamine release and photoactivated glutamate simultaneously and (2) Optimize the construction and use of a combined photoactivation probe/carbon-fiber microelectrode for measuring the impact of caged compound photoactivation on dopamine release events in vivo. This approach is innovative because it is among the first to simultaneously apply these two high temporal resolution techniques in living tissues and animals. The development and application of this proposed methodology is significant because it will enable laboratory researchers to measure sub-second timescale dopamine release in response to ms-timescale glutamate application. Moreover, this work should have a broad impact since the application of these techniques can be expanded to include the detection of other electroactive neurotransmitters and neuromodulators, such as serotonin, hydrogen peroxide (H2O2), and nitric oxide (NO), and the photoactivation of other bioactive molecules, such as GABA and specifically designed synthetic agonists/antagonists. Importantly, this research directly relates to the NIH mission of seeking fundamental knowledge about the nature and behavior of living systems and reducing the burdens of illness and disability in that applies to, but is not limited to: (1) fundamental neurobiological studies; (2) studies of dopamine-related movement disorders [e.g. Huntington's disease (HD), Parkinson's disease (PD), Tourette's syndrome (TS)]; (3) studies of addiction and depression; and (4) studies addressing the mechanisms of action of CNS-active pharmacological agents.