Neurotransmitter Interactionson Sub-Second Timescales

谷氨酸和其他神经递质/神经调节剂对多巴胺释放亚秒控制的具体机制尚不为人所知，主要是因为解决这些重要问题所需的分析方法尚未得到充分发展。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电活性生物胺（如多巴胺）的释放和摄取，但其在表征亚秒神经递质/神经调节性相互作用方面的应用尚缺乏。这一缺陷代表了一个严重的障碍，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亚秒的时间范围内。因此，本研究的中心目标是开发和应用工具，以定量研究这些神经递质/神经调节剂在组织和体内的相互作用。两种时间相容的方法，快速扫描循环伏安法和对羟基苯基笼型谷氨酸（pHP-Glu）的光激活法，将被用作模型笼系统，用于测量多巴胺释放对毫秒时间尺度暴露于外源性谷氨酸的亚秒反应。本研究的总体目标将通过成功完成两个具体目标来实现：(1)将亚秒电诱发多巴胺释放测量与脑切片中笼化化合物的光激活结合起来；(2)优化探针/碳纤维复合微电极的构建和使用，以测量笼化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，因为它是第一个同时应用这两种高时间分辨率技术在活组织和动物。该方法的发展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亚秒时间尺度多巴胺释放对谷氨酸应用的响应。此外，这项工作应该具有广泛的影响，因为这些技术的应用可以扩展到包括其他电活性神经递质和神经调节剂的检测，如血清素、过氧化氢（H{2}O{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，即寻求有关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，这适用于但不限于：(1)基础神经生物学研究；(2)多巴胺相关运动障碍的研究[如亨廷顿病（HD）、帕金森病（PD）、图雷特综合征（TS）]；(3)成瘾与抑郁的研究；(4)研究中枢神经系统活性药物的作用机制。

Ttie 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 this proposed research is to develop and apply tools that will allow for the quantitative study of these neurotransmitter/neuromodulator interactions in tissues and in vivo. Two temporally-compatible methods, fast-scan cyclic voltammetry and the photoactivation ofthe 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) integrate the sub-second measurement of electrically-evoked dopamine release with the photo-activation of caged compounds in brain slices and (2) optimize the construction and use of a combined 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 ¿mu¿s-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 (H{2}O{2}), 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.