Neurotransmitter Interactionson Sub-Second Timescales

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，很大程度上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。尽管一种技术，即碳纤维微电极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吸收，但其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尚缺乏。这种缺陷代表了一个严重的障碍，因为影响外在身体反应和认知事件的大脑信号事件发生在这个亚秒级的时间范围内。因此，这项研究的中心目标是开发和应用能够对组织和体内这些神经递质/神经调节剂相互作用进行定量研究的工具。两种时间兼容的方法，快速扫描循环伏安法和谷氨酸对羟基苯基笼形形式（pHP-Glu）的光活化（此处用作模型笼式系统）将用于测量多巴胺释放响应于毫秒时间尺度暴露于外源性谷氨酸的亚秒响应。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）将电诱发多巴胺释放的亚秒测量与脑切片中笼状化合物的光激活相结合；（2）优化组合探针/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。这种方法具有创新性，因为它是第一个在活体组织和动物中同时应用这两种高时间分辨率技术的方法。该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亚秒级多巴胺的释放，以响应“μs”时间尺度的谷氨酸应用。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展到包括其他电活性神经递质和神经调节剂的检测，例如血清素、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活化，例如GABA和专门设计的合成激动剂/拮抗剂。重要的是，这项研究与 NIH 的使命直接相关，即寻求有关生命系统的性质和行为的基础知识，并减轻疾病和残疾的负担，适用于但不限于：（1）基础神经生物学研究； (2) 多巴胺相关运动障碍的研究[例如亨廷顿病（HD）、帕金森病（PD）、抽动秽语综合征（TS）]； (3)成瘾和抑郁症的研究； (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.