Electrochemical Analysis of Dopamine Release

描述（由申请人提供）：多巴胺（DA）是大脑运动、认知和奖励通路中的关键递质，其传递功能障碍与帕金森病、精神分裂症和成瘾等重大疾病有关。本项目的长期目标是确定调节纹状体致密黑质（SNc）和腹侧被盖区（VTA） DA神经元的体树突DA释放和轴突DA释放的局部因素。在之前的资助期内，我们专注于内源性谷氨酸、GABA和Ca2+进入对DA释放的调节，使用实时伏安法记录诱发的DA释放。我们发现过氧化氢（H2O2），一种活性氧（ROS），是SNc DA神经元的细胞内信使，既调节细胞放电速率，又抑制体树突DA释放。相反，在背侧纹状体中，H2O2是一个可扩散的信使，介导谷氨酸和GABA对轴突DA释放的调节。H2O2的这些作用是通过激活atp敏感的K+ (KATP)通道介导的。提出的工作将提供H2O2调控DA传输的机制，并表明H2O2信号传导对体突和轴突DA释放的功能影响。目的1将验证H2O2通过降低通道对ATP的敏感性来激活KATP通道的假设；Aim 2将测定H2O2生成的离子依赖性；目的3将研究H2O2的产生在谷氨酸能NMDA受体调节体树突DA释放和DA细胞生理中的作用；Aim 4将评估背纹状体中谷氨酸依赖的H2O2信号的时空特征。方法包括伏安法检测DA释放，全细胞和切除膜片记录，以及H2O2、细胞内离子和线粒体膜电位的荧光成像。实验系统包括分离的DA神经元、转染的细胞和来自豚鼠和缺乏特定KATP通道亚型的小鼠的脑切片。

DESCRIPTION (provided by applicant): Dopamine (DA) is a key transmitter in motor, cognitive, and reward pathways of the brain, with dysfunction of DA transmission linked to significant disorders, including Parkinson's disease, schizophrenia, and addiction. The long-term goal of this project is to identify local factors that regulate somatodendritic DA release from DA neurons in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) and axonal DA release in striatum. In the previous funding period, we focused on DA release regulation by endogenous glutamate, GABA, and Ca2+ entry, using real-time voltammetric recording of evoked DA release. We discovered that hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is an intracellular messenger in SNc DA neurons that both modulates cell firing rate and inhibits somatodendritic DA release. By contrast, in dorsal striatum, H2O2 is a diffusible messenger that mediates regulation of axonal DA release by glutamate and GABA. These effects of H2O2 are mediated by the activation of ATP-sensitive K+ (KATP) channels. Proposed work will provide mechanistic insight into regulation of DA transmission by H2O2, as well as indicate functional consequences of H2O2 signaling on somatodendritic and axonal DA release. Aim 1 will test the hypothesis that H2O2 activates KATP channels by decreasing channel sensitivity to ATP; Aim 2 will determine the ionic dependence of H2O2 generation; Aim 3 will investigate the role of H2O2 generation in the regulation of somatodendritic DA release and DA cell physiology by glutamatergic NMDA receptors; and Aim 4 will evaluate the temporal and spatial characteristics of glutamate-dependent H2O2 signaling in dorsal striatum. Methods include voltammetric detection of DA release, whole-cell and excised patch recording, and fluorescence imaging of H2O2, intracellular ions, and mitochondrial membrane potential. Experimental systems include isolated DA neurons, transfected cells, and brain slices from guinea pigs and from mice lacking a specific KATP channel subtype. Several brain disorders that involve DA dysfunction, including Parkinson's disease and schizophrenia, have also been linked to oxidative stress. Proposed studies will clarify how endogenous H2O2 normally regulates DA release. Because unregulated H2O2 can lead to oxidative stress, however, the findings may also point to possible targets for therapeutic intervention in these debilitating disorders. This project is based on our novel finding that hydrogen peroxide is an endogenous factor that regulates the nigrostriatal dopamine pathway. Understanding factors that regulate this pathway is important, since it is nigrostriatal dopamine that is lost in Parkinson's disease, leaving individuals unable to move. Proposed studies will clarify how endogenous hydrogen peroxide normally regulates dopamine release and dopamine neuron activity in this pathway. Additionally, because unregulated peroxide can lead to oxidative stress, which is a causal factor in Parkinson's disease, the findings may also point to possible new targets for therapeutic intervention, consistent with one aspect of the mission of NINDS.