Electrochemical Analysis of Dopamine Release

描述(申请人提供)：多巴胺(DA)是大脑运动、认知和奖赏通路的关键递质，DA传递功能障碍与严重的疾病有关，包括帕金森氏症、精神分裂症和成瘾。本项目的长期目标是找出调节黑质致密部(SNC)和腹侧被盖区(VTA)的DA神经元和纹状体的轴突DA释放的局部因素。在之前的资助期间，我们重点研究了内源性谷氨酸、GABA和钙离子进入对DA释放的调节，使用实时伏安记录诱发的DA释放。我们发现，过氧化氢(H_2O_2)是一种活性氧物种(ROS)，在黑质DA神经元中是一种细胞内信使，它既调节细胞的放电速率，又抑制躯体树突状细胞DA的释放。相反，在背侧纹状体，H_2O_2是一种可扩散的信使，介导谷氨酸和GABA对轴突DA释放的调节。H_2O_2的这些作用是通过激活ATP敏感K+(KATP)通道来实现的。拟议的工作将提供对过氧化氢调节DA传递的机械性见解，以及表明过氧化氢信号对躯体树突状细胞和轴突DA释放的功能影响。目的1将验证H_2O_2通过降低通道对ATP的敏感性来激活KATP通道的假设；目的2将确定H_2O_2产生的离子依赖性；目的3将研究H_2O_2产生在谷氨酸能NMDA受体调节躯体树突状细胞DA释放和DA细胞生理中的作用；目的4将评估背侧纹状体谷氨酸依赖的H_2O_2信号的时间和空间特征。方法包括伏安法检测DA释放、全细胞和离体膜片记录、H_2O_2、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、转基因细胞和来自豚鼠和缺乏特定KATP通道亚型的小鼠的脑切片。 包括帕金森氏症和精神分裂症在内的几种涉及DA功能障碍的大脑疾病也与氧化应激有关。拟议的研究将阐明内源性过氧化氢通常如何调节DA的释放。然而，由于不受调控的过氧化氢会导致氧化应激，这一发现也可能指出治疗这些衰弱障碍的可能靶点。 这个项目是基于我们的新发现，即过氧化氢是调节黑质纹状体多巴胺途径的内源性因素。了解调节这一途径的因素很重要，因为正是黑质纹状体多巴胺在帕金森氏症中丢失，导致个人无法移动。拟议的研究将阐明内源性过氧化氢如何正常调节这一途径中的多巴胺释放和多巴胺神经元活动。此外，由于不受调控的过氧化氢会导致氧化应激，而氧化应激是帕金森氏症的一个原因，这一发现还可能指出治疗干预的新靶点，这与NINDS使命的一个方面是一致的。

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.