Electrochemical Analysis of Dopamine Release

多巴胺(DA)是大脑运动、认知和奖赏通路的关键递质，其功能障碍 DA传播与严重的疾病有关，包括帕金森氏症、精神分裂症和成瘾。 该项目的长期目标是确定调节躯体树突状DA从DA释放的局部因素 黑质致密部(SNC)、腹侧被盖区(VTA)和轴突DA的神经元 在纹状体中释放。我们之前的工作集中在内源性谷氨酸、GABA和 Ca~(2+)进入，用实时伏安记录诱发的DA释放。我们发现氢 过氧化氢(H202)是黑质多巴胺神经元胞内信使中的一种活性氧物种。 两者都调节细胞的放电速率，并抑制躯体树突状细胞DA的释放。在背侧纹状体，H202是一种 介导谷氨酸和GABA调节轴突DA释放的可扩散信使。这些影响 是通过依赖H_2O_2激活ATP敏感K+(K-ATP)通道来实现的。拟议的工作将 提供对H202调节DA传递的机械性洞察，以及指示功能 过氧化氢信号对躯体树突状细胞和轴突DA释放的影响。目标1将检验这一假设 过氧化氢通过降低通道对ATP的敏感性来激活K-ATP通道；Aim 2将决定离子 H_2O_2生成的依赖性；目标3将研究H_2O_2生成在调节 通过谷氨酸能NMDA受体的躯体树突状DA释放和DA细胞生理；AIM 4将 评估背侧纹状体谷氨酸依赖的H202信号的时间和空间特征。 方法包括伏安法检测DA释放，全细胞和切除的斑片记录，以及 过氧化氢、细胞内离子和线粒体的荧光成像！利用转基因细胞， 分离的神经元，豚鼠和K-ATP通道亚单位敲除小鼠的脑切片。 几种涉及多巴胺功能障碍的大脑疾病，包括帕金森氏症和精神分裂症， 也与氧化应激有关。拟议的研究将阐明内源性过氧化氢是如何 正常情况下调节多巴胺的释放。由于不受监管的过氧化氢会导致氧化应激，然而， 研究结果还可能为这些疾病的治疗干预指明可能的目标。

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 substantial nigra pars compacta (SNc) and ventral tegmental area (VTA) and axonal DA release in striatum. Our previous work focused on release regulation by endogenous glutamate, GABA, and Ca2+ entry, using real-time voltammetric recording of evoked DA release. We discovered that hydrogen peroxide (H202), a reactive oxygen species (ROS) is in intracellular messenger in SNc DA neurons, that both modulates cell firing rate and inhibits somatodendritic DA release. In dorsal striatum, H202 is a diffusible messenger that mediates regulation of axonal DA release by glutamate and GABA. These effects are mediated by H2O2-dependent activation of ATP-sensitive K+ (K-ATP) channels. Proposed work will provide mechanistic insight into regulation of DA transmission by H202, as well as indicate functional consequences of H2O2 signaling on somatodendritic and axonal DA release. Aim 1 will test the hypothesis that H2O2 activates K-ATP 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 reguaiton 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 H202 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 mitochondria! potential, using transfected cells, isolated neurons, and brain slices from guinea pigs and K-ATP channel-subunit knockout mice. Several brain disorders that involve dopamine dysfunction, including Parkinson's disease and schizophrenia, have also been linked to oxidative stress. Proposed studies will clarify how endogenous hydrogen peroxide normally regulates dopamine release. Because unregulated H2O2 can lead to oxidative stress, however, the findings may also point to possible targets for therapeutic intervention in these disorders.