Electrochemical Analysis of Dopamine Release

多巴胺（DA）是大脑运动、认知和奖赏通路中的关键递质， 多巴胺的传递与重大疾病有关，包括帕金森病、精神分裂症和成瘾。 本项目的长期目标是确定调节DA释放的局部因素 黑质腹侧被盖区（VTA）和轴突DA能神经元 纹状体释放。我们以前的工作集中在内源性谷氨酸，GABA， Ca 2+进入，使用诱发DA释放的实时伏安记录。我们发现氢 过氧化物（H2 O2），一种活性氧物质（ROS），在SNc DA神经元的细胞内信使中， 都调节细胞放电率并抑制体树突DA释放。在背侧纹状体中，H2 O2是一种 谷氨酸和GABA介导轴突DA释放调节的可扩散信使。这些影响 由ATP敏感性K+（K-ATP）通道的H2 O2依赖性激活介导。拟议工作将 提供H2 O2调节DA传输的机制见解，以及指示功能性 H2 O2信号对体树突和轴突DA释放的影响。目标1将检验假设 H2 O2通过降低通道对ATP的敏感性来激活K-ATP通道;目的2将确定H2 O2的离子浓度。 H2 O2生成的依赖性;目标3将研究H2 O2生成在调节 体树突DA释放和DA细胞生理学通过谷氨酸能NMDA受体;和Aim 4将 评估背侧纹状体中谷氨酸依赖性H2 O2信号传导的时间和空间特征。 方法包括DA释放的伏安检测、全细胞和切除的斑片记录，以及 H2 O2、细胞内离子和线粒体的荧光成像！潜在的，使用转染细胞， 分离的神经元和来自豚鼠和K-ATP通道亚基敲除小鼠的脑切片。 几种涉及多巴胺功能障碍的大脑疾病，包括帕金森病和精神分裂症， 也与氧化应激有关。拟议的研究将阐明内源性过氧化氢 正常调节多巴胺的释放然而，由于不受调节的H2 O2可导致氧化应激， 这些发现也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.