PRESYNAPTIC PLASTICITY OF VESICULAR DOPAMINE RELEASE

精神刺激剂药物会导致长期的行为变化，包括 依赖和精神错乱。这些行为背后的突触出现了 与心理刺激剂背后的短期机制不同 奖励。与费吉斯·凯利博士和罗伯特·爱德华兹博士合作，我们 刺激和诱导机制的研究概况 精神刺激剂诱导的多巴胺突触可塑性。 我们将测量自身受体激活对释放频率的影响 多巴胺囊泡。用电化学技术检测药物的释放 从PC12细胞中分离出单个DA囊泡，我们发现D2自身受体 激活通过降低突触前DA功能迅速抑制突触前DA功能 依赖刺激的囊泡释放频率。我们将建立 无论这些效应是由于离子电导的改变还是由于囊泡的改变 融合/对接机制和长期接触D2激动剂是否促进 心理刺激或刺激依赖的释放的敏化。 2.我们将确定VMAT表达在量子大小中的作用 从DA囊泡中释放。我们最近证明了可卡因和 安非他明和D2自身受体的激活会减少突触 囊泡通过减小量子尺寸来介导多巴胺的释放。使用PC12 由爱德华兹博士开发的线条，我们将确定是否改变 肾上腺囊泡单胺转运体的磷酸化 (VMAT1)改变量子大小，如果这是通过以下方式差分调制的 精神刺激剂、D2激动剂和VMAT抑制剂。我们会比较一下 PC12株表达高水平VMAT1的结果和 表达突触小泡VMAT(VMAT2)，它与Amph有更强的亲和力。 3.我们将检测PC12单个突触小泡的DA释放 细胞和中脑神经元。我们已经进行了第一次直接测量 中脑DA神经元的量子释放；然而，这一方案是 具有挑战性的，不容易接受实验操作的。这个 我们在PC12细胞中使用的技术测量了大型PC12中DA的释放 小泡，但不是更小的小泡，更接近于传统的 突触小泡。爱德华兹博士的发现表明，刺激 VMAT2转基因的PC12细胞从小泡中释放DA；如果是这样，我们将 观察易突变和转基因制剂的胞吐作用 操纵。我们还将确定潜在的VMAT分类信号 序列可用于将VMAT靶向小囊泡。 4.我们将确定神经元刺激、自身受体的影响 激活，以及对囊泡分类和回收的精神刺激剂。Dr。 凯利的研究表明，对回收的监管可能会控制这一数字 以及可供释放的分泌小泡的类型。我们将研究 Naive和NGF处理的PC12细胞和中脑DA神经元的再循环 大致密核颗粒与突触的差异标记成分 确定囊泡中酸性循环中间体的作用 (内吞体体)。我们将扩大观察范围，表明去极化 增加细胞内的pH梯度，这可能有助于 膜回收。我们将确定精神刺激剂和短期 长期的D2激活暴露抑制了囊泡的循环。

Psychostimulant drugs induce long-lasting behavioral changes including dependence and psychosis. The synaptic underlying these behaviors appear to be different than short-term mechanisms that underlie psychostimulant reward. In conjunction with Drs. Fegis Kelly and Robert Edwards, we outline research to elcudiate mechanisms of stimulation- and psychostimulant-induced dopamine synaptic plasticity. We will measure effects of autoreceptor activation on the release frequency of DA vesicles. Using electrochemical techniques to examine release of individual DA vesicles from PC12 cells, we have found that D2 autoreceptor activation rapidly depresses presynaptic DA function by lowering the frequency of stimulation-dependent vesicular release. We will establish whether these effects are due to altered ion conductance or altered vesicle fusion/docking mechanisms and if long-term exposure to D2 agonists promotes sensitization of psychostimulation-or stimulation-dependent release. 2. We will determine the role of VMAT expression in the quantal size of release from DA vesicles. We have recently shown that cocaine and amphetamine, as well as D2 autoreceptor activation, decreases synaptic vesicle-mediated dopamine release by reducing quantal size. Using PC12 lines developed by Dr. Edwards, we will determine if altered phosphorylation of the adrenal gland vesicular monoamine transporter (VMAT1) alters quantal size and if this is differentially modulated by psychostimulants, D2 agonists, and VMAT inhibitors. We will compare results with PC12 lines that express high levels of VMAT1 and lines that express synaptic vesicle VMAT (VMAT2), which has greater affinity for AMPH. 3. We will examine DA release from individual synaptic vesicles of PC12 cells and midbrain neurons. We have performed the first direct measurement of quantal release from midbrain DA neurons; however, this protocol is challenging and not easily amenable to experimental manipulation. The techniques we employ in pC12 cells measure DA release from large PC12 vesicles but not smaller vesicles that more closely resemble conventional synaptic vesicles. Findings by Dr. Edwards suggest that stimulation of VMAT2-transfected PC12 cells release DA from small vesicles; if so, we will observe exocytosis in a preparation amenable to mutation and transgenic manipulation. We will also determine if potential VMAT sorting signal sequences can be used to target VMAT to small vesicles. 4. We will determine effects of neuronal stimulation, autoreceptor activation, and psychostimulants on vesicle sorting and recycling. Dr. Kelly's work suggests that regulation of recycling may control the number and type of secretory vesicles available for release. We will examine recycling in naive and NGF-treated PC12 cells and midbrain DA neurons using differentially labeled components of large dense core granules and synaptic vesicles to determine the roles of acidic recycling intermediates (endosomes). We will extend observations suggesting that depolarization increases intracellular pH gradients, which may contribute tot he rate of membrane recycling. We will determine if psychostimulant and short-term and long-term D2 activation exposure inhibits vesicle recycling.