PRESYNAPTIC PLASTICITY OF VESICULAR DOPAMINE RELEASE

精神兴奋剂药物会引起持久的行为变化，包括 依赖和精神病。这些行为背后的突触 与精神兴奋剂的短期机制不同 奖励与Fegis Kelly和Robert Edwards博士一起，我们 概述研究，以阐明刺激机制-和 精神兴奋剂诱导的多巴胺突触可塑性。 我们将测量自感受器激活对释放频率的影响 DA囊泡利用电化学技术检测 从PC 12细胞的单个DA囊泡中，我们发现D2自身受体 激活通过降低突触前多巴胺的功能， 刺激依赖性囊泡释放的频率。我们将建立 这些效应是否是由于改变了离子电导或改变了囊泡 融合/对接机制，如果长期暴露于D2激动剂促进 心理刺激或刺激依赖性释放的敏化。 2.我们将确定VMAT表达的作用，在量子大小的 从DA囊泡中释放。我们最近发现可卡因和 安非他明，以及D2自身受体激活，减少突触 囊泡介导的多巴胺释放通过减少量子尺寸。使用PC 12 由爱德华兹博士开发的线，我们将确定是否改变 肾上腺囊泡单胺转运蛋白磷酸化 （VMAT 1）改变量子大小，如果这是由 精神兴奋剂、D2激动剂和VMAT抑制剂。我们将比较 用表达高水平VMAT 1的PC 12品系和 表达突触囊泡VMAT（VMAT 2），其对AMPH具有更大的亲和力。 3.我们将检查PC 12的单个突触囊泡的DA释放 细胞和中脑神经元。我们进行了首次直接测量 从中脑DA神经元的量子释放;然而，这个协议是 具有挑战性并且不容易接受实验操作。的 我们在pC 12细胞中使用的技术测量了大PC 12的DA释放 囊泡，但不是更小的囊泡，更接近于常规的 突触囊泡爱德华兹博士的研究结果表明， VMAT 2转染的PC 12细胞从小囊泡中释放DA;如果是这样，我们将 在易于突变和转基因的制剂中观察胞吐作用 操纵我们还将确定潜在的VMAT分选信号 序列可用于将VMAT靶向小囊泡。 4.我们将确定神经元刺激，自身感受器 激活和精神兴奋剂对囊泡分类和回收的影响。博士 凯利的工作表明，对回收利用的监管可能会控制数量 以及可用于释放的分泌囊泡的类型。我们将研究 在幼稚和NGF处理的PC 12细胞和中脑DA神经元中的再循环， 大致密核心颗粒和突触的差异标记成分 囊泡，以确定酸性循环中间体的作用 （内体）。我们将进一步观察， 增加细胞内pH梯度，这可能有助于 膜回收我们将确定是否精神兴奋剂和短期 而长期暴露于D2活化状态会抑制囊泡的再循环。

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.