Dopamine and Glutamate Receptor Interactions

描述（由申请人提供）：虽然多巴胺受体（DA）介导安非他明和可卡因的急性效应，但其长期给药产生的神经适应性涉及谷氨酸依赖性突触可塑性。许多这样的神经适应发生在脑桥核（NAc），这是一个对成瘾至关重要的大脑区域，并接受会聚的DA和谷氨酸输入。 最近的工作表明，调节兴奋性传递的关键机制涉及AMPA受体的磷酸化和表面表达的改变。我们开发了培养的出生后NAc神经元作为一个模型系统，在其中调查是否DA受体，在精神兴奋剂管理过程中激活，可以影响这些改变突触强度的基本机制。我们发现，D1 DA受体刺激增强AMPA受体亚基GIuR 1的磷酸化。然后，使用抗体的细胞表面表位的GluR 1和荧光显微镜，我们证明了快速下调的表面GluR 1谷氨酸和快速上调的D1激动剂。我们建议在这个模型系统中进一步表征DA和AMPA受体之间的相互作用。目的1将使用荧光显微镜检查与天气标志物突触素，其他AMPA受体亚基，和NMDA受体亚基NR 1的表面GIuR 1斑点的共定位。目的2将进一步表征谷氨酸和DA激动剂对GluR 1表面表达的调节。表面生物素化实验将用于验证定量免疫荧光试验获得的先前结果。然后，后一种方法将用于研究D1和D2激动剂、谷氨酸激动剂（谷氨酸、NMDA和AMPA）的作用以及这些药物之间的相互作用。目的3将检验D1激动剂通过蛋白激酶A（PKA）依赖性机制增加GluR 1表面表达的假设。我们将确定D1激动剂效应是否由cAMP-PKA通路的激活剂复制并被抑制剂阻止。目的4建立NAc和皮层神经元共培养的方法，保留选择性分析NAc神经元突起上GluR 1的能力。增加皮层神经元将恢复天气性谷氨酸能输入。在这些共培养物中，我们将确定NAc神经元上GluR 1定位于突触的程度，以及D1受体刺激是否会改变这一点。我们还将举例说明重复D1受体刺激的影响，因为所有最初的尝试，以模拟长期精神兴奋剂管理的影响，在一个文化系统。这些研究将提供对调控机制的深入了解， 当在慢性药物暴露过程中被破坏时，可能会导致成瘾的神经可塑性不适当。

DESCRIPTION (provided by applicant): While dopamine receptors (DA) mediate acute effects of amphetamine and cocaine, neuroadaptations produced by their chronic administration involve glutamate-dependent synaptic plasticity. Many such neuroadaptafions occur in the nucleus accumbens (NAc), a brain region that is critical to addiction and receives convergent DA and glutamate inputs. Recent work suggests that critical mechanisms for regulating excitatory transmission involve alterations in the phosphorylation and surface expression of AMPA receptors. We developed cultures of postnatal NAc neurons as a model system in which to investigate whether DA receptors, activated during psychostimulant administration, can influence these fundamental mechanisms for altering synaptic strength. We found that D1 DA receptor stimulation enhances phosphorylation of the AMPA receptor subunit GIuR1. Then, using antibody to a cell surface epitope of GluR1 and fluorescence microscopy, we demonstrated rapid down-regulation of surface GluR1 by glutamate and rapid up-regulation by a D1 agonist. We propose to further characterize interactions between DA and AMPA receptors in this model system. Aim 1 will use fluorescence microscopy to examine colocalization of surface GIuR1 puncta with the synoptic marker synaptophysin, other AMPA receptor subunits, and the NMDA receptor subunit NR1. Aim 2 will further characterize regulation of GluR1 surface expression by glutamate and DA agonists. Surface biotinylation experiments will be used to verify prior results obtained with quantitative immunofluorescence assays. Then, the latter approach will be used to study effects of D1 and D2 agonists, glutamate agonists (glutamate, NMDA and AMPA), and interactions between these agents. Aim 3 will test the hypothesis that D1 agonists increase GluR1 surface expression through a protein kinase A (PKA)-dependent mechanism. We will determine if D1 agonist effects are reproduced by activators of the cAMP-PKA pathway and prevented by inhibitors. Aim 4 will establish methods for co-culturing NAc and cortical neurons that preserve the ability to selectively analyze GluR1 on the processes of NAc neurons. Addingcortical neurons will restore synoptic glutamatcrgic input. In these co-cultures, we will determine the extent to which GluR1 on NAc neurons is localized to synapses and whether this is altered by D1 receptor stimulation. We will also examhle the effect of repeated D1 receptor stimulation, as all initial attempts to model the effects of long-term psychostimulant administration in a culture system. These studies will provide insight into regulatory mechanisms that, when disrupted during chronic drug exposure, may contribute to inappropriate neuroplasticity underlying addiction.