Dopamine and Glutamate Receptor Interactions

描述(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，但其慢性给药产生的神经适应涉及谷氨酸依赖的突触可塑性。许多这样的神经适应纤维存在于伏隔核(NAC)，这是一个对成瘾至关重要的大脑区域，接受汇聚的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变化。我们发展了新生NAC神经元的培养作为一个模型系统，在其中研究在精神刺激剂注射期间激活的DA受体是否可以影响这些改变突触强度的基本机制。我们发现D1DA受体的刺激增强了AMPA受体亚单位GIuR1的磷酸化。然后，使用细胞表面GluR1表位的抗体和荧光显微镜，我们证明了谷氨酸迅速下调细胞表面GluR1，而D1激动剂迅速上调GluR1。我们建议在这个模型系统中进一步描述DA和AMPA受体之间的相互作用。目的1利用荧光显微镜观察GIuR1点与突触体标记物突触素、其他AMPA受体亚基和NMDA受体亚基NR1的共定位。目的2将进一步表征谷氨酸和多巴胺激动剂对GluR1表面表达的调节。表面生物素化实验将被用来验证先前用定量免疫荧光分析获得的结果。然后，后一种方法将被用来研究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验证D1激动剂通过蛋白激酶A(PKA)依赖的机制增加GluR1表面表达的假说。我们将确定cAMP-PKA通路的激活剂是否复制了D1激动剂的效应，并通过抑制剂来阻止。目的4将建立共培养NAC和皮质神经元的方法，以保持选择性地分析NAC神经元突起上的GluR1的能力。添加皮质神经元将恢复天气谷氨酸能输入。在这些共培养中，我们将确定NAC神经元上GluR1定位于突触的程度，以及这种情况是否会被D1受体刺激所改变。我们还将检验重复的D1受体刺激的效果，因为所有最初的尝试都是为了在培养系统中模拟长期给予精神刺激剂的效果。这些研究将提供对监管机制的洞察， 当在慢性药物暴露期间中断时，可能会导致不适当的神经可塑性，潜在的成瘾。

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.