Selective real-time activation of ERK1/2 signaling in dopamine neurons

摘要： 可卡因是一种被广泛滥用的精神刺激剂，主要通过提高突触外多巴胺水平来发挥作用。 通过抑制多巴胺转运体功能。目前，没有可行和有效的 可用于可卡因使用障碍的治疗选择。对下游航道整治的详细了解 介导可卡因暴露后发生的生理适应的分子底物及其如何 对可卡因滥用的病理生理学的贡献对于确定潜在的治疗方法至关重要 治疗可卡因使用障碍的战略，从而影响公共健康。体外研究表明， 细胞内ERK1/2信号通路在细胞内关键蛋白调控中的中心作用 多巴胺能神经元。然而，我们对这些结果如何转化为体外实验的理解仍然存在差距。 以及可卡因使用障碍的体内模型。 我们建议采用一种新的方法，能够在时间和解剖上精确地控制ERK1/2 多巴胺神经元中的信号。这种方法涉及到我们实验室开发的带有Cre的病毒结构 该信号通路的蓝光诱导激活子依赖于重组酶的表达。病毒式传播 构建将与表达Cre重组酶的转基因大鼠在多巴胺能 特异性靶向多巴胺神经元内ERK1/2信号的神经元。 为了支持我们的方法，我们观察到蓝光暴露后ERK1/2激活增加，并 NAc中DA终末ERK1/2的激活干扰了DA的神经传递。 我们将使用这种有针对性的遗传方法来进一步理解细胞内ERK1/2的作用 多巴胺能细胞中调节多巴胺神经传递的信号。我们将使用生化分析来 确定关键多巴胺能蛋白的表达、磷酸化和亚细胞定位的变化 (目标1)和采用体内快速扫描循环伏安法(目标2)研究DA神经递质的变化 多巴胺能神经元ERK1/2信号的激活 我们相信，这个项目的成功完成将为细胞和细胞的调节带来新的见解 分子变化参与了可卡因使用障碍的发展。我们预计这些实验 本申请中描述的将有助于未来的研究，以确定这些途径的独特下游靶点 与可卡因相关的行为。我们预计这将最终导致新的治疗途径 治疗这种疾病。

Abstract: Cocaine is a widely abused psychostimulant that acts primarily by elevating extrasynaptic dopamine levels through the inhibition of dopamine transporter function. At the present time, there are no viable and effective treatment options available for cocaine use disorders. A detailed understanding of the regulation of downstream molecular substrates mediating the physiologic adaptations that occur following cocaine exposure and how they contribute to the pathophysiology of cocaine abuse is of critical importance for identifying potential therapeutic strategies for treating cocaine use disorders and thus for public health. In vitro studies have demonstrated a central role for the intracellular ERK1/2 kinase signaling pathway in the regulation of key proteins within dopaminergic neurons. However, gaps remain in our understanding of how these results translate into ex vivo and in vivo models of cocaine use disorders. We propose to employ a novel approach enabling temporally and anatomically precise control over ERK1/2 signaling in dopamine neurons. This approach involves a viral construct developed in our laboratories with Cre recombinase-dependent expression of a blue-light inducible activator of this signaling pathway. The viral construct will be employed in combination with transgenic rats that express Cre recombinase in dopaminergic neurons to specifically target intracellular ERK1/2 signaling in dopamine neurons. In support of our approach, we have observed increased ERK1/2 activation following blue light exposure and that ERK1/2 activation of DA terminals in the NAc disrupts DA neurotransmission. We will use this targeted genetic approach to further our understanding of the role of intracellular ERK1/2 signaling in dopaminergic cells in regulating dopamine neurotransmission. We will use biochemical assays to determine changes in expression, phosphorylation, and subcellular localization of key dopaminergic proteins (Aim 1) and employ in vivo fast scan cyclic voltammetry (Aim 2) to study alterations in DA neurotransmission resulting from the activation of ERK1/2 signaling in dopaminergic neurons We believe the successful completion of this project will lead to novel insights into the regulation of cellular and molecular changes involved in the development of cocaine use disorder. We anticipate that the experiments described in this application will facilitate future studies to identify unique downstream targets of these pathways that mediate cocaine-associated behaviors. We expect this will ultimately lead to novel therapeutic avenues for treating this disorder.