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

多巴胺神经元中 ERK1/2 信号的选择性实时激活

基本信息

批准号：
10706605
负责人：
Rodrigo A. España
金额：
$ 18.94万
依托单位：
DREXEL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-30 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10706605
关键词：
Anatomy Animals Biochemical Biochemistry Biological Assay Cells Cocaine Cocaine Abuse Cocaine use disorder Complex Corpus striatum structure Data Development Disease Disputes Dopamine Dopaminergic Cell Enterobacteria phage P1 Cre recombinase Functional disorder Future Genetic Goals In Vitro Intervention Laboratories Light MAPK3 gene Measures Mediating Molecular Neurobiology Nucleus Accumbens Pathway interactions Periodicity Pharmaceutical Preparations Phosphorylation Phosphotransferases Physiological Physiological Adaptation Preparation Process Proteins Psychological reinforcement Public Health Rat Transgene Rattus Regulation Resolution Rewards Role Scanning Signal Pathway Signal Transduction Slice Surface Synapses Synaptic plasticity Synaptosomes System Therapeutic Therapeutic Intervention Time Tissues Translating Tyrosine 3-Monooxygenase Tyrosine Phosphorylation Ventral Tegmental Area Viral adeno-associated viral vector cell growth regulation cocaine exposure cocaine related behaviors cocaine use dopamine system dopamine transporter dopaminergic neuron effective therapy experimental study extracellular genetic approach in vitro Model in vivo in vivo Model innovation insight mesolimbic system neurochemistry neurotransmission novel novel strategies novel therapeutics pharmacologic protein expression reuptake stimulant abuse targeted treatment tool uptake

项目摘要

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.

抽象的：可卡因是一种被广泛滥用的精神刺激剂，主要是通过提高外斜外多巴胺水平来起作用通过抑制多巴胺转运蛋白的功能。目前，没有可行有效的可卡因使用障碍的治疗选择。对下游调节的详细理解可卡因暴露后发生生理适应的分子底物以及它们如何促进可卡因滥用的病理生理学对于确定潜在的治疗至关重要治疗可卡因使用障碍的策略，从而用于公共卫生。体外研究表明细胞内ERK1/2激酶信号通路的中心作用多巴胺能神经元。但是，差距在我们对这些结果如何转化为离体中的理解中仍然存在以及可卡因使用障碍的体内模型。我们建议采用一种新颖的方法，从而实现对ERK1/2的时间和解剖学精确的控制多巴胺神经元中的信号传导。这种方法涉及在我们的CRE实验室中开发的病毒结构该信号通路的蓝光诱导激活剂的重组酶依赖性表达。病毒构造将与在多巴胺能中表达CRE重组酶的转基因大鼠结合使用神经元特异性靶向多巴胺神经元中细胞内ERK1/2信号传导。为了支持我们的方法，我们已经观察到蓝光暴露和 NAC中DA终端的ERK1/2激活破坏了DA神经传递。我们将使用这种靶向遗传方法来进一步了解细胞内ERK1/2的作用多巴胺能细胞中的信号传导调节多巴胺神经传递。我们将使用生化测定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（AIM 1）并使用体内快速扫描循环伏安法（AIM 2）研究DA神经传递的变化由多巴胺能神经元中ERK1/2信号的激活产生我们认为，该项目的成功完成将导致对细胞和细胞调节的新见解可卡因使用障碍发展涉及的分子变化。我们预计实验本应用程序中描述的将促进未来的研究，以确定这些途径的独特下游目标介导与可卡因相关的行为。我们希望这最终将导致新颖的治疗途径治疗这种疾病。