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

• 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
• 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

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.

摘要： 可卡因是一种被广泛滥用的精神兴奋剂，主要通过提高突触外多巴胺水平发挥作用 通过抑制多巴胺转运体的功能。目前，没有可行和有效的 可卡因使用障碍的现有治疗选择。详细了解下游的调控 介导可卡因暴露后发生的生理适应的分子底物以及它们如何 有助于可卡因滥用的病理生理学是至关重要的，以确定潜在的治疗 治疗可卡因使用障碍的战略，从而促进公共卫生。体外研究表明， 细胞内ERK 1/2激酶信号通路在调节细胞内关键蛋白中的中心作用 多巴胺能神经元然而，我们对这些结果如何转化为离体的理解仍然存在差距 和可卡因使用障碍的体内模型。 我们建议采用一种新的方法，使时间和解剖学上精确控制ERK 1/2 多巴胺神经元的信号。这种方法涉及我们实验室开发的Cre病毒构建体 该信号传导途径的蓝光诱导型激活剂的重组酶依赖性表达。病毒 构建体将与在多巴胺能神经元中表达Cre重组酶的转基因大鼠组合使用。 神经元特异性靶向多巴胺神经元中的细胞内ERK 1/2信号传导。 为了支持我们的方法，我们观察到蓝光暴露后ERK 1/2激活增加， ERK 1/2激活NAc中的DA末端破坏DA神经传递。 我们将使用这种靶向遗传学方法来进一步了解细胞内ERK 1/2的作用。 在调节多巴胺神经传递的多巴胺能细胞中的信号传导。我们将使用生化分析， 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）并采用体内快速扫描循环伏安法（Aim 2）研究DA神经传递的改变 由多巴胺能神经元中ERK 1/2信号传导的激活引起 我们相信，该项目的成功完成将导致对细胞调控的新见解， 分子变化参与可卡因使用障碍的发展。我们预计这些实验 在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目标 介导可卡因相关行为。我们预计这将最终导致新的治疗途径， 治疗这种疾病。