Pathway-specific Fyn signaling in the striatum and ethanol drinking

描述（由申请人提供）：本提案旨在研究背内侧纹状体（DMS）内神经元亚群对乙醇的反应中发生的信号依赖性神经适应。DMS在目标导向行为和成瘾中起着核心作用，我们发现啮齿动物自愿和被动暴露于乙醇会激活DMS中特异性的酪氨酸激酶Fyn。我们进一步发现，响应于乙醇，活化的Fyn磷酸化NMDAR的NR 2B亚基（GluN 2B），导致NMDA和AMPA受体（NMDAR/AMPAR）的突触活性增加。最后，我们发现，在DMS的Fyn信号通路起着至关重要的作用，乙醇饮用行为的机制。DMS中的神经元表达多巴胺（DA）D1或D2受体（D1 R，D2 R），形成直接的“Go”和间接的“No-Go”通路。因此，DA被认为对D1 R和D2 R神经元具有不同且可能相反的作用。D1 R与G蛋白（Gs）偶联，G蛋白激活cAMP/PKA信号传导，而D2 R与Gi偶联，Gi抑制cAMP/PKA。我们发现，Fyn激活依赖于cAMP/PKA，并且体内施用D1 R激动剂而不是D2 R激动剂激活DMS中的Fyn信号传导，特别是在D1 R DMS神经元中。总之，这些数据表明DA激活D1 R神经元中由D1 R/Gs/Fyn/GluN 2B/AMPAR组成的信号通路。乙醇增加纹状体中的DA水平，我们假设乙醇激活Fyn信号，特别是在DMS中的D1 R神经元，以促进直接“Go”通路中的神经适应，从而驱动乙醇饮用行为。我们还假设，远程激活的Gs信号在D1 R DMS神经元足以产生类似的Fyn依赖的细胞和行为的神经适应。这些假设将使用在D1 R或D2 R神经元中表达Cre的转基因小鼠进行体内测试，并将利用创新的分子和化学遗传学方法来操纵单基因（Cre-FLEX），并在特定的神经元亚群中使用由设计药物独家激活的设计受体（DREADD）方法远程激活Gs信号传导。目的1和目的2将确定乙醇诱导的Fyn依赖性适应是否发生在DMS中的D1 R神经元中，而不是D2 R神经元中，以及DMS D1 R神经元中的Gs DREADD激活是否足以产生类似的结果。目的3研究D1 R DMS神经元Fyn信号通路在乙醇饮酒行为中的作用，以及D1 R DMS神经元Fyn信号通路的Gs DREADD激活是否足以产生乙醇寻求和饮酒行为。结合分子和系统神经科学的方法，我们将解开细胞类型的特定信号适应的基础发展和维持乙醇饮用和寻求行为。

DESCRIPTION (provided by applicant): This proposal is aimed at studying signaling-dependent neuroadaptations that occur in response to ethanol in subpopulations of neurons within the dorsomedial striatum (DMS). The DMS plays a central role in goal- directed behaviors and addiction, and we found that voluntary and passive exposure of rodents to ethanol activates the tyrosine kinase Fyn specifically in the DMS. We further discovered that in response to ethanol, activated Fyn phosphorylates the NR2B subunit of the NMDARs (GluN2B) resulting in increased synaptic activity of NMDA and AMPA receptors (NMDAR/AMPAR). Finally, we showed that the Fyn-signaling pathway in the DMS plays a crucial role in mechanisms underlying ethanol-drinking behaviors. Neurons in the DMS express either dopamine (DA) D1 or D2 receptors (D1R, D2R) that form the direct "Go" and indirect "No-Go" pathways. Thus, DA is thought to have different and possibly opposite effects on D1R and D2R neurons. D1Rs are coupled to G protein �s (Gs), which activates cAMP/PKA signaling, whereas D2Rs are coupled to Gi, which inhibits cAMP/PKA. We found that Fyn activation depends on cAMP/PKA, and that in vivo administration of a D1R agonist but not D2R agonist activates Fyn signaling in the DMS, and specifically in D1R DMS neurons. Together, these data suggest the DA activates a signaling pathway consisting of D1R/Gs/Fyn/GluN2B/AMPAR in D1R neurons. Ethanol increases DA levels in the striatum, and we hypothesize that ethanol activates Fyn signaling specifically in D1R neurons in the DMS to facilitate neuroadaptations in the direct "Go" pathway that drive ethanol drinking behaviors. We also hypothesize that remote activation of Gs signaling in D1R DMS neurons is sufficient to produce similar Fyn-dependent cellular and behavioral neuroadaptations. These hypotheses will be tested in vivo using transgenic mice that express Cre in D1R or D2R neurons, and will utilize innovative molecular and chemico- genetic approaches to manipulate single genes (Cre-FLEX), and to remotely activate Gs signaling using the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) method in specific subpopulations of neurons. Aim 1 and Aim 2 will determine whether Fyn-dependent adaptations induced by ethanol occur in D1R, but not D2R, neurons in the DMS, and whether Gs DREADD activation in DMS D1R neurons is sufficient to produce similar outcomes. Aim 3 will investigate the contribution of Fyn signaling in D1R DMS neurons to ethanol drinking behaviors, and whether Gs DREADD activation of Fyn in D1R is sufficient to produce ethanol seeking and drinking. Combining Molecular and Systems Neuroscience methodologies, we will unravel cell- type specific signaling adaptations that underlie the development and maintenance of ethanol drinking and seeking behaviors.