Pathway-specific Fyn signaling in the striatum and ethanol drinking

描述（由申请人提供）：该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，我们发现啮齿类动物自愿和被动暴露于乙醇中会激活DMS中特异性的酪氨酸激酶Fyn。我们进一步发现，在乙醇的作用下，激活的Fyn磷酸化NMDARs的NR2B亚基（GluN2B），导致NMDA和AMPA受体（NMDAR/AMPAR）的突触活性增加。最后，我们发现DMS中的fyn信号通路在酒精饮酒行为的机制中起着至关重要的作用。DMS中的神经元表达多巴胺（DA） D1或D2受体（D1R, D2R），形成直接的“Go”和间接的“No-Go”通路。因此，DA被认为对D1R和D2R神经元具有不同的甚至可能相反的作用。D1Rs与G蛋白αs （Gs）偶联，激活cAMP/PKA信号，而D2Rs与Gi偶联，抑制cAMP/PKA信号。我们发现Fyn的激活依赖于cAMP/PKA，并且在体内给药D1R激动剂而不是D2R激动剂可以激活DMS中的Fyn信号，特别是D1R DMS神经元。综上所述，这些数据表明DA激活了D1R神经元中由D1R/Gs/Fyn/GluN2B/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DMS中D1R神经元中的Fyn信号，促进直接“Go”通路的神经适应，从而驱动酒精饮酒行为。我们还假设D1R DMS神经元中Gs信号的远程激活足以产生类似的fyn依赖性细胞和行为神经适应。这些假设将在D1R或D2R神经元中表达Cre的转基因小鼠体内进行测试，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），并使用设计药物独占激活的设计受体（DREADD）方法在特定的神经元亚群中远程激活Gs信号。Aim 1和Aim 2将确定乙醇诱导的fynn依赖性适应是否发生在DMS的D1R神经元中，而不是D2R神经元中，以及DMS D1R神经元中Gs - DREADD激活是否足以产生类似的结果。Aim 3将研究D1R DMS神经元中Fyn信号对乙醇饮用行为的贡献，以及D1R中Gs - DREADD激活Fyn是否足以产生乙醇寻找和饮酒。结合分子和系统神经科学方法，我们将揭示细胞类型特定的信号适应，这是乙醇饮酒和寻找行为发展和维持的基础。

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 αs (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.