Pathway-specific Fyn signaling in the striatum and ethanol drinking

描述(申请人提供)：这项建议旨在研究在背内侧纹状体(DMS)内神经元亚群中发生的响应于乙醇的信号依赖的神经适应。DMS在目标导向行为和成瘾中起着核心作用，我们发现，自愿和被动地将啮齿动物暴露在乙醇中会激活DMS中特异的酪氨酸激酶Fyn。我们进一步发现，作为对乙醇的反应，激活的Fyn磷酸化NMDAR的NR2B亚单位(GluN2B)，导致NMDA和AMPA受体(NMDAR/AMPAR)的突触活性增加。最后，我们证明了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神经元有不同的甚至可能相反的影响。D1Rs与G蛋白αS(Gs)偶联，激活cAMP/PKA信号，而D2Rs偶联Gi，抑制cAMP/PKA。我们发现Fyn的激活依赖于cAMP/PKA，并且在体内给予D1R激动剂而不是D2R激动剂可以激活DMS中的Fyn信号，尤其是D1R DMS神经元中的Fyn信号。综上所述，这些数据表明，DA激活了D1R神经元上由D1R/Gs/Fyn/GluN2B/AMPAR组成的信号通路。乙醇增加了纹状体中DA的水平，我们假设乙醇特异性地激活了DMS中D1R神经元中的Fyn信号，以促进驱动酒精饮酒行为的直接“GO”途径的神经适应。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依赖的细胞和行为神经适应。这些假说将在体内使用在D1R或D2R神经元中表达Cre的转基因小鼠进行验证，并将利用创新的分子和化学遗传学方法操纵单基因(Cre-flex)，并使用由设计药物独有激活的设计者受体(DREADD)方法在特定神经元亚群中远程激活Gs信号。目的1和目的2将确定酒精诱导的依赖Fyn的适应是否发生在DMS的D1R神经元上，而不是D2R神经元上，以及DMS D1R神经元中Gs DREADD的激活是否足以产生类似的结果。目的3研究D1R DMS神经元Fyn信号在饮酒行为中的作用，以及D1R神经元Fyn的Gs DREADD激活是否足以产生寻找酒精和饮酒。结合分子和系统神经科学方法，我们将揭示细胞类型的特定信号适应，这些信号适应是酒精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.