In Vivo Two-Photon Imaging of Cortical Activity in Alcohol-Dependent Mice

描述(申请人提供)：这个项目的目标是使用活体双光子激光扫描显微镜(2PLSM)来检查酒精依赖小鼠神经元活动的变化。导致依赖的长期饮酒与大脑功能的显着变化有关，包括皮质处理和认知的损害。这些损伤可能是向大量饮酒过渡的基础，阐明这些变化的机制对于开发恢复对行为控制的治疗和干预至关重要。酒精对脑功能的作用机制研究中的一个关键限制是依赖体外技术来监测神经元功能。这些方法虽然非常有价值，但在体外组织样本制备过程中，相关感觉输入的丢失和正常电路的中断会阻碍这些方法的实施。虽然脑成像方法可以避免这种并发症，但由于缺乏空间分辨率和神经元亚型的特异性，它们受到了影响。在这项应用中，我们将使用体内2PLSM来成像酒精依赖小鼠的神经元活动和神经血管功能。在目标1中，我们将利用最近开发的小鼠品系，当与特定的CRE驱动小鼠品系杂交时，在特定的神经元亚群中表达钙传感器蛋白GCaMP3。通过将GCaMP3小鼠分别与Wfs1-TG2-CreERT2系和Pvalb-2A-Cre系杂交，可以监测谷氨酸能锥体神经元和快速放电中间神经元的神经元钙动力学。在休息和呈现可靠地诱导视觉皮质神经元活动的视觉提示期间，活动都将受到监测。在目标2中，将通过使用Co-I最近显示的一种染料来监测神经血管耦合(例如，局部脑血流的活动依赖变化)，以有效地发出小动脉血流变化的信号。总之，这两个互补目标的结果将建立体内2PLSM技术来研究酒精的作用，并将支持未来旨在分析酒精对皮质功能影响的研究。

DESCRIPTION (provided by applicant): The goal of this project is to use in vivo two-photon laser scanning microscopy (2PLSM) to examine changes in neuronal activity in ethanol-dependent mice. Chronic drinking that leads to dependence is associated with marked changes in brain function including impairments in cortical processing and cognition. These impairments likely underlie the transition to heavy drinking and elucidating the mechanisms that underlie these changes is critical towards developing treatments and interventions that restore control over behavior. A key limitation in the study of the mechanistic actions on alcohol on brain function is the reliance upon in vitro techniques to monitor neuronal function. These approaches, while extremely valuable, are hindered by the loss of relevant sensory input and disruption of normal circuitry during preparation of the in vitro tissue sample. While brain imaging approaches can avoid this complication, they are compromised by a lack of specificity with regard to spatial resolution and neuronal sub-type. In this application, we will use in vivo 2PLSM to image neuronal activity and neurovascular function in alcohol-dependent mice. In Aim 1, we will utilize a recently developed line of mice that, when crossed with a specific Cre-driver mouse line, expresses the calcium-sensor protein GCaMP3 in a defined sub-population of neurons. Neuronal calcium dynamics will be monitored in glutamatergic pyramidal neurons and fast-spiking interneurons by crossing the GCaMP3 mice with the Wfs1-Tg2-CreERT2 line and Pvalb-2A-Cre line; respectively. Activity will be monitored both during rest and during presentation of visual cues that reliably induce activity in visual cortex neurons. In Aim 2, neurovascular coupling (e.g., activity-dependent changes in local brain blood flow) will be monitored by the use of a dye recently shown by the Co-I to effectively signal changes in arteriolar blood flow. Together, results from these two complementary aims will establish the technique of in vivo 2PLSM in the study of alcohol action and will support future studies designed to analyze the effects of alcohol on cortical function.