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

本项目的目标是使用体内双光子激光扫描显微镜（2 PLSM） 以检测酒精依赖小鼠神经元活动的变化。长期饮酒 导致依赖的大脑功能的显著变化， 大脑皮层处理和认知的损伤这些损伤可能是 过渡到大量饮酒，并阐明这些变化背后的机制 对开发治疗和干预措施至关重要， 行为酒精对大脑作用机制研究的一个关键局限 功能是依赖于体外技术来监测神经元功能。这些 这些方法虽然非常有价值，但却因失去相关的感官输入而受到阻碍 以及在体外组织样品制备过程中正常回路的破坏。而 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。在本申请中， 我们将在体内使用2 PLSM来成像神经元活动和神经血管功能， 酒精依赖小鼠在目标1中，我们将利用最近开发的小鼠品系， 当与特定的Cre-driver小鼠品系杂交时，表达钙传感器 蛋白GCaMP 3在一个确定的神经元亚群。神经元钙动力学 将通过以下方式在多巴胺能锥体神经元和快速尖峰中间神经元中监测 将GCaMP 3小鼠与Wfs 1-Tg 2-CreERT 2系和Pvalb-2A-Cre系杂交; 分别在休息期间和视觉呈现期间都将监测活动 可靠地诱导视觉皮层神经元活动的线索。在目标2中，神经血管 耦合（例如，局部脑血流的活动依赖性变化）将通过 最近Co-I显示的染料的使用有效地指示小动脉的变化， 血流这两个互补目标的结果将共同确立 在酒精作用的研究，并将支持未来的研究在体内2 PLSM技术 旨在分析酒精对大脑皮层功能的影响

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.