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

该项目的目标是使用活体双光子激光扫描显微镜(2PLSM) 检测酒精依赖小鼠神经元活性的变化。长期饮酒 这会导致依赖与大脑功能的显著变化有关，包括 大脑皮层处理和认知障碍。这些损害很可能是 过渡到大量饮酒并阐明这些变化背后的机制 对于开发治疗和干预措施以恢复对 行为。酒精对脑的作用机制研究中的一个关键局限 功能是依靠体外技术来监测神经元的功能。这些 方法，虽然非常有价值，但由于相关感官输入的丧失而受阻 以及在制备体外组织样品期间正常电路的中断。而当 脑成像方法可以避免这种并发症，但它们因缺乏 在空间分辨率和神经元亚型方面具有特异性。在此应用程序中， 我们将在体内使用2PLSM来成像神经活动和神经血管功能 酒精依赖的小鼠。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驱动小鼠品系杂交时，表达钙传感器 确定的神经元亚群中的GCaMP3蛋白。神经元钙动力学 将在谷氨酸能锥体神经元和快速放电中间神经元中被监测 将Wfs1-TG2-CreERT2系和Pvalb-2A-Cre系与GCaMP3小鼠杂交； 分别进行了分析。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，局部脑血流量依赖于活动的变化)将由 使用Co-I最近展示的一种染料来有效地发出微动脉变化的信号 血液流动。这两个相辅相成的目标的结果将共同确立 体内2PLSM技术在酒精作用研究中的应用 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.