Structural and Functional imaging with Multiphoton Microscopy in Alzheimer's Mice

描述(申请人提供)：近年来，多光子显微镜被用来更好地了解阿尔茨海默病(AD)在完整的活体小鼠脑中的病理生理学。我们将使用产生老年斑的转基因小鼠模型来研究这些斑块对体内神经元的功能和结构影响。老年斑是这种疾病的主要标志。通过监测特定细胞内的钙离子浓度，可以用多光子显微镜观察神经元的功能。在神经元激活过程中，细胞内钙呈指数级增加，并可使用钙敏感的荧光探针进行检测。这些探针增加它们的亮度，改变它们的激发/发射光谱或参与荧光共振能量转移(FRET)来表示钙浓度的变化。能够实时观察老年斑对神经元活动的功能影响，将以前所未有的空间和时间分辨率为测试治疗提供一种新的标记。为了实现这一目标，我们将开发一种基因转移技术，使我们能够将基于FRET的、对钙敏感的基因结构直接引入成年小鼠的大脑。这种结构编码的蛋白质强健地充满了胞体和神经突起。这将使我们能够研究斑块沉积引起的钙浓度的动态平衡变化。还可以使用该探针监测动态钙瞬变，从而提供在体内以脊柱水平的分辨率研究神经元激活的能力。我们还打算采用新发表的批量装载功能性小分子染料的技术，用于成年转基因小鼠。随着大量神经元被这样的钙敏感指示物染色，我们将用单细胞分辨率来确定斑块对邻近细胞的功能后果。通过结合新的成像方式、疾病神经生物学和系统水平的神经科学，我们希望对阿尔茨海默病的发病机制提供重要和独特的见解。摘要：到目前为止，科学家们还不清楚随着阿尔茨海默病的进展，活着的大脑中的单个细胞网络是如何发生故障的。我们将使用动物模型来确定神经网络是如何受到影响的，以及特定的治疗是否可以导致正常功能的恢复。

DESCRIPTION (provided by applicant): In recent years, multiphoton microscopy has been used to gain a better understanding of the pathophysiology of Alzheimer's Disease (AD) in intact live mouse brains. We will use transgenic mouse models that develop senile plaques, a dominant marker of the disease, to investigate the functional and structural consequences of these plaques on neurons in vivo. Neuronal function can be investigated with multiphoton microscopy by monitoring the concentration of calcium within a given cell. Intracellular calcium increases exponentially during neuronal activation and can be detected using calcium-sensitive fluorescent probes. These probes increase their brightness, shift their excitation/emission spectra or engage in Fluorescence Resonance Energy Transfer (FRET) to denote a change in calcium concentration. The ability to observe in real-time the functional effects of senile plaques on neuronal activity will provide a novel marker for testing therapeutics with unprecedented spatial and temporal resolution. To achieve this goal we will develop a gene transfer technique that will allow us to introduce a FRET-based, calcium-sensitive genetic construct directly into the adult mouse brain. The protein encoded by this construct robustly fills soma and neuritic processes. This will permit us to investigate homeostatic alterations in calcium concentration caused by plaque deposition. Dynamic calcium transients can also be monitored using this probe, providing the ability to investigate neuronal activation with spine-level resolution in vivo. We also aim to adapt newly published techniques in bulk loading of functional small-molecule dyes for use in adult, transgenic mice. With a large ensemble of neurons stained with such calcium-sensitive indicators, we will determine the functional consequences of plaques on neighboring cells with single-cell resolution. By combining new imaging modalities, disease neurobiology, and systems-level neuroscience, we hope to provide important and unique insight into the pathogenesis of Alzheimer's Disease. SUMMARY: To date, scientists do not understand how networks of individual cells in a living brain malfunction as Alzheimer's Disease progresses. We will use animal models to determine how neural networks are affected and whether specific therapies can lead to recovery of normal function.