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

描述（由申请人提供）：近年来，多光子显微镜已用于更好地了解完整活小鼠脑中阿尔茨海默病（AD）的病理生理学。我们将使用转基因小鼠模型，发展老年斑，一个主要的标志物的疾病，研究功能和结构的后果，这些斑块的神经元在体内。神经元功能可以用多光子显微镜通过监测给定细胞内的钙浓度来研究。细胞内钙在神经元激活期间呈指数增加，并且可以使用钙敏感的荧光探针来检测。这些探针增加它们的亮度，移动它们的激发/发射光谱或参与荧光共振能量转移（FRET）以指示钙浓度的变化。实时观察老年斑对神经元活动的功能影响的能力将为测试具有前所未有的空间和时间分辨率的疗法提供新的标记物。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的钙敏感遗传构建体直接引入成年小鼠大脑。由该构建体编码的蛋白质稳健地填充索马和神经炎过程。这将使我们能够研究斑块沉积引起的钙浓度的稳态改变。动态钙瞬变也可以使用这种探针进行监测，提供了在体内研究具有脊髓水平分辨率的神经元激活的能力。我们还旨在适应新发表的技术，在散装功能性小分子染料用于成年转基因小鼠。用这种钙敏感指示剂染色的大量神经元，我们将用单细胞分辨率确定斑块对相邻细胞的功能后果。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，我们希望为阿尔茨海默病的发病机制提供重要而独特的见解。摘要：到目前为止，科学家们还不了解随着阿尔茨海默病的进展，活脑中单个细胞的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.