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

描述（由申请人提供）：近年来，多光子显微镜已被用于更好地了解阿尔茨海默病（AD）在完整活小鼠大脑中的病理生理。我们将使用发展老年斑的转基因小鼠模型，研究这些斑块对神经元的功能和结构影响。老年斑是该疾病的主要标志。通过监测给定细胞内钙的浓度，可以用多光子显微镜研究神经元的功能。细胞内钙在神经元激活过程中呈指数增长，可以用钙敏感的荧光探针检测到。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。实时观察老年斑对神经元活动的功能影响的能力将为前所未有的空间和时间分辨率的治疗测试提供新的标记。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的钙敏感遗传结构直接引入成年小鼠的大脑。由这种结构编码的蛋白质健壮地填充了体细胞和神经突起。这将使我们能够研究由斑块沉积引起的钙浓度的稳态改变。动态钙瞬态也可以使用该探针进行监测，提供了在体内以脊柱水平分辨率研究神经元激活的能力。我们还旨在适应新发表的技术在功能性小分子染料的散装装载用于成年转基因小鼠。通过对大量神经元进行钙敏感染色，我们将以单细胞分辨率确定斑块对邻近细胞的功能影响。通过结合新的成像方式，疾病神经生物学和系统级神经科学，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：迄今为止，科学家们尚不清楚随着阿尔茨海默病的进展，活体大脑中单个细胞网络是如何发生功能障碍的。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.