Molecular probes to visualize endogenous synaptic proteins in vivo

描述(申请人提供)：这项资助中提出的研究目的是产生工具，使内源性突触蛋白能够在活体神经元中可视化。以前，我们已经产生了被称为FingRs(纤维连接蛋白内体产生的mRNA显示)的重组抗体，可以在活的神经元中表达，在那里他们以非侵入性和高保真的方式标记内源性靶蛋白。此外，转录调控系统控制FingRs的表达水平，使它们在与内源性同行完全相同的水平上表达，确保低背景。在这笔赠款中，我们将产生表达荧光蛋白融合的FingRs的转基因小鼠，这些FingRs识别突触蛋白。这些小鼠大脑中单个神经元中的突触蛋白可以在体内实时可视化。反过来，这将允许在 分子水平与细胞、回路和整个动物水平的事件相关。特别是，通过绘制神经元中突触蛋白的位置和数量，将有可能监测活神经元中突触输入和输出的强度。一种能力 监测突触强度将对研究与突触连接异常相关的疾病非常有用，包括精神分裂症、精神发育迟滞和自闭症。

DESCRIPTION (provided by applicant): The purpose of the research proposed in this grant is to generate tools that will allow endogenous synaptic proteins to be visualized in neurons in vivo. Previously we have generated recombinant antibodies known as FingRs (Fibronectin intrabodies generated with mRNA display) that can be expressed in living neurons where they label endogenous target proteins noninvasively and with high fidelity. Furthermore, a transcriptional regulation system controls the expression levels of FingRs so that they are expressed at precisely the same level as their endogenous counterparts, insuring low background. In this grant we will generate transgenic mice that express fluorescent protein-fused FingRs that recognize synaptic proteins. Synaptic proteins in individual neurons in the brains of these mice can be visualized in vivo in real time. In turn, this will allow events at the molecular level to be correlated with events at the cellular, circuit and whole animal level. In particular, by mapping the locations and amounts of synaptic proteins in neurons, it will be possible to monitor the strength of both synaptic inputs and outputs in living neurons. The ability to monitor synaptic strength will be very useful for studying diseases associated with aberrant synaptic connectivity including schizophrenia, mental retardation and autism.