A genetic strategy to record cell-cell interactions

描述(申请人提供)：要理解大脑回路中发生的计算，需要确定这些回路中的神经元是如何相互连接的。近年来，已经设计了几种新的方法(最著名的是序列电子显微镜、复制缺陷狂犬病病毒和GRAP)来识别大脑电路的接线图。为了克服现有策略的一些局限性，我们建议通过跨突触控制转录来产生一个新的遗传编码系统来追踪大脑电路，这将为研究电路连接和功能之间的关系打开新的机会。我们提出的系统是基于Notch受体的分子逻辑。在这个系统中，表达人工配体的神经元(“发送者”神经元)激活其突触伙伴(“接受者”神经元)上的转基因Notch受体。当配体-受体在突触部位相互作用时，工程设计的受体在其跨膜区被切割，并释放出一段蛋白质片段，调节突触伙伴的转录。我们的初步体外实验已经证实了这一策略的可行性，我们建议将这一设计应用于识别转基因动物中神经元电路的线路图，无论是在小鼠还是果蝇中。

DESCRIPTION (provided by applicant): Understanding the computations that take place in brain circuits requires identifying how neurons in those circuits are connected to each other. In recent years several new approaches (most notably, serial electron microscopy, replication-deficient rabies viruses, and GRASP) have been designed to identify the wiring diagrams of brain circuits. To overcome some of the limitations of the currently available strategies we propose to generate a new genetically-encoded system to trace brain circuits by transsynaptic control of transcription that will open new opportunities for investigating the relationship betwee circuit connectivity and function. The system that we propose is based on the molecular logic of the Notch receptor. In this system, neurons expressing an artificial ligand ("sender" neurons) activate a genetically-modified Notch receptor on their synaptic partners ("receiver" neurons). Upon ligand-receptor interaction in synaptic sites, the engineered receptor is cleaved in its transmembrane domain and releases a protein fragment that regulates transcription in the synaptic partners. Our initial experiments in vitro have confirmed the feasibility of this strategy and we propose to apply this design towards identifying wiring diagrams of neuronal circuits in transgenic animals, both in mice and drosophila.