Dynamic mapping of the complete synaptome using recombinant probes

描述(申请人提供)：在这里，我们建议开发一个实验范式，以允许动态监测每个谷氨酸能和GABA/甘氨酸能突触在活体大脑中的强度和位置。结合对有机体的行为操控，这一范例将允许研究大脑如何在突触结构中编码信息。这一范例将涉及三项技术的结合：1.重组探针，可在体内标记突触后兴奋性和抑制性部位，允许并行监测突触连接的位置和强度。2.2P-SPIM显微镜，它可以非常快速地成像大体积，而不会漂白，并且潜在地具有各向同性的分辨率。3.以便于操作和分析的方式计算和存储每个突触的位置和强度的软件。实验将在斑马鱼身上进行，因为它们的大脑相对较小，是半透明的。 它们能够做出相对复杂的行为。实验将被用来确定该范例的可行性，并回答有关突触在睡眠期间如何调节的基本问题，以及它们在学习范例中如何改变，如声音习惯化和位置偏好/厌恶条件反射。

DESCRIPTION (provided by applicant): Here we propose to develop an experimental paradigm to allow dynamic monitoring of the strength and location of every glutamatergic and GABA/Glycinergic synapse within the brain of a living organism. In combination with behavioral manipulation of the organism this paradigm will allow for study of how the brain encodes information in synaptic structure. This paradigm will involve combining three technologies: 1. Recombinant probes with which postsynaptic excitatory and inhibitory sites can be labeled in vivo, allowing the location and strength of synaptic connections to be monitored in parallel. 2. 2P-SPIM microscopy, which can image large volumes very quickly, without bleaching and, potentially, with isotropic resolution. 3. Software to calculate and store the location and strengt of each synapse in such a manner that it can be easily manipulated and analyzed. Experiments will be performed in zebrafish, as they have semi-transparent brains that are relatively small, yet they are capable of relatively complex behaviors. Experiments will be used both to establish the viability of the paradigm and to answer fundamental questions about how synapses are modulated during sleep, as well as how they are changed in learning paradigms such as sound habituation and place preference/aversion conditioning.