Dynamic mapping of the complete synaptome using recombinant probes

描述（由申请人提供）：在此，我们提出开发一种实验范例，以允许动态监测活生物体脑内每个谷氨酸能和GABA/甘氨酸能突触的强度和位置。结合生物体的行为操纵，这种范式将允许研究大脑如何在突触结构中编码信息。这种模式将涉及三种技术的结合：1。一种重组探针，可以在体内标记突触后兴奋和抑制位点，从而并行监测突触连接的位置和强度。2. 2 P-SPIM显微镜，可以非常快速地对大体积进行成像，没有漂白，并且可能具有各向同性分辨率。3.计算和存储每个突触的位置和距离的软件，以便于操作和分析。实验将在斑马鱼中进行，因为它们有相对较小的半透明大脑， 它们能够进行相对复杂的行为。实验将用于建立范式的可行性，并回答有关突触在睡眠期间如何调制的基本问题，以及它们如何在学习范式中改变，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.