Project 7: Dynamics of Synapse Formation and Elimination in vivo

该项目的中心假设是，大脑的长期可塑性在很大程度上是通过神经元回路的形态重组来调节的，特别是通过选择性地形成和消除突触。尽管这是一个被广泛接受的观点，但在活体内突触的翻转特征还很少，对控制突触重塑的发育、环境和活动依赖因素知之甚少，特别是在 完好无损的动物。我们将生产带有荧光标记突触的PSD-95-GFP转基因小鼠和大鼠，用于中枢神经元突触形成和消除(翻转)的时间推移成像。我们将测量体内突触更新的模式和调节，目的是将这些变化与可塑性和行为联系起来。这些转基因动物的神经元培养将被用于研究分子和生理机制 控制突触的动态。我们的具体目标是：1)在组成型启动子下建立表达PSD-95-GFP的转基因小鼠，以标记脑内兴奋性突触；2)在活性诱导启动子下建立表达不稳定PSD-95-GFP的转基因小鼠，选择性地标记最近活跃的神经元中新形成的突触；3)利用新开发的慢病毒技术，在构成启动子和活性诱导启动子下建立表达PSD-95-GFP的转基因大鼠；4)利用转基因PSD-95-GFP动物，表征出生后发育过程中视皮质突触更新的动态及其视觉输入的调节；5)以培养的PSD-95-GFP动物神经元为研究对象，研究突触翻转的时空模式和可能的机制。在与Tonegawa、Bear和Liu的合作中，这项提议使用多学科方法来探索神经元内的突触相互作用，以及皮质可塑性与学习和记忆的形态相关性。 该项目有助于实现中心的多项目标，并依赖于综合的科学专门知识和 中心的设施。

The central hypothesis of this project is that long-term plasticity of the brain is mediated in large part by morphological reorganization of neuronal circuits, and specifically by the selective formation and elimination of synapses. Although this is a widely held belief, the turnover of synapses has been minimally characterized in vivo, and little is known about the developmental, environmental and activity-dependent factors that control synapse remodeling, especially in the intact animal. We will produce PSD-95-GFP transgenic mice and rats with fluorescently-labeled synapses for time-lapse imaging of synapse formation and elimination (turnover) in central neurons. We will measure the pattern and regulation of synapse turnover in vivo, with the goal of correlating these changes to plasticity and behavior. Neuron cultures from these transgenic animals will be used to investigate the molecular and physiological mechanisms that control synapse dynamics. Our specific aims are: 1) Create transgenic mice expressing PSD-95-GFP under a constitutive promoter to label excitatory synapses in brain; 2) Create transgenic mice expressing unstable PSD-95-GFP under an activity-inducible promoter to selectively label newly formed synapses in recently active neurons; 3) Create transgenic rats expressing PSD-95-GFP under constitutive and activity-inducible promoters, using newly developed lentiviral technology; 4) Characterize the dynamics of synapse turnover in visual cortex during postnatal development and its regulation by visual input, using the transgenic PSD-95-GFP animals; 5) Characterize the spatiotemporal patterns and underlying mechanisms of synapse turnover in vitro, using cultured neurons derived from the PSD-95-GFP animals. In collaboration with Tonegawa, Bear, and Liu, this proposal uses a multidisciplinary approach to explore synaptic interactions within neurons, and the morphological correlates of cortical plasticity and learning and memory. The project contributes to multiple objectives of the Center and depends on the combined scientific expertise and facilities of the Center.