Internal Dynamics of the Postsynaptic Density

描述（由申请人提供）：突触功能的活动调节变化是学习和神经发育分子理论的核心，也是包括阿尔茨海默病、精神分裂症和癫痫在内的疾病和障碍的目标。在大脑的谷氨酸能突触中，多结构域蛋白建立了突触后密度（PSD）的核心，PSD是连接神经递质受体与肌动蛋白细胞骨架和细胞内信号通路的结构。有人提出，这种PSD蛋白通过控制突触受体的锚定或移动来控制突触强度，但关于如何在PSD内完成这一过程的直接证据一直缺乏。此外，虽然PSD的大小和形状与突触受体水平在单个时间点上的测定相关，但尚不清楚单个PSD是否在各种形态之间转换，或者这种转换是否介导了长期抑郁期间突触的减弱。我们已经建立了一种新的荧光形态测定法来检测单个活的psd，并开发了两种高分辨率的光漂白测定法来监测它们内部蛋白质的运动。我们发现psd经历了很大程度的连续形态变化，这与对简单支架的期望相反。然而，核心蛋白在复合物内的流动性是非常有限的，这表明内部结构仍然保持。因此，我们假设PSD形式的动态调整持续改变突触功能，并且长期的突触抑制需要破坏该结构以适应受体的移除。在本研究中，我们将阐明PSD内部动力学在突触功能和可塑性中的作用。在培养的大鼠海马神经元中，我们将使用定量荧光显微镜以及膜片钳电生理，光解和时间分辨电子显微镜来解决以下具体目标。首先，PSD的形态变化是否与突触前结构相协调？第二，这些变化会动态改变突触强度吗？第三，肌动蛋白是否控制突触后支架的内部稳定性？第四，在长期抑郁症中受体丧失前后，PSD是否经历了形态或动力学的调节变化？这些问题的答案将填补我们对突触结构-功能关系理解的巨大空白。此外，他们将提供一个重要的平台来测试关于破坏突触传递的疾病的分子基础的假设。

DESCRIPTION (provided by applicant): Activity-regulated changes in synapse function lie at the heart of molecular theories of learning and neural development, and are the targets of diseases and disorders including Alzheimer's Disease, schizophrenia, and epilepsy. At glutamatergic synapses of the brain, multi-domain proteins establish the core of the postsynaptic density (PSD), the structure which links neurotransmitter receptors to the actin cytoskeleton and to intracellular signaling pathways. It has been proposed that such PSD proteins control synaptic strength by controlling the anchoring or mobility of synaptic receptors, but direct evidence regarding how this might be accomplished within the PSD has been lacking. Furthermore, though PSD size and shape correlate with synaptic receptor levels when assayed as a population at single time points, it is not known whether single PSDs transition among various morphologies, or whether such transformations mediate weakening of synapses during long-term depression. We have established a novel fluorescence morphometry approach for examining single, living PSDs, and have developed two high-resolution photobleaching assays for monitoring the movement of proteins within them. We have found that PSDs undergo a large degree of continuous morphological change, contrary to expectations for a simple scaffold. However, mobility of core protein within the complex is extremely limited, indicating that internal structure is nevertheless maintained. We therefore hypothesize that dynamic adjustment of PSD form continuously alters synapse function, and that long-term synaptic depression requires the disruption of this structure to accommodate the removal of receptors. In this proposal, we will clarify the role of PSD internal dynamics in synaptic function and plasticity. In cultured neurons from rat hippocampus, we will use quantitative fluorescence microscopy along with patch-clamp electrophysiology, photolysis, and time-resolved electron microscopy to address the following specific aims. First, is PSD morphological change coordinated with presynaptic structure? Second, do these changes dynamically alter synaptic strength? Third, does actin control the internal stability of the postsynaptic scaffold? Fourth, does the PSD undergo regulated changes in morphology or dynamics before or after receptor loss in long-term depression? The answers to these questions will fill large gaps in our understanding of the synaptic structure-function relationship. Further, they will provide an important platform on which to test hypotheses regarding the molecular basis of disorders that disrupt synaptic transmission.