Trk modulation of CNS synaptic structure & function

描述(由申请人提供)：本提案旨在确定角色 神经营养因子及其受体Trks在调节突触结构中的作用 并在发育中的中枢神经系统中发挥作用。之前的工作由 Gonzalez等人。(1999)证明在神经肌肉突触，TrkB是 定位于肌纤维突触后膜及TrkB介导 信号调节突触后乙酰胆碱受体的聚集。而当 TrkB在中枢神经系统突触调制中的突触前作用 经过广泛研究，突触后TrkB介导的信号转导的作用 很大程度上是未被开发的。要检验TrkB介导的信号在 突触后神经递质的成熟与维持 中枢神经系统突触上的受体簇，影响突触的结构和功能， TrkA、B、C及其配体在游离海马区的定位 神经元和组织切片及其定位是如何被调节的 活动将被确定。确定Trk介导的信号转导如何影响 海马区突触的结构和功能重组腺病毒 而其他方法将被用来过度表达显性-负性， 海马神经元中的截短Trks或全长Trks和其他突变Trks。 将使用免疫染色和共聚焦来解决结构变化 显微镜检查和功能改变将通过电生理进行评估。这个 TrkB介导的信号调节神经递质受体的可能性 集群和集群维护非常重要，将对其进行评估。 最后，神经元活动在调节营养反应性中的作用 将通过检测Trk的运输来确定海马神经元的数量 表达荧光蛋白标签的受体和神经营养素 被引入海马神经元培养。这些实验将有助于我们的 了解Trk介导的信号在突触中的功能作用 中枢神经系统的形成和维持以及营养信号和 反应性受突触活动的调节。

DESCRIPTION (provided by applicant): This proposal aims to determine the role of neurotrophins and their receptors, the Trks, in modulating synapse structure and function in the developing central nervous system. Previous work by Gonzalez et al. (1999) demonstrated that at neuromuscular synapses, TrkB is localized to the postsynaptic membrane of muscle fibers and that TrkB mediated signaling modulates clustering of postsynaptic acetylcholine receptors. While the presynaptic role of TrkB in synaptic modulation in the CNS has been extensively studied, the role of postsynaptic TrkB-mediated signaling has been largely unexplored. To test the hypothesis that TrkB-mediated signaling plays a role in the maturation and maintenance of postsynaptic neurotransmitter receptor clusters at CNS synapses, influencing synapse structure and function, the localization of TrkA, B and C and their ligands in dissociated hippocampal neurons and histological sections and how their localization is modulated by activity will be determined. To determine how Trk-mediated signaling affects the structure and function of hippocampal synapses, recombinant adenoviruses and other methods will be used to over-express either dominant-negative, truncated Trks or full length and other mutant Trks in hippocampal neurons. Structural changes will be addressed using immunostaining and confocal microscopy, and functional changes will be assessed eletrophysiologically. The possibility that TrkB-mediated signaling modulates neurotransmitter receptor clustering and cluster maintenance is of interest and will be evaluated. Finally, the role of neuronal activity in regulating the trophic responsiveness of hippocampal neurons will be determined by examining the trafficking of Trk receptors and neurotrophins expressing fluorescent proteins tags that have been introduced into hippocampal neuron cultures. These experiments will aid in our understanding of the functional role of Trk-mediated signaling in synaptic formation and maintenance in the CNS and how trophic signaling and responsiveness are modulated by synaptic activity.