Trk modulation of CNS synaptic structure & function

描述（由申请人提供）：本提案旨在确定 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 Gonzalez等人（1999）证明，在神经肌肉突触中，TrkB是 位于肌纤维的突触后膜，TrkB介导 信号传导调节突触后乙酰胆碱受体的聚集。而 TrkB在CNS突触调节中的突触前作用已经被 广泛研究，突触后TrkB介导的信号传导的作用已经被 大部分未开发。为了验证TrkB介导的信号传导在细胞内发挥作用的假设， 在突触后神经递质的成熟和维持中的作用 CNS突触的受体簇，影响突触结构和功能， TrkA、B和C及其配体在大鼠海马神经元中定位 神经元和组织切片以及它们的定位如何被 活动将被确定。为了确定Trk介导的信号传导如何影响 海马突触结构和功能，重组腺病毒 而其它方法将用于过表达显性阴性， 截短的Trks或全长和其它突变的Trks。 将使用免疫染色和共聚焦显微镜来处理结构变化。 显微镜检查，并将电生理学评估功能变化。的 TrkB介导的信号调节神经递质受体的可能性 集群和集群维护是令人感兴趣的，将进行评估。 最后，神经元活动在调节营养反应中的作用 海马神经元的运输将通过检查Trk 受体和表达荧光蛋白标签的神经营养因子， 引入海马神经元培养物中。这些实验将有助于我们 理解Trk介导的信号传导在突触中的功能作用 在中枢神经系统中的形成和维持，以及营养信号和 反应性由突触活动调节。

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.