AMPA RECEPTOR ACTIVITY AND DENDRITE MORPHOGENESIS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。虽然NMDA受体参与出生后活动依赖的可塑性已经得到了很好的证实，但对非NMDA受体在这一过程中的功能了解较少。在运动神经元中，AMPA GluR1亚基在发育期间表达，但在神经元达到成熟表型时下调。先前的实验表明，运动神经元在发育末期过表达GluR1会导致树突分支点数量增加，这表明这些神经元中AMPA受体的表达在某些方面控制了树突可塑性。这一建议将使用实时成像的哺乳动物脊髓培养和皮层培养与单个AMPA受体亚基转染，以测量树突重组的动态速率。构成钙透性和钙不透性AMPA受体的亚单位的确切发育作用将使用双电载体转染进行研究。定量PCR将用于建立AMPA受体在发育神经元中表达的时间模式。此外，使用RNAi暂时限制单个AMPA受体的敲除将研究AMPA受体的发育调节是否限制了树突的形态可塑性。实验将研究神经元活动本身是否调节特定AMPA受体亚基的表达。对培养神经元的研究将辅以体内模型的观察，这些模型采用靶向敲除AMPA受体亚基及其细胞内信号分子，以及运动和姿势功能障碍模型。确定AMPA受体如何控制树突可塑性的特定特征是理解神经元发育过程中功能连接如何被调节的基础。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. While the involvement of NMDA receptors in postnatal activity-dependent plasticity is well established, less is understood about the function of non-NMDA receptors in this process. In motor neurons, the AMPA GluR1 subunit is expressed during development, but is downregulated when neurons have attained their mature phenotype. Previous experiments have shown that overexpression of GluR1 in motor neurons at the end of their development results in an increased number of dendrite branch-points, suggesting that some aspects of dendrite plasticity are controlled by AMPA receptor expression in these neurons. This proposal will use real-time imaging of mammalian spinal cord cultures and cortical cultures transfected with individual AMPA receptor subunits, to measure dynamic rates of dendrite reorganization. The precise developmental role of subunits which constitute calcium-permeable and calcium-impermeable AMPA receptors will be investigated using transfection of bicistronic vectors. Quantitative PCR will be used to establish the temporal pattern of AMPA receptor expression in developing neurons. Further, temporally restricted knock-down of individual AMPA receptors using RNAi will investigate whether developmental regulation of AMPA receptors limits morphological plasticity of dendrites. Experiments will investigate whether neuronal activity itself regulates expression of specific AMPA receptor subunits. Studies in cultured neurons will be complemented with observations drawn from in vivo models which employ targeted knockout of AMPA receptor subunits and their intracellular signaling molecules, and from models of locomotor and postural dysfunction. Determining how AMPA receptors govern specific features of dendritic plasticity is fundamental in comprehending how functional connectivity may be modulated during neuronal development.