AMPA RECEPTOR ACTIVITY AND DENDRITE MORPHOGENESIS

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。虽然NMDA受体在出生后活动依赖可塑性中的作用已得到充分证实，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亚单位在发育过程中表达，但在神经元达到成熟表型后表达下调。先前的实验表明，在运动神经元发育结束时，GluR1的过度表达会导致树突分支点的数量增加，这表明树突可塑性的某些方面受到这些神经元中AMPA受体表达的控制。这项建议将使用哺乳动物脊髓培养物和携带单个AMPA受体亚基的皮质培养物的实时成像，来测量树突重组的动态速率。通过双顺反子载体的转染，将研究构成钙渗透和钙不渗透AMPA受体的亚基的精确发育作用。定量聚合酶链式反应将被用来建立发育中神经元中AMPA受体表达的时间模式。此外，使用RNAi对单个AMPA受体进行时间限制的敲除将调查AMPA受体的发育调节是否限制了树突的形态可塑性。实验将调查神经元活动本身是否调节特定AMPA受体亚单位的表达。对培养神经元的研究将得到来自体内模型的观察，该模型使用定向敲除AMPA受体亚单位及其细胞内信号分子，以及来自运动和姿势功能障碍的模型。确定AMPA受体如何控制树突状可塑性的特定特征，是理解神经元发育过程中功能连接如何受到调节的基础。

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.