P2-EXPRESSION OF THE AMPA RECEPTOR SUBUNIT GLUR2 IN CHICK LUMBAR MOTONEURONS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 AMPA受体的钙通透性是由在天然通道中插入GluR2亚基决定的：至少插入一个GluR2亚基就会产生不透钙的AMPA受体。钙离子通透性的AMPA受体可以在早期胚胎发育中发现，但这一事件的功能意义尚不清楚。为了更好地了解钙通透性AMPA受体在神经元发育过程中的功能作用，我们研究了GluR2在鸡腰髓中的表达。Fura-2细胞内钙离子记录表明，在胚胎第6天，AMPA受体刺激在脊髓神经元中产生显著的钙信号。在E8至E11期间，由AMPA刺激产生的钙信号在一组大的脊髓神经元(DII标记的推定为运动神经元)中减少。在所有被测试的年龄，由AMPA产生的钙信号是特异的，可以被广泛的AMPA受体阻滞剂CNQX消除。实时定量聚合酶链式反应和蛋白质印迹分析表明，在E5和E7之间的腹侧脊髓中没有GluR2的mRNA和蛋白表达。GluR2的表达在E8时开始明显，并在整个E13期间保持高水平。这些发现表明，鸡脊髓存在GluR2表达的关键期，这一关键期与细胞程序性死亡、腹侧脊髓神经元的形态和电分化等重要发育过程相吻合。用麝香酚(一种GABA受体激动剂，通过破坏脊髓网络活动来导致发育中的胚胎瘫痪)抑制脊髓自发电活动，导致脊髓腹侧神经元GluR2基因表达显著减少。用管神经阻断神经肌肉活动后，GluR2基因的表达无明显变化。这些发现表明，持续的电活动是鸡腹侧脊髓正确表达GluR2所必需的。

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. The calcium-permeability of AMPA receptors is determined by the insertion of a GluR2 subunit in the native channel: insertion of at least one GluR2 subunit results in calcium-impermeable AMPA receptors. Calcium-permeable AMPA receptors can be found during early embryonic development, but the functional significance of this event is poorly understood. To gain a better understanding of the functional role of calcium-permeable AMPA receptors during neuronal development, we have explored GluR2 expression in the chick lumbar spinal cord. Fura-2 recordings of intracellular calcium indicate that AMPA receptor stimulation generates a significant calcium signal in spinal neurons at embryonic day (E) 6. Between E8 and E11, calcium signals generated by AMPA stimulation decrease in a population of large spinal neurons (putatively identified as motoneurons by DiI labeling). At all ages tested, calcium signals generated by AMPA are specific and can be eliminated by the broad AMPA receptor blocker CNQX. Real time PCR and western blot analyses indicate GluR2 mRNA and protein expression are absent in ventral spinal cords between E5 and E7. GluR2 expression becomes noticeable at E8 and remains high throughout E13. These findings suggest that there is a critical period for GluR2 expression in the chick spinal cord that coincides with important developmental processes like programmed cell death as well as morphological and electrical differentiation of ventral spinal neurons. Inhibition of spontaneous electrical activity in the spinal cord with muscimol (a GABA receptor agonist that induces paralysis of the developing embryos by disrupting spinal cord network activity) results in a significant reduction in GluR2 mRNA expression in ventral spinal neurons. No change in GluR2 mRNA expression was observed by blocking neuromuscular activity with tubocurare. These findings suggest that ongoing electrical activity is required for proper GluR2 expression in the chick ventral spinal cord.