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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 AMPA受体的钙渗透性由GluR 2亚基在天然通道中的插入决定：至少一个GluR 2亚基的插入导致钙不可渗透的AMPA受体。 钙渗透性AMPA受体可以在早期胚胎发育过程中发现，但这一事件的功能意义知之甚少。 为了更好地了解钙渗透性AMPA受体在神经元发育过程中的功能作用，我们研究了鸡腰髓中GluR 2的表达。 Fura-2对细胞内钙离子的记录表明，AMPA受体刺激在胚胎（E）6天的脊髓神经元中产生显著的钙信号。 在E8和E11之间，AMPA刺激产生的钙信号在大的脊髓神经元群体中减少（通过DiI标记鉴定为运动神经元的脊髓）。 在所有年龄段的测试中，AMPA产生的钙信号是特异性的，可以被广泛的AMPA受体阻断剂CNQX消除。 真实的时间PCR和蛋白质印迹分析表明，谷氨酸受体2的mRNA和蛋白质表达的腹侧脊髓之间的E5和E7的缺席。 GluR 2表达在E8时变得明显，并在整个E13期间保持高水平。 这些研究结果表明，有一个关键时期，在鸡脊髓中的GluR 2表达，符合重要的发育过程，如程序性细胞死亡和形态和电分化的腹侧脊髓神经元。 用蝇蕈醇（一种GABA受体激动剂，通过破坏脊髓网络活动诱导发育中胚胎的麻痹）抑制脊髓中的自发电活动导致腹侧脊髓神经元中GluR 2 mRNA表达显著降低。 用管箭毒阻断神经肌肉活动，没有观察到GluR 2 mRNA表达的变化。 这些研究结果表明，持续的电活动所需的适当GluR 2在鸡腹侧脊髓的表达。

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 analysis 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 and 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.