PP2-EXPRESSION OF THE AMPA RECEPTOR SUBUNIT GLUR2 IN CHICK LUMBAR MOTONEURONS

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。AMPA受体的钙通透性是由在天然通道中插入GluR2亚基决定的：至少一个GluR2亚基的插入导致钙不渗透的AMPA受体。钙渗透性AMPA受体可以在胚胎早期发育过程中发现，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GluR2在鸡腰脊髓中的表达。细胞内钙的Fura-2记录表明，AMPA受体刺激在胚胎日脊髓神经元中产生显著的钙信号(E) 6。在E8和E11之间，AMPA刺激产生的钙信号在大脊髓神经元群（通过DiI标记推定为运动神经元）中减少。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受体阻滞剂CNQX消除。Real time PCR和western blot分析显示，E5 ~ E7期脊髓腹侧无GluR2 mRNA和蛋白表达。GluR2的表达在E8变得明显，并在整个E13保持高表达。这些发现表明，鸡脊髓中存在GluR2表达的关键时期，这一时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muscimol（一种GABA受体激动剂，通过破坏脊髓网络活动诱导发育中的胚胎瘫痪）抑制脊髓自发电活动导致腹侧脊髓神经元GluR2 mRNA表达显著降低。用管状管阻断神经肌肉活动未观察到GluR2 mRNA表达的变化。这些发现表明，在小鸡腹侧脊髓中，GluR2的正常表达需要持续的电活动。

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.