Cristóvão Armando J, Oliveira Catarina R, Carvalho Caetana M
Center for Neuroscience of Coimbra, Department of Zoology, Faculty of Science and Technology, University of Coimbra, 3004-517, Portugal.
Brain Res Mol Brain Res. 2002 Mar 28;99(2):125-33. doi: 10.1016/s0169-328x(02)00105-5.
The N-methyl-D-aspartate (NMDA) ionotropic glutamate receptors were studied in retina cells developing in chick embryos and in retina cells cultured as retinospheroids, at the same stages of development. In the retinospheroids, the activity of the NMDA receptors was followed by monitoring the changes in the intracellular free calcium concentration (Ca2+), in response to NMDA or to L-glutamate. The expression of the subunits NMDAR1, NMDAR2A/B and NMDAR2C in the retinospheroids and in chick retinas were determined by Western blot analyses. The changes in Ca2+ in response to 400 microM NMDA increased from 5 h in vitro to 3 days in vitro (DIV) and remained constant until 14 DIV, whereas the Ca2+ response to 500 microM L-glutamate increased from 5 h in vitro to 3 DIV and decreased slightly until 14 DIV. In the retinospheroids, the expression of the NMDAR1 and NMDAR2A/B subunits increased from 5 h in vitro until 14 DIV, whereas the NMDAR2C subunit increased from 5 h in vitro until 10 DIV and remained constant until 14 DIV. In the retinas, the expression of NMDAR1 increased from embryonic day 8 (E8) until E15, decreased until E18, and increased again until day 22 (post-hatched 1, PH1). The NMDAR2A/B increased from E8 until E18 and decreased slightly until PH1, whereas the NMDAR2C subunit increased from E8 until E15, remained constant until E18, and increased again until PH1. The results suggest that NMDA receptors are expressed and functionally active at early embryonic stages in the retina and in retinospheroids, before synapse formation (E12). However, the calcium responses to NMDA were relatively constant from 3 DIV until 14 DIV, showing no correlation with the increase in the expression of the studied NMDA receptor subunit during the same period. Also, the patterns of NMDA receptor subunits expressed in chick embryo retina cells cultured in vitro and in retina cells developing in vivo were similar.
在鸡胚发育中的视网膜细胞以及在相同发育阶段培养成视网膜球状体的视网膜细胞中，对N-甲基-D-天冬氨酸（NMDA）离子型谷氨酸受体进行了研究。在视网膜球状体中，通过监测细胞内游离钙浓度（Ca2+）的变化来跟踪NMDA受体的活性，该变化是对NMDA或L-谷氨酸的反应。通过蛋白质免疫印迹分析确定视网膜球状体和鸡视网膜中NMDAR1、NMDAR2A/B和NMDAR2C亚基的表达。对400微摩尔NMDA的Ca2+反应从体外培养5小时增加到体外培养3天（DIV），并一直保持恒定直到14 DIV，而对500微摩尔L-谷氨酸的Ca2+反应从体外培养5小时增加到3 DIV，并在14 DIV之前略有下降。在视网膜球状体中，NMDAR1和NMDAR2A/B亚基的表达从体外培养5小时增加到14 DIV，而NMDAR2C亚基从体外培养5小时增加到10 DIV，并在14 DIV之前保持恒定。在视网膜中，NMDAR1的表达从胚胎第8天（E8）增加到E15，在E18之前下降，然后在第22天（孵化后1天，PH1）再次增加。NMDAR2A/B从E8增加到E18，并在PH1之前略有下降，而NMDAR2C亚基从E8增加到E15，在E18之前保持恒定，并在PH1之前再次增加。结果表明，在视网膜和视网膜球状体的早期胚胎阶段，即在突触形成（E12）之前，NMDA受体就已表达并具有功能活性。然而，从3 DIV到14 DIV，对NMDA的钙反应相对恒定，与同期所研究的NMDA受体亚基表达的增加没有相关性。此外，体外培养的鸡胚视网膜细胞和体内发育的视网膜细胞中表达的NMDA受体亚基模式相似。
