Neurophysiological study for excitatory amino acids-induced neurotoxicity and apoptosis in cultured retinal neurons.

1.血管活性肠肽(VIP)对谷氨酸诱导的视网膜神经细胞毒性的保护作用VIP(10 nM~(-1)µm)可浓度依赖性地减轻谷氨酸诱导的神经毒性。这种保护作用可被VIP受体拮抗剂VIP6-28和蛋白激酶A(PKA)抑制剂H-89所拮抗。VIP对谷氨酸诱导的电流和钙离子内流无影响。提示VIP可通过VIP受体上调cAMP水平，起到保护谷氨酸诱导的视网膜神经元神经毒性的作用。激活的PKA可能在谷氨酸神经毒性中钙离子内流的下游起作用。垂体腺苷环化酶激活肽(PACAP)对谷氨酸诱导的视网膜神经细胞毒性的保护作用。PACAP27(10 nM~(-1)µm)和PACAP38(10 nM~(-1)um)以浓度依赖的方式减弱谷氨酸诱导的神经毒性。H-89可抑制PACAPs对丝裂原活化蛋白(MAP)的激活。PACAP可能通过激活PKA受体激活MAP，作为神经保护因子对抗谷氨酸诱导的神经毒性。用磁性细胞分选器(MACS)富集大鼠视网膜神经节细胞(RGCs)。我们提出了一种利用MACS培养RGC的新技术，使RGCs的富集率从0.55%提高到31.0%，并提高了RGCs的存活率，可持续2周以上。全细胞膜片钳检测结果表明，MACs分离的RGCs上存在NMDA、海人藻酸和AMIPA三种离子亲性谷氨酸受体亚型。该培养体系被认为是研究谷氨酸诱导的RGC神经毒性的合适模型。

1. Protection of vasoactive intestinal peptide (VIP) against glutamate-induced neurotoxicity in cultured retinal neurons.VIP (10 nM - 1 muM) was attenuated glutamate-induced neurotoxicity in a concentration-dependent manner. This protective effect was antagonized by VIP6-28, a VIP recptor antagonist, and H-89, a protein kinase A (PKA) inhibitor. VIP did not affect glutamate-induced current and Ca^<2+> influx. VIP is suggested to act as a protective factor against glutamate-induced neurotoxicity of retinal neurons by elevating the cAMP level via VIP receptors. Activated PKA might have an effect downstream of the Ca^<2+> influx in glutamate neurotoxicity.2. Protection of pituitary adenylate cyclase activating peptide (PACAP) against glutamate-induced neurotoxicity in cultured retinal neurons.PACAP27 (10 nM - 1 muM) and PACAP38 (10 nM - 1 muM) were attenuated glutamate-induced neurotoxicity in a concentration-dependent manner. Activation of mitogen activated protein (MAP) kinase by PACAPs was inhibited by H-89. PACAPs are suggested to act as neuroprotective factors against glutamate-induced neurotoxicity by activating MAP kinase, which is due to PKA activation via PACAP receptors.3. Rat retinal ganglion cells (RGCs) culture enriched with the magnetic cell sorter (MACS).We demonstrated a new RGC-culture technique using MACS.It enriched RGCs from 0.55 % to 31.0 % of all retinal neurons and enhanced the survival of enriched RGCs for over 2 weeks. The whole-cell patch clamp evaluation was suggested that three ionotropic glutamate receptor subtypes such as NMDA, kainate and AMIPA receptors existed on MACS-separated RGCs. This culture system is thought to be an appropriate model for studying glutamate-induced neurotoxicity in RGC.