ANALYSES OF CHANGES IN GLUTAMATE RECEPTOR SUBUNITS IN CULTURED RETINAL GANGLION CELLS DEATH

我们检测了α-amino-3-hydroxy-5-methylisoxazole-4-propionate受体亚单位蛋白在纯化的视网膜神经节细胞(RGC)中的发育表达。从分离的大鼠(出生后6-8天)视网膜细胞中分离纯化RGCs，采用改良的两步淘洗法，在含有神经营养因子和Forsklin的无血清培养液中进行培养。分别于培养第1、3、7天(L DIV、3 DIV、7 DIV)，用抗AMPA受体亚基GluR1to GluR4和微管相关蛋白(MAP)2的抗体对培养的RGCs进行免疫组织化学染色，用Fura-2荧光法检测细胞内[Ca^&lt；2+&Gt；]i对谷氨酸的影响。从1DIV开始，AMPA受体和MAP2在RGCs的体树突区均有表达，7DIV时在体树突区的表达逐渐增强。在所有发育阶段，AMPA受体和MAP2的表达都是共存的，GluR2的免疫反应性与其他亚单位相同。在3DIV和7DIV上，谷氨酸对[Ca~(2+)&t；~(2+)]_i的累积相当。这些结果表明，在培养的视网膜神经节细胞中，所有AMPA受体亚基的表达几乎相同，而降低GluR2的表达并不是视网膜神经节细胞通透性所必需的。

We examined the developmental expression of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptor subunit proteins in purified retinal ganglion cells (RGCs). RGCs were purified from dissociated rat retinal cells (postnatal days 6-8), using a modified two-step panning method and cultured in serum-free medium containing neurotrophic factors and forskolin. Immunohistochemistry was performed on the cultured RGCs on days 1, 3, and 7 in vitro (l DIV, 3 DIV, and 7 DIV) using specific antibodies against AMPA receptor subunits, GluR1 to GluR4, and microtubule-associated protein (MAP) 2. Changes in the level of intracellular Ca^<2+> ([Ca^<2+>]_i) to glutamate were measured with fura-2 fluorescence. Immunoreactivity of both AMPA receptors and MAP2 was expressed in the somatodendritic area of the RGCs from 1 DIV on, and expression gradually increased in the somatodendritic area on 7 DIV. In all developmental stages, the expression of both AMPA receptors and MAP2 was colocalized, and GluR2 immunoreactivity was equal to that of the other subunits. Accumulation of [Ca^<2+>]_i to glutamate was equal on 3 DIV and 7 DIV. These results suggest that all AMPA receptor subunits are almost equally expressed in cultured RGCs and that reduction of GluR2 expression is not required for Ca^<2+> permeability in RGCs.