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

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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.