Expression of AMPA receptor subunit proteins in purified retinal ganglion cells

Purpose: To determine whether alpha-amino-3-hydroxy-5-methylisoxazole-4-propioate (AMPA) receptor (AMPAR) subunit proteins are expressed in cultured retinal ganglion cells (RGCs). Methods: 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 cultured RGCs on days 1, 3, and 7 in vitro (1 DIV, 3 DIV, and 7 DIV) using specific antibodies against AMPAR subunits GluR1 to 4 and microtubule-associated protein (MAP) 2, which is a neuronal market. Glutamate-induced Ca2+ influx was measured with fura-2 acetoxymethyl ester fluorescence. Results: GluR1 to 4 proteins were expressed in the cell body of RGCs on I DIV. RGCs showed strong GluR1 to 4 immunoreactivity in both cell bodies and processes on 3 DIV and 7 DIV, with the gradual spreading of expression and the growth of processes. At all time points examined, GluR2 immunoreactivity was equal to that of the other subunits. Accumulation of intracellular Ca2+ levels in RGCs induced by glutamate occurred equally on both 3 DIV and 7 DIV. Conclusion: All AMPAR subunits are almost equally expressed in cultured RGCs.