STIMULATION OF THE AMPA RECEPTOR IN RETINAL GANGLION CELLS INCREASES PHOSPHORYLATION OF CREB

Purpose: Activation of the ɑ-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor through glutamate or other agonists allows influx of cations including calcium, potassium and sodium. The purpose of this study was to investigate the neuroprotective role of the AMPA receptor in-vitro, in purified retinal ganglion cells (RGCs) and retinal mixed culture lacking RGCs (mixed retinal culture), by assessing the phosphorylation of cAMP response element-binding protein (p-CREB) stimulated by calcium influx. Methods: Purified rat RGCs were isolated from P3-P7 Sprague-Dawley rats and cultured by a double immunopanning technique using an antibody to Thy 1.1. The residual neurons in the retina following RGC isolation (supernates of the panning procedure) were used as the mixed retinal culture (lacking RGCs). Calcium imaging was used to identify the functionality of the AMPA receptors and selectivity of the AMPA agonist. RGCs and mixed retinal neurons were cultured for 7 days before AMPA treatment. Following treatment with AMPA for 6 hours, proteins were extracted and western blot analysis was carried out to determine changes in expression of the p-CREB and p-ERK1/2, which were normalized to total CREB, total ERK1/2, and beta tubulin. Results: AMPA receptors were stimulated through administration of AMPA (100μM), which depolarized the purified RGCs and increased intracellular calcium. The AMPA mediated calcium ion influx was significantly attenuated by approximately 87.8% (p<0.001) following pre-treatment with 20μM of NBQX (AMPA receptor antagonist). Pretreatment with a NMDA receptor (I μM MK801) or Kainate receptor (50μM UBP301) antagonists simultaneous with AMPA administration did not significantly decrease calcium influx. AMPA increased p-CREB by 4.3 ± 1.1 fold in purified RGCs (p=0.004), however, in mixed culture p-CREB did not change appreciably compared to control. ERK1/2 phosphorylation was significantly decreased in mixed retina culture (p<0.05), but not in purified RGC culture treated with AMPA. Conclusions: The data demonstrate distinct differences in the response to AMPA between RGCs and other neuronal populations in the retina. In particular, the lack of significant changes in the phosphorylation of ERK1/2 in purified RGCs following AMPA treatments suggests that an alternative pathway for phosphorylation of CREB maybe more important in RGC cell survival.