Characterization of Rat Retinal Ganglion Cells with a Differentiated Morphology

Abstract

Abstract: : Purpose: Previously we have shown that transformed rat retinal ganglion cells (RGC–5) undergo a morphological change after co–culture with human non–pigmented cilliary epithelial (HNPE) cells. The purpose of this study was to characterize these morphologically different RGC–5 cells using functional assays and immunocytochemistry. Methods: HNPE cells were seeded on collagen inserts in DMEM complete medium and added to RGC–5 cells seeded on glass coverslips in 6–well plates. After 3 days of co–culture the wells were observed by light microscopy and pictures were taken. Changes in intracellular calcium concentrations were measured in response to 50µM glutamate using Fura–2 calcium imaging in the presence and absence of MK–801, a NMDA receptor antagonist. Whole cell patch clamp technique was employed to detect changes in current following glutamate treatment. Immunocytochemistry was performed using antibodies against Thy–1. Results: There was an increase in intracellular calcium concentrations in response to 50µM glutamate. Pretreatment with MK–801 inhibited the glutamate–induced increase in intracellular calcium. Treatment of glutamate caused an inward current across the membrane in RGC–5 co–cultured with HNPE. The morphologically different RGC–5 cells expressed Thy–1. Conclusions:RGC–5 cells upon co–culture with HNPE develop a differentiated phenotype which is responsive to glutamate. The NMDA receptor may be involved due to the inhibition of the calcium response with the treatment of MK–801. Although these cells change morphology they continue to express Thy–1 which is a characteristic retinal ganglion cell marker. Further characterization of these cells and the channels involved are currently being conducted using whole cell patch clamping, western blotting and immunocytochemistry.

Keywords: ganglion cells • retinal culture • excitatory neurotransmitters