Transformed Rat Retinal Ganglion Cells (RGC–5) Respond to Glutamate After Co–Culture With Human Non–Pigmented Cilliary Epithelial (HNPE) Cells

Abstract

Abstract: : Purpose: Previously we have shown that transformed rat retinal ganglion (RGC–5) cells undergo a morphological change indicative of a differentiated phenotype, after co–culture with human non–pigmented cilliary epithelial (HNPE) cells. These differentiated RGC–5 cells have exhibited an consistent increase in intracellular calcium ([Ca2+]i) in response to 100 µM NMDA as shown by Fura–2 imaging and whole cell patch clamping. The purpose of this study was to determine if the differentiated RGC–5 cells respond to glutamate treatment and to evaluate the expression of Thy–1 and Brn–3b, which are known markers of retinal ganglion cells. Methods: HNPE cells were seeded on collagen inserts and placed over RGC–5 cells seeded on glass coverslips in 6–well plates. After 3 days of co–culture the wells were observed by light microscopy for morphological changes. Changes in [Ca2+]i were measured in response to 250µM and 500 µM glutamate using Fura–2 imaging in the presence or absence of 10µM MK–801, a NMDA receptor antagonist. Immunocytochemistry was performed using antibodies against Thy–1 and Brn–3b. Results: After co–culture the [Ca2+]i of RGC–5 cells increased in response to both 250 µM and 500 µM glutamate. Pretreatment with 10µM MK–801 inhibited the glutamate–induced increase in [Ca2+]i. The morphologically different RGC–5 cells continued to express Thy–1 and Brn–3b. Conclusions: RGC–5 cells upon co–culture with HNPE cells develop a differentiated phenotype which are responsive to glutamate. The glutamate–induced increase in [Ca2+]i was inhibited by MK–801, a NMDA receptor antagonist. Although these cells change morphology, they continue to express Thy–1 and Brn–3b. Further characterization of these cells and the channels involved are currently being conducted using whole cell patch clamping.

Keywords: ganglion cells • retinal culture • excitatory neurotransmitters