Expression of Metabotropic Glutamate Receptors in Primary Cultured Retinal Ganglion Cells

Abstract

Abstract: : Purpose: Retinal ganglion cell (RGC) death is the end stage of many diseases of the optic nerve, including glaucoma. The mechanisms of RGC death are not fully elucidated. Chronic elevation of glutamate may contribute to the pathophysiology of RGC death. This mechanism has been studied in vitro via ionotropic and metabotropic receptors. However, little is known of the expression of metabotropic glutamate receptors (mGluRs) in primary cultured cells. The purpose of this study was to examine the expression of the eight mGluRs in cultured rat RGCs under normal conditions. Methods: RGCs were purified from retinas of day–10 postnatal Sprague–Dawley rats using Thy–1 immunopanning technique. The cells were seeded into 35mm dishes (150K cells per dish). Immunocytochemistry was performed on the RGCs cultured for two weeks. Polyclonal antibodies specific to all the eight subtype–mGluRs were used to label the cultured cells with a standard technique of fluorescence immunocytochemistry. Each antibody was tested on RGCs derived from at least three different animals. Negative control for each of the antibodies was performed by omitting the primary antibody. Immunoreactivity was evaluated by fluorescence microscopy and compared with negative controls. Results: RGC purification was >95% confirmed by co–labeling with the selective marker Thy–1. All mGluR subtypes were expressed in primary cultured RGCs. The Group I mGluRs (1, 5) and Group II (2, 3) were localized on somas and processes, and appeared on every RGC (100%). Whereas the Group III mGluRs (4, 6, 7, and 8) were predominantly localized on somas. However, mGluR6 was only detected in a very small number (approximately 5%) of the RGCs, which may represent specific expression to one RGC type. Conclusions: We have demonstrated the expression of all eight mGluRs in cultured RGCs. Future experiments will be directed at examining the expression of each mGluR in cells subjected to modeled ischemia (oxygen and glucose deprivation) or increased pressure (high pressure chamber) to determine their involvement in retinal ganglion cell pathophysiology.

Keywords: ganglion cells • receptors