Pure culture of adult mammalian cone photoreceptors: electrophysiological characterization.

Abstract

Abstract: : Purpose: Although most forms of retinitis pigmentosa are related to mutations of rod photoreceptor (PR) specific genes, cone degeneration leads to complete blindness. Preservation of cone PRs in human diseases would thus limit functional deficits and permit a near normal activity in diurnal conditions. Since no pure culture of adult cone PRs has been described that could help at screening both cone–promoting survival factors and pharmacological compounds, we developed a new procedure allowing specific isolation of cone PR. Methods: Pig eyes were obtained from a local slaughterhouse. Cone PRs were purified from a total retinal cell suspension obtained by enzymatic dissociation of the retinal tissue. In culture, cone PR cells were identified and quantified by immunolabeling with a human cone–arrestin (hCAR) antibody and opsin antibodies. Cellular identity was confirmed by single cell RT–PCR for cone arrestin. Cell function was assessed by patch–clamp recordings in the whole cell mode. Results: Immediately after cell seeding, most cells exhibited cone PR morphology with a typical large inner segment and a short outer segment. Immunostaining with antibodies directed against either S–opsin or M/L–opsin showed that both cone populations were present in the preparation. The hCAR–positive cells accounted for 93 % of cells observed under transmitted light. Few rod bipolar cells were observed among contaminating cells (< 5 %). Cone PR survived for long period in vitro although 36 % of cultured cells degenerated over the first week. Single–cell RT–PCR assays confirmed that 95 % of purified cells were cones when tested immediately after purification or after 48h in vitro. Patch–clamp recordings confirmed the homogeneity of the preparation since recorded cells expressed highly similar voltage–dependant conductances. However, with time, functional changes appeared in the electrophysiological properties of the cultured cells with respect to in situ recorded–cones. Conclusions: This highly purified preparation should provide an adequate model to screen neurotrophic or pharmacological molecules promoting cone PR survival. It should further open the way to comparative studies of cone PR in normal and pathological conditions.

Keywords: photoreceptors • retinal culture • pharmacology