Generation of enriched populations of cultured photoreceptor cells

This laboratory has recently described a monolayer culture system that allows the survival and differentiation of chick embryo retinal neurons and photoreceptors in the absence of contamination by connective tissue, endothelial, glial, or pigmented epithelial cells. The authors report here that the photoreceptor cells can be further purified by exploiting the selective toxicity of kainic acid (KA) and beta-bungarotoxin (BBT), which are known to destroy many retinal neurons without affecting photoreceptors. When added to retinal cultures, both KA and BBT caused developmental stage- and concentration-dependent degeneration without affecting the number or qualitative properties of photoreceptors. Some retinal neurons were already sensitive to KA after only 2 days in vitro, and their number increased as the cultures matured. The maximum effect of KA (a loss of 60% of the neurons) was obtained with an 8-24 hr treatment with 2 mM KA after 6 days in vitro. Neuronal sensitivity to BBT showed a developmental pattern similar to KA. However, maximum neuronal losses produced by BBT were higher (70% of the neurons) and occurred at lower concentrations (1-2 nM). Combined treatments with KA and BBT did not show additive effects or potentiation between toxins. KA- or BBT-induced neuronal degeneration was accompanied by a greater than 90% loss of neuronal markers such as choline acetyltransferase activity or high-affinity GABA uptake. Transmission electron microscopy showed the integrity of photoreceptor cells in KA- and BBT-treated cultures. These results show that both KA and BBT are useful tools for the generation of enriched photoreceptor populations in vitro.