Rod photoreceptors dissociated from the adult rabbit retina

Abstract

Rod photoreceptors have been isolated from the adult rabbit retina using enzymatic and mechanical dissociation procedures; their fine structure, synaptic activity, and long-term viability were examined using conventional electron-microscopic, quick-freezing, and cell culture techniques. Freshly dissociated photoreceptors were well-preserved compared to their counterparts in the intact retina. About half of the cells, however, exhibited broad continuity between inner and outer segments. Quick-frozen, freeze-substituted rods differed from chemically fixed cells in 3 respects: (1) there was an increased amount of granular matrix in the cytoplasm, mitochondria, and rough endoplasmic reticulum; (2) branching and anastomosing profiles of smooth endoplasmic reticulum had disappeared from the inner segment; and (3) the number of synaptic vesicles within the spherule was highly variable, in some cases leaving synaptic ribbons completely denuded of their halo of vesicles. Light-adapted, solitary rod cells continued to be synaptically active: their endings were capable of endocytosis when placed in the dark in the presence of extracellular ferritin and tracer was incorporated into vesicles and vacuoles; this uptake was much reduced when the cells were incubated with the tracer in the light. Thus, synaptic vesicle regeneration was stimulated in the dark, suggesting that vesicles underwent exocytosis in the dark. Isolated rod cells adhered poorly to most standard substrates; without proper adhesion, cells deteriorated in 2-4 hr. However, photoreceptors did adhere to glutaraldehyde-fixed Vitrogen gels and could be maintained for over 48 hr on this substrate if kept in a complete medium at 22 degrees C. In contrast, Müller cells adhered quickly to a laminin substrate with their endfoot processes. The differential adhesion properties of Müller and photoreceptor cells may be useful in obtaining pure populations of glial cells or neurons from the adult mammalian retina.