Identification and characterisation of cell types accumulating GABA in rat retinal cultures using cell type specific monoclonal antibodies

Abstract

Monolayer and reaggregate cultures have been established from neonatal rat retina. After 7 days in culture, 60 nM [ 3H]γ-aminobutyric acid (GABA) was used to identify cells with a high affinity uptake mechanism for GABA. Approximately 80% of the process-bearing cells were found to be labelled. These cells were identified as amacrine cells by double-labelling experiments combining [ 3H]GABA uptake with immunocytochemical labelling with monoclonal antibody HPC-1 which in retina is specific for amacrine cells. The ability of cultures to synthesize GABA from glutamate was investigated at various times. Little synthesis was observed during the first few days in culture. This lag was followed by an increase in the amount of synthesis until 3 weeks of culture. When clumps and reaggregate cultures of retinal cells were examined by [ 3H]GABA uptake, a time-dependent redistribution of labelled cells was observed. After 20 h in culture, GABA-positive cells were distributed over the whole cell mass. Over the next few days, the labelled cells became more common on the outer edge of the aggregates and less common in inner regions. By 7 days of culture, no labelled cell bodies were found on the inside of the aggregates, although such cells could be labelled by [ 3H] d-aspartate. The results provide positive identification of a subclass of retinal cells in culture, and show that at least one aspect of retinal histogenesis is not dependent upon extra-retinal tissues or the position imposed by the temporal order of retinal cell birth.