IN-VITRO IDENTIFICATION OF RETINAL GANGLION-CELLS IN CULTURE WITHOUT THE NEED OF DYE LABELING

We here describe a method for the identification of a distinct neuronal phenotype at all stages of development in culture without the need of any staining procedure. Based purely on a size criterion we can rapidly select vital retinal ganglion cells (RGCs) for further studies out of a mixed culture of rat retinal cells. In order to establish a size criterion for retinal cells of various age, RGCs were first labeled immunocytochemically with an antibody against the ganglion cell-specific surface glycoprotein Thy-1. Soma diameters were then determined for labeled and unlabeled cells between embryonic day 16 (E16) and postnatal day 90 (P90). Unlabeled neurons of all ages had soma diameters between 3.6 mu m and 12 mu m (mean diameter: 6.3 mu m). In contrast, soma diameters of RGCs ranged from 8.4 mu m to 28 mu m and the number of RGCs with large soma diameters increased with age. Thus, in a mixed retinal cell culture only RGCs are larger than 12 mu m and can be selected solely based on their size. The validity of the size criterion during the whole period of retinal cell differentiation offers the possibility to study the development of cellular functions and ion channel properties in a distinct type of cell without the risque of artifacts introduced by staining.