Development of ganglion cells and their axons in organized cultures of fetal mouse retinal explants

Abstract

Retinas from 13-15 day fetal mice were explanted alone, with adjacent eyeball tissue, or with nearby superior colliculus explants. The organotypic structure of the retina developed in situ, including photoreceptors, interneurons, plexiform layers, ganglion cells, and an optic fibre layer. Electrophysiologic recordings demonstrated that functional synaptic networks developed resembling bioelectric response patterns seen in situ. Within half-retinas, arrays of optic fibers converged to the optic nerve head; in co-cultures with tectum they could become myelinated. Large bundles of long, naked neurites--1 degree primary retinal fibers--emerged from the explant in the first few days in vitro; these could often be traced back to the optic nerve head and a detailed survey of their properties using horseradish peroxidase (HRP) tracing methods identified tham as ganglion cell axons. When growing upon collagen substrata, 1 degree fibers began to disintegrate during the second week in vitro; however, many 1 degree fibers that grew into superior colliculus explants were maintained for at least 5 weeks in vitro, where they formed elaborate, functional terminal arborizations. In a few cases, 1 degree fibers grew across skeletal muscle fibers and appeared to induce them to contract. A second type of neuritic outgrowth pattern appeared after the first week in vitro: 2 degrees retinal fibers. This was composed of a mixed population of interneuronal neurites; a small percentage was catecholaminergic. Our characterization of the morphologic properties of retinal ganglion cells and their axons in organotypic cultures provides the necessary background to interpret electrophysiologic mapping and neural-specificity analyses of retino-CNS co-cultures. This in vitro model system may have biological relevance to understanding the cues that control the development of the retinotectal projection in situ.