Isolation of ganglion cells from the retina

Abstract

A microdissection technique was developed to isolate cellular layers of the goldfish retina. Three regions of the neural retina, the photoreceptor-interneuron layer (PL), the vascular layer (VL), and the ganglion cell layer (GCL) were recovered. A cleavage plane between the PL and GCL occurred at the level of the inner plexiform region as shown by light microscopy. Scanning electron microscopy found the predominant cells of the PL to include rods and cones while the VL contained a capillary network that arose from the retinal artery. The GCL consisted of pear-shaped ganglion cells about 5 μm in size with single, unbranched axons with diameters of about 0.5 μm. This preparation was virtually free of other cell types or debris. Transmission electron microscopy demonstrated that axons of the GCL were unmyelinated in contrast to those fibers distal to the optic nerve head. On the basis of morphology, microdissection could isolate a single population of retinal neurons free of glia. Biochemical indicators were used to determine the degree of separation of the GCL from the PL and VL. Markers of the cholinergic system, acetylcholinesterase and α-bungarotoxin binding, predominate only within the PL. These findings are consistent with known histochemical features of the retina and suggest that the GCL is not contaminated by the neighboring photoreceptor-interneuron region. Ganglion cell axons distal to the optic nerve head are myelinated and contain high levels of the marker enzyme 2′,3′-cyclic nucleotide-3′-phosphodiesterase. In contrast, this enzymatic activity is not detected within the unmyelinated axons of the glia free GCL preparation. To examine retinal events during axonal regeneration, transection of ganglion cell axons was carried out at the level of the optic tract. Axotomy led to stimulated amino acid incorporation detected within the GCL and not other regions of the retina. This specific response of the GCL was observed both in vivo and in vitro indicating that isolated ganglion cells remain intact and biologically active. Microdissection provides, therefore, a population of retinal ganglion cells that may be used to analyze the biochemical events of neuronal regeneration.