Autoradiographic identification of acetylcholine in the rabbit retina.

Abstract

Rabbit retinas were studied in vitro under conditions known to maintain their physiological function. Retinas incubated in the presence of [3H]choline synthesized substantial amounts of [3H]phosphorylcholine and [3H]acetylcholine. [3H]Phosphorylcholine eventually proceeded into phospholipids, primarily phosphatidylcholine. Retinas pulse-labeled by a 15 min exposure to 0.3 .mu.M [3H]choline were incubated for an hour under chase conditions designed to retain newly synthesized acetylcholine within synapses or to promote its release. After incubation the 2 groups of retinas contained equal amounts of radioactivity in the phospholipid pathway, but only the retinas incubated under the acetylcholine-protecting conditions contained [3H]acetylcholine. Freeze-dried, vacuum-embedded tissue from each retina was autoradiographed on dry emulsion. All retinas showed Ag grains over the photoreceptor cells and faint labeling of all ganglion cells. In the retinas that contained [3H]acetylcholine, Ag grains also accumulated densely over a few cells with the position of amacrine cells, over a subset of cells in the ganglion layer and in 2 bands over the inner plexiform layer. Fixation of the retina with aqueous osmium tetroxide retained only the radioactive compounds located in the photoreceptor and ganglion cells. Sections from freeze-dried tissue lost their water-soluble choline metabolites when exposed to water and autoradiography of such sections again revealed radioactivity primarily in the photoreceptor and ganglion cells. Radioactive compounds extracted from the sections reflected those present in the tissue before processing; analysis of the compounds eluted from sections microdissected along the outer plexiform layer showed [3H]acetylcholine was synthesized only by cells of the inner retina. Photoreceptors and ganglion cells are apparently distinguished by a rapid synthesis of choline-containing phospholipids, while acetylcholine synthesis is restricted to a few cells at both margins of the inner plexiform layer. The only neurons to release acetylcholine within the rabbit retina are probably a small group of amacrine cells.