Co-release of acetylcholine and gamma-aminobutyric acid by a retinal neuron

Rabbit retinas were vitally stained with 4',6-diamidino-2-phenylindole (DAPI), a fluorescent compound that selectively accumulates within the cholinergic amacrine cells. The retinas were then incubated in vitro in the presence of radioactive gamma-aminobutyric acid (GABA) and autoradiographed. The cells that accumulated DAPI were found to accumulate GABA, confirming immunohistochemical evidence that the cholinergic amacrine cells contain GABA. Incubation of retinas in the presence of elevated concentrations of K+ caused them to release acetylcholine and GABA, and autoradiography showed depletion of radioactive GABA from the cholinergic amacrine cells. This indicates that the cholinergic amacrine cells can secrete acetylcholine and GABA. Retinas were double-labeled with [14C]GABA and [3H]acetylcholine, allowing simultaneous measurement of their release. The release of [14C]GABA was found to be independent of extracellular Ca2+. Radioactive GABA synthesized endogenously from [14C]glutamate behaved the same way as radioactive GABA accumulated from the medium. In the same experiments the simultaneously measured release of [3H]acetylcholine was strongly Ca2+-dependent, indicating that the releases of acetylcholine and GABA are controlled by different mechanisms. Synaptic vesicles immunologically isolated from double-labeled retinas contained much [3H]acetylcholine and little or no [14C]GABA. These results suggest that the cholinergic amacrine cells release acetylcholine primarily by vesicle exocytosis and release GABA primarily by means of a carrier.