Acetylcholine-synthesizing amacrine cells: identification and selective staining by using radioautography and fluorescent markers

The fluorescent DNA stain 4, 6, diamidino-2 phenylindole (DAPI) was applied to the cut axons of the rabbit optic tract, from which it was retrogradely transported to the retinal ganglion cell bodies. The labelled retinas were isolated from the eye and maintained in vitro in the presence of [3H]choline. They were then quick-frozen, freeze-dried, vacuum-embedded, and radioautographed on dry emulsion for identification of the acetylcholine-synthesizing cells. Inspection of the radioautographs by fluorescence microscopy showed the two labels not to co-exist: the cells that contained the transported fluorescence did not contain radioactive acetylcholine. In other animals the optic nerve was sectioned, causing retrograde degeneration of a large fraction of the ganglion cells. A population of small, round neurons in the ganglion cell layer was spared. These retinas synthesized [3H]acetylcholine at the same rate as control tissues; and radioautography showed an identical distribution of the acetylcholine-synthesizing cells. We conclude that the acetylcholine-synthesizing neurons of the ganglion cell layer are displaced amacrine cells. When DAPI was injected intraocularly instead of being applied to the optic tract, a regular mosaic of neurons in the ganglion cell layer was selectively stained, and two bands of fluorescence were observed in the inner plexiform layer, at the level where two bands of radioactive acetylcholine were observed in radioautographs. Quantitative analysis showed that the DAPI-stained cells were the same size as those that survive optic nerve section. Like the acetylcholine-synthesizing cells, they appear to be displaced amacrines; when wheatgerm agglutinin labelled by Evans blue was applied to the optic tract and DAPI was injected intraocularly, the red fluorescence of Evans blue and the blue fluorescence of DAPI accumulated in different cells. When DAPI was injected intraocularly and radioautography for acetylcholine was carried out, the cells brightly labelled by DAPI were found to have synthesized acetylcholine. We conclude that topically applied DAPI selectively labels the acetylcholine-synthesizing neurons of the ganglion cell layer. The distribution of the acetylcholine-synthesizing cells was established by counting the DAPI-labelled cells in whole-mounts. Their peak density was 790 cells per square millimetre in the visual streak; it declined to a near-plateau of about 175 cells per square millimetre in the dorsal and ventral periphery. The morphology and distribution of the cells indicate that they are the same population previously stained by neurofibrillar methods in the peripheral rabbit retina.