Nicotinic acetylcholine receptors are synthesized in chick embryo retina before synapses appear. Most of the receptors are found associated with neurites in the inner synaptic layer of the retina; later in development the receptors appear in the outer synaptic laver.Cells dissociated from retina and cultured in vitro form aggregates and also sort out into regions comprised either of neurites or cell bodies. Most of the nicotinic acetylcholine receptors of aggregates are found associated with neurite regions. The receptor distribution of cultured retina cells thus resembles that of intact retina.125I-Labeled a-bungarotoxin, a specific ligand of the nicotinic acetylcholine (AcCh) receptor, was used to detect AcCh receptors in the developing chick embryo retina. This species of AcCh receptor is a synaptic marker, since the AcCh receptors of striated muscle cells (1, 2) and certain parasympathetic neurons (3) are found in abundance only at the site of the synapse. After denervation, the receptors are found over the entire surface of the cell.Most of the recent information regarding the nicotinic AcCh receptor has been obtained with the receptors of the electric organ of fish, and striated muscle (for reviews see refs. 4-6). a-Bungarotoxin has been used to detect AcCh receptors in sympathetic ganglia (7) and brain (8-10), but the nicotinic AcCh receptors of retina have not been studied in this manner. The vertebrate retina is a model system for studies on synaptic communication, since relatively few types of neurons are present and cells dissociated from retina form synapses in vitro (11,12). The retina contains acetylcholine and enzymes that catalyze the synthesis and the hydrolysis of this compound (13-17). In addition, electrophysiologic studies show that some neurons in retina respond to acetylcholine and that both nicotinic and muscarinic AcCh receptors are present in the retina of certain organisms (18)(19)(20)(21)(22).In this report, the concentration of nicotinic AcCh receptors and their location in chick embryo retina are described as a function of the developmental age of the retina. H-21) with 20 mM Hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.4, instead of NaHCO3, and 2 mg of serum albumin per ml of medium]. Pieces of retina were incubated for 60 min at 370 in 0.5-1.0 ml of solution A with 10 nM 125I-aBT and then were washed five times with 5 ml of cold solution A and twice with solution A without serum albumin. The tissue was fixed with 10% (vol/vol) Abbreviations: aBT, a-bungarotoxin; l25I-aBT, a-bungarotoxin labeled with 2 atoms of 125I per toxin molecule; AcCh, acetylcholine.
MATERIALS AND METHODS
Assay of