In vitro conditions and muscarinic signaling influence the ratio of type-I to type-II cholinergic amacrine cells. E7 retinas were dissociated, plated and grown in culture for 24 hours. After 24 hours the cells were added with atropine (1, 10 or 50 μM), carbachol (1, 10 or 50 μM), or saline as a control, and maintained in culture for an additional 48 hours. Cells were labeled with antibodies to CRABP (red) and ChAT (green). Arrows (a-c) indicate type-II cholinergic cells that are immunoreactive for ChAT and CRABP, and arrow-heads indicate type-I cholinergic cells that are immunoreactive for ChAT alone. Cholinergic cells (arrows, d and e) did not accumulate BrdU after 3 DIV. The calibration bar (50 μm) in panel c applies to a-c and in e applies to d and e. Panel f shows the percentage of cholinergic cells also positive for CRABP (type-II cells). Asterisks indicate p-values < 0.05. Abbreviations: CRABP – cellular retinoic acid binding protein; ChAT – choline acetyltransferase; BrdU – Bromodeoxyuridine.