Fig. 3. Direct application of ATP to isolated neonatal retinas induces death of cholinergic neurons. (A-C) A P7 rat central retina cholinergic cell labeled with Alexa Fluor 488 dextran is illustrated at different times after application of 1 mM ATP. Small blebs in the dendritic tree are already apparent after a few minutes of ATP application (A), and become more conspicuous within 30 minutes. The cell soma (located in a different focal plane) displayed blebbing (left inset in C), and exposed phosphatidylserine on the outer membrane, as evidenced by Cy3-Annexin labeling (right inset in C). (D,E) Extensive soma blebbing was apparent in cholinergic neurons 30 minutes after application of 1 mM ATP (D) or 0.1 mM ATP (E). (F) In addition to blebbing (left) the cholinergic cells became permeable to propidium iodide (right), which displays a stronger fluorescence in the cell nucleus as a consequence of DNA binding (30 minutes after application of 1 mM ATP). (G) Between 2 hours and 12 hours after ATP application the soma of most cholinergic neurons lost their blebs and appeared shrunken (right panel) with respect to their original size (left panel). The small dark spot visible in D-G is the gene-gun bullet. Scale bar: 30 µm (A-C); 15 µm (insets in C); 10 µm (D-G). Images were acquired in black and white. By convention acquisition with the 488 nm emission filter (Alexa Fluo 488 dextran) are shown in yellow, acquisitions with the 568 nm emission filter (propidium iodide, Cy3-annexin) in red.