MT disassembly in puffing-induced initiation of axonal varicosities. (A and B) Stimulating NMDA receptors (100 µM l-glutamate, 10 µM l-glycine, and 0.5 µM TTX) for 20 min induced varicosities in dendrites (A) but not axons (B) in mature neurons (23 DIV). (C and D) Depolymerizing MTs by Noco (10 µg/ml for 15 min; C) but not actin filaments by LatA (2.5 µM for 2.5 h; D) induced varicosities in axons. (E–G) The onset time (E), density (F), and size (G) of induced varicosities along dendrites or axons under the conditions in A–D. In the drug treatment experiments, the onset time was defined as the time needed to form clear varicosities. Error bars indicate means ± SEM. Unpaired t test: **, P < 0.05. (H) Long axonal segments under control conditions (top) or developing multiple varicosities induced by puffing (bottom) were imaged by TEM. TEM experiments showed ultrastructural changes of axons (top) and dendrites (bottom) under control (7 DIV; I), puffing (7 DIV; J), and Glu treatment (17 DIV; K) conditions. Bars: (A–D) 25 µm; (H–K) 1 µm.