Figure 2. Synaptic Activity Promotes New Spine Growth in a Proteasome- and NMDA Receptor-Dependent Manner.

(A) Images of dendrites from EGFP-expressing neurons at 5–10 DIV before and after the addition of vehicle or bicuculline (30 μM) at t = 0 (black arrow). Yellow arrows indicate new spines. (B) Elevated neural activity in response to bicuculline increased the rate of new spine addition (blue bar; 116 spines, 6 cells) as compared to vehicle control (black bar; 68 spines, 6 cells; p < 0.01). Inhibiting the proteasome with MG132 blocked the bicu-culline-induced increase in spine outgrowth and decreased baseline new spine growth (light green bar; 46 spines, 7 cells; p < 0.05) to a level similar to that in the presence of MG132 alone (dark green bar; data from Figure 1; p = 0.4). (C) Schematic of uncaging-induced spine outgrowth. A new spine (red arrow) was induced by focal photolysis of MNI-caged glutamate (red dot) adjacent to a section of dendrite devoid of spines. (D) Images of dendrites from EGFP-expressing neurons at 7–8 DIV before and after focal photolysis of MNI-glutamate (red dots) at t = 0 (black arrow). Red arrows indicate new spines. (E) Uncaging-induced spine outgrowth (blue bar; 16 successes out of 44 trials on 15 cells) was significantly reduced in the presence of lactacystin (10 μM; green bar; 6 successes out of 40 trials on 12 cells; p < 0.05). (F) Images of dendrites from EGFP-expressing neurons at 7–11 DIV before and after the addition of CPP (30 μM) or CPP + lactacystin (10 μM) at t = 0 (black arrow). Yellow arrows indicate new spines. (G) The rate of new spine addition decreased in the presence of CPP (light green bar; 39 spines, 8 cells) as compared to vehicle control (black bar; 105 spines, 8 cells; p < 0.001). No further decrease in spine outgrowth was observed with simultaneous blockade of the proteasome with lactacystin (dark green bar; 45 spines, 7 cells; p = 0.4). Error bars represent SEM.