Figure 5. GlyR activation regulates the formation, but not the maintenance, of GlyR clustering. A, Schematic timelines of strychnine treatments on mature neurons. Neurons cultured over 14 DIV without previous application of strychnine were applied for the administration of 1 μm strychnine for 48 h (+STR 48h), 36 h (+STR 36h), 24 h (+STR 24h), 1 h (+STR 1 h), or immediate (+STR 0 h) before and during imaging. B, Normalized fluorescence curves at focal spots with mCherry-GPHN signals in Ctrl (n = 14 cells, black), Ctrl with acute (+STR 0h; n = 6 cells, orange), with 1 h (+STR 1h; n = 9 cells, green), with 24 h (+STR 24h; n = 15 cells, cyan), with 36 h (+STR 36h; n = 11 cells, magenta), and with 48 h (+STR 48h; n = 10 cells, purple) of STR treatment before each experiment. Each point and bar represents mean and ±SEM, respectively. C, Average mobile fractions (mean ± SEM) shown in B. D, Fluorescence recovery curves after photobleaching at focal spots expressing mCherry-GPHN in WASH (n = 15 cells, black, shown in Fig. 4D), WASH with BAPTA-AM (n = 5 cells, orange), WASH with KN62 (n = 6 cells, green), WASH with Rp-cAMP (n = 5 cells, cyan), and WASH with GFX (n = 11 cells, magenta) conditions. Each point and bar represents mean and ±SEM, respectively. E, Averaged mobile fractions at focal spots expressing mCherry-GPHN in WASH (n = 15 cells, shown in Fig. 4E), WASH with BAPTA-AM (n = 5 cells), WASH with KN62 (n = 6 cells), WASH with Rp-cAMP (n = 5 cells), and WASH with GFX (n = 11 cells) conditions (mean ± SEM), for the data shown in D. The significance of difference was tested by the Kruskal–Wallis test followed by post hoc comparisons using the Mann–Whitney U test with Bonferroni’s correction. **p < 0.01/15 and p < 0.01/10 after Bonferroni’s correction for the 15 and 10 tests, respectively. n.s., Not significant.