Fig. 2: Adeno-associated virus exposure of human iPSC-differentiated glutamatergic cortical neurons with AAV9-MeCP2-GFP-huHTR2A-shRNA leads to a decrease in spontaneous electrical activity.

Following plating of neurons, three different conditions were tested starting at Day 5: 1) Vehicle control consisting of PBS only (green circles, labeled “control”); 2) Scrambled AAV9 shRNA at MOI of 3 × 10⁵ (blue circles, labeled “scrambled”); 3) HTR2A hu-shRNA at MOI 3 × 10⁵ (red triangles, labeled “AAV9”). Electrophysiological recordings were acquired three times per week following day 5 in vitro. As noted in the text, recording anomalies observed on day 14 were deemed unreliable and were excluded from further analysis. By inspection, it was evident from the AAV9-MeCP2-GFP-huHTR2A-shRNA results that the largest differences noted between the three conditions were on days 12, 16, and 18, and especially so for the synchrony index. Thus, data on these three days were examined for each MEA metric using a repeated measures ANOVA to determine the main effect of “condition,” and then a post hoc comparison was performed comparing the three conditions, with p-values adjusted via the Fisher LSD method. A, B Significant differences occurred in mean burst duration between the AAV9 and both of the other two conditions (p < 0.001). C, D Significant differences in average burst frequency were observed between AAV9 vs. the control (p < 0.01) and scrambled conditions (p < 0.05). E, F A significant difference in average network bursts occurred between AAV9 and the control condition (p < 0.01). G, H Significant differences in the synchrony index were observed between both conditions (p < 0.001). I, J Significant differences in the number of spikes per burst were observed between AAV9 vs. the control (p < 0.01) and scrambled conditions (p < 0.05). All data are expressed as the mean ± S.E. Asterisks denote: *p < 0.05; **p < 0.01; ***p < 0.001.