Figure 5. CB1 antagonism suppresses network activities before and after adaptation to BCC-induced hyperexcitation. A, Experimental design. Neurons DIV21–DIV28 were treated with ctrl media (Ctrl, or naive) or 20 μm BCC (BCC, or downscaled) for 40 h before baseline was recorded for 10 min. After resting for 1 h, naive or downscaled neurons were recorded and quantified with MEA in 10-min bins once every 30 min over 6 h in the absence (Ctrl, BCC) or presence (Ctrl+AM251, BCC+AM251) of 50 nm AM251, a potent CB1 inverse agonist. Media were allowed to equilibrate for 10 min after the drug treatment (Time 0) before the first time point was taken. The same experimental design was used for live-cell imaging, but those cells were only recorded for 1 h in the absence or presence of 500 nm AM251. B, Quantification of mean firing rate, burst frequency and network burst frequency from naive neurons untreated (Ctrl) or treated for 6 h with 50 nm AM251 (Ctrl+AM251), and from downscaled neurons untreated (BCC) or treated for 6 h with 50 nm AM251 (BCC+AM251), as illustrated in A. Note that AM251 strongly suppresses network activities in naive cells which remains significantly depressed for 1.5 h, but the response is greatly attenuated in downscaled cells. Results were normalized to a within-well control recorded for 10 min at ∼1 h before treatment (A, baseline), and presented as mean ± SEM from four to six independent culture preparations with quadruplicate wells. Two-way ANOVA with Tukey’s multiple comparison test; #p ≤ 0.05 Ctrl versus Ctrl+AM251, *p ≤ 0.05 Ctrl+AM251 versus BCC+AM251 throughout the duration indicated by the bracket and/or at time points denoted by the symbols. C, Quantification of average mean firing rate, burst frequency and network burst frequency for the first 1.5 h of treatment, during which AM251-induced suppression of network activities remains statistically significant in naive cells. Note that attenuation of AM251-induced suppression of network activities is particularly prominent during the first 1.5 h. D, Double derivative images showing spontaneous changes in iGluSnFR fluorescence corresponding to a single synchronous glutamate event under control condition. E, Representative traces of iGluSnFR fluorescence corresponding to multiple synchronous glutamate events recorded in DIV21–DIV25 culture cortical neurons naive and untreated (Ctrl, top left), naive and treated with 500 nm AM251 for up to 1 h (Ctrl+AM251, bottom left), downscaled and untreated (BCC, top right) or downscaled and treated with 500 nm AM251 for up to 1 h (BCC+AM251, bottom right), similar to MEA experimental design illustrated in A. Results are presented as change in fluorescence for each recording normalized to its minimum fluorescence (ΔF/F, dotted line represent minimum fluorescence). Scale bar represents 10 s (horizontal) and 10% change from minimum fluorescence (vertical). Ctrl data in panel are from the same set of data as in Extended Data Figure 5-1. F, Quantification of interevent intervals and area under curve for data shown in E. Acute AM251 application causes reduction of glutamatergic event amplitude in naive and downscaled cells but leads to increased frequency in downscaled and reduced frequency in naive cells. Results are presented as mean ± SEM from three to six independent culture preparations with four to eight wells in each culture. One-way ANOVA with Šidák’s multiple comparison test; *p ≤ 0.05, **p ≤ 0.01. ns = not significant.