Figure 2
CB1 antagonist SR141716A (SR) increases excitatory synaptogenesis in a cortical spheroid model of human brain development. (a) Work flow of synapse analysis. (b) Representative confocal images of cortical spheroids stained with excitatory presynaptic marker VGLUT1 and excitatory postsynaptic marker PSD95. (c) Representative confocal images of cortical spheroids stained with inhibitory presynaptic marker VGAT and inhibitory postsynaptic marker gephyrin. All cortical spheroids endogenously express GFP-linked β-actin (ACTIN-GFP). This was used as the internal control for synaptic area measurements. (d) Excitatory presynaptic (VGLUT1) and postsynaptic (PSD95) area after SR treatment. Both high and low doses of SR increased VGLUT1 area compared to vehicle control (0 vs 30 and 0 vs 300: p = 0.0001). SR increased PSD95 area in a dose dependent manner (30 vs 300: p = 0.0001). (e) Box plot of inhibitory presynaptic (VGAT) and postsynaptic (gephyrin) area after SR treatment. The low dose but not the high dose of SR increased VGAT area (0 vs 30: p = 0.0035). (f) SR increases excitatory synapse area in a dose dependent manner (30 vs 300: p =  < 0.0001). SR also increased inhibitory synapse area when compared to control (0 vs 30: p = 0.0121, 0 vs 300: p = 0.0301) (g) SR increases the number of excitatory synapses in a dose dependent manner (30 vs 300: p = 0.0030) and increases the number of inhibitory synapses at the low dose when compared to vehicle control (0 vs 30: p = 0.0011). (h) SR increases the area of CB1-positive excitatory synapses at the low dose when compared to vehicle control (0 vs 30: p = 0.0001) but has no effect on the ratio of CB1-positive inhibitory synapses. (i) Cumulative distribution plot of individual excitatory synapse size after treatment with SR. Analyzed by Kolmogorov–Smirnov tests. Data from graphs d-h was analyzed by ordinary one-way ANOVA with multiple comparisons. Data represented as mean ± SEM. n = 90 areas analyzed per dose group. Significance (*) defined as p < 0.05.
