Supplementary Figure 3: dVenus expression after behavioral exploration of Arc-dVenus mice.

(a) Schematic illustration of experimental procedures. SWs were monitored in hippocampal slices prepared from Arc-dVenus transgenic mice that had been allowed to explore a novel enriched environment for 30 min. (b) Representative control images of dVenus fluorescence (green) and Hoechst counterstain (blue) in control naïve mice (Home cage), animals that explored the novel enriched environment A for 30 min (30 min in A), animals that explored the novel but less-enriched environment B (30 min in B), and animals that sequentially explored environments A and B for 15 min each (15 min in A/15 min in B). Scale bar = 20 μm. (c) Percentages of dVenus(+) neurons to the total neurons were compared in home-cage mice (n = 3 mice), animals that explored environment A for 30 min (n = 4 mice), animals that explored environment B (30 min in B, n = 4 mice), and animals that explored sequentially explored A and B for 15 min each (15 min in A/15 min in B, n = 3 mice). Three 200-μm-thick sections were prepared per mouse from a middle part (2,600 μm ventral from bregma) of the hippocampus 5 h after the exploration, and 21 confocal sections were taken at a Z interval of 1 μm. Cells were considered dVenus(+) when the mean fluorescence intensity in the soma (ROI Φ = 5 μm) was higher than the background fluorescence intensity by twice its SD. The background intensities were measured from 50 ROIs (Φ = 5 μm) selected randomly from cell body-absent regions in the stratum radiatum in the same histological sections, and their mean and SD were used to detect dVenus(+) neurons. Neurons were identified by their nuclear morphology. Error bars represent the SEM. *P = 0.011, Q = 4.04 versus Home cage; **P = 0.0052, Q = 4.50 versus Home cage; Tukey's multiple comparison test after one-way ANOVA.