a) Simplified diagram of the constitutively active Rheb (Rheb CA) upstream of mTORC1. ( b) Diagram of the electrode placement around an E15±0.5 fetal brain (left). Photograph of a P0 mouse head with GFP fluorescence in the medial prefrontal cortex (mPFC) following electroporation at E15 (right). The ellipse highlights the location of the somatosensory cortex (SSC). ( c) Confocal images of pS6 immunostaining (red) and GFP and tdTomato fluorescence (green) in coronal sections from 2-month-old mice containing neurons electroporated with either Rheb CA+tdTomato CAG or GFP CAG alone in the mPFC. Scale bar, 40 μm. ( d) Bar graphs of total pS6 immunoreactivity per cell (FU: arbitrary fluorescence unit, N=4 per condition) from data shown in ( c). ** P<0.01 (Student’s t-test). ( e– g) Images of coronal sections (top) containing neurons electroporated with control plasmid in the mPFC ( e), with Rheb CA in the mPFC ( f) or in the SSC ( g), and corresponding representative examples of EEG (black) and EMG (grey) traces (bottom). Scale bar, 1 mm. ( h) Expanded time window (10 s) view of events found in the EEG trace shown in f, numbered correspondingly to the blue boxes. ( i) Bar graphs show the number (#) of seizures/day and seizure duration in animals with seizures ( N=8) from the mPFC Rheb CA-expressing cohort. Control animals ( N=9) electroporated with a control plasmid did not exhibit any seizures. * P<0.05 (Student’s t-test). ( j) Three-dimensional reconstruction of mouse brains containing neurons electroporated with either Rheb CA+tdTomato CAG or a control plasmid GFP CAG, both pseudocolored green to ease visualization. ACC, anterior cingulate cortex; CC, corpus callosum; MC, motor cortex. Error bars, s.e.m.