Overexpression of caspase-3-resistant triple mutant of Akt1 (D108A/D119A/D462N) blocks LTD in CA1 neurons of organotypic hippocampal slice cultures. (A) Cleavage of wildtype or mutant Akt1 proteins by caspase-3 in vitro. In vitro translated WT Akt1, D108A/D119A/D462N triple mutant or Quadruple mutant were incubated with increasing amounts of recombinant active caspase-3 (10, 20, and 40 ng) at 37°C for 18 h. The caspase-3 inhibitor DEVD-fmk was either omitted or included in the reactions containing 40 ng caspase-3. Position of full-length Akt1 is indicated. (B) Pairwise analysis of wildtype Akt1 (Akt1) transfected cells and nearby untransfected cells on basal AMPA receptor-mediated EPSCs (left panel, EPSCAMPA, recorded at a holding potential of −70mV) and NMDA receptor-mediated EPSCs (right panel, EPSCNMDA, recorded at +40mV). Red symbols show mean ± SEM. (C) Overexpression of WT Akt1 had no effect on LTD compared to neighboring untransfected cells (p<0.01, n=6). (D) Pairwise analysis of Akt1triple mutant (D108A/D119A/D462N)-transfected cells and nearby untransfected cells on basal AMPA receptor-mediated EPSCs and NMDA receptor-mediated EPSCs. (E) Induction of LTD was blocked in Akt1 triple mutant transfected cells (p>0.05, n=6) but LTD was intact in neighboring untransfected cells. (F) LTP was unaffected by overexpression of Akt1 triple mutant. Graphs show mean ± SEM. See also Figure S6.