Supplementary Figure 1: Experimental design to investigate the effect of cue-specific trauma to an F0 generation on subsequently conceived generations.

Novel experimental paradigm that uses olfactory fear conditioning to examine the structural and functional changes in the nervous systems of adult mice (F1 and F2) conceived after the F0 generation was trained to associate specific odorant presentations with mild foot-shocks. Briefly, F0 adult male mice were trained to associate Acetophenone or Propanol presentation with mild-footshocks (5 odor-shock pairings/session, 3 sessions, 1 session/day). Ten days after this conditioning, these F0 males were mated with naïve females. Ten days after the mating was setup, the F0 males were separated from the females. F1 offspring born were tested at 2-months of age. For studies of the F2 generation, F1 males that had no previous exposure to either Acetophenone or Propanol were mated with naïve females for 10 days, and resulting F2 offspring were used for analyses. Our experimental design minimized the possibility of a “social transmission” mode of information transfer. Specifically, the F0 male has absolutely no contact with the F1 offspring, is placed with the female 10 days after the last conditioning day, should not have any trace of the conditioned odor on his skin or hair to transfer to the mother, and is separated from the female after a 10 day period to minimize any in utero exposure of the pups to the conditioned male.