Figure 1: Salient Beginning Safety Conditioning is the most effective safety training for in context fear inhibition.

(a) Timeline of behavioral testing. Mice were first habituated to the conditioning context for 2 days (Days 1–2), and then either fear or safety conditioned (5 trials per day, Days 3–4). Fear conditioning consisted of CS1 (50 ms long 4kHz tone-pips, once a second, lasting 30 sec) co-terminating with a shock US (0.6mA, 1 s). Safety conditioning consisted of CS1 presentations that were explicitly unpaired with the US (mean interval between US and all proximal CSs, 49 sec; range, 40 to 60 sec). The Salient Beginning Safety Conditioning was structured in the same way as the Safety conditioning, the only difference being a CS2 (houselight) that was co-presented for 1 sec at the beginning of CS1. The Salient End Safety Conditioning group was presented with CS2 co-terminating with the end of CS1 for 1 sec. The next day, mice were tested for in-context fear retrieval (Day 5), and on Day 6, mice were placed in the Open Field to test for the effects of fear or safety conditioning in Phase I on exploration of a novel anxiogenic context. After a 3 day break (Day 7–9), mice underwent differential fear conditioning for 3 days (12 trials per day, Days 10–12), and then were placed in a new context for fear discrimination retrieval (Day 13). (b) Defensive freezing for all groups during conditioning (Day 3). (c) In-context retrieval of fear and safety learning. Pre-tone freezing for all groups in the 30s preceding the first tone and during the 30s of the first tone. Post-hoc contrasts: Fear Conditioned group pre-tone vs. tone, p < 0.0001, Salient Beginning safety pre-tone vs tone, p = 0.0002, Salient End safety group pre-tone vs. tone, = 45.21±7.03 %, tone = 74.78±5.65%, p = 0.057. (d) In-context retrieval behavior across all trials. Left, defensive freezing for all groups during the 30s pre-tone period prior to each tone. Note that the data for Trial 1 refers to a pre-cue (contextual) recall period, whereas data for all other trials refers to the ITI periods once cue retrieval began. Post-hoc contrasts: Less pre-tone 1 (contextual) freezing in the Fear Conditioned than in Safety group (p < 0.0001), less pre-tone 1 freezing in Fear Conditioned group than the Salient Beginning safety group (p = 0.041). Right, defensive freezing for all groups on each trial. Post-hoc contrasts: Salient Beginning safety group froze significantly less on trials 1 (p = 0.0086), and 4 (p = 0.04). ***, p<0.0001; **, p<0.001, *p<0.05.