To more fully elucidate the specificity of AM3506′s effects, we conducted a number of experiments in which 1.0 mg/kg AM3506 was systemically administered at different phases of conditioning, fear retrieval and extinction. We found that AM3506 given before conditioning did not affect fear or extinction the next day (Supplementary Figure S2b), or retrieval 10 days later (t-test: P>0.05, n = 8) (Figure 1d), indicating a lack of effect on fear acquisition and fear retrieval, as well as post-extinction fear. The latter negative effect is particularly interesting given that FAAH levels remain inhibited by ∼75% at the time of extinction training (that is, 1 day after pre-conditioning injection). This suggests that a certain threshold level of inhibition may be required at the time of extinction to enable long-term reductions in fear or, alternatively, that the timing of the injection, and the stress associated with it, may influence the efficacy of the drug's fear-reducing effects.52
