Time determines the neural circuit underlying associative fear learning.

Ultimately associative learning is a function of the temporal features and relationships between experienced stimuli. Nevertheless how time affects the neural circuit underlying this form of learning remains largely unknown. To address this issue, we used single-trial auditory trace fear conditioning and varied the length of the interval between tone and foot-shock. Through temporary inactivation of the amygdala, medial-prefrontal cortex (mPFC) and dorsal-hippocampus in rats, we tested the hypothesis that different temporal intervals between the tone and the shock influence the neuronal structures necessary for learning. Here, we show for the first time that the amygdala is critically involved in the acquisition of auditory fear learning when there is a temporal gap between the tone and the shock. Moreover, imposing a short interval (5 s) between the two stimuli also recruits the medial pre-frontal cortex (mPFC), while learning the association across a longer interval (40 s) becomes additionally dependent on a third structure, the dorsal-hippocampus. Thus, our results show that increasing the interval length between tone and shock leads to the requirement of an increasing number of brain areas for the association between the two stimuli to be acquired. These findings demonstrate that the temporal relationship between events is a key factor in determining the neuronal mechanisms underlying associative fear learning