In trace fear conditioning, the prelimbic cortex exhibits persistent activity during the interval between the conditioned and unconditioned stimuli, which maintains a conditioned stimulus representation. Regions cooperating for this function or encoding the conditioned stimulus before the interval could send inputs to the prelimbic cortex, supporting learning. The basolateral amygdala has conditioned stimulus- and unconditioned stimulus-responsive neurons, convergently activated. The prelimbic cortex could directly project to the basolateral amygdala to associate the transient memory of the conditioned stimulus with the unconditioned stimulus. We investigated the neuronal circuit supporting temporal associations using contextual fear conditioning with a 5-s interval, in which 5 s separates the contextual conditioned stimulus from the unconditioned stimulus. Injecting retrobeads, we quantified c-Fos in prelimbic cortex- or basolateral amygdala-projecting neurons from 9 regions after contextual fear conditioning with a 5-s interval or contextual fear conditioning, in which the conditioned and unconditioned stimuli overlap. The contextual fear conditioning with a 5-s interval activated ventral CA1 and perirhinal cortex neurons projecting to the prelimbic cortex and prelimbic cortex neurons projecting to basolateral amygdala. Both fear conditioning activated ventral CA1 and lateral entorhinal cortex neurons projecting to basolateral amygdala and basolateral amygdala neurons projecting to prelimbic cortex. The perirhinal cortex → prelimbic cortex and ventral CA1 → prelimbic cortex connections are the first identified prelimbic cortex afferent projections participating in temporal associations. These results help to understand time-linked memories, a process required in episodic and working memories.
