TRACE BUT NOT DELAY FEAR MEMORY IN MICE IS SUSTAINED BY PERSISTENT HIPPOCAMPAL NEURONAL ACTIVITY

The neural mechanisms supporting temporal associations across delays remain poorly understood. We investigated whether persistent hippocampal activity underlies trace fear conditioning, where a conditioned stimulus (CS) predicts an aversive unconditioned stimulus (US) after a stimulus-free interval. Using and miniscope calcium imaging in freely behaving mice, we recorded dorsal hippocampal CA1 activity during trace conditioning with 20-s or 60-s trace intervals. Mice successfully acquired fear memory with a 20-s trace interval but failed at 60 s, despite intact delay conditioning. Hippocampal neurons exhibited CS-evoked activity that persisted beyond stimulus offset; however, this sustained activity decayed rapidly over time. At 20 s post-CS offset, about 10% of initially responsive neurons remained active, whereas at 60 s only 0.4% maintained elevated activity. The sharp decline in persistent activity between 20 and 60 s directly paralleled the behavioral deficit in trace memory formation. These findings demonstrate that successful trace conditioning depends on maintained hippocampal ensemble activity bridging the CS-US interval, supporting a working memory model wherein transient neural persistence encodes recent events for associative learning. When this activity decays below a critical threshold, temporal associations cannot form, explaining the failure of long-interval trace conditioning. Our results establish persistent hippocampal firing as a physiological substrate for bridging temporal gaps in episodic memory. This work was supported by RSF grant #23-78-00010 and Non-Commercial Foundation for Support of Science and Education “INTELLECT”.