Neural substrate of conditioned stimulus for associative learning in the hippocampus

Associative learning allows animals to learn the predictive relationships between events. Presentation of a conditioned stimulus (CS) preceding an unconditioned stimulus (US) within a short interval associates these events. However, little is known about the neural substrates that represent CSs. By combining in vivo calcium imaging with retrospective identification of dorsal hippocampal CA1 neurons, in which <i>c-fos</i> is induced during contextual fear conditioning in mice, we found that prospective <i>c-fos</i> (+) neurons were activated upon novel environmental exposure. Importantly, a part of them was transiently reactivated 1 to 2 s before the shock US as well as during memory retrieval, and these activities were correlated with memory expressions. Optogenetic silencing demonstrated that timing-dependent neuronal activity is crucial for memory formation. Our study identifies a cellular substrate for the CS that underlies the CS-US temporal contiguity rule under physiological conditions and suggests how the internal representation of context serves as a CS, demonstrating the role of prospective engram cells at the moment of conditioned learning.