Hippocampal CA1 neuronal ensembles efficiently discriminate routes during foraging- and homing-like task

In natural conditions, rodents continuously explore their environment and form spatial representations that enable them to navigate and locate relevant sites. Additionally, rodents rely on a route-based navigation strategy, consisting of well-defined paths that enhance efficiency and confer survival advantages. However, the contribution of the hippocampus to route-based navigation during continuous exploration—such as searching for resources and returning home—remains largely unexplored, particularly regarding its onset and the mechanisms of route discrimination. Here, we address this gap by investigating when and how hippocampal CA1 activity differentiates between overlapping trajectories in a continuously explored environment. Using calcium imaging in freely moving mice, we tracked neural activity as animals self-initiated routes in search of water. Our findings show that CA1 neurons rapidly establish an accurate global spatial map upon second exposure to the task. With increasing familiarity, route-specific representations progressively emerge and are maintained over the long term. In parallel with the refinement of these spatial representations, hippocampal involvement in route-choice prediction also develops with experience. Finally, our study provides insight into the neural basis of spatially flexible, ethologically relevant navigational strategies.