Distinct developmental trajectories of externally and internally generated hippocampal sequences and assemblies

The hippocampus generates cognitive maps through two distinct mechanisms: external representations anchored to environmental landmarks and internal sequences integrating self-referenced information. In adult CA1, these functions segregate radially, with deep cells processing external cues and superficial cells handling internal representations. This anatomical organization suggests that developmental processes constrain the formation of these functionally distinct circuits. However, the developmental timeline for the emergence of these two types of hippocampal representations remains unknown. Using longitudinal two-photon calcium imaging in head-fixed mice during the fourth postnatal week, we investigated when internal vs. external hippocampal representations emerge. We recorded from deep and superficial CA1 layers while mice ran on treadmills that were bare or enriched with tactile cues, addressing the challenge of tracking cells across sessions. Internal sequences emerged during the mid-fourth postnatal week, following the development of cue-based representations. These sequences initially encoded elapsed time before evolving to integrate run distance, arising after rest cell assemblies, as spatial coding and functional connectivity networks stabilized. These findings identify the fourth postnatal week as a critical developmental period marking the establishment of a balanced integration between external landmark-based and internal self-referenced spatial representations. This transition period may constitute a vulnerable developmental window during which disruptions could predispose to disorders characterized by altered cognitive maps.