The perforant pathway and CA3-Schaffer collateral afferents coordinate to regulate spatial navigation

The entorhinal-hippocampal system constitutes a pivotal neural circuit in the central nervous system, critically involved in processing spatial learning and navigation-associated memory. However, the specific neural interactions between entorhinal inputs and intra-hippocampal subcircuits that underlie navigation learning remain elusive. To address this gap, we integrated multimodal approaches including in vivo calcium imaging, dual-color optogenetic manipulation, chemogenetic intervention, electrophysiological recordings, immunohistochemistry, and navigation-based behavioral assays to dissect how entorhinal-hippocampal afferents modulate hippocampal computations. Intriguingly, Schaffer collateral projections from CA3 to CA1 neurons (CA3-SC afferents) demonstrated sustained hyperactivity during navigation learning. Pharmacogenetic suppression of medial entorhinal-hippocampal afferents via the perforant pathway (PP) attenuated both CA3-SC neural responses and navigation memory performance. By implementing dual-color theta-burst stimulation (DL-TBS) to co-activate ChrimsonR-expressing CA3-SC afferents and Chronos-expressing PP terminals, we observed robust heterosynaptic long-term potentiation (hetero-LTP) in the dorsal CA1 region. This LTP induction was mediated synergistically by NMDA receptor activation and voltage-gated calcium channel signaling. Importantly, PP afferent activity was found to be indispensable for both task execution and memory consolidation during navigation. Our findings establish that PP-mediated entorhinal inputs exert multilevel regulatory control over hippocampal function, spanning from synaptic plasticity to behavioral output, thereby advancing mechanistic understanding of memory-related neurological pathologies.