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

The entorhinal-hippocampal system constitutes a pivotal neural circuit in the central nervous system. It is critically involved in processing spatial learning and memory. However, the specific neural interactions between entorhinal inputs and intra-hippocampal subcircuits that underlie spatial coding 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 Morris water maze (MWM) behavior to dissect how entorhinal-hippocampal afferents modulate hippocampal computations. Intriguingly, CA1-projecting CA3 neurons exhibited pronounced hyperactivity during early spatial learning, with activity gradually declining after sustained task performance. Chemogenetic inactivation of medial entorhinal-hippocampal afferents attenuated both neural responses of CA1-projecting CA3 neurons and the performance of spatial learning, hinting that medial entorhinal cortex (MEC) inputs to the hippocampus are essential for animals to execute spatial tasks precisely. By implementing dual-light theta-burst stimulation to co-activate ChrimsonR-expressing CA3-CA1 afferents and Chronos-expressing MEC-CA1 terminals, we observed robust heterosynaptic long-term potentiation in the dorsal CA1 region in vitro brain slice. This neuroplasticity was mediated synergistically by activating both NMDA receptors and voltage-gated calcium channels. Our findings establish that entorhinohippocampal afferents exert multilevel regulatory control over hippocampal function, thereby advancing mechanistic understanding of memory-related neurological pathologies.