Parallel pathways provide hippocampal spatial information to prefrontal cortex.

Long range synaptic connections define how information flows through neuronal networks. Here we combined retrograde and anterograde transsynaptic viruses to delineate areas that exert direct and indirect influence over the dorsal and ventral prefrontal cortex (PFC) of the rat (both sexes). Notably, retrograde tracing using pseudorabies virus (PRV) revealed that both dorsal and ventral areas of the PFC receive prominent disynaptic input from the dorsal CA3 (dCA3) region of the hippocampus. The PRV experiments also identified candidate anatomical relays for this disynaptic pathway, namely the ventral hippocampus, lateral septum, thalamus, amygdala, and basal forebrain. To determine the viability of each of these relays, we performed three additional experiments. In the first, we injected the retrograde monosynaptic tracer Fluoro-Gold (FG) into the PFC and the anterograde monosynaptic tracer Fluoro-Ruby (FR) into the dCA3 to confirm the first-order connecting areas, and revealed several potential relays regions between the PFC and dCA3. In the second, we combined PRV injection in the PFC with polysynaptic anterograde viral tracer (HSV-1) in the dCA3 to reveal co-labeled connecting neurons, which were evident only in the ventral hippocampus. In the third, we combined retrograde AAV injections in the PFC with an anterograde AAV in the dCA3 to reveal anatomical relay neurons in the ventral hippocampus and dorsal lateral septum. Together, these findings reveal parallel disynaptic pathways from the dCA3 to the PFC illuminating a new anatomical framework for understanding hippocampal-prefrontal interactions. We suggest that the representation of context and space may be a universal feature of prefrontal function. Significance statement: The known functions of the prefrontal cortex are shaped by input from multiple brain areas. We used transneuronal viral tracing to discover multiple prominent disynaptic pathways through which the dorsal hippocampus (specifically the dorsal CA3) has the potential to shape the actions of the prefrontal cortex. The demonstration of neuronal relays in the ventral hippocampus and lateral septum presents a new foundation for understanding long-range influences over prefrontal interactions, including the specific contribution of the dorsal CA3 to prefrontal function.