The CA1, an important subregion of the hippocampus, is anatomically and functionally heterogeneous in the dorsal and ventral hippocampus. Here, to dissect the distinctions between the dorsal (dCA1) and ventral CA1 (vCA1) in anatomical connections, we systematically analyzed the direct inputs to dCA1 and vCA1 projection neurons (PNs) with the rabies virus-mediated retrograde trans-monosynaptic tracing system in Thy1-Cre mice. Our mapping results revealed that the input proportions and distributions of dCA1 and vCA1 PNs varied significantly. Inside the hippocampal region, dCA1 and vCA1 PNs shared the same upstream brain regions, but with distinctive distribution patterns along the rostrocaudal axis. The intrahippocampal inputs to the dCA1 and vCA1 exhibited opposite trends, decreasing and increasing gradually along the dorsoventral axis, respectively. For extrahippocampal inputs, dCA1 and vCA1 shared some monosynaptic projections from certain regions such as pallidum, striatum, hypothalamus, and thalamus. However, vCA1, not dCA1, received innervations from the subregions of olfactory areas and amygdala nuclei. Characterization of the direct input networks of dCA1 and vCA1 PNs may provide a structural basis to understand the differential functions of dCA1 and vCA1.

Despite significant progresses in dissecting neural circuits with recombinant adenoassociated viruses (rAAVs), compatible and efficient gene expressions of multiple vectors within the same cell still remains challenging. Here, we developed MAP-ENVIVIDERS, a recombinase system-dependent (including Cre-lox/Flp-FRT recombinase systems) viral copackaging strategy to ameliorate mutual suppression and enhance compatibility among different rAAVs to improve neurocircuit tracing.For neuron labeling with wild-type and Cre-driver line mice, MAP-ENVIVIDERS was ~5-fold and over 10-fold more sensitive than the mixture of independently packaged rAAVs, respectively, enabling us to identify new type of neurons and capture massive morphological details of individual neurons (for example, a cholinergic neuron with 9,500+ axonal branches). For trans-monosynaptic retrograde tracing with rabies virus-mediated systems, MAP-ENVIVIDERS achieved 10-to 70fold enhancement of tracing efficiency in 40% of all input brain regions compared with the previous strategies. Therefore, MAP-ENVIVIDERS provides powerful tools for elucidating morphological detail and organization of neurocircuits.