Fig 4. Cre-dependent retrograde monosynaptic rabies tracing reveals semiquantitative noncanonical CA3 inputs from vCA1, SUBv, Prh, and SUBtr.

(A) Schematic of retrograde monosynaptic rabies tracing from dCA3 subregions in Cre transgenic mice. Injections of rabies virus (RV-DsRed: EnvA-SADΔG-RV-DsRed, red) and AAV helper virus (AAV8-hSyn-DIO-TC66T-2A-eGFP-2A-OG, green) are administered into specific subregions of dCA3. From top to bottom, the schematics of CA3a, CA3b, and CA3c injection sites are shown. (B–D) Images of injection sites of the 3 CA3 subregions (B1 to B3 for CA3a, C1-C3 for CA3b and D1 to D3 for CA3c) and corresponding rabies virus–mediated labeling of presynaptic neurons in contralateral CA3 regions with DAPI staining (B4, C4, and D4). Injection sites are shown in white boxes along the transverse axis. The enlarged views of the white box regions are shown for CA3a (B2 and B3), CA3b (C2 and C3), and CA3c (D2 and D3). Images in B2, C2, and D2 show EGFP-labeled excitatory cells (green), while the images of B3, C3, and D3 allow for visualization of DsRed and EGFP double-labeled starter neurons (white arrowheads). The images of B4, C4, and D4 show contralateral CA3 inputs for the corresponding dCA3 subregions. The star indicates the border of CA3a and CA2 determined by PCP4 staining (S3 Fig). The boundaries of 3 CA3 subregions are indicated by white lines. The scale bar (400 μm) applies to B1, B4, C1, C4, D1, and D4. The scale bar (50 μm) applies to B2, B3, C2, C3, D2, and D3. (E) Illustration of noncanonical inputs to the CA3a subregion (E1 to E3). The boxed regions at vCA1 and Prh in the E1 left panel are shown in the middle and right panels, respectively, at a higher magnification. The cell labels in the SUBv and SUBtr are shown in E2 and E3. (F) and (G) are formatted similarly to E, to illustrate noncanonical inputs to CA3b and CA3c subregions, respectively, from vCA1, Prh, SUBv, and SUBtr. The scale bar (800 μm) applies to E1, F1, and G1. The scale bar (400 μm) applies to E2, F2, G2, E3, F3, and G3. The scale bar (100 μm) applies to all the magnified input regions. (H1) Semiquantitative analyses of input connection strengths measured by the CSI across Prh, vCA1, SUBv, and SUBtr following rabies tracing in CA3 subregions. vCA1 inputs are organized by the spatial location at the pyramidal layer (vCA1 py.) and oriens layer (vCA1 or.). n = 6 mice per CA3 subregion. Data are from 8 Camk2α-Cre; TVA mice and 10 Camk2α-Cre mice. All data are presented as mean ± SE; *, ** indicate the CSI statistical significance level of p ≤ 0.05 and p ≤ 0.01, respectively (Kruskal–Wallis test followed by Dunn comparison test). The results of the Kruskal–Wallis tests are indicated by the lines, and the results of Dunn tests are indicated by the brackets above the bars. See S2 and S3 Tables for more statistical information. (H2) Semiquantitative measurements of the PI for specific brain regions following the same format as in H1. The same abbreviations as in Fig 1. The raw data for Fig 4H1 and 4H2 are included in S2 Data. CSI, connection strength index; dCA3, dorsal CA3; DG, dentate gyrus; Prh, perirhinal cortex; SUBtr, subiculum transition area; SUBv, ventral subiculum; vCA1, ventral CA1.