Fig. 4. Strategy for selective deletions of both Tenm3 and Tenm4 from the MEC or CA1 in Tenm3/4 double cKO mice to achieve exclusively pre- or postsynaptic ablation of Tenm3 and Tenm4 expression.

a Design of AAVs used for Tenm3/4 deletions. The synapsin promoter drives bicistronic expression of Cre or ∆Cre (control) and soluble tdTomato, enabling visualization of infected neurons and their axonal projections. b Experimental approach. Tenm3/4 DcKO mice were stereotactically infected with AAVs at P0, and analyzed at P13, P21, or P35. c Representative image of a horizontal section of the hippocampal formation of a control mouse at P13 whose MEC was infected with ΔCre- and tdTomato-expressing AAVs at P0 (asterisk = infected area). Note that MEC neurons project abundant tdTomato-positive axons to the S. lacunosum-moleculare of the proximal CA1 (p-CA1) and the molecular layer of distal subiculum (d-sub), but not to the distal CA1 (d-CA1) or proximal subiculum (p-sub). Dotted boxes indicate areas that were quantitatively analyzed in Fig. 5a–c, Supplementary Figs. 4, 6. d Same as c, except that the CA1 region was infected stereotactically with ΔCre- and tdTomato-expressing AAVs. Note that the CA1 region neurons, as visualized in horizontal sections, send sparse axons to distal subiculum. Dotted boxes indicate areas that were quantitatively analyzed in Fig. 5g–i, Supplementary Figs. 5, 7. Scale bars: 0.5 mm (c, d).