Fig. 4.

Mapping the direct projection target regions of the OB with our monosynaptic anterograde tracing system (H129-dTK-tdT + helper AAV2/9-TK-GFP). A Timeline for mapping the direct OB output pathways using H129-dTK-tdT along with AAV2/9-TK-GFP helper. Immunostaining against tdTomato is required for robust detection of H129-labeled postsynaptic neurons. B Schema of the simplified OB projection pathways. AAV2/9-TK-GFP, 1.0 × 10¹² vg/mL, 450 nL, H129-dTK-tdT, 5.0 × 10⁸ pfu/mL, 400 nL, perfusion time: day 32. C Representative image of the virus injection site in the OB. The boxed areas are presented in the right panels at a higher magnification. The starter neurons labeled by both GFP and tdTomato are indicated with white arrows. D–F Representative tracing images of the regions innervated by the OB. To visualize the labeled post-synaptic neuron, a brain slice was immunostained with tdTomato antibody. The boxed areas are presented in the right panels at a higher magnification. Coronal brain slices were counterstained with DAPI, and tdTomato signals were amplified by immunostaining with the anti-DsRed polyclonal antibody and Alexa Fluor 594-conjugated goat anti-rabbit IgG (H+L). LEnt, lateral entorhinal cortex.