Fig. 2: Comparison of the efficiency of 4 retrograde vectors when combined with anterograde vector AAVDJ-DIO-ChR2-EYFP.

The EYFP expression in neurons double infected by anterograde AAVDJ-DIO-ChR2-EYFP and a retrograde vector was visualized with immunostaining with Ni-DAB. Each image is shown in a sagittal plane. a–d EYFP-immunopositive neurons were dominantly found in the VTA, though a few EYFP-immunopositive neurons were found along the trajectory of a glass pipet used for the injection of viral vector (arrows in b and d). e–h In the VTA, the cell bodies and neuropils of EYFP-immunopositive neurons were observed. i–l In the M1, EYFP-immunopositive axons were observed. Arrowhead in l indicates a layer 5 pyramidal neuron that exhibits EYFP immunoreactivity. m–t The EYFP-immunopositive axons in the cortical superficial layers (m–p) as well as in the deeper layers (q–t) of the cerebral cortex are shown in higher magnification images. The location of high magnification images (m–t) was taken from the black squares with corresponding letters in the lower magnification images (i–l). Scale bar = 1 mm in (d) (applies to a–c); 100 µm in (h) (applies to e–g); 500 µm in (l) (applies to i–k); 50 µm in (t) (applies to m–s).