Fig. 1: Mapping of ACC-ACC projection pathway.

a The procedure of the retrograde trans-monosynaptic rabies virus (RV) tracing strategy. b A schematic of the AAV helper and RV separately micro-injected into the right ACC. c Fluorescence image of the whole brain after 3D reconstruction. The horizontal view for one 3D-reconstructed whole brain (red: DsRed⁺; green: EGFP⁺). Inside the dotted white box is the ACC. Scale bar, 800 μm. d Fluorescence images of the brain slice containing ACC (dotted white box) after injection of AAV helper and RV into the right ACC. Box 1 (ipsilateral ACC) and 2 (contralateral ACC) were augmented in (e). Scale bar, 500 μm. e Representative images of EGFP⁺ and DsRed⁺ neurons in ipsilateral (upper) and contralateral ACC (bottom) with the unilateral ACC injection of AAV helper and RV. The white arrows point to the projection fibers from the contralateral ACC. Scale bar, 50 μm. f Cell numbers analysis of DsRed⁺ neurons at different layers of the contralateral ACC slices of the 30 μm thickness. g Schematic of left ACC injection of AAV2/9-CMV-Cre and right ACC injection of AAV2/R-EF1α-DIO-EYFP-EYFP in mice. h Fluorescence images of the brain slices from ACC-ACC sparsely labeled. The white box was augmented in Fig. 1i. Scale bar, 500 μm. i Representative image of labeled EYFP⁺ neurons in the ipsilateral ACC and EYFP⁺ projection fibers in the contralateral ACC. Scale bar, 200 μm. j A schematic of the left ACC injection of AAV-CaMKIIα-EGFP and right ACC injection of AAV-CaMKIIα-mCherry in mice. k Fluorescence images of the brain slices containing ACC after injection of AAV-CaMKIIα-EGFP and AAV-CaMKIIα-mCherry into ACC. White boxes were augmented in (l). Scale bar, 200 μm. l Representative images of labeled EGFP⁺ neurons and mCherry⁺ fibers in the left ACC (upper, box 4) and labeled mCherry⁺ neurons and EGFP⁺ fibers in the right ACC (bottom, box 5). Scale bar, 50 μm. Error bars represent SEM. ACC anterior cingulate cortex, Ispi ipsilateral, Contra contralateral.