Extended Data Fig. 3: Identification of anatomical projections to the ADn.

a, WT mice were micro-infused with Retrobeads in the ADn, and b, neurons in anterior cingulate cortex (ACC), retrosplenial cortex (RSP), and presubiculum (PRE) were retrogradely labeled (n = 3). Examples from additional mice are included. c, WT mice were micro-infused with the retrograde tracer cholera toxin subunit B (CTB) in the ADn, and d, neurons in retrosplenial cortex (RSP), and presubiculum (PRE) were retrogradely labeled (n = 3). Additional examples of ADn infusion site and retrogradely labeled CA3 are also shown. e, WT mice were micro-infused with the anterograde tracer PHA-L in the CA3, and f, in addition to ipsilateral ADn (Fig. 2f), ipsilateral CA1, contralateral CA1 and CA3 were anterogradely labeled, while much less labeling was observed in contralateral ADn (n = 4). An additional example of anterograde labeling in ADn is shown. g-h, To visualize specifically CA3-ADn projections, WT mice were micro-infused with AAV-DIO-mCherry bilaterally in CA3 and AAVrg-Cre-EGFP unilaterally in ADn (n = 4). Majority of the CA3-ADn (mCherry+) cells were visualized in CA3 ipsilateral to the site of ADn infusion. To see whether anatomical projections from other regions to ADn are inhibitory, different VGAT-Cre mice were micro-infused with AAV-DIO-EYFP in i-j, retrosplenial cortex (RSP), k-l, anterior cingulate cortex (ACC), or m-n, presubiculum (PRE) (n = 3 for each region). Unlike the CA3 (Fig. 3c,d), none of these regions showed terminal projections in the ADn (blue scale bars: 100 μm, white scale bars: 50 μm).