Supplementary Figure 1: RVΔGL-Cre can be used in combination with Cre-dependent AAV for intersectional targeting of projection neurons in mice.

(a) RVΔGL-Cre injection site from a paired injection experiment in an Ai75 (Cre-dependent nuclear tdTomato) mouse. Center of injection site is enlarged in panel b. RVΔGL-Cre was injected into the retrosplenial cortex (RSP) so that cells across the brain which provide input to RSP were labeled via Cre-induced nuclear tdTomato expression (magenta). (c) AAV-FLEX-EGFP was injected into anterior cingulate cortex (ACA) so cells that were co-infected with RVΔGL-Cre also express EGFP. Several EGFP+ cell bodies in cortical layers 2/3 and 5 are visible in this section, as well as axonal projections in contralateral ACA and the striatum (STR). EGFP-labeled projections from ACA can be seen in the lateral dorsal (LD) and central lateral (CL) thalamic nuclei while cells retrogradely labeled from RSP are present only in LD (a). Axonal projections from ACA neurons are visible in the RVΔGL-Cre injection site (arrowheads in b indicate some axons). (d) Segmented pixels from this experiment projected onto a cortical surface view of the Allen Institute Common Coordinate Framework show the extent and ___location of cortical projections labeled with this paired injection strategy. The approximate locations of the AAV-FLEX-EGFP and RVΔGL-Cre injections are indicated, as well as the rostral-caudal ___location of the coronal sections in panels (a) and (c). Images were acquired by serial 2-photon tomography. Scale bars: a, c = 1 mm; b = 500 μm. This experiment was repeated with similar results for 8 different pairs of injection coordinates (n = 2 for the specific ACA-RSP coordinates shown) in a total of 15 mice.