Supplementary Figure 1 Distribution of starter cells for RV-mediated retrograde tracing. Parcellation of cortical areas is based on Allen Mouse Brain Atlas and drawn to scale. Thick white curves, outlines of VIS, SS, AUD, ACA and MO. Thin gray curves, outlines of primary visual cortex (VISp), barrel field of somatosensory cortex (SSbfd) and secondary motor cortex (MOs). Color scale, normalized starter cell density. Scale bar, 1 mm.
Supplementary Figure 2 Laminar distribution of starter cells for RV-mediated retrograde tracing from VIS, SS and AUD.
Supplementary Figure 3 Whole-brain distributions of inputs to VIS, SS and AUD. (a) Viral vectors and injection procedure for RV-mediated trans-synaptic retrograde tracing. (b) Fluorescence images of LGd, VP and MG (yellow box in coronal diagram), showing RV-labeled input neurons (red) to VIS, SS and AUD, respectively (scale bar, 100 µm). Red, tdtomato; blue, DAPI. (c-e) RV-labeled neurons detected in all samples of each group (VIS, blue; SS, red; AUD, green; scale bar, 1 mm). White masks indicate injection sites excluded from data analysis. (f) Percentages of retrogradely labeled input neurons in selected cortical and subcortical brain regions (VIS, n = 3 mice; SS, n = 3; AUD, n = 3). Included are cortical areas with > 2% labeling and thalamic structures with > 1% labeling. Each circle represents one mouse. Error bar, ± s.e.m.
Supplementary Figure 4 Frontal cortical inputs to VIS, SS and AUD.
Supplementary Figure 5 Whole-brain distributions of axonal projections from VIS, SS and AUD. (a) Viral vector and injection procedure for tracing axonal projections. (b) Fluorescence images of LGd/LGv, VP and MG (yellow box in coronal diagram), showing the axons (red) from VIS, SS and AUD, respectively (scale bar, 200 µm). Red, mcherry; blue, DAPI. (c-e) Axons detected in all samples of each group (VIS, blue; SS, red; AUD, green; scale bar, 1 mm). White masks indicate injection sites excluded from data analysis. (f) Percentages of labeled axons in cortical and subcortical brain structures (VIS, n = 3 mice; SS, n = 3; AUD, n = 3). Included are cortical areas with > 1% labeling and thalamic structures with > 0.8% labeling. Each circle represents one mouse. Error bar, ±s.e.m.
Supplementary Figure 6 Example experiments showing reciprocal connections between the frontal cortices and the sensory cortices. (a) Fluorescence images of VIS, SS and AUD (red box in coronal diagram), showing the axons from ACA or MO (scale bar, 200 µm). Red, mcherry; blue, DAPI. (b) Right, fluorescence images of VIS, SS and AUD (red box in coronal diagram), showing RV-labeled input neurons (red) to ACA or MO (scale bar, 200 µm). Inset, enlarged view of the region in white box (scale bar, 10 µm). Red, tdtomato; blue, DAPI.
Supplementary Figure 7 ACA innervates Pvalb +, Sst + and Vip + interneurons and glutamatergic neurons (Camk2α + ) in VIS, PTLp and RSP.
(a) Viral vectors and injection procedure for RV-mediated transsynaptic retrograde tracing. (b) Upper panel, viral injection sites in VIS (red box in coronal diagram) of Pvalb-Cre, Sst-Cre and Vip-Cre mice (scale bar, 200 µm). Inset, enlarged view of the region in white box showing starter cells (yellow; scale bar, 20 µm). Lower panel, fluorescence images of ACA and MO (red box in coronal diagram) showing RV-labeled input neurons (green) to each subtype of interneurons in VIS (scale bar, 200 µm). Green, EGFP; red, mcherry; blue, DAPI. (c) Similar to (b), for ACA inputs to PV+, SOM+, VIP+ interneurons and glutamatergic neurons in PTLp. (d) Similar to (b), for ACA inputs to Pvalb+, Sst+, Vip+ interneurons and glutamatergic neurons in RSP. (e) Percentages of input neurons in ACA retrogradely labeled from VIS, PTLp and RSP in Camk2α-Cre (yellow), Pvalb-Cre (blue), Sst-Cre (red) and Vip-Cre mice (green). n = 3 mice in each group. Each circle represents one mouse. Error bar, ± s.e.m.
Supplementary Figure 8 ACA interneurons (Pval +, Sst + and Vip + ) are innervated by VIS, PTLp and RSP.
(a) Left, viral injection sites in ACA (red box in coronal diagram) of Pvalb-Cre, Sst-Cre and Vip-Cre mice (scale bar, 200 µm). Inset, enlarged view of the region in white box showing starter cells (yellow; scale bar, 20 µm). Right, fluorescence images of VIS, PTLp and RSP (red box in coronal diagram) showing RV-labeled input neurons (green) to each subtype of interneurons in ACA (scale bar, 200 µm). Green, EGFP; red, mcherry; blue, DAPI. (b) Percentages of retrogradely labeled input neurons in selected cortical and subcortical brain regions (n = 3 mice in each group). Each circle represents one mouse. Included are the same structures as in Fig. 2h.
Supplementary Figure 9 Whole-brain distributions of axonal projections from MO SS neurons. (a) Viral vectors and injection procedure for tracing the axonal projections from MO SS neurons. (b) Left, bright field image of ACA and MO showing RV-labeled neurons from SS (scale bar, 200 µm). Inset, enlarged view of the region in red box (scale bar, 20 µm). Immunostaining for tdtomato was performed to convert the fluorescence signal (tdtomato expressed by RV) into nickel-enhanced DAB signal; Middle, image of detected axon signals in SS (scale bar, 200 µm); right, bright field image of the region in green box, showing RV-labeled axons of MO SS neurons (scale bar, 100 µm). (c) Axons detected in all MO SS samples (scale bar, 1 mm). White masks indicate injection sites excluded from data analysis. (d) Percentages of labeled axons in selected cortical and subcortical brain structures (n = 3 mice). Each circle represents one mouse. Error bar, ±s.e.m. Data for MO axons are the same as in Figure 2D, shown here to facilitate comparison.
Supplementary Figure 10 ACA VIS and ACA SC neurons form synapses in the identified cortical and subcortical areas. (a) Viral vectors and injection procedure for labeling the presynaptic sites of ACA VIS and ACA SC neurons. (b) Upper panel, fluorescence images of VIS, PTLp, RSP and STR (red box in coronal diagram) showing axons (green) and putative presynaptic sites (red) from ACA VIS neurons. Lower panel, similar to upper panel, showing axons (green) and putative presynaptic sites (red) from ACA SC neurons in RSP, PrL/ILA, LP/LD, STR and SC. Green, mgfp; red, mruby. Scale bar, 20 µm.
Supplementary Figure 11 WGA-Cre-induced EYFP expression in ACA VIS and ACA SC neurons. (a) Viral vectors and injection procedure for retrograde labeling of ACA VIS and ACA SC neurons with EYFP. (b) Left, fluorescence images of AAV-EF1α-mCherry-IRES-WGA-Cre injection site (red) in VIS (upper panel) and SC (lower panel). Right, WGA-Cre-induced EYFP expression (green) in ACA. Note that mcherry fluorescence is located in neuronal somatas at the injection site (left), but not in the ACA (right). White arrowheads, neurons expressing EYFP. Blue, DAPI.
Supplementary Figure 12 WGA-Cre-labeled ACA VIS and ACA SC neurons (EYFP + ) largely overlapped with Retrobead-labeled ACA VIS and ACA SC neurons, respectively. (a) Viral vectors and retrobead injection procedure for retrograde labeling of ACA VIS and ACA SC neurons with EYFP (mediated by WGA-Cre) or red retrobeads. (b) Left, fluorescence images of AAV-EF1α-mCherry-IRES-WGA-Cre and red retrobeads injection site (red) in VIS (upper panel) and SC (lower panel). Middle, retrobeads (red) and WGA-Cre-induced EYFP expression (green) in ACA. Right, enlarged view of the region in white box. White arrowheads, neurons containing both EYFP and retrobeads. Blue, DAPI. Note that most EYFP+ neurons in ACA were also labeled by red retrobeads (VIS injection, 83 ± 4% (s.e.m.), n = 3 mice; SC injection, 80 ± 4%, n = 3 mice).
Supplementary Figure 13 WGA-Cre-labeled ACA VIS and ACA SC neurons (EYFP + ) showed no overlap with Retrobead-labeled ACA SC and ACA VIS neurons, respectively. (a) Viral vectors and retrobead injection procedure for retrograde labeling of ACA VIS and ACA SC neurons with EYFP (mediated by WGA-Cre) or red retrobeads. (b) Upper panel: Left, fluorescence image of AAV-EF1α-mCherry-IRES-WGA-Cre injection site (red) in VIS. Middle, red retrobead injection site in SC. Right, retrobeads (red) and WGA-Cre-induced EYFP expression (green) in ACA. Enlarged view is from the region in white box. Lower panel, similar to upper panel, but the AAV was injected in SC and red retrobeads were injected in VIS. Green arrowheads, neurons expressing EYFP. Red arrowheads, neurons containing red retrobeads. Blue, DAPI. Note that the EYFP+ neurons in ACA were not labeled by red retrobeads (0%, n = 3 mice in each group).
Supplementary Figure 14 Control experiments for WGA-Cre-mediated trans-synaptic tracing in ACA. (a) Viral vectors and injection procedure for negative control. AAV expressing WGA-Cre was injected into VIS or SC of wild-type mice and AAV with Cre-dependent expression of TVA receptors was injected into the ACA, followed by injection of RV expressing EGFP into ACA. Note that AAV with Cre-dependent expression of rabies glycoprotein was omitted in this control experiment. (b) Left, fluorescence images of AAV-EF1α-mCherry-IRES-WGA-Cre injection site (red) in VIS (upper panel) and SC (lower panel). Middle, injection site of other AAV and RV in ACA containing AAV/RV infected neurons (green). Right, without rabies glycoprotein, no RV-labeled neurons were found in frontal cortical regions such as PL/ILA. Note that due to the leaky TVA expression (Cre-independent) 19,21, the RV labeled neurons were not restricted in the layer 5 of ACA in the group with WGA-Cre expression in SC. Since leaky expression of rabies glycoprotein is unlikely sufficient to reconstitute infectious RV particles, this does not compromise long-distance tracing. (c) Schematic of the whole-brain distribution of RV-labeled neurons without rabies glycoprotein. RV-labeled neurons were located in the regions within 700 mm anterior and posterior to the center of injection site in ACA (Bregma +0.3 mm), indicated by green shading.
Supplementary Figure 15 Whole-brain distributions of inputs to MO SS neurons. (a) Viral vectors and injection procedure for RV-mediated transsynaptic retrograde tracing from MO SS neurons. (b) Left, fluorescence images of AAV-EF1α-mCherry-IRES-WGA-Cre injection site in SS (scale bar, 1 mm). Middle, injection site of other AAVs and RV in MO (scale bar, 1 mm). Inset, enlarged view of the region in white box showing AAV/RV infected neurons (green, scale bar, 20 µm). Right, retrogradely labeled neurons (green) in SS (scale bar, 200 µm). Inset, enlarged view of the region in white box (scale bar, 20 µm). Green, EGFP; red, mcherry; blue, DAPI. (c) RV-labeled neurons detected in all MO SS samples. White masks indicated the injection sites excluded from data analysis (scale bar, 1 mm). (d) Percentages of retrogradely labeled neurons in selected cortical and subcortical brain structures (MO SS, n = 4 mice). Each circle represents one mouse. Error bar, ±s.e.m. Data for MO inputs are the same as in Figure 3D.