Widespread and bilaterally symmetrical cortical projections to VISp, SSp-bfd and MOp.

(A) Retrograde nuclear retro-AAV was injected across all layers of the primary visual cortex (VISp), somatosensory barrel field (SSp-bfd) and the motor cortex (MOp) of mice. The locations of the centroids on the injection bolus is shown on the CCFv3 cortical flat map. Injections performed in the right hemisphere injection (n = 11) are flipped to their corresponding location in the left hemisphere. (B) Two photon images showing examples of injection sites and labeled nuclei of retro-AAV labeled cells. Visualization of detected projection neurons for three individual mice warped into the 3D rendered space of the two hemispheres. (C) Plot showing the fraction of cortical cells per hemisphere detected for each mouse and pooled according to each injection site (mean ± sem). Asterisks indicate statistical significance (p < 0.05). (D) Bar graphs indicating the mean number (± sem) of cortical areas in which labeled neurons were observed. Text indicates the range of areas. (E) Flat maps indicating the relative cell counts normalized for each hemisphere excluding both the injection target (X) area its homotopic counterpart (#). Right, Bar graphs indicating the fraction of cells in the contralateral hemisphere located in the homotopic versus all other cortical areas (mean ± sem). Note that areas ILA (5), ENTl (44) and ENTm (45) are not represented in the cortical flat map on the left (see also Fig.S1C). (F) Bar graphs showing the average correlation (R2 ± sem) of the ipsi-versus contra-lateral fractional count superimposed on the individual R2 values for each experiment. (G) Plot of the ipsi-versus contra-lateral fractional count for each of the three target areas. Each circle represents the average of six fractional count values (± sem) for a given area. Open circles indicate instances where the difference of the ipsilateral values are not significant from the contralateral values. Black circles indicate a significant contralateral bias while gray circles indicate an ipsilateral bias. Dashed line indicates the unity line.

Modular organization of the intra and interhemispheric projection onto VISp, SSp-bfd and Mop

(A) Schematic of the cortical flat map highlighting the six cortical modules and their corresponding areas. (B) Bar graphs of the normalized fractional cell counts for each of the six modules for ipsilateral and contralateral hemispheres ranked by decreasing ipsilateral weights. For the contralateral hemisphere the homotopic target region is not included. (C) Rank plot based on the relative weights of connectivity onto the target areas VISp, SSp-bfd and MOp for all cortical brain areas. The size of the circle indicates relative fractional count on a logarithmic scale. Corresponding areas on the ipsilateral and contralateral hemisphere are connected with a line.

Layer 6 is a main source of input to VISp, SSp-bfd and MOp.

(A1) A segmented image of the cortex used to assign the laminar position of detected neurons. Image of segmented Enthorinal (ENT), Ectorhinal (ECT), and lateral visual area (VISl) on the contralateral hemisphere showing local dominance by L2/3 (A2), L5 (A3) and L6a (A4), respectively. (B) Bar plot showing average fraction of cells (± sem, n = 6 mice) in L2/3, L4, L5, L6a and L6b across the ipsilateral and contralateral hemisphere for the three injection targets. (C) Average heat maps (n = 6 mice) based on the fractional count across laminae for each cortical area separated according to injection target and local dominance for ipsilateral (C1) and contralateral (C2) hemispheres. Dominance quantification is based on average laminar fractional count across animals. Crosses indicate cortical areas that do not contain L4. (D) Bar graphs showing the average percentage of areas (± sem, n = 6 mice) in which L2/3, L5 and L6 dominate the fractional cell count for ipsilateral (D1) and contralateral (D2) hemispheres. Unless indicated, all comparisons reach statistical significance (n.s. = non-significant). Dominance quantification is based on laminar fractional count per animal. (E) Stacked bar plots showing average laminar dominance for all cortical areas (± sem) pooled across all injection targets. Asterisks indicate statistical significance (p < 0.05).

Cortical hierarchy onto VISp, SSp-bfd and MOp.

(A) Cartoon showing the laminar basis of the anatomical determination of cortical hierarchy (Top). As the fraction of supragranular (L2/3) neurons decrease and/or the number of L5 and L6 neurons increase, the proportion of infragranular neurons (fILN) increases reflecting a predominance of feedback input (Bottom). (B) Segmented image of the ipsilateral and contralateral anterolateral visual area (VISal) projecting onto VISp showing the laminar position of detected cells. (C) Plots showing fILN values for all neurons within each hemisphere (blind to individual cortical areas) for each mouse for the three target injections. Average fILN values (± sem) are depicted by filled circles. (D) Plot showing the average fILN values (± sem) for each of the 45 areas (pooled across different injection experiments) and ranked according to the ipsilateral fILN values. (E) Scatter plot showing the correlation between the ipsilateral fILN values and the difference between the contralateral and ipsilateral fILN values for the three target injections. Average correlation values (± sem, n = 6 mice) for each target area are displayed (top right). Highlighted in red are the primary (VISp), rostrolateral (VISrl), lateral (VISl) and anterolateral (VISal) visual areas projecting to SSp-bfd that display low ipsilateral fILN and high contralateral fILN values. (F) Heatmap plot displaying the ipsilateral and contralateral fILN values for each cortical brain area for the three injection targets. Open circles denote injection areas. Gray circles indicate cortical areas with no projections to VISp, SSp-bfd or MOp. Numbers 1-45 indicate different brain areas, see Fig. S1.

Sensory and motor but not lateral, prefrontal and medial projections explain asymmetric cortical hierarchy.

(A) Plot showing the average fILN values for all areas in a given module for n = 18 mice irrespective of target area. Red circles indicate the sensory and motor modules (visual, auditory and somatomotor; v-a-sm) while grey circles indicate prefrontal, medial and lateral (pf-m-l) modules. (B) Bar graphs comparing cross hemispheric fILN values (mean ± sem) for prefrontal, medial and lateral (pf-m-l) and sensory and motor (v-a-sm) modules. Each circle represents a single animal (n = 18). (C) Bar graphs (mean ± sem) showing the interhemispheric difference in fILN values between the two modular groupings for each target projectome. Each circle represents a single animal (n = 6, respectively).

L6 dominates cortical hierarchy in sensory and motor areas within and between the two hemispheres.

(A) Schematic illustrating at least three hypothetical ways in which changes in the relative distribution of L2/3, L5 and L6 neurons can give rise to increases in the fILN and reflect a predominantly feedback network. (B) Bar graphs showing the average (± sem) normalized fractional count (excluding L4 and 6b) of cells for the sensory and motor modules projecting to each target area from both hemispheres (ipsi, gray; contra, black), (C) Heat map displaying the fractional change of projections for L2/3, L5 and L6a between the contralateral and ipsilateral hemisphere. Displayed are the 24 individual brain areas in the sensory and motor modules for the three target areas VISp, SSp-bfd and MOp. Asterisks denote areas that significantly change between the ipsilateral and the contralateral hemispheres. Crosses indicate excluded homotopic areas. (D) Flat map indicating the three sensory and motor modules (top). Stacked horizontal bar plots displaying the percentage of the total number cortical areas in the sensory and motor modules that significantly decrease (blue) or increase (orange; indicated by the asterisks in C) for the three target areas. (E) (Left) Plots showing the average fILN (± sem) for each sensory and motor area (pooled across all target areas, n = 18 mice) and ranked according to the ipsilateral values for the default network (gray filled circles), the default network excluding L5 (open circles) and the default network excluding L6 (black filled circles). (Right) Scatter plots showing the average ipsilateral fILN scores for each of the twenty four areas of the default network, the default network excluding L5 and the default network excluding L6. Filled triangles represent the mean (± sem) of the average fILN values. Red line indicates the mean of the ipsi default network. (F) (Left) Plots showing the average fILN (± sem) for each sensory and motor area (pooled across all target areas, n = 18 mice) and ranked according to the ipsilateral values for the default network (gray filled triangles), the contralateral values for the default network (gray filled circles), the contralateral values for the default network excluding L5 (open circles) and the default network excluding L6 (black filled circles). (Right) Scatter plots showing the average fILN scores for each of the twenty four areas of the ipsi and contra default network, the default contra network excluding L5 and the default contra network excluding L6. Filled triangles represent the mean (± sem) of the average fILN values. Red line indicates the mean of the ipsi default network.