Cortical layer 6 mediates intra- and inter-hemispheric connectivity and its hierarchical organisation

Cortico-cortical projection neurons bridge functionally distinct brain regions within and between the cortical hemispheres. Determining the logic of this macrocircuitry is necessary for understanding how cortical integration enables high level brain function involving multiple sensory, motor and cognitive processes. To begin to address this, we have performed a systematic anatomical analysis of the interareal organization of the cortex-wide projectome onto the primary visual, somatosensory and motor cortices of adult mice. Using brain-wide retrograde viral tracing followed by serial 2-photon tomography and automated 3D detection of nucleus-labelled cells we have identified both homotopic and more than twenty heterotopic cortical areas projecting to each of these three target areas. Contralateral projections are predominantly mediated by a specific subset of cortico-cortical neurons in layer 6a whereby in almost 60% of contralateral cases the projection from the source regions was dominated by L6 input, followed by layers 5 and 2/3. Based on cortical hierarchy analysis that compares the relative contribution of inputs from infra- and supra-granular layers we find that contra-hemispheric projections exhibit high levels of hierarchy compared to their ipsi-cortical counterpart, independent of the targeted injection area. The magnitude of the interhemispheric difference in hierarchy was largest for sensory and motor areas compared to frontal, medial or lateral brain areas and can be explained by a proportional increase in L6 projection neurons. Taken together, this highlights a dominant role for L6 in global cortical communication and a generalizable feature of both intra- and inter-hemispheric hierarchical organization.