Highly branched and complementary distributions of layer 5 and layer 6 auditory corticofugal axons in mouse

The auditory cortex (AC) exerts a powerful, yet heterogeneous, effect on its subcortical targets. Auditory corticofugal projections emanate from distinct bands in layers 5 (L5) and 6 (L6), which have complementary anatomical and physiological properties. While several studies have suggested that corticofugal projections from L5 branch widely, others have suggested that there are multiple, mostly independent sets of L5 corticofugal projections. Even less is known about L6; no studies have examined whether the various L6 corticofugal projections are independent. Therefore, we examined branching patterns of L5 and L6 auditory corticofugal neurons, using the corticocollicular system as an index projection, using both traditional and novel approaches. We first confirmed that dual retrograde injections into the mouse inferior colliculus and auditory thalamus co-labeled subpopulations of L5 and L6 AC neurons. We then used an intersectional approach to selectively re-label L5 or L6 corticocollicular somata and found that both layers sent extensive branches to striatum, amygdala, superior colliculus, thalamus and nuclei of the lateral lemniscus. L5 corticocollicular axons also sent an unpaired projection to the superior olivary complex. Using a novel approach to separately label L5 and L6 axons in the same mouse, we found that L5/6 terminal distributions partially spatially overlapped and that a subset of giant terminals was only found in L5-derived axons. Overall, the high degree of branching and complementarity in the distributions of L5 vs. L6 axons suggest that corticofugal projections should be considered as two widespread systems of projections, rather than a collection of individual projections.