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

Abstract

The auditory cortex exerts a powerful, yet heterogeneous, effect on subcortical targets. Auditory corticofugal projections emanate from layers 5 and 6 and have complementary physiological properties. While several studies suggested that layer 5 corticofugal projections branch widely, others suggested that multiple independent projections exist. Less is known about layer 6; no studies have examined whether the various layer 6 corticofugal projections are independent. Therefore, we examined branching patterns of layers 5 and 6 auditory corticofugal neurons, using the corticocollicular system as an index, using traditional and novel approaches. We confirmed that dual retrograde injections into the mouse inferior colliculus and auditory thalamus co-labeled subpopulations of layers 5 and 6 auditory cortex neurons. We then used an intersectional approach to relabel layer 5 or 6 corticocollicular somata and found that both layers sent extensive branches to multiple subcortical structures. Using a novel approach to separately label layers 5 and 6 axons in individual mice, we found that layers 5 and 6 terminal distributions partially spatially overlapped and that giant terminals were only found in layer 5-derived axons. Overall, the high degree of branching and complementarity in layers 5 and 6 axonal distributions suggest that corticofugal projections should be considered as 2 widespread systems, rather than collections of individual projections.

Microprism-based two-photon imaging of the mouse inferior colliculus reveals novel organizational principles of the auditory midbrain

“…The brain was then fixed by a subsequent perfusion of 4% PFA. Selected 40 μm coronal sections at the level of the IC were immunostained for NeuN using (NeuN (D4G40) XP® Rabbit mAB, Cell Signaling, Cat# 62994, 1:100) primary antibody and (goat anti-rabbit IgG conjugated to Alexa 405, 1:200) secondary antibody following the immunostaining procedures that were previously described (Issa, Sekaran, & Llano, 2023; Vaithiyalingam Chandra Sekaran et al, 2021). Confocal images were obtained using a Confocal Zeiss LSM 710 Microscope using 10 and 40x objectives and tile scan functions.…”

“…The brain was then fixed by a subsequent perfusion of 4% PFA. Selected 40 m coronal sections at the level of the IC were immunostained for NeuN using (NeuN (D4G40) XP® Rabbit mAB, Cell Signaling, Cat# 62994, 1:100) primary antibody and (goat anti-rabbit IgG conjugated to Alexa 405, 1:200) secondary antibody following the immunostaining procedures that were previously described (Issa, Sekaran, & Llano, 2023;Vaithiyalingam Chandra Sekaran et al, 2021). Confocal images were obtained using a Confocal Zeiss LSM 710 Microscope f(1,4,16) = 173.0, 0, and 1.8 -p = 5.3x10 -10 , 1.0, and 0.17 for the region, sound level, and interaction, respectively, % of spectrally modulated cells ± SEM = 23±2% vs 76±2% for modules vs matrix -3 animals, **p<0.05 vs modules) or temporally (two-way ANOVA: f(1,4,16) = 65.9, 0, and 2.2 -p = 4.6x10 -7 , 1.0, and 0.11 for the region, sound level, and interaction, respectively, % of temporally modulated cells ± SEM = 23±2% vs 76±2% for modules vs matrix, n = 3 animals, **p<0.05 vs modules) modulated cells, respectively, across different sound levels in modules (green bars) vs. matrix (blue bars) imaged via microprism, (**p<0.05, matrix vs. modules).…”