Axon terminal distribution in layer 1 of the mouse auditory cortex: differential projections from the dorsal and medial subdivisions of the medial geniculate body and the marginal zone of the posterior thalamic nuclei

Layer 1 (L1) of the neocortex integrates bottom-up and top-down signals. Inputs to L1, however, remain incompletely characterized. L1 of the auditory cortex (ACX) receives ascending inputs from the medial geniculate body (MGB) subdivisions and the surrounding posterior thalamic nuclei (PTN). The precise manner in which these structures innervate L1 is not fully understood. Here we examined the distribution of axon terminals from MGB/PTN subdivisions in L1 of the mouse ACX using virus-based axonal labeling. A bulk injection into the entire MGB and its adjacent PTN (referred to as W) confirmed their projection to upper L1, in addition to other layers. However, we observed multiple vertical axon bundles with irregular inter-bundle intervals in L2 in coronal sections. To identify their origin, we first applied a retrograde tracer to the surface of the ACX and found labeled cell bodies across MGB/PTN subdivisions. The distribution of labeled cells could be dichotomously divided into a dorsomedial (DM) region, primarily encompassing the dorsal and medial nuclei of MGB, and a ventrolateral (VL) region, primarily containing the marginal zone (MZ) of PTN. Sparsely labeled neurons in the caudal part of the ventral MGB (MGv) were also observed. We then injected the virus tracer into the DM region containing the dorsomedial subdivisions of MGB and the dorsomedial MGv (dmMGB), and into the VL region containing the MZ and the ventrolateral MGv, for anterograde labeling of axons. A DM injection resulted in strong, uniform labeling of axons in upper L1, without apparent axon bundles in L2, while a VL injection produced clear axon bundles in L2, as well as labeling in upper L1. The bundle density and inter-bundle interval were not significantly different between the W and VL injection cases, suggesting that the MZ is the primary origin of the axon bundles in L2. Interestingly, axons labeled by VL injections had a higher density at locations where the axon bundles reached upper L1, resulting in a clustered distribution of axons in this layer. Coherence analyses confirmed that axon density in upper L1 varied in phase with that in L2 for the VL injection cases. In tangential sections, axons labeled by W injections in lower L1 appeared to distribute in a square grid-like pattern, with expanded nodes. Quantitative analysis revealed that the axon bundles in coronal sections predominantly corresponded to the grid nodes in the tangential sections. Taken together, our results suggest a strong, uniform distribution of dmMGB axon terminals and a square grid-like distribution of MZ axon terminals in cortical upper L1. These two ascending inputs may exert differential influences on the function of L1 in the ACX.