GluN3A is required for coordinated postnatal development of axonal and dendritic branching patterns in mouse L2/3 callosal projection neurons

Callosal projections connect both cortical hemispheres via the corpus callosum, allowing bilateral integration of sensory information. Callosal axons originate mainly from layer (L)2/3 pyramidal neurons in primary sensory areas and project to homotopic contralateral regions. The projections display a stereotyped layer-specific pattern, targeting distinct dendritic domains of contralateral L2/3 neurons. In mouse somatosensory cortex, such precise innervation emerges in an activity-dependent manner during the second and third postnatal weeks but the molecular determinants are largely unknown. Using in utero electroporation of fluorescent reporters to label axonal and dendritic arbors of L2/3 neurons through postnatal development, we show that loss-of-function of Grin3a (gene encoding the non-conventional NMDA receptor GluN3A subunit) disrupts region and layer-specific contralateral targeting by callosal axons without affecting early axonal navigation or midline crossing. Rather than concentrating at the border between primary/secondary somatosensory cortex (S1/S2), callosal axons in male and female GluN3A knockout mice form a second column laterally in S2. Within the S1/S2 border, axonal arbors fail to innervate their normal destinations in L1 and outer 2/3 and shift towards inner L2/3 regions. Analysis of dendritic architecture revealed that GluN3A deletion drives proximal bifurcation and premature branching of apical dendrites of L2/3 neurons, with inward expansion of recipient dendritic trees matching callosal axon profiles in Grin3a knockouts. Together with conditional loss of function experiments, our results suggest that the dendritic patterning of postsynaptic L2/3 neurons directs the position of callosal axons within their target fields and implicate GluN3A in the postnatal timing and specificity of this process. Significance statement The formation of functional neural circuits requires a precise spatiotemporal overlap of incoming axons with specific dendritic domains of the target postsynaptic neurons, along with plasticity mechanisms that will stabilize some of the connections formed. Tight regulation of these processes during critical postnatal windows ensures input selectivity across brain regions, neuronal subtypes, and within distinct subcellular compartments, ultimately giving rise to highly stereotyped neural circuits that support accurate information transfer. Our study uncovers a previously unrecognized role for non-conventional N-methyl-D-aspartate (NMDA) receptor GluN3A subunits in coordinating the segregation of callosal axon projections onto defined dendritic domains of layer 2/3 neurons and specific areas of the mouse somatosensory cortex.