Mapping prefrontal afferents along development.

Introduction: The medial prefrontal cortex (mPFC) serves as a central hub for cognitive, emotional, and social processes by integrating inputs from numerous brain regions. Understanding the mechanisms underlying the formation and myelination of prefrontal afferents is essential for elucidating the emergence, maturation, and refinement of mPFC-dependent behaviors. However, despite extensive characterization of long-range projections to the mPFC in the adult brain, their developmental trajectories remain poorly understood. Methods: In this study, we combine retrograde labeling, light-sheet imaging, and automated cell segmentation to quantitatively map afferent projections to the mPFC from neonatal stages to adulthood. Results: We show that ipsi- and contralateral afferent densities undergo marked, region-specific developmental changes. Quantitative laterality analysis revealed that many afferent pathways develop an increasing ipsilateral bias after neonatal age, arising through distinct combinations of ipsilateral and contralateral changes rather than a uniform loss of contralateral inputs. In addition, we identify region-specific developmental patterns, including increased projection densities from limbic regions during early adolescence and a progressive decline in inputs from the mediodorsal thalamus toward adulthood. Notably, myelination of interhemispheric prefrontal afferents begins only after the reduction of neonatal contralateral projections. Conclusion: Together, these findings reveal distinct, region-specific developmental dynamics of prefrontal afferents, with important implications for the maturation of mPFC-dependent functional and behavioral outputs.