BMP4 Restricts Axonal Ramification and Bouton Density in Cholinergic Projections from the Basal Forebrain

Abstract Axonal projections and precise synapse formation are essential for the establishment of functional neural circuits, and their dysregulation contributes to various psychiatric and neurodegenerative disorders. Although numerous extracellular cues secreted from surrounding tissues regulate axon guidance and synapse formation, the mechanisms by which neurons locally regulate terminal patterning within their projection fields remain poorly understood. Bone morphogenetic protein 4 (BMP4) is a classical morphogen that is involved in early neural tube patterning during development. However, its postnatal function remains unclear. Previous in vitro studies using hippocampal glutamatergic neurons demonstrated that BMP4 can act locally on axons to suppress synapse formation, raising the possibility that BMP4 regulates terminal differentiation after target innervation. Here, we investigated the role of BMP4 in postnatal cholinergic circuit development using cholinergic neuron-specific Bmp4 conditional knockout (Bmp4 cKO) mice, with adult analyses performed in males. Basal forebrain cholinergic neurons project extensively throughout the neocortex and are essential for higher brain functions, including arousal and attention. Bmp4 cKO mice exhibited increased cholinergic axon terminals in the neocortex, hippocampus, and amygdala, as well as increased axonal ramification in the neocortex without affecting cytoarchitectonic organization or cholinergic neuron numbers in the basal forebrain. These findings demonstrate that BMP4 functions as a negative regulator of cholinergic terminal formation and axonal arborization, suggesting that BMP4 may act locally to restrict cholinergic axonal expansion during neural circuit maturation. Visual Abstract Significance Statement Cholinergic projections from the basal forebrain are critical for cortical function, yet the mechanisms that regulate their terminal architecture remain poorly understood. Here, we show that BMP4 restricts cholinergic axonal ramification and bouton density using cholinergic neuron-specific Bmp4 conditional knockout mice. Bmp4 deletion increased axonal ramification and bouton density without altering cholinergic neuron number in the basal forebrain. These findings reveal a previously unrecognized role for BMP4 in regulating cholinergic terminal architecture during postnatal neural circuit development. Together, our results identify BMP4 as a regulator that limits excessive cholinergic innervation and reveal a previously underappreciated role of BMP4 signaling in postnatal neural circuit refinement.