Tunneling Nanotube–like Connections in the Developing Cerebellum: Distinct from Cytokinetic and Intercellular Bridges

Intercellular communication is essential for brain development. While classical communication modes—such as paracrine, juxtacrine and synaptic signaling—are well characterized, emerging evidence suggests that tunneling nanotubes (TNTs), membranous bridges primarily observed in vitro, may also contribute. However, their presence and function in vivo remain unclear, partly due to the challenge of distinguishing them from other types of intercellular connections (ICs). Building on prior evidence of ICs in the external granule layer (EGL) of the developing cerebellum, we investigated their nature in postnatal day 7 (P7) mice. Using immunofluorescence and genetically sparse labeling, we distinguished cytokinetic bridges (CBs), transient connections formed between daughter cells during the final stage of cell division, and intercellular bridges (IBs), more stable connections that persist after division, from TNT-like structures, which form independently of cell division. We observed CBs but not IBs in the EGL. We also observed TNT-like connections throughout the EGL, which appeared to connect both clonally and non-clonally related cells. The presence of these TNT-like structures in the developing EGL suggests that TNT-like connections may contribute to intercellular communication, paving the way for further study of a previously unrecognized mechanism that could influence neuronal fate, migration, and network formation during cerebellar development.