Activity‐induced conversion from dendritic filopodia to spines mediated by NMDA receptor‐dependent calcium transients and vesicular exocytosis

Synapses are essential for neural information processing by gating the propagation of activity in synaptically connected neuronal circuits. During the development of such circuits, neuronal activity contributes to synaptic formation and stabilization, or synaptogenesis. However, the precise cellular mechanisms underlying the signalling and structural changes during synaptogenesis remain incompletely understood. In cultured hippocampal neurons during early development, we found that chronic activity blockade with tetrodotoxin (TTX) significantly reduced dendritic spine formation while simultaneously increasing the number of dendritic filopodia. Notably, many of these filopodia expressed GluA1-containing AMPA receptors and GluN2B-containing NMDA receptors but lacked corresponding specialized presynaptic counterparts. Intriguingly, glycine stimulation commonly used for inducing chemical long-term potentiation (LTP) led to a significant decrease in the density of dendritic filopodia and an increase in spines, suggesting an induced conversion from filopodia to spines. Live-cell imaging revealed the dynamic process in which dendritic filopodium-axon contact developed into specialized spine synapse after glycine stimulation. In neurons transfected with GCaMP6f, this transformation was associated with a marked increase in the occurrence of calcium transients within individual filopodia. Blocking NMDA receptor-mediated calcium influx with AP5 or disrupting vesicular exocytosis by cleaving VAMP family SNARE proteins with tetanus toxin strongly inhibited the filopodium-spine conversion. These findings, together with the observation of recycling endosomes within or near filopodia under light and electron microscopy, suggest that calcium influx through filopodial NMDA receptors signals activity-dependent conversion of dendritic filopodia into spines, probably by triggering VAMP-dependent exocytosis of recycling endosomes. KEY POINTS: Activity deprivation during early neuronal development shifts dendritic protrusions toward filopodia. Dendritic filopodia with NMDA receptors act as activity-sensitive precursors for postsynaptic spines prior to presynaptic partner recruitment. NMDA receptor-dependent calcium transients within filopodia drive their structural conversion into spines. VAMP-mediated exocytosis is the downstream effector linking calcium signalling to spine morphogenesis.