Endogenous β-neurexins on axons and within synapses show regulated dynamic behavior

Neurexins are key organizer molecules that regulate synaptic function and are implicated in autism and schizophrenia. β-neurexins interact with numerous cell adhesion and receptor molecules, but their neuronal localization remains elusive. Using single-molecule tracking and high-resolution microscopy to detect neurexin1β and neurexin3β in primary hippocampal neurons from knockin mice, we demonstrate that endogenous β-neurexins are present in fewer than half of excitatory and inhibitory synapses. Moreover, we observe a large extrasynaptic pool of β-neurexins on axons and show that axonal β-neurexins diffuse with higher surface mobility than those transiently confined within synapses. Stimulation of neuronal activity further increases the mobility of synaptic and axonal β-neurexins, whereas inhibition causes the opposite. Blocking ectodomain cleavage by metalloproteases also reduces β-neurexin mobility and enhances glutamate release. These findings suggest that the surface mobility of endogenous β-neurexins inside and outside of synapses is dynamically regulated and linked to neuronal activity.