Light and electron microscopic imaging of synaptic vesicle endocytosis at mouse hippocampal cultures

Following the release of neurotransmitters at synaptic vesicles via exocytosis, endocytosis is initiated to retrieve vesicles that have fused with the plasma membrane of nerve terminals and recycle them, thus sustaining synaptic transmission. Here, we describe imaging-based protocols for quantitative measurements of endocytosis at cultured synapses. These protocols include (1) primary culture of mouse hippocampal neurons, (2) studying endocytosis at neurons transfected with a pH-sensitive synaptophysin-pHluorin2× using fluorescent microscopy, and (3) imaging endocytosis at fixed neurons with electron microscopy. For complete details on the use and execution of this protocol, please refer to Wu et al. (2016) and Wu et al. (2021).