Abstract
Acute perturbations of clathrin and associated proteins at synapses have provided a wealth of knowledge on the molecular mechanisms underlying clathrin-mediated endocytosis (CME). The basic approach entails presynaptic microinjection of an inhibitory reagent targeted to the CME pathway, followed by a detailed ultrastructural analysis to identify how the perturbation affects the number and distribution of synaptic vesicles, plasma membrane, clathrin-coated pits, and clathrin-coated vesicles. This chapter describes the methodology for acutely perturbing CME at the lamprey giant reticulospinal synapse, a model vertebrate synapse that has been instrumental for identifying key protein-protein interactions that regulate CME in presynaptic nerve terminals with broader extension to nonneuronal cell types.