Rapid analysis of synaptic vesicle endocytosis in synaptosomes

Abstract

Synaptic vesicle endocytosis (SVE) is a critical mechanism by which synaptic transmission is regulated and maintained at presynaptic boutons. Small molecules that target SVE may prove to be useful not only for basic research into synaptic function but also in treating diseases that involve pathological neurotransmission. However, there are relatively few small molecules targeting SVE that have known molecular targets, and the development of these compounds has been a slow process. The lack of development on this front has been, in part, due to the laborious nature of traditional methods to study SVE, which are unsuited to the screening of compounds. This chapter describes a recently developed high-throughput approach to the analysis of SVE, which uses synaptosomes and high-content imaging. Rat forebrain synaptosomes are attached to glass microplates, and depolarization-induced SVE is measured by the uptake of the fluorescent styryl dye FM4-64. The synaptosomes may be used fresh or frozen and stored in liquid nitrogen, with the latter option greatly expanding the utility of the assay, by reducing the animal numbers required and allowing experiments to be done "on demand." The assay provides a rapid approach to screening small molecules for effects on SVE that will hopefully see an increase in the number of available small molecules that can target this important process at synapses. © Springer Science+Business Media New York 2014.