Preparation of P2 or percoll-purified synaptosomes from mammalian brain tissue

Abstract

Isolated nerve endings have been widely used as a model for studying synaptic functions in the brain. These nerve endings can be separated from the axons and postsynaptic connections when neuronal tissue is homogenized in an isotonic buffer. The plasma membrane of the sheared nerve endings reseals to form pinched-off nerve terminals called synaptosomes. Synaptosomes show similar morphology and contain all the intact molecular machinery found in nerve terminals in vivo, including the proteins, synaptic vesicles, and mitochondria, necessary for regulating synaptic function for the release, uptake, and storage of neurotransmitters upon different stimulations. The methods for making synaptosomes have varied and been modified for different experimental applications. The goal of this chapter is to describe optimized procedures used in our laboratories for isolation of intact and functional synaptosomes from whole mammalian brain tissue, at two levels of purity: the “crude” P2 and the Dunkley S1 method of Percoll-purified fractions, for molecular, biochemical, and functional assays. These represent the shortest experimental methods to date. The protocol involves mild homogenization of the brain tissue in isotonic conditions, with medium centrifugation speeds to minimize the mechanical damage to the synaptosomes and the preparation time.