Synaptosomes are obtained by mild homogenization of nervous tissue under isotonic conditions and subsequent fractionation using differential and density gradient centrifugation.

Synaptosomes were first isolated in an attempt to identify the subcellular compartment corresponding to the fraction of so-called bound acetylcholine that remains when brain tissue is homogenized in iso-osmotic sucrose.

Particles containing acetylcholine and its synthesizing enzyme choline acetyltransferase were originally isolated by Hebb and Whittaker (1958)[1] at the Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge, UK.

In a collaborative study with the electron microscopist George Gray from University College London, Victor P. Whittaker eventually showed that the acetylcholine-rich particles derived from guinea-pig cerebral cortex were synaptic vesicle-rich pinched-off nerve terminals.

They maintain a normal membrane potential, contain presynaptic receptors, translocate metabolites and ions, and when depolarized, release multiple neurotransmitters (including acetylcholine, amino acids, catecholamines, and peptides) in a Ca2+-dependent manner.