Comparison of simple sucrose and percoll based methodologies for synaptosome enrichment

Abstract

Synaptosomes are isolated nerve terminals. They represent an extremely attractive in vitro model system to study synaptic physiology since they preserve morphological and functional characteristics of the synapse. As such they have been used to investigate synaptic dysfunctions associated with neuropathologies like Alzheimer's disease. In the present work two simple methodologies for isolating synaptosomal-enriched fractions were compared for the first time. The starting points of both protocols were rat cortical or hippocampal homogenized tissues that underwent several differential centrifugation steps followed by a final purification of synaptosomal-enriched fractions using either a Percoll gradient or a Sucrose gradient. Comparison of the fractions obtained was carried out, using both biochemical and electron microscopy approaches. In the biochemical analysis the protein levels of pre-synaptic, post-synaptic, nuclear and mitochondrial markers were evaluated. Additional characterization of the synaptosomal-enriched fractions was performed using transmission electron microscopy. In summary, the results indicate that under the conditions tested the Sucrose based protocol is more efficient for the isolation of synaptosomal-enriched fractions from both neuronal tissues, being particularly efficient for hippocampus that is a less abundant brain tissue. Further, the sucrose protocol apparently results in a higher yield of viable synaptosomes suitable for further assays, including structural and functional studies of synapses; making this an attractive procedure to study processes associated with neuropathologies.

Keywords: Electron microscopy; Percoll; Presynaptic terminals; Sucrose; Synaptosomal-enriched fractions.