Ultrafast Isolation of Synaptic Terminals From Rat Brain for Cryo-Electron Tomography Analysis.

Abstract

Understanding the nanoscale organization and molecular rearrangement of synaptic components is critical for elucidating the mechanisms of synaptic transmission and plasticity. Traditional synaptosome isolation protocols involve multiple centrifugation and resuspension steps, which may cause structural damage or alter the synaptosomal fraction, compromising their suitability for cryo-electron tomography (cryo-ET). Here, we present an ultrafast isolation method optimized for cryo-ET that yields two types of synaptosomal fractions: synaptosomes and synaptoneurosomes. This streamlined protocol preserves intact postsynaptic membranes apposed to presynaptic active zones and produces thin, high-quality samples suitable for in situ structural studies. The entire procedure, from tissue homogenization to vitrification, takes less than 15 min, offering a significant advantage for high-resolution cryo-ET analysis of synaptic architecture. Key features • Ultrafast synaptic terminal isolation from tissue homogenization to vitrification completed within 15 min. • Retention of postsynaptic membranes with synaptic receptors and postsynaptic density (PSD) proteins. • The thickness of the samples is suitable for in situ cryo-ET analysis. • Enables cryo-ET studies of synaptic structures and postsynaptic membrane proteins such as AMPA receptors.