Isolating synapses: synaptosome and postsynaptic density fractionation

Many questions about synapses are questions of place: is a receptor at the synapse or elsewhere in the cell? A blot of the whole tissue measures only the total. Subcellular fractionation separates the tissue into parts first. When brain tissue is homogenised gently in an isotonic medium, nerve terminals are torn from their axons and reseal into small sealed particles, synaptosomes, which a fast gradient procedure can isolate in approximately an hour (Dunkley et al., 2008); the name was introduced for these nerve-ending particles in the 1960s (Whittaker et al., 1964).

The classic preparation uses centrifugation twice over. Differential centrifugation separates particles by how fast they sediment: a low-speed spin removes nuclei and debris, and a faster one pellets crude synaptosomes together with free mitochondria and myelin fragments. A discontinuous sucrose gradient then separates them by density: myelin floats on 0.85 M sucrose, small membranes collect at 0.85/1.0 M, synaptosomes at 1.0/1.2 M, and mitochondria sediment to the bottom (Cohen et al., 1977). Extracting the synaptosomes with the mild detergent Triton X-100 dissolves their membranes but leaves the postsynaptic density, a dense protein network beneath the postsynaptic membrane, which pellets (Cohen et al., 1977; Carlin et al., 1980). The method has changed little in forty years (Bermejo et al., 2014).

Every fraction is only enriched, never pure, so it is validated with marker proteins on a Western blot. PSD-95, a scaffold protein that is highly enriched in the PSD fraction (Cho et al., 1992), should rise; presynaptic proteins such as synaptophysin should largely disappear from it (Hahn et al., 2009); and nuclear, myelin and mitochondrial markers show contamination from the wrong particles. PSD fractions can also be prepared from human post-mortem brain tissue (Hahn et al., 2009).

The target is Rx, a hypothetical receptor subunit of approximately 100 kDa. The simulator records the true contents of every fraction, so the decisions that changed the result are shown at the end.

Learning objectives

• Plan a fractionation study with the animal or donor as the unit, coded samples and, for human tissue, donors matched for post-mortem interval
• Homogenise tissue so that synaptosomes survive, and explain why the buffer must be isotonic, cold and contain inhibitors
• Choose centrifugation forces and times for P1 and P2, and collect the synaptosome band from a sucrose gradient
• Isolate the postsynaptic density as the Triton X-100-insoluble fraction, and explain what a too weak or too strong detergent does
• Validate fractions with enrichment and depletion markers, calculate enrichment per microgram relative to the homogenate, and recognise contamination
• Compare groups with Welch's t-test, a 95% confidence interval and an effect size, and distinguish a redistribution of a protein from a change in its total amount

Ethics and safety

Mouse tissue comes from animals used under a protocol approved by an Institutional Animal Ethics Committee (in India, the CCSEA under the Prevention of Cruelty to Animals Act 1960; elsewhere, frameworks such as EU Directive 2010/63/EU, the US Animal Welfare Act and PHS Policy, and the UK Animals (Scientific Procedures) Act 1986); group sizes are justified in advance, and one brain can provide many fractions for several questions (Reduction, one of the 3Rs). Experiments are reported following the ARRIVE guidelines, developed by the NC3Rs in the United Kingdom and endorsed by journals internationally (Percie du Sert et al., 2020). Human brain tissue comes from brain banks, donated with the informed consent of the donor or their family, and is used under ethics committee approval and national rules (in India, the ICMR National Ethical Guidelines of 2017; in England, Wales and Northern Ireland, the Human Tissue Act 2004); the groups are diagnoses, with no experimental manipulation. At the bench, ultracentrifuges need training and balanced rotors, detergents and inhibitor cocktails are irritants, and gloves and eye protection are worn.

Key terms

• Homogenate: Tissue broken up in buffer with a homogeniser; the starting fraction (H), against which every enrichment is measured.
• Dounce homogeniser: A glass tube with a close-fitting glass or Teflon pestle; strokes of the pestle shear tissue apart gently.
• Isotonic: Having the same osmotic strength as the inside of cells (here 0.32 M sucrose), so that sealed particles neither swell nor shrink.
• Synaptosome: A resealed nerve terminal, often with a piece of the postsynaptic membrane and density attached, formed when brain tissue is homogenised gently.
• Postsynaptic density (PSD): A dense, protein-rich plate under the postsynaptic membrane of excitatory synapses that anchors receptors and signalling proteins.
• Differential centrifugation: Successive spins of increasing force (in multiples of gravity, g) that pellet particles in order of how fast they sediment.
• P1, S1, P2, S2: Pellet and supernatant of the first spin (nuclei and debris; everything else), then of the second spin of S1 (crude synaptosomes; cytosol and light membranes).
• Sucrose density gradient: Layers of sucrose solutions of increasing density; in a long fast spin particles collect at the interface matching their own density.
• Triton X-100: A mild nonionic detergent that dissolves lipid membranes but leaves dense protein networks such as the PSD insoluble.
• Marker protein: A protein of known location used to show what a fraction contains: PSD-95 for the PSD, synaptophysin for synaptic vesicles, histone H3 for nuclei.
• Enrichment: Signal per microgram of protein in a fraction divided by signal per microgram in the homogenate; above 1 is enriched, below 1 depleted.
• Post-mortem interval (PMI): The time between death and freezing of donated tissue; proteins degrade during it.
• Pseudoreplication: Treating repeated measurements of one animal or preparation as independent observations, which overstates the evidence.