Abstract
This book outlines various techniques for the isolation and application of synaptosomes for neuroscience research. One of the simplest techniques involves fractionation of synaptoneurosomes through filtration and low-speed centrifugation (Hollingsworth J Neurosci 5:2240–2253, 1985). This approach however is limited by two essential stages of the synaptoneurosome tissue preparation: the manual homogenization and filtrations are both laborious and slow. We have updated this traditional technique to include modern benchtop homogenizers and centrifugal filter units to simplify these labor-intensive stages, to make each stage faster and to reduce the variability between samples. Here we outline our protocol to produce filtered synaptoneurosomes that reduce sample preparation time, increase the amount of tissue recovered, and, most importantly, increase protein enrichment.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Suzdak PD et al (1986) A selective imidazobenzodiazepine antagonist of ethanol in the rat. Science 234:1243–1247
Suzdak PD, Schwartz RD, Skolnick P, Paul SM (1986) Ethanol stimulates gamma-aminobutyric acid receptor-mediated chloride transport in rat brain synaptoneurosomes. Proc Natl Acad Sci U S A 83:4071–4075
Morrow AL, Pace JR, Purdy RH, Paul SM (1990) Characterization of steroid interactions with gamma-aminobutyric acid receptor-gated chloride ion channels: evidence for multiple steroid recognition sites. Mol Pharmacol 37:263–270
Weiler IJ et al (1997) Fragile X mental retardation protein is translated near synapses in response to neurotransmitter activation. Proc Natl Acad Sci U S A 94:5395–5400
Wu L et al (1998) CPEB-mediated cytoplasmic polyadenylation and the regulation of experience-dependent translation of α-CaMKII mRNA at synapses. Neuron 21:1129–1139
Quinlan EM, Olstein DH, Bear MF (1999) Bidirectional, experience-dependent regulation of N-methyl-D-aspartate receptor subunit composition in the rat visual cortex during postnatal development. Proc Natl Acad Sci U S A 96:12876–12880
Quinlan EM, Philpot BD, Huganir RL, Bear MF (1999) Rapid, experience-dependent expression of synaptic NMDA receptors in visual cortex in vivo. Nat Neurosci 2:352–357
Hebb CO, Whittaker VP (1958) Intracellular distributions of acetylcholine and choline acetylase. J Physiol 142:187–196
Cohen RS, Blomberg F, Berzins K, Siekevitz P (1977) The structure of postsynaptic densities isolated from dog cerebral cortex: I. Overall morphology and protein composition. J Cell Biol 74:181–203
Hollingsworth EB et al (1985) Biochemical characterization of a filtered synaptoneurosome preparation from Guinea pig cerebral cortex: cyclic adenosine 3′:5′-monophosphate- generating systems, receptors, and enzymes. J Neurosci 5:2240–2253
Weiler IJ, Greenough WT (1991) Potassium ion stimulation triggers protein translation in synaptoneurosomal polyribosomes. Mol Cell Neurosci 2:305
Heynen AJ, Quinlan EM, Bae DC, Bear MF (2000) Bidirectional, activity-dependent regulation of glutamate receptors in the adult hippocampus in vivo. Neuron 28:1–10
Murphy KM, Tcharnaia L, Beshara SP, Jones DG (2012) Cortical development of AMPA receptor trafficking proteins. Front Mol Neurosci 5:65
Murphy KM, Beston BR, Boley PM, Jones DG (2005) Development of human visual cortex: a balance between excitatory and inhibitory plasticity mechanisms. Dev Psychobiol 46:209–221
Beston BR, Jones DG, Murphy KM (2010) Experience-dependent changes in excitatory and inhibitory receptor subunit expression in visual cortex. Front Synaptic Neurosci 2:138
Williams K, Irwin DA, Jones DG, Murphy KM (2010) Dramatic loss of Ube3A expression during aging of the mammalian cortex. Front Aging Neurosci 2:18
Pinto JGA, Hornby KR, Jones DG, Murphy KM (2010) Developmental changes in GABAergic mechanisms in human visual cortex across the lifespan. Front Cell Neurosci 4:16
Murphy KM, Balsor J, Beshara S, Siu C, Pinto JGA (2014) A high-throughput semi-automated preparation for filtered synaptoneurosomes. J Neurosci Methods 235:35–40
Pinto JGA, Jones DG, Williams CK, Murphy KM (2015) Characterizing synaptic protein development in human visual cortex enables alignment of synaptic age with rat visual cortex. Front Neural Circuits 9:3
Beshara S, Beston BR, Pinto JGA, Murphy KM (2015) Effects of fluoxetine and visual experience on glutamatergic and GABAergic synaptic proteins in adult rat visual cortex. eNeuro 2 ENEURO0126–15.2015. https://doi.org/10.1523/ENEURO.0126-15.2015
Siu CR, Beshara SP, Jones DG, Murphy KM (2017) Development of glutamatergic proteins in human visual cortex across the lifespan. J Neurosci 37:6031–6042
Chandler LJ, Crews FT (1990) Calcium- versus G protein-mediated phosphoinositide. Hydrolysis in rat cerebral cortical synaptoneurosomes. J Neurochem 55:1022–1030
Villasana LE, Klann E, Tejada-Simon MV (2006) Rapid isolation of synaptoneurosomes and postsynaptic densities from adult mouse hippocampus. J Neurosci Methods 158:30–36
Chang JW et al (2012) Synaptoneurosome micromethod for fractionation of mouse and human brain, and primary neuronal cultures. J Neurosci Methods 211:289–295
Titulaer MN, Ghijsen WE (1997) Synaptoneurosomes. A preparation for studying subhippocampal GABA receptor activity. Methods A Mol Biol 72:49–59
Acknowledgements
This work was supported by NSERC Grant RGPIN-2015-06215 awarded to KM, and an OGS awarded to JB.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Balsor, J.L., Murphy, K.M. (2018). Protocol for a High-Throughput Semiautomated Preparation for Filtered Synaptoneurosomes. In: Murphy, K. (eds) Synaptosomes. Neuromethods, vol 141. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8739-9_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8739-9_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8738-2
Online ISBN: 978-1-4939-8739-9
eBook Packages: Springer Protocols