Isolation of synaptic vesicles from genetically engineered cultured neurons

• Published in: Journal of neuroscience methods 2019-01, Vol.312, p.114-121
• Main Authors: McKenzie, Catherine, Spanova, Miroslava, Johnson, Alexander, Kainrath, Stephanie, Zheden, Vanessa, Sitte, Harald H., Janovjak, Harald
• Format: Article
• Language: English
• Subjects: Animals; Cell Fractionation - methods; Cells, Cultured; Genetic Engineering; Glutamic Acid - metabolism; Lentivirus - physiology; Neuronal culture; Neurons - metabolism; Neurotransmitter uptake; Rats, Wistar; Release; Synaptic vesicle; Synaptic Vesicles - metabolism
• ISSN: 0165-0270; 1872-678X
• DOI: 10.1016/j.jneumeth.2018.11.018

•A procedure to isolate of SVs from cultured neurons and limited neuronal samples.•SVs from culture exhibit neurotransmitter uptake comparable to SVs from brain tissue.•Visualization of a tagged synaptophysin demonstrates efficient SV modification.•SVs from genetically engineered neurons are accessible without transgenic animals. Synaptic vesicles (SVs) are an integral part of the neurotransmission machinery, and isolation of SVs from their host neuron is necessary to reveal their most fundamental biochemical and functional properties in in vitro assays. Isolated SVs from neurons that have been genetically engineered, e.g. to introduce genetically encoded indicators, are not readily available but would permit new insights into SV structure and function. Furthermore, it is unclear if cultured neurons can provide sufficient starting material for SV isolation procedures. Here, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. We show that ∼108 plated cortical neurons allow isolation of suitable SV amounts for functional analysis and imaging. We found that SVs isolated from cultured neurons have neurotransmitter uptake comparable to that of SVs isolated from intact cortex. Using total internal reflection fluorescence (TIRF) microscopy, we visualized an exogenous SV-targeted marker protein and demonstrated the high efficiency of SV modification. Obtaining SVs from genetically engineered neurons currently generally requires the availability of transgenic animals, which is constrained by technical (e.g. cost and time) and biological (e.g. developmental defects and lethality) limitations. These results demonstrate the modification and isolation of functional SVs using cultured neurons and viral transduction. The ability to readily obtain SVs from genetically engineered neurons will permit linking in situ studies to in vitro experiments in a variety of genetic contexts.