Isolation Of Synaptic Vesicles Research Articles

Isolation of Synaptic Vesicles from Mammalian Brain.

Synaptic vesicles (SVs) store neurotransmitters and undergo a fine-tuned regulatory and dynamic cycle of exo- and endocytosis, which is essential for neurotransmission at chemical synapses. The development of protocols for isolating SVs from biological extracts was a fundamental accomplishment since it allowed for characterizing the molecular properties of SVs using biochemical methods. In this chapter, we describe a modified procedure for isolating SVs from a few g of rodent brain and that can be completed within ~12h. The protocol involves the preparation of isolated nerve terminals from which SVs are released by osmotic shock and then enriched via various centrifugation steps, followed by size exclusion chromatography as final purification step. The final vesicle fraction is 22-fold enriched in SVs over the starting material, and the final yield of SVs obtained using this protocol is approximately 20μg of protein per gram of mouse brain. The degree of contamination by other organelles and particles monitored by morphology and immunolabeling compares well with that of the classical protocols.

Isolation of synaptic vesicles from genetically engineered cultured neurons

BackgroundSynaptic 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. New methodHere, we demonstrate an efficient ex vivo procedure to obtain functional SVs from cultured rat cortical neurons after genetic engineering with a lentivirus. ResultsWe 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. Comparison with existing methodsObtaining 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. ConclusionsThese 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.

Isolation of Synaptic Vesicles

Synaptic vesicles are the most abundant secretory organelle in eukaryotic neural cells. Synaptic vesicles are physically distinct from other membrane-bound organelles because they are small, spherical, and highly uniform in size with a diameter of about 40 nm. Synaptic vesicles also have a characteristically low density because water and phospholipids account for 88% of an individual synaptic vesicle's weight. The homogeneous size and density of the synaptic vesicle are characteristics that are exploited in most synaptic vesicle isolation procedures. Synaptic vesicles are purified by isopycnic/velocity sedimentation and size-based purification schemes. However, protocols differ in the tissue source of vesicles, the way the tissue is homogenized, and the way the vesicles are fractionated. This unit describes two well-characterized and widely used synaptic vesicle isolation procedures that are capable of generating membrane fractions that are 20 to 30 times enriched in synaptic vesicles.

SV2 and o‐rab3 Remain Associated with Recycling Synaptic Vesicles

o-rab3 is an electric ray homologue of low molecular weight of GTP-binding proteins thought to be involved in targeting of secretory vesicles to sites of exocytosis. The stimulation-dependent association of o-rab3 with synaptic vesicles was compared with that of the membrane-integral synaptic vesicle protein 2 (SV2). On application of immunoelectron microscopy and the colloidal gold technique, antibodies against either protein labeled the synaptic vesicle membrane compartment. Synaptic vesicles recycled under conditions of low frequency stimulation (0.1 Hz) retained their complement of both SV2 and o-rab3. Isolation of synaptic vesicles by density-gradient centrifugation and subsequent column chromatography yielded no indication of a stimulation-dependent release of o-rab3 from synaptic vesicles. In contrast, multivesicular bodies and vacuoles occasionally observed in the nerve terminals contained SV2 but little if any o-rab3. It is concluded that o-rab3 remains associated with the synaptic vesicle membrane compartment during stimulation-induced cycles of repeated exo- and endocytosis. o-rab3 may be lost once the vesicle enters the prelysosomal pathway.

Rab3C is a synaptic vesicle protein that dissociates from synaptic vesicles after stimulation of exocytosis

Rab3 proteins are small GTP-binding proteins of the Ras superfamily. Four highly homologous Rab3 proteins termed Rab3A, Rab3B, Rab3C, and Rab3D have been described. Rab3A has previously been shown to be a constituent of synaptic vesicles in neurons that undergoes membrane dissociation-association cycles during synaptic vesicle recycling. Here we report that Rab3C copurifies with Rab3A during the isolation of synaptic vesicles. Organelles immunoisolated with monoclonal antibodies directed against Rab3A led to a coenrichment of Rab3A and Rab3C, demonstrating that both Rab3 proteins are colocalized on the same organelle. In isolated nerve terminals, stimulation of neurotransmitter release resulted in a dissociation of Rab3C from synaptic vesicle and/or recycling membranes. This dissociation parallels that of Rab3A observed under the same conditions. In contrast, no change was observed in the membrane-association of Rab5, a Rab protein localized on early endosomes. We conclude that in the nervous system Rab3C is localized on synaptic vesicles and, like Rab3A, cycles on and off the synaptic vesicle membrane in parallel with exocytotic release of neurotransmitter.

Purification and characterization of synaptic vesicles from the electric organ of Torpedo ocellata.

Highly purified synaptic vesicles have been isolated from the electric organ of Torpedo ocellata by a rapid procedure which enables the concurrent isolation of synaptic vesicles and of intact presynaptic nerve endings (synaptosomes). The purification procedure consists of homogenization of fresh electric tissue in iso-osmotic glycine in the presence of EGTA, differential and density gradient centrifugation, and gel permeation on a glass beads column of 2500 a pore size. The purity of the vesicles was evaluated both biochemically and morphologically. The vesicles contain acetylcholine (ACh) and ATP in a ratio of 3:1 and at specific concentrations of 2,100nmol ACh/mg protein and 1,010nmol ACh/mg phospholipid. They are associated with Ca+2/Mg+2 ATPase activity and are devoid of the ouabain sensitive Na+/K+ ATPase. The relatively high yields as well as the short preparation time (about 9h for the vesicles and 4h for the synaptosomes) enables the employment of large samples of the isolated material on the day of preparation.

Isolation of synaptic vesicles from Narcine brasiliensis electric organ: some influences on release of vesicular acetylcholine and ATP

A simple density gradient centrifugation technique for separating electric organ cholinergic synaptic vesicles from other organelles and membrane fragments is described. Frozen, ground electric organ is extracted with a solution of similar density to the vesicles; during the subsequent centrifugation, vesicles remain suspended in the extraction medium and heavier contaminating structures sediment out. In confirmation of results obtained with mammalian central nervous system vesicles, a biphasic pattern of efflux of bound ACh is demonstrated. Low levels of phospholipase A 2 (EC 3.1.1.4.) induce efflux of ACh from the vesicle fraction; it is shown that the concomitant fall in vesicle bound ATP is due to ATP efflux rather than ATP hydrolysis within the vesicle.

Cyclic AMP and phosphodiesterase in synaptic vesicles from mouse brain.

AbstractThe isolation of synaptic vesicles from mouse brain by a modification of previously reported methodology is described. Homogeneity of the preparations was ascertained by electron microscopy. Vesicles thus isolated contained cyclic AMP (365 ± 44 pmol/mg of protein; mean ± S.E.M.) and adenosine 3′,5′‐monophosphate phosphodiesterase activity (500 pmol/mg of protein per 30 min). The number of vesicles in three of the vesicle preparations was determined by a visual count of grid fields under the electron microscope. The content of cyclic AMP in the vesicles was calculated at 5000‐7500 molecules/vesiclc. The presence of cyclic AMP in the synaptic vesicles from presynaptic nerve endings would support a role for this nucleotide in nerve transmission.

Isolation of synaptic vesicles and structural organization of the acetycholine system within brain nerve endings.

Journal of NeurochemistryVolume 10, Issue 4 p. 225-235 ISOLATION OF SYNAPTIC VESICLES AND STRUCTURAL ORGANIZATION OF THE ACETYLCHOLINE SYSTEM WITHIN BRAIN NERVE ENDINGS* E. de Robertis, E. de Robertis Instituto de Anatomía General y Embriologia, Facultad de Ciencias Médicas, Buenos Aires, ArgentinaSearch for more papers by this authorGeorgina Rodriguez de Lores Arnaiz, Georgina Rodriguez de Lores Arnaiz Instituto de Anatomía General y Embriologia, Facultad de Ciencias Médicas, Buenos Aires, ArgentinaSearch for more papers by this authorL. Salganicoff Amanda Pellegrino de Iraldi, L. Salganicoff Amanda Pellegrino de Iraldi Instituto de Anatomía General y Embriologia, Facultad de Ciencias Médicas, Buenos Aires, ArgentinaSearch for more papers by this authorL. M. Zieher, L. M. Zieher Instituto de Anatomía General y Embriologia, Facultad de Ciencias Médicas, Buenos Aires, ArgentinaSearch for more papers by this author E. de Robertis, E. de Robertis Instituto de Anatomía General y Embriologia, Facultad de Ciencias Médicas, Buenos Aires, ArgentinaSearch for more papers by this authorGeorgina Rodriguez de Lores Arnaiz, Georgina Rodriguez de Lores Arnaiz Instituto de Anatomía General y Embriologia, Facultad de Ciencias Médicas, Buenos Aires, ArgentinaSearch for more papers by this authorL. Salganicoff Amanda Pellegrino de Iraldi, L. Salganicoff Amanda Pellegrino de Iraldi Instituto de Anatomía General y Embriologia, Facultad de Ciencias Médicas, Buenos Aires, ArgentinaSearch for more papers by this authorL. M. Zieher, L. M. Zieher Instituto de Anatomía General y Embriologia, Facultad de Ciencias Médicas, Buenos Aires, ArgentinaSearch for more papers by this author First published: April 1963 https://doi.org/10.1111/j.1471-4159.1963.tb05038.xCitations: 365 * This paper was supported by the Consejo Nacional de Investigaciones Cientificas y Técnicas, Buenos Aires, Argentina and Grant B 1549 of the National Institute of Health. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume10, Issue4April 1963Pages 225-235 RelatedInformation

Isolation of synaptic vesicles from nerve endings of the rat brain.

SINCE the discovery of ‘synaptic vesicles’ as a specific component of synapses1,2 it was suggested that they could be associated with the storage and possibly with the synthesis of transmitter substances. This concept was supported by degeneration experiments3 and specially by the stimulation of some cholinergic terminals4, which suggested that within the ending a balance exists between the formation and release of synaptic vesicles. However, the isolation of such a sub-cellular component remained as the most direct approach to the elucidation of the physiological significance and chemical nature of these vesicles.