A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions

Dunkley, Peter R.; Heath, John; Harrison, Steven M.; Jarvie, Paula E.; Glenfield, Pamela J.; Rostas, John A. P.

doi:10.1016/0006-8993(88)91383-2

Cited by 362 publications

(231 citation statements)

References 16 publications

Supporting

Mentioning

223

Contrasting

Order By: Relevance

“…Animals were euthanatized by decapitation and their brains were removed and the hippocampi excised on ice. Percoll gradient‐purified synaptosomes were prepared from individual hippocampi as previously described (Dunkley, Jarvie, Heath, Kidd, & Rostas, 1986; Dunkley et al., 1988). Synaptosome extracts were prepared by incubating equal quantities of synaptosomal protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) sample buffer for 30 min at 37°C; under these conditions monomeric SNAREs are denatured, but the 7SC is thermally stable and fails to disassemble (Matveeva et al., 2003).…”

Section: Methodsmentioning

confidence: 99%

Linking kindling to increased glutamate release in the dentate gyrus of the hippocampus through the STXBP5/tomosyn‐1 gene

et al. 2017

View full text Add to dashboard Cite

IntroductionIn kindling, repeated electrical stimulation of certain brain areas causes progressive and permanent intensification of epileptiform activity resulting in generalized seizures. We focused on the role(s) of glutamate and a negative regulator of glutamate release, STXBP5/tomosyn‐1, in kindling.MethodsStimulating electrodes were implanted in the amygdala and progression to two successive Racine stage 5 seizures was measured in wild‐type and STXBP5/tomosyn‐1−/− (Tom−/−) animals. Glutamate release measurements were performed in distinct brain regions using a glutamate‐selective microelectrode array (MEA).ResultsNaïve Tom−/− mice had significant increases in KCl‐evoked glutamate release compared to naïve wild type as measured by MEA of presynaptic release in the hippocampal dentate gyrus (DG). Kindling progression was considerably accelerated in Tom−/− mice, requiring fewer stimuli to reach a fully kindled state. Following full kindling, MEA measurements of both kindled Tom+/+ and Tom−/− mice showed significant increases in KCl‐evoked and spontaneous glutamate release in the DG, indicating a correlation with the fully kindled state independent of genotype. Resting glutamate levels in all hippocampal subregions were significantly lower in the kindled Tom−/− mice, suggesting possible changes in basal control of glutamate circuitry in the kindled Tom−/− mice.ConclusionsOur studies demonstrate that increased glutamate release in the hippocampal DG correlates with acceleration of the kindling process. Although STXBP5/tomosyn‐1 loss increased evoked glutamate release in naïve animals contributing to their prokindling phenotype, the kindling process can override any attenuating effect of STXBP5/tomosyn‐1. Loss of this “braking” effect of STXBP5/tomosyn‐1 on kindling progression may set in motion an alternative but ultimately equally ineffective compensatory response, detected here as reduced basal glutamate release.

show abstract

Section: Methodsmentioning

confidence: 99%

Linking kindling to increased glutamate release in the dentate gyrus of the hippocampus through the STXBP5/tomosyn‐1 gene

et al. 2017

View full text Add to dashboard Cite

show abstract

“…Synaptic mitochondria were suspended in isolation medium containing 1 mg/mL BSA to remove free fatty acid from mitochondrial membranes. The purity of synaptosome and mitochondria isolated from the Percoll gradient was examined with electron microscopy and demonstrated in our previous studies [40][41][42][43].…”

Section: Isolation Of Synaptic Mouse Brain Mitochondriamentioning

confidence: 95%

“…Mice were decapitated, and brains were removed and homogenized in ice-cold isolation medium containing 225 mM mannitol, 75 mM sucrose, 5 mM HEPES, and 1 mM EGTA, pH 7.4, at 4°C. As described previously [40], a slightly modified procedure by Dunkley et al [41] was employed to separate synaptosomes and non-synaptic mitochondria. Briefly, after low-speed spin (1300 × g for 3 min) of the brain homogenate, the supernatant was centrifuged at 21 000 × g for 10 min.…”

Section: Isolation Of Synaptic Mouse Brain Mitochondriamentioning

confidence: 99%

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

et al. 2008

View full text Add to dashboard Cite

By employing a capillary ITP (CITP)/CZE-based proteomic technology, a total of 1795 distinct mouse Swiss-Prot protein entries (or 1705 nonredundant proteins) are identified from synaptic mitochondria isolated from mouse brain. The ultrahigh resolving power of CITP/CZE is evidenced by the large number of distinct peptide identifications measured from each CITP fraction together with the low peptide fraction overlapping among identified peptides. The degree of peptide overlapping among CITP fractions is even lower than that achieved using combined CIEF/nano-RP LC separations for the analysis of the same mitochondrial sample. When evaluating the protein sequence coverage by the number of distinct peptides mapping to each mitochondrial protein identification, CITP/CZE similarly achieves superior performance with 1041 proteins (58%) having 3 or more distinct peptides, 233 (13%) having 2 distinct peptides, and 521 (29%) having a single distinct peptide. The reproducibility of protein identifications is found to be around 86% by comparing proteins identified from repeated runs of the same mitochondrial sample. The analysis of the mouse mitochondrial proteome by two CITP/CZE runs results in the detection of 2095 distinct mouse Swiss-Prot protein entries (or 1992 nonredundant proteins), corresponding to 59% coverage of the updated Maestro mitochondrial reference set. The collective analysis from combined CITP/CZE and CIEF-based proteomic studies yields the identification of 2191 distinct mitochondrial protein entries (or 2082 nonredundant proteins), corresponding to 76% coverage of the MitoP2-database reference set.

show abstract

“…Tissue was homogenized (0.1 g of tissue\ml) in gradient solution (0.32 M sucrose\1 mM EDTA\0.25 mM dithiothreitol, adjusted to pH 7.4 with 1 M NaOH). Synaptosomes were isolated by Percoll2 gradient centrifugation [20] and resuspended in HBSS (124 mM NaCl\4 mM KCl\1.2 mM MgSO % \10 mM glucose\25 mM Hepes, adjusted to pH 7.4 with 5 M NaOH) at approx. 5.0 mg\ml protein, as described previously [11].…”

Section: Preparation Of Synaptosomesmentioning

confidence: 99%

Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer

Reis

Prado

Kalapothakis

et al. 1999

Biochemical Journal

View full text Add to dashboard Cite

Glutamate concentration increases significantly in the extracellular compartment during brain ischaemia and anoxia. This increase has an important Ca2+-independent component, which is due in part to the reversal of glutamate transporters of the plasma membrane of neurons and glia. The toxin phoneutriatoxin 3-4 (Tx3-4) from the spider Phoneutria nigriventer has been reported to decrease the evoked glutamate release from synaptosomes by inhibiting Ca2+ entry via voltage-dependent Ca2+ channels. However, we report here that Tx3-4 is also able to inhibit the uptake of glutamate by synaptosomes in a time-dependent manner and that this inhibition in turn leads to a decrease in the Ca2+-independent release of glutamate. No other polypeptide toxin so far described has this effect. Our results suggest that Tx3-4 can be a valuable tool in the investigation of function and dysfunction of glutamatergic neurotransmission in diseases such as ischaemia.

show abstract

A rapid Percoll gradient procedure for isolation of synaptosomes directly from an S1 fraction: homogeneity and morphology of subcellular fractions

Cited by 362 publications

References 16 publications

Linking kindling to increased glutamate release in the dentate gyrus of the hippocampus through the STXBP5/tomosyn‐1 gene

Linking kindling to increased glutamate release in the dentate gyrus of the hippocampus through the STXBP5/tomosyn‐1 gene

Application of capillary isotachophoresis‐based multidimensional separations coupled with electrospray ionization‐tandem mass spectrometry for characterization of mouse brain mitochondrial proteome

Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer

Contact Info

Product

Resources

About