Preparation of Synaptosomes

Decapitate two rats, remove the cerebral cortices and place them in 30ml of ice cold 320 mM sucrose (solution 1). Scraping off the white matter helps by reducing myelin, one of the main contaminants of synaptosomal preparations. All the following steps should be carried out at 4°C. 

Homogenize the tissue in a glass-Teflon homogenizer, with 10 strokes (up and down) at 800 r.p.m. Avoid formation of foam. 

Decant the supernatant into a fresh tube and spin for 12 min at 14 000 g (e.g., SS34 rotor at 11 000 r.p.m). A pellet is generated in which three layers can be distinguished a dark brown bottom part (mostly mitochondria), a lighter brown middle part (synap-tosomes) and a whitish top layer(mostly myelin). 

Resuspend the pellet in approximately 7-8 ml of solution 2. Try to avoid resuspending too many of the mitochondria. 

This fraction (P2) can be used for poisoning and release experiments if high purity of the synaptosomal fraction is not required. In this case the protein concentration should be determined. Then, the sample should be divided into aliquots containing the desired amount of protein (usually 1-3 mg), recentrifuged at 14 000 g for 12 min and stored as pellets on ice until use. 

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Mouledous, L., Hunt, S., Harcourt, R., Harry, J.L., Williams, K.L. and Gutstein, H.B. (2003) Proteomic analysis of immunostained, laser-capture microdissected brain samples. Electrophoresis 24,296-302. Nagy, A. and Delgado-Escueta, A.V. (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll). J. Neurochem. 43, 1114-1123. 

The procedure for the preparation of synaptosomes and small synaptic vesicles (SSV) from rat brain cortex follows established methods with minor modifications (Schiavo and Montecucco, 1995). 

Preparation of synaptosomes. Synaptosomes were prepared from the brains of male mice (20-30 g Blue Spruce Farms, Altamont, NY) either by a modification of the method of Hajos (8) or by the method of Dodd et al. (9). Both preparations gave qualitatively similar results, but the magnitude of all sodium fluxes per mg of synaptosomal protein was much greater with the latter preparation. A preparation enriched in synaptosomes was prepared from the brains of juvenile rainbow trout (Salmo gairdneri obtained from the New York State Fish Hatchery, Bath, NY) by homogenization in 14 volumes of 0.7 M sucrose, and centrifugation first at 2000 for 10 min and then at 31000 for 30 min. The pellet from the second centrifugation was resuspended in sodium-free buffer identical to that used in previous studies (7) except that it also contained 370 mM sucrose. 

Nagy, A., and Delgado-Escueta, A. V., (1984). Rapid Preparation of Synaptosomes from Mammalian Brain Using Nontoxic Isoosmotic Gradient Material (Percoll), ,/ Neurochem. 43 1114-1123. 

Nagy, A. and Delgado-Escueta, A V (1984) Rapid preparation of synaptosomes from mammalian brain using nontoxic isoosmotic gradient material (Percoll) J Neurochem. 43, 1114—1123. 

Preparation of Synaptosomal Plasma Membranes by Subcellular Fractionation... 

In this chapter, the preparation of synaptosomal plasma membranes using centrifugation techniques will be described in detail. The method here is based on that described previously by Kristjansson et al. (14) with only slight modifications. Section 3.1. outlines protocols for dissection and homogenization of the brain, indicating the parameters most important to obtain synaptosomal plasma membrane preparations of reproducibly high quality. Section 3.2. describes the subcellular fractionation procedure itself, and Section 3.3. outlines a protocol for assessment of yield of the synaptosomal plasma membranes employing a protein determination assay described by Bradford (15). 

Fig. 1. Preparation of synaptosomal plasma membranes. See Section 3.2. for complete details.

Haios, R, Improved method for preparation of synaptosomal fractions of high purity. Brain Res., 93, 485-489 (1975). 

Whittaker, V. P., and Barker, L. A., 1972, The subcellular fractionation of brain tissue with special reference to the preparation of synaptosomes and their component organelles, in "Methods of Neurochemistry", Vol. 2, R. Fried, ed.,... 

Koe B. Molecular geometry of inhibitors of the uptake of catecholamines and serotonin in synaptosomal preparations of rat brain. J. Pharmacol. Exp. Ther. 199 649, 1976. 

Gylys KH, Fein JA, Yang F, Cole CM. 2004. Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human... 

Annunziato L, LeBlanc P, Kordon C, Weiner RI (1980) Differences in kinetics of dopamine uptake in synaptosome preparations of the median eminence relative to other dopaminergically innervated brain regions. 

Booth, R.F. and Clark, J.B. (1978) A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176, 365-370. 

It is interesting to notice that the characterization of the cellular and molecular mechanisms of the star fruit intoxication needs to pass through a group of different experimental protocols among them in vivo and the in vitro bioassays. The correlation between in vivo and in vitro models is then more complex that we should think it is [53]. Thus, the particular case of synaptosomes and GABA and glutamate release and re-uptake, shows neurochemical dynamics associated to star fruit intoxication mechanisms in a preparation which consists of isolated synaptic terminals (44). However, additional studies are needed with brain slices from control brains treated with the AcTx and even the use of ex vivo models in vitro bioassays from tissue after in vivo experiments), for example, in our case, brain slices from treated animals. 

An important aspect of the preparation and isolation of subcellular particles from brain regions is the criteria by which purity is assessed. Electron microscopy of the various subcellular fractions can provide among the best pieces of evidence for the presence in the preparation of the organelles or subcellular fragments of interest. However, a number of biochemical markers (usually enzymes) that have been established to be present in certain fractions can also be assayed to demonstrate the enrichment of the organelle of interest. For instance, acetylcholinesterase is a common marker for synap-tosomes dopamine-P-hydroxylase is a marker for catecholamine storage vesicles within the synaptosome and cytochrome c oxidase is a marker for mitochondria. Most of the enzymatic markers can be assayed routinely. 

Incubation of synaptosome preparations with labelled choline and isolation of the different constituents of the synaptosome by hypo-osmotic rupture and density gradient centrifugation showed labelling of the cytoplasmic ACh but very little labelling of the vesicular ACh (< 2%) (Marchbanks, 1969). Incubation of synapto-somes with labelled ACh showed that the cytoplasmic ACh but not the vesicular ACh was readily exchanged with external ACh. 

Fluvoxamine and clovoxamine are nontricyclic monoamine uptake inhibitors structurally related to the tricyclic smtidepressant noxiptiline (fig. 3). Both fluvoxamine and dovoxamine are trans isomers. Clovoxamine is a potent but nonselective inhibitor of the 5-HT and NE transporter in a synaptosomal preparation of the rat brain frontal cortex (Kj=5.9 and 7.0 nM respectively) and a weak (KjS 720 nM) inhibitor of the DA transporter in synaptosomes of the corpus striatum [26]. 

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