A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain.
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ABSTRACT:
SUBMITTER: Booth RF
PROVIDER: S-EPMC1186243 | biostudies-other | 1978 Nov
REPOSITORIES: biostudies-other
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Project description:In order to measure the activity of neuronal mitochondria, a representative proof of neuronal processes, physiologically relevant mitochondrial samples need to be gained as simply as possible. Existing methods are, however, either for tissue samples of large size and/or homogenous microstructures only, or are not tested for mitochondrial function measurements. In the present article we describe a gradient fractionation method to isolate viable and well-coupled mitochondria from relatively heterogeneous histological microstructures such as the hypothalamus. With this new method, we are able to isolate a sufficient amount of functional mitochondria for determination of respiratory activity, in a short period of time, using affordable equipment. •Verified by electron microscopy, our method separates highly enriched and well-preserved perikaryal and synaptosomal mitochondria. Both fractions contain minimal cell debris and no myelin. Respiratory measurements (carried out by Clark-type electrode) confirmed undisturbed mitochondrial function providing well-evaluable records. The demonstrated protocol yields highly viable mitochondrial subfractions within 3 h from small brain areas for high-precision examinations. Using this procedure, brain regions with relatively heterogeneous histological microstructure (hypothalamus) can also be efficiently sampled.•Up to our present knowledge, our method is the shortest available procedure with the lowest sample size to gain debris-free, fully-viable mitochondria.
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Project description:Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.
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Project description:RationaleNovel synthetic "bath salt" cathinones continue to appear on the street as abused and addictive drugs. The range of subjective experiences produced by different cathinones suggests that some compounds have primarily dopaminergic activity (possible stimulants) while others have primarily serotonergic activity (possible empathogenics). An understanding of the structure activity relationships (SARs) of these compounds will help in assessing the likely behavioral effects of future novel structures, and to define potential therapeutic strategies to reverse any reinforcing effects.ObjectivesA series of methcathinone analogs was systematically studied for their activity at the dopamine and serotonin transporters. Compound structures varied at the aromatic group, either by substituent or by replacement of the phenyl ring with a naphthalene or indole ring.MethodsA novel, high-yielding synthesis of methcathinone hydrochlorides was developed which avoids isolation of the unstable free bases. Neurotransmitter transporter release activity was determined in rat brain synaptosomes as previously reported. Compounds were also screened for activity at the norepinephrine transporter.ResultsTwenty-eight methcathinone analogs were analyzed and fully characterized in dopamine and serotonin transporter release assays. Compounds substituted at the 2-position (ortho) were primarily dopaminergic. Compounds substituted at the 3-position (meta) were found to be much less dopaminergic, with some substituents favoring serotonergic activity. Compounds substituted at the 4-position (para) were found to be far more serotonergic, as were disubstituted compounds and other large aromatic groups. One exception was the fluoro-substituted analogs which seem to favor the dopamine transporter.ConclusionsThe dopaminergic to serotonergic ratio can be manipulated by choice of substituent and location on the aromatic ring. It is therefore likely possible to tweak the subjective and reinforcing effects of these compounds by adjusting their structure. Certain substituents like a fluoro group tend to favor the dopamine transporter, while others like a trifluoromethyl group favor the serotonin transporter.
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