High-Affinity Binding of [3H]5-Hydroxytryptamine to Brain Synaptosomal Membranes: Comparison with [3H]Lysergic Acid Diethylamide Binding
G. FillionJean-Claude RousselleMarie‐Paule FillionDominique BeaudoinM. GoinyJean‐Michel DeniauJoseph Jacob
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Abstract:
[3H]5-Hydroxytryptamine ([3H]5-HT) binds to crude brain membrane preparations at two different sites (Kd = 1-3 nM and 10-30 nM). These two sites are present in a limited number as saturable populations and selectively bind 5-HT and related structures. In the same crude membrane preparations, lysergic acid diethylamide (LSD) also binds at two different sites (Kd = 3-4 nM and 20-30 nM). 5-HT binding is found mostly in fractions enriched in synaptosomal and microsomal membranes; fractions rich in mitochondria or in synaptic vesicles have a low binding capacity. The two serotoninergic sites are physically separable; only high-affinity binding sites are found on purified synaptosomal membranes, whereas both types of sites are present in fractions enriched in microsomal membranes. The interaction between LSD and 5-HT shows that high-affinity binding sites for 5-HT are not identical with those for LSD, since the inhibition of binding of one substance by the other is complex.Keywords:
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A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy
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The human brain is estimated to contain trillions of synaptic nerve terminals. These are the connections between neurons that are responsible for transmitting information and are modified as a result of learning. A valuable tool for studying synapses is the isolated nerve terminal, or synaptosome, which is obtained by homogenizing the brain in such a way that individual synapses pinch off to form metabolically active compartments that can recapitulate neurotransmitter release. This protocol describes the stepwise fractionation of rat brain tissue to yield synaptosomes and synaptic vesicles, which can be used in many different experimental approaches to study the structure and protein composition of the synapse and even dissect the molecular mechanisms of neurotransmission.
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Abstract —Antigen‐antibody crossed electrophoresis has been applied to the study of rat brain synaptosomes and synaptic vesicles. Several antigens could be visualized. By comparison with previously describéd water‐soluble antigens from rat brain, some of the antigens in the synaptosome and the synaptic vesicle preparations were identified; among these were antigens which have been determined as brain‐specific. Furthermore, the antisera against the two subcellular fractions were compared with the anti‐serum against water‐soluble antigens from rat brain.
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Unique Lipid Chemistry of Synaptic Vesicle and Synaptosome Membrane Revealed Using Mass Spectrometry
Synaptic vesicles measuring 30-50 nm in diameter containing neurotransmitters either completely collapse at the presynaptic membrane or dock and transiently fuse at the base of specialized 15 nm cup-shaped lipoprotein structures called porosomes at the presynaptic membrane of synaptosomes to release neurotransmitters. Recent study reports the unique composition of major lipids associated with neuronal porosomes. Given that lipids greatly influence the association and functions of membrane proteins, differences in lipid composition of synaptic vesicle and the synaptosome membrane was hypothesized. To test this hypothesis, the lipidome of isolated synaptosome, synaptosome membrane, and synaptic vesicle preparation were determined by using mass spectrometry in the current study. Results from the study demonstrate the enriched presence of triacyl glycerols and sphingomyelins in synaptic vesicles, as opposed to the enriched presence of phospholipids in the synaptosome membrane fraction, reflecting on the tight regulation of nerve cells in compartmentalization of membrane lipids at the nerve terminal.
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In this paper, our protocol for preparation of brain synaptosomes is described. Synaptosomes are a valuable model system for analysis of structural components of the synapse as well as for investigation of synaptic function. Synaptosomal preparations are necessary for understanding molecular changes at synapses where critical post-translational modifications of synaptic proteins may occur. Not only are synaptosomes rich in synaptic proteins, but they can be used for analyzing uptake of neurotransmitters into synaptic vesicles and for analysis of the involvement of neurotransmitter synthesis and release. Synaptosomes can be stimulated with increased calcium influx to release neurotransmitters. Synaptosomal preparations have been used in characterizing calcium dependent phosphorylation and activation of the GABA synthesizing enzyme GAD65 (L-glutamic acid decarboxylase with molecular weight of 65 kDa). By examining protein complexes on the membrane of synaptic vesicles obtained from synaptosomal preparations, it was possible to characterize the role of GAD65 in the coupled synthesis and vesicular uptake of GABA (γ-aminobutyric acid) culminating in GABA vesicular release, which contributes in an important way to fine-tuning of GABAergic neurotransmission.
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The activity of acetylcholinesterase (ACE) and Na, K-ATPase is distributed among three subfractions of synaptic membranes isolated from light (C) and heavy (D) synaptosomes of the optic area of the rabbit cerebral cortex. The levels of specific activity of both enzymes in C subfractions are similar to those in D subfractions. At the same time the specific activity of ACE and Na, K-ATPase in membrane fractions from both light and heavy synaptosomes is different. Such a biochemistry of subsynaptic components from certain brain structures favors studying a fine morphochemical organization of an isolated nerve terminal and its relationship with the activity of CNS in different functional states.
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Ethanol was shown to cause a redistribution of synaptic vesicles in incubated synaptosomes. While the number of synaptosomes containing synaptic vesicles attached to the presynaptic membrane decreased markedly, an increase in the number of synaptosomes lacking membrane-vesicle associations was observed. The findings support the possibility of a presynaptic action of ethanol and point to the role of membrane-attached vesicles in synaptic transmission.
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