Isolation of Synaptic Vesicles from Nerve Endings of the Rat Brain

Robertis, E. De; Arnaiz, Georgina Rodrı́guez de Lores; Iraldi, Amanda Pellegrino de

doi:10.1038/194794a0

Cited by 111 publications

(23 citation statements)

References 7 publications

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“…The dimensions and appearance of the small vesicles, which are particularly numerous in microsomal pellets obtained by means of the "fast" centrifugation procedure (Fig. 3), suggest their identity with synaptic vesicles, de Robertis et al (15), Whittaker (17), and Johnson and Whittaker (37) have recently studied the effect of hypotonicity on preparations of isolated nerve ending particles and have found that the synaptic vesicles contained within the nerve ending particles are resistant to rupture even after particles have been exposed for 1 hour to 0.06 M sucrose. This hypotonic treatment did result, however, in the total rupture of the nerve ending particles themselves.…”

Section: Discussionmentioning

confidence: 99%

“…These studies showed that the maximal degree of attachment of glutamine synthetase to the membranes of the microsomal fraction was obtained when sucrose-free media were used for the isolated of the fraction. Recently de Robertis et al (15,16) and Whittaker et al (17,18) have subjected cerebral fractions containing nerve ending particles to osmotic shock by suspension in distilled water. This has led to the isolation of relatively clean preparations of synaptic vesicles.…”

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Morphological and Biochemical Correlates of Cerebral Microsomes

Verster

Sellinger

Harkin

1965

The Journal of Cell Biology

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Microsomal fractions, both homogeneous in appearance and functionally operative, were isolated from a homogenate of rat cerebral cortex by fractionation in water. The preparations thus obtained contain the membranous elements of the endoplasmic reticulum, synaptic vesicles, and ribosomes. Esterase, ATPase, and glutamine synthetase were found to be present and fully functional in the microsomal fractions isolated in water. The contamination of the water-isolated microsomal fractions by mitochondria and lysosomes was found to be considerably lower than in microsomal pellets isolated in sucrose. The contamination by nerve ending particles, as judged by electron microscopy and by the levels of soluble lactic dehydrogenase entrapped in the cytoplasm of the particles, was also low. Most of the contamination by mitochondria and nerve ending particles could be removed by treatment of the microsomal pellet with 150 mM NaC1. Resistant to elution by this treatment is the lysosomal contamination as well as microsomal esterase and ATPase. Glutamine synthetase, on the other hand, was almost totally solubilized. Microsomal preparations isolated in water are also shown to contain amounts of protein, RNA, phospholipid, and ganglioside comparable to those found in microsomal preparations isolated in sucrose.The microsomal fraction isolated from mammalian tissues by high speed centrifugation has not been unequivocally characterized, for it has often been found to consist of disrupted fragments of intracellular materials, including the cellular envelope, that connot readily be compared with, or identified with, intact structures (1, 2). This is particularly true for fractions isolated from brain, owing to the complex and heterogeneous organization of this organ. In contrast to brain is the liver, for instance, from which fractions can be isolated which are much more homogeneous as a eonsequence of the less complex organization of this organ. Palade and Siekevitz (3) have demonstrated that in rat liver the microsomal fraction consists preponderantly of vesicular and tubular fragments of the intracellular endoplasmic reticulum and of ribosomal granules. Hanzon and Toschi (4) described a similar composition for the microsomal fraction of brain tissue. However, more recent studies by de Robertis et al. (5) and by Whittaker (6) have revealed that the microsomal fraction of brain, as usually isolated in sucrose media, consists of a rather heterogeneous popula-69

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Morphological and Biochemical Correlates of Cerebral Microsomes

Verster

Sellinger

Harkin

1965

The Journal of Cell Biology

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“…The synaptic vesicles, as well as the cell itself, are surrounded with lipoprotein membranes (de Robertis, de Lores Arnaiz & de Iraldi, 1962;Whittaker, Michaelson & Kirkland, 1964), and there is probably a common physico-chemical mechanism which underlies the release of transmitter substances at different synapses.…”

Section: Introductionmentioning

confidence: 99%

A study of the mechanism of quantal transmitter release at a chemical synapse

Blioch¹,

Glagoleva²,

Liberman³

et al. 1968

The Journal of Physiology

189

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SUMMARY1. The nerve-muscle preparation of the cutaneous pectoris of the frog has been used to study quantal transmitter release.2. When the osmotic pressure of the external solution is raised 15-2 fold, the frequency of miniature end-plate potentials (m.e.p.p.s) rises by 1-5-2 orders of magnitude. This effect is independent of the presence of Ca2+ ions and of the nature of the substances by which the osmotic pressure has been increased.3. In Ca2+ free hypertonic solution the nerve impulse still invades the nerve terminals but does not alter the frequency of the m.e.p.p.s.4. The arrival of the impulse in the terminals causes an immediate increase in the rate of quantal release, provided divalent cations are present whose passage through the axon membrane is facilitated by excitation (Ca2+, Sr2+, Ba2+).5. Divalent cations which penetrate only slightly (Mg2+, Be2+) lower the frequency of m.e.p.p.s and suppress the end-plate potential (e.p.p.) evoked by an impulse, in the presence of Ca2+ ions. Be2+ is a more effective inhibitor than Mg2+.6. In Ca2+ free solutions, adding Mg2+ causes an increase in the frequency of m.e.p.p.s evoked by depolarization of the nerve endings or by treatment with ethanol.7. The trivalent cation La3+ is more effective than divalent cations are in increasing the frequency of m.e.p.p.s. The tetravalent cation T-4+ also raises the m.e.p.p. frequency.8. The observations summarized in paragraphs 2-7 indicate that the frequency of m.e.p.p.s at a constant temperature depends only on the concentration of uni-, di-and trivalent cations inside the nerve ending. It ZHANNA L. BLIOCH AND OTHERS is suggested that the internal cation concentration influences the adhesion between synaptic vesicles and the membrane of the nerve ending.9. For a model experiment, artificial phospholipid membranes have been used to study the effect of uni-, di-, tri-and tetravalent cations on the adhesion process. At pH 7-7*4, the time required for adhesion to take place decreases with increasing cation concentration in the bath. Ca2+ ions are 100-1000 times more effective than K+ ions; La3+ and Th4+ ions are still more effective. The 'adhesion time' decreases when the pH is lowered; it increases greatly with lowering of temperature.10. The hypothesis is put forward that the mutual adhesion of artificial vesicles made of phospholipid membranes, and the adhesion between synaptic vesicles and the membrane of the nerve ending arise by a common mechanism. In both cases, the important factor is the influence of cations on the electric double layer at the membrane surface.

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“…However Segawa's report (17) in which when MAGI was injected into the reserpine treated rabbit, remarkable restora tion of increase in 5-HT concentration after 5-HTP administration was obtained in P2 fraction but not in P3-fraction might suggest that some different mechanism exists. The effect of medium pH on 5-HT uptake by P2-fraction has been discussed by many Electron microscopic observations revealed that synaptic vesicles have vesicle mem brane (1,19,20). There are two possible mechanisms for the binding of 5-HT to sv: 1) 5-I1T is solely bound to the outer surface of vesicle membrane electrically or chemically.…”

Section: Ef L'ct Of Reserpinementioning

confidence: 99%

Uptake and Storage Mechanism of 5-Hydro-Xytryptamine in Rabbit Brain Stem and Effect of Reserpine

1971

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In a previous paper (1) to clarify the physiological significance of endogenous 5-hy droxytryptamine (5-HT) the subcellular distribution of 5-HT in rabbit brain stem has been studied and the electron microscopical structures of the fractions which contain 5-HT have been examined. 5-HT was principally found in P2-fraction which consisted largely of mitochondria and nerve-ending particles (NEPs). After density-gradient centrifuga tion 5-HT was found to be comparatively highly concentrated in NEPs-fraction (P2B fraction). On the other hand suspension of P2-fraction in hypo-osmotic medium and subsequent differential centrifugation resulted in high concentration of 5-HT in synaptic vesicles (sv) fraction although considerable amount of 5-HT was also found in P2D and P,S-fraction. In general 5-HT in brain stem was not so highly concentrated in specific fraction as with the distribution of acetylcholine (ACh) (2, 3) but rather diffusibly distrib uted among several fractions.In this paper the role of 5-HT in synaptic transmission, especially in central nervous system was investigated mainly through the experiments on 5-HT uptake by subcellular fraction of rabbit brain stem. The chemical transmission at synapses consists of the fol lowing consecutive steps: 1) release of transmitter by synaptic impulses. 2) combination of transmitter with postsynaptic receptor. 3) generation of impulses by postsynaptic po tential. It appears that the study into the formation, uptake, storage, release and in activation of 5-HT is necessary for full and accurate understanding of mechanism of chemi cal transmission, especially when there is no direct evidence for that 5-HT is synaptic trans mitter in central nervous system. Segawa and Kuruma (4), Segawa et al. (5) in this la boratory have already shown that NEPs or sv of brain, when incubated in a medium con taining 5-HT, could take up 5-HT from the medium and some drugs could inhibit the uptake of 5-HT. However we have as yet very little information as to whether NEPs or sv are specific uptake and storage site for 5-HT in situ. Also the mechanisms with which amine can be taken up by synapses and drug inhibits the uptake are poorly under Reprints requests and enquiries should be sent to Tomio Segawa,

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Isolation of Synaptic Vesicles from Nerve Endings of the Rat Brain

Cited by 111 publications

References 7 publications

Morphological and Biochemical Correlates of Cerebral Microsomes

Morphological and Biochemical Correlates of Cerebral Microsomes

A study of the mechanism of quantal transmitter release at a chemical synapse

Uptake and Storage Mechanism of 5-Hydro-Xytryptamine in Rabbit Brain Stem and Effect of Reserpine

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