Pinched-off Nerve Endings Research Articles

Inhibition of vesicular glutamate storage and exocytotic release by Rose Bengal.

It had been thought that quantal size in synaptic transmission is invariable. Evidence has been emerging, however, that quantal size can be varied under certain conditions. We present evidence that alteration in vesicular [(3)H]L-glutamate (Glu) content within the synaptosome (a pinched-off nerve ending preparation) leads to a change in the amount of exocytotically released [(3)H]Glu. We found that Rose Bengal, a polyhalogenated fluorescein derivative, is a quite potent membrane-permeant inhibitor (K(i) = 19 nM) of glutamate uptake into isolated synaptic vesicles. This vesicular Glu uptake inhibition was achieved largely without affecting H(+)-pump ATPase. We show that various degrees of reduction elicited by Rose Bengal in [(3)H]Glu in synaptic vesicles inside the synaptosome result in a corresponding decrease in the amount of [(3)H]Glu released in a depolarization- (induced by 4-aminopyridine) and Ca(2+)-dependent manner. In contrast, fluorescein, the halogen-free analog of Rose Bengal, which is devoid of inhibitory activity on vesicular [(3)H]Glu uptake, failed to change the amount of exocytotically released [(3)H]Glu. These observations suggest that glutamate synaptic transmission could be altered by pharmacological intervention of glutamate uptake into synaptic vesicles in the nerve terminal, a new mode of synaptic manipulation for glutamate transmission.

Human brain N-methyl-D-aspartate receptors regulating noradrenaline release are positively modulated by HIV-1 coat protein gp120.

To investigate the effect of HIV-1 gp120 on the function of glutamate receptors of the N-methyl-D-aspartate (NMDA) type in the human brain. The monitoring of neurotransmitter release from superfused isolated nerve endings is widely recognized as a technique appropriate for the study of neurotransmitter release and to attribute a precise localization to the site(s) of action of drugs able to modulate release. Synaptosomes (pinched-off nerve endings) were prepared from fresh human brain tissue samples removed during neurosurgery, labelled with [3H]-noradrenaline and superfused at a rate of 0.5 ml/min with NMDA in the presence of gp41, gp160, gp120 or the V3 loop, with or without NMDA receptor antagonists. Fractions of superfusate were collected and measured for radioactivity. NMDA elicited a glycine-sensitive release of [3H]-noradrenaline from human brain synaptosomes. HIV-1 gp120 potentiated the NMDA (1 mM)-evoked [3H]-noradrenaline release (maximal effect approximately 110% at 1 nM). The release elicited by NMDA plus gp120 was prevented by the classical NMDA receptor antagonists dizocilpine or 7-chlorokynurenic acid, as well as by memantine. The potentiation by gp120 of the NMDA-evoked [3H]-noradrenaline release was mimicked by gp160 but not by gp41. The effect of gp120 was retained by the V3 loop. Finally, gp120 reversed (1 nM) and surmounted (10nM) the antagonism by 10 microM 7-chlorokynurenate of the NMDA-evoked [3H]-noradrenaline release. gp 120 binds directly through the V3 loop at noradrenergic axon terminals in human brain neocortex and may alter the function of presynaptic NMDA receptors mediating regulation of noradrenaline release.

Cholinergic properties of pinched-off synaptic nerve endings from the central nervous system of the cockroachPeriplaneta americana

The preparation and cholinergic properties of a subcellular fraction, enriched in pinched-off nerve-endings (synaptosomes) from the central nervous system of the cockroach Periplaneta americana, are described. The endings retained their cytoplasmic components, as shown by the presence of marker enzymes and by ultrastructural examination. A carrier-mediated, high-affinity uptake of [3H] choline into the synaptosomes was demonstrated, and this uptake was saturable, depended on a Na+-gradient, and was inhibited by hemicholinium-3. It had an apparent Km value of 0.6 (±0.1) μM, and a Vmax of 20.5 (±2.5) pmol min−1 per mg of protein. The high-affinity [3H]choline uptake was associated with the synthesis of [3H]phosphocholine and [3H]O-acetylcholine. The rate of [3H]choline uptake in synaptosomes was increased by DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane] at 100 nM concentration, and this increase was inhibited by tetrodotoxin, while neostigmine appeared to be a potent inhibitor (I50 = 10 pM) of the DDT-activated uptake of [3H]choline. The site of action of the insecticides was specifically on the pre-synaptic nerve terminals because the synaptosomes preparation did not retain the post-synaptic membrane of the original nerve-endings. Cockroach synaptosomes provided a useful in-vitro preparation for studying the effects of insecticides on the pre-synaptic nerve endings.

A cytoskeleton from synaptosomes which is rich in a spectrin-like protein

There is an an increasing amount of evidence for the linking of intracellular cytoskeletal structures to membrane proteins of the plasma membrane in both the lymphocyte and erythrocyte and such complexes can be separated from other membrane components by extraction with non-ionic detergents (Koch & Smith, 1978; Branton et al., 1981; Mescher et al., 1981; Geiger, 1983). The purpose of the present work is to identify and characterize cytoskeletonmembrane links in synaptosomes, pinched-off nerveendings which can be prepared from brain by subcellular fractionation (Whittaker, 1969). Snaptosomes were prepared from rat cerebral cortex as described previously (Hesketh et al., 1977), harvested by centrifugation and resuspended in 0.32~-sucrose. After incubation in 10m-Tris/O. 15~-NaCl/I% Nonidet P40/1 mM-MgC12/ 1 mM-EGTA/l m-phenylmethylsulphonyl fluoride for 30min at M 0 C (Hesketh et al., 1983), the samples were centrifuged at 35000g for 30min and the pellet of insoluble cytoskeletal proteins was either resuspended in a small volume of l0m-Tris buffer, pH8.0, or fixed in 2.5% glutaraldehyde in 30mwPipes (14piperazinediethanesulphonic acid), pH 7.2, and processed for electron microscopy. SDS/polyacrylamide-gel electrophoresis was carried out using 7.5% acrylamide gels in a discontinuous system with Tris/glycine buffer (Laemmli, 1970). After electrophoresis gels were either stained with Coomassie Brilliant Blue to reveal proteins or the proteins were transferred to nitrocellulose paper (Towbin et al., 1979) for either detection of glycoproteins using a biotin/ avidin system (Gordon-Weeks, 1983) or immunological detection of spectrin-like protein. SDS/polyacrylamide-gel electrophoresis showed the Nonidet residue to contain seven major polypeptide bands, the approximate molecular weights of which were 225, 158, 78,70, 56,40 and 45 kilodaltons. Densitometry showed the 225-kilodalton component to comprise about 50% of the protein content. Transfer of the protein to nitrocellulose sheets and incubation with antibodies against spectrin showed this component to be a spectrin-like protein (Hesketh er al., 1983). This protein is presumably identical to fodrin, a spectrin-like protein recently isolated from brain (Burridge et ul., 1982). PSD contain a doublet of polypeptides of molecular weight very similar to fodrin and which react with anti-fodrin antibodies (Carlin et al., 1983). However, the Nonidet synaptosome cytoskeleton appears quite distinct from such PSD; the polypeptide composition is quite different, there being much more spectrin present in the Nonidet residue and very little of the 51-kilodalton PSDspecific protein. In addition the residue contained very little PSD material as judged by electron microscopy but consisted of fine filaments and amorphous, electron-dense material Using the biotinfavidin blotting technique several concanavalin A-binding glycoproteins were found present in the Nonidet residue; the major binding protein was of 178 kilodaltons, with other components of approximate molecular weight 200, 157, 141, 116, 104 and 58 kilodaltons. In conclusion Nonidet treatment of synaptosomes has allowed the preparation of a synaptosome cytoskeleton which contains primarily fodrin, a spectrin-like protein, and also actin. By comparison with the erythrocyte, it is

Giant synaptosomes.

Investigations using synaptosomes, pinched-off nerve ending particles from brain, have greatly improved our knowledge of presynaptic function. However, these structures, like most nerve endings, are too small to be penetrated with microelectrodes. We have treated synaptosomal preparations from rat brain with a neutral protease and obtained fused structures large enough to be recorded from directly with microelectrodes; we report here that these particles (30-250 microM in diameter) contain mitochondria and structures resembling synaptic vesicles, morphological features characteristic of synaptosomes. These 'giant synaptosomes' have resting membrane potentials in the range -45 to -76 mV, are depolarized by increasing concentrations of K+, show responses to a variety of neuroactive substances and exhibit active membrane responses to depolarizing current pulses. These results suggest that this preparation will be of value in further studies of nerve terminal function.

Sodium channels in presynaptic nerve terminals. Regulation by neurotoxins.

Regulation of Na+ channels by neurotoxins has been studied in pinched-off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle.

The effects of harmaline on GABA fluxes in pinched-off nerve endings

The effects of harmaline on GABA fluxes in pinched-off nerve endings

Depletion of Synaptosomal Neurotransmitter Pool by Sudden Cooling

An increasing number of reports deal with the uptake, storage and release of putative neurotransmitters by synaptosomes (pinched-off nerve endings)1–6. In most of these, incubated synaptosomes were rapidly cooled to 0°–4° C and washed, in order to measure the uptake or to study the release of the accumulated neurotransmitter1,6,7–10. Synaptosomes are usually collected by filtration through cellulose ester filters (‘Millipore’), followed by washing with ice-cold medium7–10. We noted previously a large loss of accumulated and of endogenous GABA and glutamate during collection of synaptosomes on filters11 and here we suggest that this loss is caused by an alteration of the membrane structure induced by sudden cooling and that the different depletion observed with various neurotransmitters may depend on their intrasynaptosomal compartmentation.

Specific glycine--accumulating synaptosomes in the spinal cord of rats.

Subcellular fractionation of rat spinal cord on continuous sucrose density gradients provides evidence for the existence of a specific synaptosomal fraction (enriched in pinched-off nerve endings) that accumulates glycine selectively by way of a high-affinity transport system. The particles in this fraction sediment to a less-dense portion of sucrose gradients than do particles that accumulate neutral, basic, aromatic, and acidic amino acids. Particles accumulating gamma-aminobutyric acid are even less-dense than those storing exogenous glycine. The glycine-specific synaptosomal fraction also exists in the brain stem but not in the cerebral cortex. These findings provide neurochemical support for the suggestion that glycine has a specialized synaptic function, perhaps as neurotransmitter, in mammalian spinal cord.

A unique synaptosomal fraction, which accumulates glutamic and aspartic acids, in brain tissue.

Subcellular fractionation of rat cerebral cortical slices on sucrose density gradients provides evidence for the existence of a unique synaptosomal fraction (enriched in pinched-off nerve endings) that selectively accumulates glutamic and aspartic acids. The particles in this fraction sediment to a less dense portion of sucrose gradients than do particles that accumulate aromatic, basic, and neutral (large and small) amino acids. Particles that store gamma-aminobutyric acid are even less dense than those that contain exogenous glutamic and aspartic acids. The distribution of endogenous glutamic acid encompasses both that of exogenous glutamic acid and that of the neutral and basic amino acids. These findings provide neurochemical support for the suggestion that glutamic and/or aspartic acid has a specialized synaptic function, perhaps as a neurotransmitter, in the mammalian brain.

Effects of L-dopa on efflux of cerebral monoamines from synaptosomes.

THE mechanism of the effects of large doses of L-dopa on brain function is ill-defined. This form of precursor loading produces marked increases in brain dopamine, little or no change in noradrenaline, and a substantial decrease in serotonin with an initial increase in 5-H indolyl-3-acetic acid (5-HIAA)1–3. These observations suggest that serotonin may be released by L-dopa or its metabolites. Isolated synaptosomes (pinched-off nerve endings) actively accumulate exogenously labelled amines into structures which normally contain these amines4–6. Although the distribution of the labelled amine in the endogenous stores is not clearly defined, these structures seem to provide valid models for nerve endings in brain and may afford a direct and sensitive means of assessing the influence of drugs on the release of neurohumours. Using this technique, we have found that L-dopa profoundly affects the efflux of cerebral monoamines.

Effects of lithium and of pH on synaptosomal metabolism of noradrenaline.

CURRENT hypotheses about mania and depression are that they correspond to greater or lesser availability of catecholamines at central synapses1, or to changes in electrolyte and water distribution2. The mode of action of lithium salts, which seem to be effective at least in mania, has been related to both hypotheses3. There are several findings regarding catecholamine metabolism. They include a disappearance of brain noradrenaline after inhibition of tyrosine hydroxylase which is more rapid following Li+ treatment4; a shift in brain metabolism of noradrenaline from extracellular methylation (by catechol-O-methyl transferase), toward intraneuronal deamination (by monoamine oxidase)5; and a decreased rate of release of 3H-noradrenaline during electrical stimulation of brain slices in vitro6. Evidence regarding effects of Li+ on active uptake of noradrenaline into neurones, a major means of inactivation of catecholamine released at synapses, is somewhat in conflict. One study7 reported enhanced uptake in vitro of 3H-noradrenaline into synaptosomes (“pinched-off nerve endings”) prepared after the administration of large amounts of unneutralized Li2CO3. This finding has not been confirmed, and there is evidence that uptake of 3H-noradrenaline into intact brain5, or brain slices6, may not be altered by prior treatment of rats with LiCl. Thus questions arise as to the effect of carbonate ion or pH on amine uptake. These findings, together, suggest that lithium may tend to keep noradrenaline in a pre-synaptic location, while increasing catabolism, particularly by oxidative deamination. The net effect would be decreased availability of this putative transmitter at central synapses, an interpretation consistent with the antimanic effects of lithium.

Isolated nerve endings (neurosecretosomes) from the posterior pituitary. Partial separation of vasopressin and oxytocin and the isolation of microvesicles.

Subcellular fractions of the bovine posterior pituitary, including one composed almost exclusively of pinched-off nerve endings (neurosecretosomes), were characterized electron microscopically, hormonally, and enzymically. 15% of the nerve terminals in the gland were isolated as neurosecretosomes, as estimated from determinations of lactic dehydrogenase, a soluble, cytoplasmic enzyme. Neurosecretosomes were subdivided into three fractions by density-gradient centrifugation. The three subfractions, each shown to be nearly homogeneous populations of neurosecretosomes by means of electron microscopic and enzymic criteria, differed from each other in their vasopressin/oxytocin (VP/OT) ratios. The VP/OT ratio increased from the lightest to the densest fraction, indicating that VP is localized to denser and OT to lighter neurosecretosomes; similar results have been obtained previously for subfractions of neurosecretory granules (NSG). No morphological differences were apparent in neurosecretosomes among the three subfractions. Although complete separation of VP and OT was not achieved, the findings suggest that VP and OT are each stored in a different species of nerve ending and support the hypothesis that a given neurosecretory cell synthesizes, stores, and secretes only one of the peptide hormones. Microvesicles, 40-80 mmicro diameter and contained in typical neurosecretory cell terminals, are believed to be degradation products of membrane ghosts of depleted NSG; electron micrographs indicative of this transformation are presented. A fraction rich in microvesicles, but containing some NSG membranes, was prepared by density-gradient centrifugation of an osmolysate of neurosecretosomes. Smaller, apparently nonneurosecretory nerve endings, lacking NSG but filled with small vesicles, are occasionally seen in sections from whole gland. The vesicles in these atypical posterior pituitary nerve endings may be true neurohumor-containing, "synaptic" vesicles.

The yield of synaptosomes from the cerebral cortex of guinea pigs estimated by a polystyrene bead "tagging" procedure.

The number of synaptosomes (pinched-off nerve endings) produced/g guinea pig cortex on homogenizing this tissue under defined conditions is estimated to be in the region of 4×1011 using two different polystyrene bead “tagging” procedures. This is the same order of magnitude as the number of nerve endings/g cortex calculated from histological estimates given in the literature of the number of neurones in the cortex and the extent of their cortical connexions.

Excitatory and depressant properties of certain brain fractions.

ALTHOUGH it is generally believed that at most synapses in the brain ‘transmitter’ substances are released from nerve endings to excite or inhibit the post-synaptic cells, the identity of the transmitters involved is still very much in doubt. Gray and Whittaker1 have shown that a fraction of brain homogenates can be prepared containing mainly pinched-off nerve endings. More recently it has been possible to release apparently intact synaptic vesicles from such a nerve-ending fraction and to separate them from other membrane structures by density-gradient centrifugation2. The purified vesicle fraction contains acetylcholine but not the other components of the acetylcholine system. Since nerve endings can be expected to contain substantial amounts of various transmitter substances, we have examined the excitatory and depressant effects produced by various subtractions of guinea-pig brain, when applied from micropipettes to single units in the guinea-pig's cerebral cortex.

The seperation of catecholamine storage vesicles from rat heart

The concept that acetylcholine is released from certain presynaptic nerve terminals in packets or quanta of uniform size led to the speculation that the transmitter might be stored in synaptic vesicles (1,2). The isolation of acetylcholine-containing pinched-off nerve endings (synaptosomes) from brain (3) and more recently the synaptic vesicles themselves (4,5) have stimulated investigations of the subcellular localization of biogenic amines in peripheral and central nervous tissue. In guinea pig brain the storage particles that contain norepinephrine (NE) and 5-hydroxytryptamine have been found to differ from those containing acetylcholine in that the first two have sedimentation characteristics more dense than the acetylcholine containing vesicles (6). Recent studies have shown that catecholamines in heart (7,8), brain (6,9) and splenic nerve (10) are stored in subcellular vesicles. Potter and Axelrod (7,11) reported that 30 minutes after the intravenous administration of DL-norepinephrine-7-H 3 to rats a sizable fraction of the labeled compound in several peripheral tissues appeared in a fraction associated with microsomes. These authors suggested that the radioactive NE is taken up from the circulation and fixed at the anatomical sites which bind endogenous NE. The morphological characterization of these sites was not possible with the Potter and Axelrod preparation. A modification of the density gradient centrifugation procedure of Potter and Axelrod (11) has made it possible to isolate NE containing particles of considerably greater homogeneity. Both endogenous NE and administered H 3NE are localized in a particulate fraction which equilibrates in that part of the gradient which corresponds in density ot 0.45–0.5 M sucrose. Electronmicroscopic examination of this fraction has revealed numerous small vesicles approximately 500 Å in diameter, some microsomal fragments but essentially no other subcellular organelles.

Gangliosides and acetylcholine of the central nervous system

Both gangliosides are bound acetycholine are higher in grey matter than in white. The subcellular distributions of these two compounds appear to be parallel in rat brain, occuring primarily in the crude mitochondrial fraction, and upon further fractionation both gangliosides and bound acetycholine appear in the pinched-off nerve ending fraction. The synaptic vesicle isolated after disruption of the nerve endings by osmotic shock contains both acetylcholine and gangliosides. This parallel distribution and the physical properties of gangliosides suggest a functional role for gangliosides in the transport of acetylcholine from synaptic vesicles through the presynaptic membrane.

Gangliosides and acetylcholine of the central nervous system-III the binding of radioactive acetylcholine by subcellular particles of the brain

The mitochondrial, pinched-off nerve endings, and synaptic vesicle fractions prepared from rat brain have been shown to take-up and bind radioactive acetylcholine in vitro. This in vitro binding of acetylcholine by brain particles appears to be a passive proces;. A linear response exists between the amount of acetylcholine bound and the concentration of acetylcholine in the suspending sucrose solution. The slope of the response indicates 6μ moles of acetylcholine bound per g of nerve endings in 30 min in 0.1 M acetylcholine solution. The radioactive acetylcholine bound to the particles may be released by acid, heat, osmotic shock, freezing and thawing, exposure to lipid solvents, and incubation with neuraminidase. Using particles labelled with radioactive acetylcholine, it has been possible to show that gangliosides and acetylcholine are contained in the same subfraction obtained by sedimentation of the nerve endings fraction in a continuous sucrose gradient. A possible role for gangliosides in the transport of acetylcholine at the synapse is outlined.

THE SUBCELLULAR LOCALIZATION OF DOPAMINE AND ACETYLCHOLINE IN THE DOG CAUDATE NUCLEUS.

The distribution of dopamine (3,4-dihydroxyphenylethylamine) between the subcellular fractions from homogenates of dog caudate nucleus was compared with the distributions of acetylcholine, 5-hydroxytryptamine and lactate dehydrogenase activity. The distributions of noradrenaline and 5-hydroxytryptamine between the subcellular fractions from homogenates of dog hypothalamus were also determined. Most of the dopamine, in contrast to acetylcholine, occurred in the soluble supernatant fraction; the remainder was associated with the fractions rich in pinched-off nerve ending particles, but localization in any one fraction was not as sharp as that of acetylcholine. Evidence is presented that suggests that the dopamine occurs in a free or easily released form throughout cell cytoplasm.

The subcellular localization of 5-hydroxytryptamine in guinea pig brain

The subcellular distribution of 5-HT in subfractions from guinea pig brain homogenates was assayed by fluorimetric techniques and agrees with that of earlier studies employing the rat fundus strip bioassay method. The present study reaffirms the finding that 5-HT is principally found in a particulate fraction free of myelin and mitochondria and identifiable as consisting largely of pinched-off nerve endings (NEPs). The forces binding 5-HT to the storage sites are quite labile relative to those which bind acetylcholine. Iproniazid raises the level of 5-HT in brain tissue but does not alter the subcellular distribution. Density gradient experiments were carried out in order to separate the 5-HT containing particles from those containing acetylcholine. Evidence is presented suggesting that acetylcholine is preferentially associated with the lighter, smaller nerve ending particles and that 5-HT is associated with the larger, denser NEPs; however, complete overlap occurs.