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A morphological comparison of neuromuscular and nerve-electroplaque synapses of torpedo was performed. Synaptic vesicles are much smaller at the neuromuscular synapse. The question of the respective role of these populations is raised.
We used fluorescence recovery after photobleaching (FRAP) to measure the mobility of synaptic vesicles in frog motor nerve terminals. Vesicles belonging to the recycling pool or to the reserve pool were selectively labeled with FM1-43. In resting terminals, vesicles in the reserve pool were immobile, while vesicles in the recycling pool were mobile. Nerve stimulation increased the mobility of reserve pool vesicles. Treatment with latrunculin A, which destroyed actin filaments, had no significant effect on mobility, and reducing the temperature likewise had little effect, suggesting that recycling pool vesicles move by simple diffusion. Application of okadaic acid caused vesicle mobility in both pools to increase to the same level. We could model these and others' results quantitatively by taking into account the relative numbers of mobile and immobile vesicles in each pool, and vesicle packing density, which has a large effect on mobility.
Neurons have at least two pathways of regulated secretion, which involve two classes of secretory organelles: typical synaptic vesicles (SVs) and large dense-core vesicles. Large dense-core vesicles store and secrete peptide neurotransmitters and amines, and may be seen as the neuronal counterpart of secretory granules of endocrine cells. SVs are highly specialized secretory organelles, which store and secrete non-peptide hormones and play a dominant role in the fast, point-to-point signalling typical of the nervous system. Microvesicles that share a variety of biochemical and functional similarities with SVs (synaptic-like microvesicles) have recently been described in endocrine cells. SVs and synaptic-like microvesicles are closely related to vesicular carriers of the receptor-mediated recycling pathway. They undergo repeated cycles of exo-endocytosis, which are thought to involve endosomal intermediates. In mature neurons, SVs are concentrated in axon endings. To gain insight into the mechanisms responsible for SV targeting, we have studied the traffic of SV proteins in both endocrine cells and developing hippocampal neurons in primary culture at different stages of differentiation. Additionally, the distribution of the SV protein synaptophysin, when expressed by transfection in fibroblastic cells or in polarized epithelial cells (MDCK cells), was investigated. SV proteins are already present in developing neurons at stages preceding the establishment of neuronal polarity. As axons and dendrites form, SV proteins are found in both types of processes, although they become progressively more concentrated in the axon. Throughout these developmental stages SVs undergo active exo-endocytotic recycling.(ABSTRACT TRUNCATED AT 250 WORDS)
The "kiss-and-run" model of exocytosis and endocytosis predicts that synaptic vesicles can undergo fast and efficient recycling, after fusion with the plasmalemma, without intermixing of membranes. Evidence is mounting from several new experimental approaches that kiss-and-run occurs at synapses. Distinct vesicle pools, which initially were identified in morphological terms, are now being characterized in biochemical and functional terms. In addition, at least two functional recycling pathways, operating on different time scales (from milliseconds to tens of seconds), have been shown to coexist in the same synaptic system, and the two pathways appear to be differentially regulated. Taken together, these data suggest that kiss-and-run operates in parallel with the classical, coated-vesicle recycling. Here, we review recent evidence for kiss-and-run recycling and discuss whether it is a distinct process, dependent on the molecular organization of the fusing vesicle. We propose that vesicles undergo a process of "competence maturation". According to this view, the specific molecular make-up of the vesicles, their location and their interactions with nerve terminal proteins might determine not only the differential availability of the vesicles for fusion and neurotransmitter release but also the recycling path that they will follow.
In a search for biochemical markers of modified synaptic function following training of day-old chicks on a passive avoidance task, we have assayed two monoclonal antibodies to synaptic vesicle proteins (anti-p65 and anti-SV2) and one raised to postsynaptic densities (411B). We have also measured total acetylcholine (ACh) content. Measurements were made on three forebrain regions known to show metabolic and morphological change consequent on training--the lobus parolfactorius (LPO), paleostriatum augmentatum (PA), and medial hyperstriatum ventrale (MHV)--in the right and left hemispheres 2 and 24 h after training chicks on a passive avoidance task, in which they learn to avoid pecking a bead coated with methylanthranilate [methylanthranilate-trained (M-trained)]. Control chicks were trained on a water-coated bead [water-trained (W-trained)]. Twenty-four hours after training, 411B levels showed no differences between W-trained and M-trained chicks in any region. M-training reduced the titre of anti-p65 by 16% in the left PA and 15% in the left MHV and that of anti-SV2 by 19% in the left PA. M-trained chicks showed reduced total ACh content in the LPO by up to 40% and in the PA by up to 48% but had no change in ACh level in the MHV. The decreases in antibody titre were not seen in forebrains analysed 2 h after training, but tendencies toward increases in levels in the right PA and MHV were observed with all three antibodies. Significant differences between right and left hemispheric regions, independent of training, were observed for all the antibodies and for ACh content.(ABSTRACT TRUNCATED AT 250 WORDS)
In sympathetic ganglia stimulated in the presence of HC-3, the reduction in number of synaptic vesicles was observed to be accompanied by a significant decrease of the ganglionic phosphatidylcholine content.
We describe the design and operation of a machine that freezes biological tissues by contact with a cold metal block, which incorporates a timing circuit that stimulates frog neuromuscular junctions in the last few milliseconds before thay are frozen. We show freeze-fracture replicas of nerve terminals frozen during transmitter discharge, which display synpatic vesicles caught in the act of exocytosis. We use 4-aminopyridine (4-AP) to increase the number of transmitter quanta discharged with each nerve impulse, and show that the number of exocytotic vesicles caught by quick-freezing increases commensurately, indicating that one vesicle undergoes exocytosis for each quantum that is discharged. We perform statistical analyses on the spatial distribution of synaptic vesicle discharge sites along the "active zones" that mark the secretory regions of these nerves, and show that individual vesicles fuse with the plasma membrane independent of one another, as expected from physiological demonstrations that quanta are discharged independently. Thus, the utility of quick-freezing as a technique to capture biological processes as evanescent as synaptic transmission has been established. An appendix describes a new capacitance method to measure freezing rates, which shows that the "temporal resolution" of our quick-freezing technique is 2 ms or better.
An antiserum against a specific component (a glycosamino glycan) of the cholinergic synaptic-vesicle of Torpedo marmorata has been used to investigate the localization of the component in the cell body, its movement within the electromotor axon and its fate within the nerve terminal upon electrical stimulation. After immunofluorescent staining, spots are observed throughout the cytoplasm of the lobe perikarya, although they are concentrated in the region of the axon hillock. Ligation of the electromotor nerves leading from the lobe to electric organ produces a proximal build-up of material which stains readily with the antivesicle antiserum, indicating that the vesicle antigen is transported from the cell body to the nerve terminal. A marked increase in indirect immunofluorescent staining of the electric organ is observed in the nerve ending upon electrical stimulation. We interpret this result as fusion of the vesicles with the presynaptic plasma membrane and exteriorization of the vesicle antigen to the extracellular space, thereby facilitating its staining. After recovery of the system the fluorescence declines, a result that is consistent with the reinternalization of the vesicle antigen into the core of reformed vesicles. The results support a mechanism whereby vesicles recycle within the nerve terminal and transmitter is released by exocytosis.
GBR 12909 and 12935, commonly used as potent neuronal dopamine uptake blockers, also inhibit dopamine uptake into brain synaptic vesicles. The concentrations required for the latter activity (34-45 nM) are one order of magnitude higher than those required for inhibiting neuronal uptake of dopamine (1-6 nM). In contrast, the two activities differ by three orders of magnitude for cocaine (137 microM versus 0.35 microM). We propose that the vesicular effect of GBR-type dopamine uptake blockers should be taken into account when interpreting in vivo experiments.
The ligand binding relationship between the acetylcholine transporter (AcChT) and the vesamicol receptor (VR) and the kinetics of active transport were studied in synaptic vesicles purified from the Torpedo electric organ using analogues of AcCh and vesamicol. Methoxyvesamicol, which should exhibit better equilibration properties for kinetics measurements than the more potent parent, inhibits active transport in a nonlinear noncompetitive manner. AcCh analogues competitively inhibit binding of [3H]vesamicol with higher affinity in hyposmotically lysed vesicle ghosts than in intact vesicles, apparently due to removal of a competing internal, osmotically active factor. AcCh and actively transported analogues of AcCh that are up to 57% larger in van der Waals volume exhibit up to a 200-fold ratio for the dissociation constant measured by inhibition of vesamicol binding to ghosts (KIAg) compared to the Michaelis constant for transport (KM) or the IC50 value for inhibition of [3H]AcCh active transport. In contrast, two AcCh analogues that are about 120% larger and that almost surely are not transported exhibit a KIAg/IC50 ratio of about 1. The data demonstrate that the vesamicol family of compounds binds to an allosteric site in the AcChT. Initiation of active transport has no apparent effect on the affinities of vesamicol and AcCh analogues, which suggests that most of the AcChT-VR in purified vesicles is transport incompetent. Vesicle ghosts actively transport [3H]AcCh nearly as well as intact vesicles, which suggests that internal factor does not affect transport-competent AcChT-VR. A kinetics model is proposed that predicts that AcCh analogues exhibiting a KIAg/IC50 ratio significantly greater than 1 are actively transported. Some of the microscopic constants in the model are estimated. The AcChT binds AcCh very weakly with a dissociation constant of about 20-50 mM, but it transports substrates rapidly in a process exhibiting remarkably little selectivity for the detailed shape and volume of the transported ion.
We have characterized the morphological and functional properties of the readily releasable pool (RRP) and the reserve pool of synaptic vesicles in frog motor nerve terminals using fluorescence microscopy, electron microscopy, and electrophysiology. At rest, about 20% of vesicles reside in the RRP, which is depleted in about 10 s by high-frequency nerve stimulation (30 Hz); the RRP refills in about 1 min, and surprisingly, refilling occurs almost entirely by recycling, not mobilization from the reserve pool. The reserve pool is depleted during 30 Hz stimulation with a time constant of about 40 s, and it refills slowly (half-time about 8 min) as nascent vesicles bud from randomly distributed cisternae and surface membrane infoldings and enter vesicle clusters spaced at regular intervals along the terminal. Transmitter output during low-frequency stimulation (2-5 Hz) is maintained entirely by RRP recycling; few if any vesicles are mobilized from the reserve pool.
We aimed to ascertain whether pyrethroid insecticides could influence the vesicular transport of the excitatory amino acid glutamate. The incubation of rat cortical synaptic vesicles with resmethrin and permethrin, consistently stimulated both ATP-dependent and -independent uptake of [3H]glutamate, while not evoking depletion of its vesicular content. Both processes were counteracted by valinomycin, a dissipator of the transmembrane potential gradient (deltapsi(sv)). Meanwhile, the vesicular influx of 36Cl- anions was impaired by pyrethroid concentrations which did not affect the ATP-dependent uptake of [14C]methylamine, as a marker for the proton gradient (deltapH). Thus, the stimulation of glutamate transport appeared to involve mainly the deltapsi(sv). A self-attenuating effect of selected pyrethroids on putatively enhanced excitatory transmission in severe intoxication is suggested.
Mg-ATPase (1 mM) induces a decrease in the intensity of light scattering (I1) at 620 nm of rat brain synaptic vesicles (SV) suspended in sucrose, with this decrease being indicative of the swelling of the vesicles. The Mg-ATPase-induced swelling appears to be associated with the function of H+-ATPase of SV membranes, since it is completely abolished by the proton pump blocker dicyclohexylcarbodiimide and the protonophore carbonylcyanide m-chlorophenylhydrazone. The Mg-ATPase-induced swelling was enhanced in the presence of the permeable anion Cl- (in the range of 25-50 mM KCl). Ca2+ (and Mg2+) at high concentrations (0.1-1.0 mM) cause aggregation of the SV as measured by changes in the I1. Colchicine and cytochalasin do not affect SV swelling and aggregation whereas Mg-ATP (1 mM) lowers aggregation caused by Ca2+ (1 mM).
Cone photoreceptors tonically release neurotransmitter in the dark through a continuous cycle of exocytosis and endocytosis. Here, using the synaptic vesicle marker FM1-43, we elucidate specialized features of the vesicle cycle. Unlike retinal bipolar cell terminals, where stimulation triggers bulk membrane retrieval, cone terminals appear to exclusively endocytose small vesicles. These retain their integrity until exocytosis, without pooling their membranes in endosomes. Endocytosed vesicles rapidly disperse through the terminal and are reused with no apparent delay. Unlike other synapses where most vesicles are immobilized and held in reserve, only a small fraction (<15%) becomes immobilized in cones. Photobleaching experiments suggest that vesicles move by diffusion and not by molecular motors on the cytoskeleton and that vesicle movement is not rate limiting for release. The huge reservoir of vesicles that move rapidly throughout cone terminals and the lack of a reserve pool are unique features, providing cones with a steady supply for continuous release.
We have employed sulfhydryl group reagents in an attempt to determine the mechanism by which the transport of nucleotides into synaptic vesicles is controlled. Transport proved to be sensitive to N-ethylmaleimide; radiolabelled N-ethylmaleimide was used to locate the sulfhydryl group to the translocase-associated molecule previously identified as a polypeptide of Mr 34,000 [Lee and Witzemann (1983) Biochemistry 22, 6123-6130]. The nucleotide uptake was 75% inhibited by the mercurials rho-hydroxymercuribenzoate and rho-chloromercuriphenylsulfonate. Uptake was also sensitive to the reagents phenylarsine oxide and iodosobenzoic acid, which are specific for dithiols. These results indicate that a readily accessible dithiol is critical for nucleotide transport. Using the lipophilic oxidants iodosobenzoic acid and plumbagin, we demonstrated that nucleotide uptake was inhibited upon oxidation of the dithiol but that this did not involve an alteration in the affinity of the translocase for its substrate.
In vertebrate motor nerve terminals and in the electromotor nerve terminals of Torpedo there are two major pools of synaptic vesicles: readily releasable and reserve. The electromotor terminals differ in that the reserve vesicles are twice the diameter of the readily releasable vesicles. The vesicles contain high concentrations of ACh and ATP. Part of the ACh is brought into the vesicle by the vesicular ACh transporter, VAChT, which exchanges two protons for each ACh, but a fraction of the ACh seems to be accumulated by different, unexplored mechanisms. Most of the vesicles in the terminals do not exchange ACh or ATP with the axoplasm, although ACh and ATP are free in the vesicle interior. The VAChT is controlled by a multifaceted regulatory complex, which includes the proteoglycans that characterize the cholinergic vesicles. The drug (-)-vesamicol binds to a site on the complex and blocks ACh exchange. Only 10-20% of the vesicles are in the readily releasable pool, which therefore is turned over fairly rapidly by spontaneous quantal release. The turnover can be followed by the incorporation of false transmitters into the recycling vesicles, and by the rate of uptake of FM dyes, which have some selectivity for the two recycling pathways. The amount of ACh loaded into recycling vesicles in the readily releasable pool decreases during stimulation. The ACh content of the vesicles can be varied over eight-fold range without changing vesicle size.
The effect of chronic administration of chlorpromazine (CPZ) on the striatal synaptic vesicles was quantitatively investigated with an electron microscope. Six rats, three controls and three experimentals, which received 20 mg/kg b.wt. of CPZ daily for 15 months, were sacrificed and a total of 300 axo-dendritic spine synapses (50 synapses per animal) were randomly taken and divided into three zones with a width of 160-467 nm on electron micrographs (final magnification 60,000). The first zone was adjacent to the presynaptic membrane in the region of the synaptic cleft. The second and third were farther away from the first zone. In these zones we measured the vesicle density, vesicle size and vesicle elongation ratio (the shortest diameter/the longest diameter) with the following results: In both the CPZ-administered rats and the controls the vesicle number per unit area of axoplasm (vesicle density) was smaller in Zone III than in Zones I and II, while those in the latter two zones did not differ from each other. However, there was no significant difference in the vesicle number in any zone between the control and experimental animals. The vesicles in every zone were significantly larger in the CPZ-administered animals than in the controls. There were no conclusive findings on the vesicle elongation ratio (vesicle shape).
Sorting of secreted proteins into dense-core secretory granules may involve selective aggregation of regulated secretory proteins, rather than a conventional sortase. Synaptic vesicles, which mediate paracrine communication between adjacent cells, appear to arise by a modification of the early endosome pathway. Targeting to the cell surface involves the actin-based cytoskeleton and small GTP-binding proteins.