Recycling of synaptic vesicles in the cholinergic synapses of the torpedo electric organ during induced transmitter release

doi:10.1016/0306-4522(77)90024-0

Neuroscience

Volume 2, Issue 5, October 1977, Pages 695-714

https://doi.org/10.1016/0306-4522(77)90024-0 Get rights and content

Abstract

Blocks of innervated Torpedo electric tissue can be perfused in vitro and remain functioning for more than 24 h. Low frequency stimulation (0.1 Hz) of the attached nerve leads to a decay in electrical response and tissue content of acetylcholine although the number of vesicles counted in terminals does not fall. Stimulation leads to the appearance of a population of vesicles about 25% smaller in diameter than normal. If dextran (Molecular weight 10,000–40,000) is added to the perfusate an increasing number of vesicles becomes labelled during stimulation. After 720–1800 impulses 74% of all vesicles are found to contain dextran in their lumen. The label is contained mainly in the new small vesicle population. Perfusion with dextran per se does not lead to significant fine structural changes or uptake of dextran. After stimulation, dextran-containing vesicles are also found in the unmyelinated part of the terminal axon.

It is concluded that stimulation-induced transmitter release is accompanied by a recycling of synaptic vesicles which leads to uptake of extracellular marker into the lumen of the vesicles. Thus synaptic vesicles become heterogeneous as a result of their previous exo- and endocytotic activity.

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The Neurotransmitter Cycle and Quantal Size
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Citation Excerpt :
Because biological membranes are generally not considered elastic, this is very surprising. Variation in vesicle size was originally appreciated in the Torpedo electric organ, which contains two biochemically distinct populations of synaptic vesicles that can be separated by density gradient fractionation (Zimmermann and Denston, 1977). The larger vesicles, termed VP1, contain 3- to 5-fold more ACh than the smaller VP2 vesicles, but VP2 exhibit substantially higher VAChT activity (Gracz et al., 1988).
Changes in the response to release of a single synaptic vesicle have generally been attributed to postsynaptic modification of receptor sensitivity, but considerable evidence now demonstrates that alterations in vesicle filling also contribute to changes in quantal size. Receptors are not saturated at many synapses, and changes in the amount of transmitter per vesicle contribute to the physiological regulation of release. On the other hand, the presynaptic factors that determine quantal size remain poorly understood. Aside from regulation of the fusion pore, these mechanisms fall into two general categories: those that affect the accumulation of transmitter inside a vesicle and those that affect vesicle size. This review will summarize current understanding of the neurotransmitter cycle and indicate basic, unanswered questions about the presynaptic regulation of quantal size.
Loading and recycling of synaptic vesicles in the Torpedo electric organ and the vertebrate neuromuscular junction
2003, Progress in Neurobiology
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The recycling vesicles are smaller than those that have not been released (Fig. 3). Cumulative distributions of vesicle sizes (Zimmermann and Denston, 1977a; Fox and Kriebel, 1997) are fit better by lognormal probability distribution functions (PDFs) than by normal PDFs (Fig. 3). Lognormal PDFs are not symmetrical; there is an excess of larger occurrences.
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.
Limited numbers of recycling vesicles in small CNS nerve terminals: Implications for neural signaling and vesicular cycling
2001, Trends in Neurosciences
The tiny nerve terminals of central synapses contain far fewer vesicles than preparations commonly used for analysis of neurosecretion. Photoconversion of vesicles rendered fluorescent with the dye FM1-43 directly identified vesicles capable of engaging in exo–endocytotic recycling following stimulated Ca²⁺ entry. This recycling pool typically contained 30–45 vesicles, only a minority fraction (15–20% on average) of the total vesicle population. The smallness of the recycling pool would severely constrain rates of quantal neurotransmission if classical pathways were solely responsible for vesicle recycling. Fortunately, vesicles can undergo rapid retrieval and reuse in addition to conventional slow recycling, to the benefit of synaptic information flow and neuronal signaling.
Exocytosis of Catecholamine (CA)-containing and CA-free Granules in Chromaffin Cells
2001, Journal of Biological Chemistry
Citation Excerpt :
Membrane transfer from the plasma membrane to secretory granules during reversible fusion events has been reported in mast cells (25). In the electric organ of the marine ray Torpedo,two populations of vesicles can be distinguished after stimulation (26,27). One has the same size as synaptic vesicles found at rest, whereas the other has a smaller diameter (25%).
Recent evidence suggests that endocytosis in neuroendocrine cells and neurons can be tightly coupled to exocytosis, allowing rapid retrieval from the plasma membrane of fused vesicles for future use. This can be a much faster mechanism for membrane recycling than classical clathrin-mediated endocytosis. During a fast exo-endocytotic cycle, the vesicle membrane does not fully collapse into the plasma membrane; nevertheless, it releases the vesicular contents through the fusion pore. Once the vesicle is depleted of transmitter, its membrane is recovered without renouncing its identity. In this report, we show that chromaffin cells contain catecholamine-free granules that retain their ability to fuse with the plasma membrane. These catecholamine-free granules represent 7% of the total population of fused vesicles, but they contributed to 47% of the fusion events when the cells were treated with reserpine for several hours. We propose that rat chromaffin granules that transiently fuse with the plasma membrane preserve their exocytotic machinery, allowing another round of exocytosis.
Purification of active synaptic vesicles from the electric organ of Torpedo californica and comparison to reserve vesicles
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At least two distinguishable forms of synaptic vesicles exist, the active and reserve, but the reserve form is studied most because it has been difficult to purify the active vesicles. In the work reported here the active vesicles (termed VP₂) were highly enriched from the electric organ of Torpedo californica by an improved method developed for the reserve vesicles (termed VP₁) with the addition of density gradient centrifugation based on Percoll. No significant differences between the vesicular types were found in the amounts of SV1, SV2, and SV4 epitopes and P-type and V-type ATPase activities. The buoyant densities (g/ml) of VP₁ and VP₂ vesicles were determined by centrifugation in isosmotic sucrose (1.051, 1.069), Percoll (1.034, 1.040), and glycerol (1.087, 1.090) gradients. The radii were determined by dynamic quasi-elastic laser light-scattering to be (56.6 ± 10.8) nm and (55.0 ± 12.7) nm. For both vesicular types the volume of excluded sucrose is only about 37% of the volume of excluded Percoll, indicating that the surfaces are rough. Approx. 51% of the VP₁ and 32% of the VP₂ vesicular volumes are ‘osmotically active’ water that is exchangeable with glycerol. The different buoyant densities and amounts of osmotically active water in VP₁ and VP₂ vesicles probably are due to the different internal solutes. Previously observed differences in acetylcholine active transport and vesamicol binding by VP₁ and VP₂ synaptic vesicles cannot be explained by major alterations in the protein composition or conformation of the membranes in the two types of vesicles.
The heterogeneity of vesicular acetylcholine storage in cholinergic nerve terminals
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The vesicular hypothesis of quantal acetylcholine release describes the process by which discrete packages (or quanta) of the transmitter are released from nerve terminals through the exocytosis of the content of synaptic vesicles. However, cholinergic synaptic vesicles can no longer be vaguely regarded as simple membrane bound ‘sacks’ of the transmitter. Modern molecular, biochemical, morphological and electrophysiological research has revealed them to be complex cellular structures with a heterogeneous mixture of functions. Thus, not all synaptic vesicle populations are formed under the same circumstances and there are variations in the releasability of synaptic vesicle populations. This review briefly outlines some of the experimental research that has lead to our current thinking on the heterogeneity of vesicular acetylcholine storage in cholinergic nerve terminals. In addition, a model for vesicular acetylcholine storage and release is presented that attempts to accommodate many of the modern ideas concerning cholinergic synaptic vesicle function and interaction.

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Recycling of synaptic vesicles in the cholinergic synapses of the torpedo electric organ during induced transmitter release

Abstract

Neuroscience

J. biol. Chem.

Brain Res.

Brain Res.

Brain Res.

Brain Res.

Neuroscience

Synaptic vesicles: selective depletion in crayfish excitatory and inhibitory axons

Science, N.Y.

Métabolisme du calcium et libération de l'acetylcholine dans l'organeélectrique de la torpille

J. Neurochem.

Electron dense particles in cholinergic synaptic vesicles

Nature, New Biol.

Effects of calcium-containing fixation solutions on cholinergic synaptic vesicles

J. Cell Biol.

Changes in cholinergic synaptic vesicle populations and the ultrastructure of the nerve terminal membranes of Narcine brasiliensis electric organ stimulated to fatigue in vivo

J. Cell Biol.

The effects of prolonged repetitive stimulation in hemicholinium on the frog neuromuscular junction

J. Physiol., Lond.

Depletion of vesicles from frog neuromuscular junctions by prolonged tetanic stimulation

J. Cell Biol.

Turnover of transmitter and synaptic vesicles at the frog neuromuscular junction

J. Cell Biol.

Remarks on the organization of axon terminals in relation to secretory processes at synapses

The preparation and properties of ‘nerve terminal sacs’ from the electric organ of Torpedo

Expl Brain Res.

Acetylcholine compartments in stimulated electric organ of Torpedo marmorata

J. Neurochem.

Loss of vesicular acetylcholine in the Torpedo electric organ on discharge against high electrical resistance

J. Neurochem.

Synaptic vesicles in synaptosomes recycling in vitro

Nature, New Biol.

Dense particles within synaptic vesicles fixed with acid-aldehyde

J. Neurocytol.

Structural and functional changes of frog neuromuscular junctions in high calcium solutions

Evidence for recycling of synaptic vesicle membrane during transmitter release at the frog neuromuscular junction

J. Cell Biol.

Stimulation dependent alterations in peroxidase uptake at lobster neuromuscular junctions

Science, N.Y.

Evidence for the vesicle hypothesis

J. Physiol., Lond.

Transmitter release and recycling of synaptic vesicle membrane at the neuro-muscular junction

The effects of nerve stimulation and hemicholinium on synaptic vesicles at the mammalian neuromuscular junction

J. Physiol., Lond.