scholarly journals Regeneration of the active zone at the frog neuromuscular junction.

1984 ◽  
Vol 98 (5) ◽  
pp. 1685-1695 ◽  
Author(s):  
C P Ko
Keyword(s):  
Active Zone ◽  
Transmitter Release ◽  
Neuromuscular Junctions ◽  
Freeze Fracture ◽  
Frog Neuromuscular Junction ◽  
Normal Pattern ◽  
Intramembrane Particles ◽  
Total Length ◽  
Secondary Stage ◽  
Active Zones

The active zone is a unique specialization of the presynaptic membrane and is believed to be the site of transmitter release. The formation of the active zone and the relationship of this process to transmitter release were studied at reinnervated neuromuscular junctions in the frog. At different times after a nerve crush, the cutaneous pectoris muscles were examined with intracellular recording recording and freeze-fracture electron microscopy. The P face of a normal active zone typically consists of two double rows of particles lined up in a continuous segment located opposite a junctional fold. In the initial stage of reinnervation, clusters of large intramembrane particles surrounding membrane elevations appeared on the P face of nerve terminals. Like normal active zones, these clusters were aligned with junctional folds. Vesicle openings, which indicate transmitter release, were seen at these primitive active zones, even though intramembrane particles were not yet organized into the normal pattern of two double rows. The length of active zones at this stage was only approximately 15% of normal. During the secondary stage, every junction was reinnervated and most active zones had begun to organize into the normal pattern with normal orientation. Unlike normal, there were often two or more discontinuous short segments of active zone aligned with the same junctional fold. The total length of active zone per junctional fold increased to one-third of normal, mainly because of the greater number of segments. In the third stage, the number of active zone segments per junctional fold showed almost no change when compared with the secondary stage. However, individual segments elongated and increased the total length of all active zone segments per junctional fold to about two-thirds of the normal length. The dynamic process culminated in the final stage, during which elongating active zones appeared to join together and the number of active zone segments per junctional fold decreased to normal. Thus, in most regions, regeneration of the active zones was complete. These results suggest that the normal organization of two double rows is not necessary for the active zone to be functional. Furthermore, localization of regenerating active zones is related to junctional folds and/or their associated structures.

scholarly journals Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. I. Effects of black widow spider venom and Ca2+-free solutions on the structure of the active zone.

1979 ◽  
Vol 81 (1) ◽  
pp. 163-177 ◽  
Author(s):  
B Ceccarelli ◽  
F Grohovaz ◽  
W P Hurlbut
Keyword(s):  
Neuromuscular Junctions ◽  
Freeze Fracture ◽  
Spider Venom ◽  
Presynaptic Membrane ◽  
Black Widow ◽  
Black Widow Spider ◽  
Intramembrane Particles ◽  
Black Widow Spider Venom ◽  
Active Zones ◽  
Widow Spider

Black widow spider venom (BWSV) was applied to frog nerve-muscle preparations bathed in Ca2+-containing, or Ca2+-free, solutions and the neuromuscular junctions were studied by the freeze-fracture technique. When BWSV was applied for short periods (10-15 min) in the presence of Ca2+, numerous dimples (P face) or protuberances (E face) appeared on the presynaptive membrane and approximately 86% were located immediately adjacent to the double rows of large intramembrane particles that line the active zones. When BWSV was applied for 1 h in the presence of Ca2+, the nerve terminals were depleted of vesicles, few dimples or protuberances were seen, and the active zones were almost completely disorganized. The P face of the presynaptic membrane still contained large intramembrane particles. When muscles were soaked for 2-3 h in Ca2+-free solutions, the active zones became disorganized, and isolated remnants of the double rows of particles were found scattered over the P face of the presynaptic membrane. When BWSV was applied to these preparations, dimples or protuberances occurred almost exclusively alongside disorganized active zones or alongside dispersed fragments of the active zones. The loss of synaptic vesicles from terminals treated with BWSV probably occurs because BWSV interferes with the endocytosis of vesicle membrane. Therefore, we assume that the dimples or protuberances seen on these terminals identify the sites of exocytosis, and we conclude that exocytosis can occur mostly in the immediate vicinity of the large intramembrane particles. Extracellular Ca2+ seems to be required to maintain the grouping of the large particles into double rows at the active zones, but is not required for these particles to specify the sites of exocytosis.

scholarly journals Changes in miniature endplate potential frequency during repetitive nerve stimulation in the presence of Ca2+, Ba2+, and Sr2+ at the frog neuromuscular junction.

1981 ◽  
Vol 77 (5) ◽  
pp. 503-529 ◽  
Author(s):  
J E Zengel ◽  
K L Magleby
Keyword(s):  
Nerve Stimulation ◽  
Transmitter Release ◽  
Neuromuscular Junctions ◽  
Fourth Power ◽  
Bathing Solution ◽  
Frog Neuromuscular Junction ◽  
Repetitive Nerve Stimulation ◽  
Time Constants ◽  
Endplate Potential ◽  
Induced Changes

Miniature endplate potentials (MEPPs) were recorded from frog sartorious neuromuscular junctions under conditions of reduced quantal contents to study the effect of repetitive nerve stimulation on asynchronous (tonic) quantal transmitter release. MEPP frequency increased during repetitive stimulation and then decayed back to the control level after the conditioning trains. The decay of the increased MEPP frequency after 100-to 200-impulse conditioning trains can be described by four components that decayed exponentially with time constants of about 50 ms, 500 ms, 7 s, and 80 s. These time constants are similar to those for the decay of stimulation-induced changes in synchronous (phasic) transmitter release, as measured by endplate potential (EPP) amplitudes, corresponding, respectively, to the first and second components of facilitation, augmentation, and potentiation. The addition of small amounts of Ca2+ or Ba2+ to the Ca2+-containing bathing solution, or the replacement of Ca2+ with Sr2+, led to a greater increase in the stimulation-induced increases in MEPP frequency. The Sr-induced increase in MEPP frequency was associated with an increase in the second component of facilitation of MEPP frequency; the Ba-induced increase with an increase in augmentation. These effects of Sr2+ and Ba2+ on stimulation-induced changes in MEPP frequency are similar to the effects of these ions on stimulation-induced changes in EPP amplitude. These ionic similarities and the similar kinetics of decay suggest that stimulation induced changes in MEPP frequency and EPP amplitude have some similar underlying mechanisms. Calculations are presented which show that a fourth power residual calcium model for stimulation-induced changes in transmitter release cannot readily account for the observation that stimulation-induced changes in MEPP frequency and EPP amplitude have similar time-courses.

Release and Recycling of the Readily Releasable Vesicle Population in a Synapse Possessing No Reserve Population

2007 ◽  
Vol 97 (6) ◽  
pp. 4048-4057 ◽  
Author(s):  
J. H. Koenig ◽  
Kazuo Ikeda
Keyword(s):  
High Frequency ◽  
Active Zone ◽  
Neuromuscular Junctions ◽  
Recycling Rate ◽  
Spontaneous Release ◽  
Electron Microscopic ◽  
Single Action ◽  
Active Zones ◽  
Evoked Activity ◽  
Microscopic Techniques

We previously demonstrated that the tergotrochanteral muscle (TTM) of Drosophila is innervated by unique synapses that possess a small readily releasable/recycling vesicle population (active zone population), but not the larger reserve vesicle population observed at other neuromuscular junctions in this animal. Using light and electron microscopic techniques and intracellular recording from the G1 muscle fiber of the TTM, the release and recycling characteristics of the readily releasable/recycling population were observed without any possible contribution from a reserve population. Our results indicate 1) the total number of vesicles in synapses presynaptic to the G1 fiber correlates with the total number of quanta that can be released onto this fiber; 2) the number of quanta released by a single action potential onto the G1 fiber is about one half the number of morphologically “docked” vesicles in active zones onto the G1, and this ratio decreases in a partially depleted state; 3) the recycling rate at 1-Hz stimulation, a frequency that does not cause any depression, is 0.24 recycled vesicle/active zone/s; and 4) normal-appearing spontaneous release occurs from the active zone vesicle population and, unlike synapses that possess a reserve population, the frequency of this release is reduced after high-frequency evoked activity.

scholarly journals Endocytosis of synaptic vesicle membrane at the frog neuromuscular junction.

1984 ◽  
Vol 98 (2) ◽  
pp. 685-698 ◽  
Author(s):  
T M Miller ◽  
J E Heuser
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Thin Section ◽  
Nerve Terminal ◽  
Motor Nerve ◽  
Freeze Fracture ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Coated Pits ◽  
Active Zones

Frog nerve-muscle preparations were quick-frozen at various times after a single electrical stimulus in the presence of 4-aminopyridine (4-AP), after which motor nerve terminals were visualized by freeze-fracture. Previous studies have shown that such stimulation causes prompt discharge of 3,000-6,000 synaptic vesicles from each nerve terminal and, as a result, adds a large amount of synaptic vesicle membrane to its plasmalemma. In the current experiments, we sought to visualize the endocytic retrieval of this vesicle membrane back into the terminal, during the interval between 1 s and 2 min after stimulation. Two distinct types of endocytosis were observed. The first appeared to be rapid and nonselective. Within the first few seconds after stimulation, relatively large vacuoles (approximately 0.1 micron) pinched off from the plasma membrane, both near to and far away from the active zones. Previous thin-section studies have shown that such vacuoles are not coated with clathrin at any stage during their formation. The second endocytic process was slower and appeared to be selective, because it internalized large intramembrane particles. This process was manifest first by the formation of relatively small (approximately 0.05 micron) indentations in the plasma membrane, which occurred everywhere except at the active zones. These indentations first appeared at 1 s, reached a peak abundance of 5.5/micron2 by 30 s after the stimulus, and disappeared almost completely by 90 s. Previous thin-section studies indicate that these indentations correspond to clathrin-coated pits. Their total abundance is comparable with the number of vesicles that were discharged initially. These endocytic structures could be classified into four intermediate forms, whose relative abundance over time suggests that, at this type of nerve terminal, endocytosis of coated vesicles has the following characteristics: (a) the single endocytotic event is short lived relative to the time scale of two minutes; (b) earlier forms last longer than later forms; and (c) a single event spends a smaller portion of its lifetime in the flat configuration soon after the stimulus than it does later on.

scholarly journals Synaptic structural complexity as a factor enhancing probability of calcium-mediated transmitter release

1996 ◽  
Vol 75 (6) ◽  
pp. 2451-2466 ◽  
Author(s):  
R. L. Cooper ◽  
J. L. Winslow ◽  
C. K. Govind ◽  
H. L. Atwood
Keyword(s):  
Nerve Terminal ◽  
Calcium Ion ◽  
Active Zone ◽  
Transmitter Release ◽  
Structural Complexity ◽  
Ion Concentration ◽  
Freshwater Crayfish ◽  
Channel Current ◽  
Active Zones ◽  
Calcium Ion Concentration

1. In a model synaptic system, the excitatory neuromuscular junction of the freshwater crayfish, the nerve terminals possess synapses that vary in structural complexity, with numbers of active zones ranging from zero to five. Active zones on individual synapses show a wide range of separation distances. We tested the hypothesis that two active zones of a single synapse in close proximity can enhance the localized increase in free calcium ion concentration, thus enhancing the probability of neurotransmission at that synapse. We evaluated the increase in calcium ion concentration as a function of distance between adjacent active zones. 2. To test this hypothesis, a reaction-diffusion model for Ca2+ entering the presynaptic terminals was used. This test was used because 1) present measurement techniques are inadequate to resolve quantitatively the highly localized, transient calcium microdomains at synaptic active zones; and 2) there is presently no suitable preparation for physiological recording from isolated synapses with varying distances between active zones. Included in the model were intracellular buffer and a typical distribution of voltage-activated Ca2+ channels for an active zone, estimated from freeze-fracture micrographs. 3. The model indicated that localized Ca2+ clouds from discrete active zones can overlap to create spatial enhancement of Ca2+ concentration. The degree of interaction between two active zones depends on the distance between them. When two typical active zones are separated by < or = 200 nm, the maximum intracellular Ca2+ concentration ([Ca2+]i) is greater at 1) the midpoint between them, and 2) the center of each one, than at the corresponding positions for a single isolated active zone. Enhanced [Ca2+]i at the edge of the active zone where “docked” synaptic vesicles occur would be expected to have an effect on transmitter release. 4. When the model includes no intracellular buffer, the increase in [Ca2+]i is a linear function of calcium channel current, but is a nonlinear function of the number of conducting calcium channels in an active zone. With immobile buffer included, the increase in [Ca2+]i is nonlinear with respect to both channel current and number of conducting channels. 5. Inclusion of immobile buffer in the model provides “released” residual calcium that slowly accumulates during a train of current pulses. Released residual calcium accumulates more rapidly at paired active zones separated by < or = 200 nm that at single isolated active zones. 6. We propose that the probability of release is enhanced at synapses with closely associated active zones. Synapses of this type (“complex” synapses) could be selectively recruited when the neuron is active at low frequencies. At higher frequencies of neuronal activity, more distant active zones may interact and acquire a greater probability of releasing quanta. This would provide the nerve terminal with one component of a mechanism for frequency facilitation, because the number of quanta released by the terminal as a whole would increase with frequency. Thus variation in synaptic complexity in a nerve terminal provides a mechanism for short-term plasticity of transmitter release.

scholarly journals Absence of filipin-sterol complexes from the membranes of active zones and acetylcholine receptor aggregates at frog neuromuscular junctions.

1981 ◽  
Vol 88 (2) ◽  
pp. 453-458 ◽  
Author(s):  
Y Nakajima ◽  
P C Bridgman
Keyword(s):  
Acetylcholine Receptor ◽  
Acetylcholine Receptors ◽  
Neuromuscular Junctions ◽  
Freeze Fracture ◽  
Cholesterol Content ◽  
Postsynaptic Membrane ◽  
Large Particles ◽  
Glutaraldehyde Solution ◽  
Active Zones ◽  
Low Cholesterol

The polyene antibiotic filipin reacts specifically with membrane cholesterol and produces distinctive membrane lesions. We treated frog cutaneous and sartorius muscles with 0.04% filipin in a glutaraldehyde solution with or without prefixation with glutaraldehyde. Freeze-fracture of these muscles revealed numerous 19 to 38-nm protuberances and depressions (filipin-sterol complexes) in most areas of muscle, axon, and Schwann cell membranes. In the presynaptic membrane, however, these filipin-sterol complexes were absent from active zones consisting of ridges bordered with double rows of particles. In the postsynaptic membrane, filipin-sterol complexes were also virtually absent from the areas occupied by aggregates of large particles representing acetylcholine receptors. These results suggest that the membrane regions of active zones and acetylcholine receptor aggregates have a low cholesterol content.

scholarly journals Freeze-fracture studies of frog neuromuscular junctions during intense release of neurotransmitter. II. Effects of electrical stimulation and high potassium.

1979 ◽  
Vol 81 (1) ◽  
pp. 178-192 ◽  
Author(s):  
B Ceccarelli ◽  
F Grohovaz ◽  
W P Hurlbut
Keyword(s):  
Nerve Terminal ◽  
Neuromuscular Junctions ◽  
Freeze Fracture ◽  
Presynaptic Membrane ◽  
High Potassium ◽  
Unique Distribution ◽  
Active Zones ◽  
Nerve Muscle ◽  
Indirect Stimulation ◽  
Quantal Release Of Neurotransmitter

Frog cutaneous pectoris nerve muscle preparations were studied by the freeze-fracture technique under the following conditions: (a) during repetitive indirect stimulation for 20 min, 10/s; (b) during recovery from this stimulation; and (c) during treatment with 20 mM K+. Indirect stimulation causes numerous dimples or protuberances to appear on the presynaptic membrane of nerve terminal, and most are located near the active zones. Deep infoldings of the axolemma often develop between the active zones. Neither the number nor the distribution of dimples, protuberances, of infoldings changes markedly during the first minute of recovery. The number of dimples, protuberances, and infoldings is greatly reduced after 10 min of recovery. Since endocytosis proceeds vigorously during the recovery periods, we conclude that endocytosis occurs mostly at the active zones, close to the sites of exocytosis. 20 mM K+ also causes many dimples or protuberances to appear on the axolemma of the nerve terminal but they are distributed almost uniformly along the presynaptic membrane. Experiments with horseradish peroxidase (HRP) show that recycling of synaptic vesicles occurs in 20 mM K+. This recycling is not accompanied by changes in the number of coated vesicles. Since both exocytosis and endocytosis occur in 20 mM K+, it is difficult to account for this unique distribution. However, we suggest that K+ causes dimples or protuberances to appear between the active zones because it activates latent sites of exocytosis specified by small numbers of large intramembrane particles located between active zones. The activation of latent release sites may be related to the complex effects that K+ has on the quantal release of neurotransmitter.

scholarly journals Structural changes after transmitter release at the frog neuromuscular junction.

1981 ◽  
Vol 88 (3) ◽  
pp. 564-580 ◽  
Author(s):  
J E Heuser ◽  
T S Reese
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Structural Changes ◽  
Frog Neuromuscular Junction ◽  
Vesicle Membrane ◽  
Intramembrane Particles ◽  
Synaptic Vesicle Exocytosis ◽  
Quick Freezing ◽  
Vesicle Exocytosis ◽  
Active Zones

The sequence of structural changes that occur during synaptic vesicle exocytosis was studied by quick-freezing muscles at different intervals after stimulating their nerves, in the presence of 4-aminopyridine to increase the number of transmitter quanta released by each stimulus. Vesicle openings began to appear at the active zones of the intramuscular nerves within 3-4 ms after a single stimulus. The concentration of these openings peaked at 5-6 ms, and then declined to zero 50-100 ms late. At the later times, vesicle openings tended to be larger. Left behind at the active zones, after the vesicle openings disappeared, were clusters of large intramembrane particles. The larger particles in these clusters were the same size as intramembrane particles in undischarged vesicles, and were slightly larger than the particles which form the rows delineating active zones. Because previous tracer work had shown that new vesicles do not pinch off from the plasma membrane at these early times, we concluded that the particle clusters originate from membranes of discharged vesicles which collapse into the plasmalemma after exocytosis. The rate of vesicle collapse appeared to be variable because different stages occurred simultaneously at most times after stimulation; this asynchrony was taken to indicate that the collapse of each exocytotic vesicle is slowed by previous nearby collapses. The ultimate fate of synaptic vesicle membrane after collapse appeared to be coalescence with the plasma membrane, as the clusters of particles gradually dispersed into surrounding areas during the first second after a stimulus. The membrane retrieval and recycling that reverse this exocytotic sequence have a slower onset, as has been described in previous reports.

scholarly journals Hypothesis Relating the Structure, Biochemistry and Function of Active Zone Material Macromolecules at a Neuromuscular Junction

2022 ◽  
Vol 13 ◽  
Author(s):  
Joseph A. Szule
Keyword(s):  
Membrane Fusion ◽  
Synaptic Vesicle ◽  
Active Zone ◽  
Structural Integrity ◽  
Neuromuscular Junctions ◽  
Vesicle Trafficking ◽  
Material Structure ◽  
Neurotransmitter Secretion ◽  
Active Zones ◽  
And Function

This report integrates knowledge of in situ macromolecular structures and synaptic protein biochemistry to propose a unified hypothesis for the regulation of certain vesicle trafficking events (i.e., docking, priming, Ca2+-triggering, and membrane fusion) that lead to neurotransmitter secretion from specialized “active zones” of presynaptic axon terminals. Advancements in electron tomography, to image tissue sections in 3D at nanometer scale resolution, have led to structural characterizations of a network of different classes of macromolecules at the active zone, called “Active Zone Material’. At frog neuromuscular junctions, the classes of Active Zone Material macromolecules “top-masts”, “booms”, “spars”, “ribs” and “pins” direct synaptic vesicle docking while “pins”, “ribs” and “pegs” regulate priming to influence Ca2+-triggering and membrane fusion. Other classes, “beams”, “steps”, “masts”, and “synaptic vesicle luminal filaments’ likely help organize and maintain the structural integrity of active zones. Extensive studies on the biochemistry that regulates secretion have led to comprehensive characterizations of the many conserved proteins universally involved in these trafficking events. Here, a hypothesis including a partial proteomic atlas of Active Zone Material is presented which considers the common roles, binding partners, physical features/structure, and relative positioning in the axon terminal of both the proteins and classes of macromolecules involved in the vesicle trafficking events. The hypothesis designates voltage-gated Ca2+ channels and Ca2+-gated K+ channels to ribs and pegs that are connected to macromolecules that span the presynaptic membrane at the active zone. SNARE proteins (Syntaxin, SNAP25, and Synaptobrevin), SNARE-interacting proteins Synaptotagmin, Munc13, Munc18, Complexin, and NSF are designated to ribs and/or pins. Rab3A and Rabphillin-3A are designated to top-masts and/or booms and/or spars. RIM, Bassoon, and Piccolo are designated to beams, steps, masts, ribs, spars, booms, and top-masts. Spectrin is designated to beams. Lastly, the luminal portions of SV2 are thought to form the bulk of the observed synaptic vesicle luminal filaments. The goal here is to help direct future studies that aim to bridge Active Zone Material structure, biochemistry, and function to ultimately determine how it regulates the trafficking events in vivo that lead to neurotransmitter secretion.

The presynaptic active zones in three different types of fibres in frog muscle

1984 ◽  
Vol 221 (1224) ◽  
pp. 369-373 ◽  
Keyword(s):  
Active Zone ◽  
Freeze Fracture ◽  
Longitudinal Axis ◽  
Frog Muscle ◽  
Muscle Fibres ◽  
Single Muscle Fibre ◽  
Single Muscle ◽  
Intermediate Type ◽  
Different Types ◽  
Active Zones

Presynaptic active zones were studied in slow, fast and intermediate types of frog muscle fibres in freeze-fracture replicas. In fast fibres, the double rows of paired particles are present on active zone ridges perperdicular to the longitudinal axis of the nerve whereas in slow fibres active zone ridges are rudimentary or absent and double rows of particles occur in all directions, mostly paired, sometimes single. In the intermediate type of muscle fibres both types of active zone deployment coexist on a single muscle fibre.

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