Peculiarities of synaptic vesicle recycling in frog and mouse motor nerve terminals

Using electrophysiology and fluorescence microscopy with dye FM 1-43, a comparative study of peculiarities of neurotransmitter secretion, synaptic vesicle exo-endocytosis and recycling has been carried out in nerve terminals (NT) of the skin-sternal muscle of the frog Rana ridibunda and of the white mouse diaphragm muscle during a long-term high-frequency stimulation (20 imp/s). The obtained data have allowed identifying three synaptic vesicle pools and two recycling ways in the motor NT. In the frog NT, the long-term high-frequency stimulation induced consecutive expenditure of the pool ready to release, the mobilizational, and reserve vesicle pools. The exocytosis rate exceeded markedly the endocytosis rate; the slow synaptic vesicle recycling with replenishment of the reserve pool was predominant. In the mouse NT, only the vesicles of the ready to release and the mobilizational pools, which are replenished predominantly by fast recycling, were exocytosed. The exo- and endocytosis occurred practically in parallel, while vesicles of the reserve pool did not participate in the neurotransmitter secretion. It is suggested that evolution of the motor NT from the poikilothermal to homoiothermal animals went by the way of a decrease of the vesicle pool size, the more economic expenditure and the more effective reuse of synaptic vesicles owing to the high rates of endocytosis and recycling. These peculiarities can provide in NT of homoiothermal animals a long maintenance of neurotransmitter secretion at the steady and sufficiently high level to preserve reliability of synaptic transmission in the process of the high-frequency activity.     

Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals

The fluorophore FM1-43 appears to stain membranes of recycled synaptic vesicles. We used FM1-43 to study mechanisms of synaptic vesicle clustering and mobilization in living frog motor nerve terminals. FM1- 43 staining of these terminals produces a linear series of fluorescent spots, each spot marking the cluster of several hundred synaptic vesicles at an active zone. Most agents we tested did not affect staining, but the phosphatase inhibitor okadaic acid (OA) disrupted the fluorescent spots, causing dye to spread throughout the terminal. Consistent with this, electron microscopy showed that vesicle clusters were disrupted by OA treatment. However, dye did not spread passively to a uniform spatial distribution. Instead, time lapse movies showed clear evidence of active dye movements, as if synaptic vesicles were being swept along by an active translocation mechanism. Large dye accumulations sometimes occurred at sites of Schwann cell nuclei. These effects of OA were not significantly affected by pretreatment with colchicine or cytochalasin D. Electrophysiological recordings showed that OA treatment reduced the amount of acetylcholine released in response to nerve stimulation. The results suggest that an increased level of protein phosphorylation induced by OA treatment mobilizes synaptic vesicles and unmasks a powerful vesicle translocation mechanism, which may function normally to distribute synaptic vesicles between active zones.     

Monitoring synaptic vesicle recycling in frog motor nerve terminals with FM dyes

Ultrastructural observations made in the study of the frog neuromuscular junction (NMJ) almost three decades ago showed that synaptic vesicle cycling functions through a slow pathway, requiring the use of clathrin-coated vesicles and an endosomal compartment. Simultaneously, a conceptually simpler model emerged, postulating rapid retrieval of vesicle membrane through a mechanism similar to a reversal of vesicle fusion. With the advent of fluorescence imaging which allows the investigator to monitor recycling in living nerve-muscle preparations, new data appeared which reconcile at least in part the two models, indicating that both may be important at this synapse. Two different synaptic vesicle pools can be defined, a readily releasable pool (RRP), consisting of quanta that are immediately available for release, and a reserve pool (RP) that is exocytosed only after prolonged stimulation. Vesicles in the RRP recycle through a fast endocytic pathway, which does not rely on an endosomal compartment, while vesicles in the RP cycle more slowly through formation of infoldings and endosomes and their subsequent severance into vesicles. The two pools mix slowly, and their recycling may be regulated by different mechanisms.     

Intracellular movements of fluorescently labeled synaptic vesicles in frog motor nerve terminals during nerve stimulation.

We stained synaptic vesicles in frog motor nerve terminals with FM1-43 and studied changes in the shape and position of vesicle clusters during nerve stimulation. Each stained vesicle cluster appeared as a fluorescent spot. During repetitive nerve stimulation the spots gradually dimmed, most without changing shape or position. Occasionally, however, a spot moved, appearing in some cases to stream toward and coalesce with a neighboring spot. This suggests the existence of translocation mechanisms that can actively move vesicles in a coordinated fashion between vesicle clusters. Within single clusters, we saw no signs of such directed vesicle movements. Fluorescent spots in terminals viewed from the side with a confocal microscope did not shrink toward the presynaptic membrane during nerve stimulation, but dimmed uniformly. This suggests that vesicles continuously mix within a cluster during destaining and provides no evidence of active vesicle translocators within single vesicle clusters for moving vesicles to the presynaptic membrane.     

Mobility of synaptic vesicles in different pools in resting and stimulated frog motor nerve terminals

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.     

Transmitter release in proximal and distal parts of a nerve terminal of the frog sartorius muscle

Evoked synaptic potential were recorded extracellularly in experiments on a nervemuscle preparation of the frog sartorius muscle. A decrease in evoked transmitter release was found from the proximal to the distal parts of the nerve ending, due to a decrease in the probability of transmitter quantum release. The terminal portions of the synapse are less sensitive than the proximal parts to changes in Ca⁺⁺ concentration, they show less marked facilitation of transmitter release during paired and repetitive stimulation, and exhibit deeper and more rapidly developing depression. It is concluded that differences in transmitter release in the terminal parts of the synapse are due to the low reserves of transmitter and the lower premeability of the presynaptic membrane to Ca⁺⁺.     

Two endocytic recycling routes selectively fill two vesicle pools in frog motor nerve terminals

We have identified and characterized two vesicle recycling pathways in frog motor nerve terminals. We exploited the differential staining properties of FM dyes of varying hydrophobicity to label selectively two different vesicle pools, using optical imaging and electron microscopy of photoconverted dyes. During a 1 min tetanus, a rapidly recycling route places vesicles selectively into a small readily releasable pool comprising about 20% of vesicles. After the tetanus, a much slower pathway (from which FM2-10 but not FM1-43 can be rinsed) delivers vesicles via infoldings and cisternae selectively to a reserve pool with a halftime of about 8 min. Mixing between the two pools is slow. During stimulation at 30 Hz, 10-15 s is required to mobilize and release dye from the reserve pool.     

Dense-core vesicles in motor nerve terminals

Summary Motor nerve terminals on white and intermediate muscle fibers of the Atlantic hagfish (Myxine glutinosa, L.) contain translucent synpatic vesicles and about 1–2% dense-core vesicles. Terminals on red muscle fibers contain up to 40% dense-core vesicles with diameter 800–1100 Å. Examinations for formaldehyde-induced fluorescence indicate yellow fluorescence (5-HT ?) apparently corresponding with terminal axons on red muscle fibers in craniovelar muscles. Possibly red muscle fibers of Myxine receive monoaminergic innervation.The author is indebted to Dr. Finn Walvig, Biological station, University of Oslo, Drøbak, for supply of hagfishes.     

Ionic currents in nerve terminals of the frog sartorius muscle

Nerve terminal responses produced by stimulating the motor nerve were recorded extracellularly from the nerve endings of the frog sartorius muscle. A triphasic response occurred in the proximal areas of the nerve ending, beginning with a positive phase. Ionotophoretic application of tetrodotoxin, tetraethylammonium, and 4-aminopyridine indicated that the negative phase reflected inward sodium current and the third (positive) phase indicated outward potassium current. A late slow negative component was recorded using CaCl₂-filled electrodes during perfusion of erve-muscle preparations with a calcium-free solution containing tubocurarine. This component was dependent on the Ca²⁺ concentration present in the electrode, increasing when tetraethylammonium and 4-aminopyridine were added and disappearing under the effects of Co²⁺. Similar components were recorded using microelectrodes containing Sr²⁺ and Ba²⁺. It was deduced that the slow components in the response indicate currents passing through voltage-dependent calcium channels in the presynaptic membrane of the nerve ending. The time course of the calcium current is compared with that of transmitter release at the synapse.S. V. Kurashov Medical Institute, Ministry of Health of the RSFSR, Kazan'. V. I. Ulanov-Lenin Kazan' State University. Translated from Neirofiziologiya, Vol. 17, No. 6, pp. 770–779, November–December, 1985.     

Kinetics of synaptic depression and vesicle recycling after tetanic stimulation of frog motor nerve terminals.

We measured the time courses of two key components of the synaptic vesicle cycle during recovery from synaptic depression under different conditions, and used this and other information to create a kinetic model of the vesicle cycle. End plate potential (EPP) amplitudes were used to follow recovery from synaptic depression after different amounts of tetanic stimulation. This provided an estimate of the time course of vesicle mobilization from the reserve pool to the docked (readily releasable) pool. In addition, FM1-43 was used to measure the rate of membrane retrieval after tetanic stimulation, and the amount of membrane transferred to the surface membrane. This provided a measure of the rate of refilling of the reserve pool with recycled vesicles. The time courses of both synaptic depression and endocytosis were slowed by prolonged tetanic stimulation. This behavior could be fitted by a simple model, assuming a first-order kinetics for both vesicle endocytosis and mobilization. The results show that a nearly 20-fold decrease in the rate constant of endocytosis greatly delays refilling of the depleted reserve pool. However, to fully account for the slower recovery of depression, a decrease in the rate constant of vesicle mobilization from the reserve pool of about sixfold is also required.     

Vesicle cycle in mouse diaphragm motor nerve terminals

In our research on mouse diaphragm muscles the dynamic of neurotransmitter secretion and synaptic vesicles recycling (exo-endocytosis cycle) at the long-term rhythmic stimulation (20Hz) are explored using an intracellular microelectrode registration and a fluorescent microscopy. It have been shown, thate change of end plant potentials (EPP) amplitude at the rhythmic training occurs in three phases: initial transient decrease, long amplitude stabilization (1-2 min)--the plateau and secondary slow decrease. After 3 minute stimulations the EPP amplitude recovery observed during several seconds. Loading the synaptic vesicle by fluorescent endocytic dye FM 1-43 had shown that the rhythmic stimulation results to gradual (during 5-6 mines) fluorescence decrease in NT, indicating the synaptic vesicle exocytosis. The quantum analysis of the electrophysiological data and their comparison to the fluorescent researches date has allowed to assume, that mouse motor nerve terminals are characterized by high rate of endocytosis and fast synaptic vesicle reuse (average recycling time about 50 sec) that can provide effective maintenance of synaptic transmission at long high-frequency activity. Sizes of ready releasable and recycling synaptic vesicle pools are quantitatively determined. It is assumed, that vesicle recycling occurs on a short fast way to inclusion in recycling pool. So, in the stimulation protocol that were used the synaptic vesicles from reserve pool remain unused. Thus in our conditions recycling pool vesicles cycle repeatedly without reserve pool release.     

Electrical activity at motor nerve terminals of the mouse

1. Extracellular recording of the electrical activity of mammalian end-plate made it possible to distinguish three different parts on the presynaptic terminal. Bath or ionophoretic application of ionic channel blockers induced specific alterations of electric signals which revealed the localization of classical ionic channels. 2. Sodium channels are restricted to a small area, sharply located after the end of the last myelinated segment and potassium channels are distributed over the rest of the terminal branches. 3. The first direct evidence of the presence of calcium channels at the terminal part of the motor endings was obtained, when potassium channel activity was suppressed. They occupy the same region as potassium channels. 4. Finally, the differential distribution of ionic channels over the terminal and the time-course of calcium current are discussed in relation to transmitter release.     

Effect of exogenous acetylcholine on potassium currents in the frog motor nerve ending during acetylcholinesterase inhibition

The effect of exogenous acetylcholine (ACh) on potassium currents in the motor nerve ending (NE) has been studied in neuromuscular preparations of the frog cutaneous-sternal muscle by extracellular recording of evoked electrical potentials from the NE. The investigation was performed during inhibition of acetylcholinesterase (AChE) activity by specific inhibitors and AChE removal from the synaptic cleft by collagenase. After AChE inhibition by either armine or proserine, or after treatment of the preparation with collagenase, no effect of exogenous ACh in concentrations of 1·10^–4–6·^–4 mole/liter was observed, in contrast to results from preparations with intact AChE. However, under the same conditions, as in the case of active AChE, ACh in concentrations of 7·10^–4–2·10^–3 mole/liter inhibited Ca-activated potassium current of the NE membrane. Experiments with dipyroxim, a synaptic AChE reactivator, have shown that the ACh effect on the potential-dependent potassium current is mediated by specific AChE. The role of AChE is discussed in respect to its significance for realization of the ACh action on potential-dependent potassium current in NE.Translated from Neirofiziologiya, Vol. 25, No. 2, pp. 146–149, March–April, 1993.     

Possible functional role of substance P on the mammalian motor nerve terminals

The effect of substance P (sP) on mammalian skeletal myoneural transmission was studied employing innervated and denervated isolated rat diaphragm preparations. sP at a concentration of 3.7 nM facilitated the indirect twitch responses of the rat diaphragm and antagonised the paralytic effect of d-tubocurarine (d-Tc). sP failed to affect the direct twitch responses as well as the contractures induced by acetylcholine (ACh) and potassium chloride (KCl) in the denervated diaphragm. The amount of ACh released into the bathing medium in response to tetanic stimulation of the phrenic nerve was doubled in presence of sP. The study illustrates a presynaptic facilitatory involvement of sP on mammalian myoneural transmission.