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

doi:10.1016/0896-6273(92)90235-6

Neuron

Volume 9, Issue 5, November 1992, Pages 805-813

https://doi.org/10.1016/0896-6273(92)90235-6 Get rights and content

Abstract

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. Flourescent 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.

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F(t) is the absolute fluorescence at time t; RMAX is the absolute fluorescence after maximum loading. Under these experimental conditions, nerve-evoked FM4-64 intensity decay reflects synaptic vesicle exocytosis at the nerve terminal (see Betz et al., 1992; Perissinotti et al., 2008; Noronha-Matos et al., 2011; Correia-de-Sá et al., 2013). For further details on the technique, please report to Noronha-Matos and Correia-de-Sá (2014).
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Furthermore, we investigated the size of the RRP using FM1-43 imaging in PFC slices (WT n = 15 cells, 7 animals and dys −/− n = 17cells, 9 animals). FM1-43 dye is much more fluorescent when partitioned in membranes, so dye release from synaptic vesicles can be followed as a decrease in fluorescence (Betz et al., 1992). This protocol is unable to discriminate glutamatergic from GABAergic synapses, but gives insights into the changes in vesicle dynamics after loss of dysbindin-1.
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Dysbindin-1 null mice showed a decrease in the ready releasable pool of synaptic vesicles, decreases in quantal size, decreases in the probability of release and deficits in the rate of endo- and exocytosis compared with wild-type controls. Moreover, the dysbindin-1 null mice show decreases in the [Ca^2 +]_i,expression of L- and N-type Ca^2 +channels and several proteins involved in synaptic vesicle trafficking and priming. Our results provide new insights into the mechanisms of action of dysbindin-1.
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Images were acquired in the real-time mode with a high-resolution cooled CCD camera (CoolSnap HQ, Roper Scientific Photometrics, Tucson, AZ, USA). Absolute fluorescence measurements were converted to a percentage of the maximum fluorescence detected after staining, by the following equation: %F(t) = 100 × [F(t) − FN-V]/[FMAX − FN-V], where F(t) is the absolute fluorescence at time t, FMAX is the absolute fluorescence after maximum loading, and FN-V is the non-vesicular fluorescence background (i.e. fluorescence remaining at the end of the stimulation) (Betz et al., 1992; Noronha-Matos et al., 2011; Perissinotti et al., 2008). The area (μm2) of nerve terminals loaded with FM4-64, before and after the exposure to the toxin, was measured in a blind manner by an external observer after anonymizing the data using the software package MetaFluor 6.3 (Molecular Devices Inc., Sunnyvale, California, USA); images were exported to Image J 1.37c software (NIH, Bethesda, MD, USA) for mathematical analysis.
Understanding the biological activity profile of the snake venom components is fundamental for improving the treatment of snakebite envenomings and may also contribute for the development of new potential therapeutic agents. In this work, we tested the effects of BthTX-I, a Lys49 PLA₂ homologue from the Bothrops jararacussu snake venom. While this toxin induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies support the hypothesis that BthTX-I might also exert a neuromuscular blocking activity due to its ability to alter the integrity of muscle cell membranes. To gain insight into the mechanisms of this inhibitory neuromuscular effect, for the first time, the influence of BthTX-I on nerve-evoked ACh release was directly quantified by radiochemical and real-time video-microscopy methods. Our results show that the neuromuscular blockade produced by in vitro exposure to BthTX-I (1 μM) results from the summation of both pre- and postsynaptic effects. Modifications affecting the presynaptic apparatus were revealed by the significant reduction of nerve-evoked [³H]-ACh release; real-time measurements of transmitter exocytosis using the FM4-64 fluorescent dye fully supported radiochemical data. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers, increase in the outflow of the intracellular lactate dehydrogenase enzyme and progressive depolarization of the muscle resting membrane potential. In conclusion, these findings suggest that the neuromuscular blockade produced by BthTX-I results from transient depolarization of skeletal muscle fibers, consequent to its general membrane-destabilizing effect, and subsequent decrease of evoked ACh release from motor nerve terminals.
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^∗: Present address: Muscular Dystrophy Research Laboratories, Newcastle General Hospital, Westgate Road, Newcastle-Upon-Tyne, NE4 6BE, U. K.

^†: Present address: Department of Physiology, University of Bristol School of Medical Sciences, University Walk, Bristol BS81TD, U. K.

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Neuron

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

Abstract

Neuron

Neuroscience

Neuroscience

Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction

Science

Optical monitoring of transmitter release and synaptic vesicle recycling at the frog neuromuscular junction

J. Physiol.

Activity-dependent staining and destaining of vertebrate motor nerve terminals

J. Neurosci.

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