Real-time video exocytosis in rat neuromuscular … video exocytosis in rat neuromuscular junction: ... this presentation may help setting up experiments ... By cooling the CCD to

Real-time video exocytosis in rat neuromuscular junction: a powerful live cell imaging technique

J.B. Noronha-Matos1,2 and P. Correia-de-Sá1,2,* 1 Laboratório de Farmacologia e Neurobiologia/UMIB 2 Center for Drug Discovery and Innovative Medicines (MedInUP), Instituto de Ciências Biomédicas de Abel Salazar

(ICBAS), Universidade do Porto, Rua de Jorge Viterbo Ferreira nº 228, 4050-313 Porto, Portugal * Author for correspondence.

Combining fluorescence microscopy to the use of membrane-selective FM-dyes (e.g. FM4-64) has become an emerging technique that allows the study of vesicle trafficking and organelle organization in cells. Real-time vesicle exocytosis and endocytosis in living motor nerve terminals is also a possibility. Evidence is provided on how high-frequency stimulation of motor nerve terminals in the presence of FM4-64 evokes hotspots of FM4-64 fluorescence, which co-localize with preferred sites of exocytosis. These dissipate during electrical nerve stimulation, which can be taken as a measure of synaptic vesicle exocytosis. This technique has allowed the study of neuromuscular exocytosis in disease models such as Myasthenia Gravis (an auto-immune disease) and the biological effects of snake venoms in this cell mechanism. Here, focus will be given to the performance of such experiments and the outcome data (both real-time exocytosis and endocytosis experiments), reinforcing the possibility to understand the mechanisms behind diseases affecting neuromuscular transmission.

Keywords: neuromuscular junction; live cell imaging; real-time transmitter endocytosis/exocytosis; snake venoms; Myasthenia Gravis

1. Introduction

Real time fluorescence microscopy provides a non-invasive way to explore cell mechanisms in living tissues. Tissues can be imaged in its naïve state either in vivo or freshly excised (ex vivo). Dynamic processes can be non-invasively monitored within the cell, or eventually in a group of cells (cell-to-cell interactions). Evaluation of neurotransmitters and neuromodulator’s exocytosis is of emerging importance in both health and disease conditions. One such example is the functional study of the neuromuscular junction (NMJ), the most studied of all synapses, which provides the link between myelinated motor nerves and skeletal muscle. In healthy conditions, this is an integral part of an impressively efficient biological amplification system, which converts minute nerve action potentials into muscle contraction. The study of such processes has stimulated the development of a number of methods (e.g. electrophysiology with sharp intracellular microelectrodes, radioisotope neurochemical techniques) including, more recently, those that involve fluorescence video microscopy to pin-point endocytosis and exocytosis from minute motor nerve endings. Measuring phenomena in the real-time mode brings several advantages and valuable information as compared to previous techniques lacking time resolution (e.g., neurochemistry-based techniques). With fluorescence video microscopy, it is possible to monitor the time required for a certain drug to produce a physiological effect and to follow the exact moment a stimulus (electric or chemical) is more active and when it loses effectiveness. On the other hand, microscopy-based methods add spatial resolution to the evaluation of transmitter release, since one can target a specific motor endplate type. Compared to electrophysiological methods, video microscopy with fluorescent dyes has certain advantages besides direct observation of the release sites, particularly because evaluation of transmitter exocytosis does not require the reduction of the safety factor of neuromuscular transmission and it does not rely on the function of the postsynaptic cell, although muscle paralysis is advisable to keep the motor endplate within focus for the time required to perform the experiments. So, in general terms, monitoring transmitter exocytosis in the real time mode, allows a better understanding of the molecular mechanisms underlying the dynamic changes necessary for transmitter release, thus contributing to a better understanding of synaptic plasticity. Real-time vesicle exocytosis and endocytosis in living motor nerve terminals during electrical stimulation has been explored using fluorescent dyes. The membrane-selective FM-dyes (e.g. FM4-64 and FM1-43) belong to a class of amphiphilic styryl dyes developed by Betz and co-workers [1, 2]. FM-dyes fluoresce significantly only when they are within a hydrophobic environment, such as lipid-rich plasma membranes, but not when remaining in solution. Besides having been extensively used in studies on animal cells, FM-dyes are being increasingly used for studying vesicle trafficking and organelle organization. One such example is the FM4-64 fluorescent dye, a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated. Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles but not when in the extracellular fluid. A washout step is used to help removal of the external dye from the plasma membrane, so that the remaining FM dye concentrates within synaptic vesicles waiting to be expelled by another round of nerve stimulation. The rate of vesicle exocytosis is measured from the resulting decay in the fluorescence signal. Since FM

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dyes can be applied external and transiently, it is a useful tool for determining the rate of exocytosis in both cell cultures and more complex tissue samples [1, 2]. These dyes have a number of advantages: they reversibly stain membranes; they do not cross freely membranes, ensuring that dye molecules do not escape from the membrane-bound organelles formed during endocytosis. Additionally, as mentioned previously, they are more fluorescent in a hydrophobic environment (cellular membrane) than in aqueous solutions. Thus, during exocytosis, the dye released from vesicles immediately becomes significantly less fluorescent, ensuring accurate measurements of the loss of dye from the cells [1, 2].

Fig. 1 Hardware setup for real-time video-microscopy experiment. (A) The epifluorescence microscope system with its various components tagged. (B) Phrenic nerve-hemidiaphragm preparation mounted in a Perspex chamber (3 ml capacity) on the stage of an upright epifluorescence microscope. Preparations are superfused with Tyrode‘s solution via an automatic perfusion system connected to a fast solution heating device. A pair of extracellular platinum electrodes is used to stimulate the phrenic nerve; care should be taken to avoid direct contact of the electrodes with muscle fibers. Images of the labeled nerve terminals (see below) are taken using a 63x water-immersion objective lens. See text for details.

Recently, Gaffield and co-workers provided evidence on how high- but not lower-frequency stimulation of motor nerve terminals in the presence of FM4-64 evokes fluorescence hotspots, which co-localize with preferred sites of exocytosis detected using transgenic mice motor nerve terminals expressing synaptopHluorin [3]. However, while synaptopHluorin spots disappear quickly after stimulation ends (because of internalization and re-acidification of endosomes, which quenches synaptopHluorin fluorescence), FM4-64 fluorescence remains high long after the loading stimulation ceases and, therefore, are more manageable. Dissipation of FM4-64 hotspots during electrical nerve stimulation can be taken as a measure of synaptic vesicle exocytosis. According to Betz and co-workers [1], this assumption is valid during approximately the first minute of continuous stimulation (the ‘dead time’ for endocytosis), which gives enough time to accommodate the unloading stimulation pattern. With this in mind, this chapter pertains to primarily target scientists who do not have prior experience in real-time video microscopy designed to monitor both endocytosis and exocytosis at the skeletal neuromuscular junction, although this presentation may help setting up experiments in other preparations. A description of the setup used as well as examples of outcome data will be presented, so the reader may her/himself be able to execute similar experiments.


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2. Setting up a real-time video microscopy experiment

For real time video microscopy experiments, our laboratory uses an upright epifluorescence microscope (Zeiss Axiophot, Oberkochen, Germany) equipped with a XBO 75W Xenon arc lamp. Xenon lamps are ideal for fluorescence measurements from the ultraviolet through the visible and into the near infrared region. This particular illumination source has been widely used due to its favorable characteristics, namely the ability to deliver high energy output over a wide wavelength range. FM4-64 fluorescence is detected using a BP 546/12 nm excitation filter, coupled to a LP 590 nm filter for fluorescence emission. Images are acquired in the real-time mode with a high-resolution cooled CCD camera (CoolSnap HQ, Roper Scientific Photometrics, Tucson, AZ, USA). Such cameras present very low noise characteristics. By cooling the CCD to approximately 30 degrees below the ambient temperature, these cameras are able to produce higher quality images than low cost uncooled cameras, an aspect that is essential during fast acquisition experiments. The CCD camera is connected to a computer running a digital image acquisition software (MetaFluor 6.3; Molecular Devices Inc., Sunnyvale, CA, USA) [Fig. 1(A)].

Fig. 2 Nerve-evoked transmitter endocytosis measured by video microscopy (loading protocol) using the FM4-64 fluorescent dye in rat neuromuscular junction. (A) A schematic diagram of the loading protocol. After a 30 min equilibration period, phrenic nerve-hemidiaphragm preparations are incubated with α-bungarotoxin (α-BTX, 4 µM during 15–20 min) to prevent nerve-evoked muscle fiber contractions that would otherwise complicate the analysis of fluorescence signals under the microscope. The preparations are then incubated for 10 min with FM4-64 (5 µM) before stimulation. Then, the phrenic nerve trunk is stimulated for 5 s at 50 Hz frequency. Following a 10 min period with the dye at rest, the FM4-64 is washed vigorously during 30 min. (B) Schematic diagram of the nerve terminal during the FM4-64 dye uptake. Nerve stimulation (5 s at 50 Hz frequency) in the presence of the styryl dye FM4-64 increases the fluorescence intensity in the whole terminal including a fast delivery pool of vesicles during the following endocytosis process, which co-localize with preferred sites of exocytosis, named hotspots. (C) Average FM4-64 fluorescence (arbitrary units) plotted versus time during FM4-64 loading in rat neuromuscular junction, measured in hotspot regions and in the whole nerve terminal. Stimulation (50 Hz for 5 s) is applied at zero time. In these circumstances, there is a lag time of 1–2 min before fluorescence increase (endocytosis) becomes significant. The micrograph at the figure right hand-side represents a typical nerve terminal during the loading protocol with FM4-64 at the indicated time. FM4-64 hotspots are evidenced with white arrows. Zoom is 630x.


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To perform these experiments in living tissues an immersion objective with a high magnification power is required. To detect and photograph motor endplates, which are small spots (average area of 500-1000 um2) in rodent hemidiaphragm preparations, we use a 63x/0.90 n.a. water immersion objective lens (Achroplan; Zeiss) [4]. Either the left or the right rat phrenic nerve-hemidiaphragm preparations (4–6 mm width) are isolated. The preparations are then mounted on the stage of the upright epifluorescence microscope [Fig. 1(B)]. The phrenic nerve is passed through a pair of extracellular platinum electrodes for further stimulation. Pulses are generated via a Grass S48 (Quincy, MA, USA) stimulator coupled to a stimulus isolation unit (Grass SIU5) operating in current constant mode [Fig. 1(B)]. Each muscle is superfused with gassed (95% O2 + 5% CO2) Tyrode solution (pH 7.4) containing (mM): NaCl 137, KCl 2.7, CaCl2 1.8, MgCl2 1, NaH2PO4 0.4, NaHCO3 11.9 and glucose 11.2 [4, 5, 6]. Temperature is adjusted to 230C in order to preserve FM4-64 fluorescent dye [4]. We use an automatic perfusion system (ValveLink8.2; Digitimer, Welwyn Garden City, UK) connected to a fast solution heating device (TC-344B; Harvard Apparatus, March-Hugstetten, Germany) to deliver both the Tyrode’s solution and test drugs at 1 ml/min. After a 30-min equilibration period, phrenic nerve-hemidiaphragm preparations are incubated with α-bungarotoxin (α-BTX 4 µM, during 15–20 min) to prevent nerve-evoked muscle fiber contractions that would otherwise complicate the analysis of fluorescence signals under the microscope. Under these circumstances, α-BTX blocks irreversibly muscle-type nAChRs containing α1 subunits with no action on nAChRs present on motor nerve terminals [7, 8]. This procedure, which is devoid of effect on neurotransmitter exocytosis [8], allows the identification of the associated nerve terminal without the interference of adjacent membranes non-selectively labeled with the FM4-64 dye. A final washout of 10 min is performed to remove unbound toxin [Fig. 2(A)]. At this point, no muscle contraction should be observed upon electrical nerve stimulation. It is important to perform this test before the loading protocol, focusing on muscle fibers under the microscope. Otherwise, as mentioned previously, visualization of motor nerve endplates will be impossible due to muscle twitching [4].

Fig. 3 Nerve-evoked transmitter exocytosis measured by video microscopy (unloading protocol) using the FM4-64 fluorescent dye in rat neuromuscular junction. (A) A schematic diagram of the unloading protocol. (B) Schematic diagram of transmitter exocytosis elicited by stimulation of motor nerve terminals loaded with the FM4-64 fluorescent dye. Using high-frequency (50 Hz) nerve stimuli, vesicles from the fast releasable pool fuse with the plasma membrane allowing the FM4-64 release (exocytosis). (C) A typical motor endplate labeled with FM4-64. Images were taken at the indicated times just before and after phrenic nerve stimulation at 50 Hz (zoom is 630x). Three regions of interest (ROIs) are shown: ROI 1, whole nerve terminal; ROI 2, background; ROI 3, nerve terminal. Right hand-side panel represents FM4-64 fluorescence intensity changes (arbitrary units) plotted versus time for the three indicated ROIs during the unloading protocol. Please note that fluorescence decays for ROI 1 but not for ROI 2 and 3. The vertical dashed lines represent starting and ending of the stimulus. Stimulation (50 Hz) started at zero time.


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3. Imaging of nerve terminals: loading (endocytosis) and unloading (exocytosis) protocols

3.1 Monitoring Endocytosis

In order to follow fluorescent dye staining of nerve terminals, we use the protocol presented in Fig. 2(A). The loading protocol is based on the fact that the efficacy of a nerve terminal depends on its ability to sustain vesicle supply from a reserve pool as well as on its ability to recycle spent vesicles for reuse [9]. So, after a 10 min incubation period with FM4-64 (5 µM) made up in Tyrode’s solution, loading of synaptic vesicles is achieved by stimulating the phrenic nerve trunk with 250 pulses of supramaximal intensity (0.04 ms duration, 8 mA) applied at a frequency of 50 Hz, followed by an additional 10 min period of rest with the dye [Fig. 2(A)]. Nerve stimulation promotes exocytosis that is followed by vesicles recycling (endocytosis). Consequently, incorporation of the FM4-64 dye is made possible in high-probability release or fast destaining pool of vesicles [9] [Fig. 2(B)]. During the incubation period with FM4-64, we observe a gradual increase in FM4-64 fluorescence in the whole nerve terminal, which is faster in the preferred sites of exocytosis (hotspots) [Fig. 2(C)]. This increase is more evident after starting the phrenic nerve stimulation (250 pulses delivered at 50-Hz frequency) at zero time. Fluorescence staining reaches a plateau, ensuring full loading of nerve terminals several minutes after stimulus application [Fig. 2(C)]. To remove the excess of unloaded fluorescent dye from the incubation fluid, the preparations are washed at a rapid flow rate during 30 min [Fig. 2(A)]. The major concern in FM-dye imaging is phototoxicity, which appears usually before any clear signs of photobleaching and alters the ability of the preparations to recycle vesicles. One way to avoid this constrain is to keep the illumination to a minimum. For example, images may be acquired once every 10 s for both loading and unloading protocols, with an exposure time of 150 ms (see below). To this end, it is mandatory to synchronize the excitation beam with image acquisition, so that photobleaching due to repetitive image acquisition of FM4-64-loaded nerve terminals is kept to a minimum (less than 3%); this procedure also avoids phototoxicity.

Fig. 4 FM4-64 fluorescence decay during electrical stimulation of the phrenic nerve with 50 Hz-bursts: dependence of action potentials generation and extracellular Ca2+. Experiments were performed in a control situation and (left graph) after removing external Ca2+ (+ EGTA 1 mM), in the presence (middle graph) of CdCl2 (500 µM, a non-specific voltage sensitive calcium channel blocker) or (right graph) of tetrodotoxin (TTX 1 µM, an action potential generation blocker). Fluorescence decay is expressed as a percentage of maximal loading considering that 100% is the fluorescence intensity at zero time. The vertical dashed lines represent starting and ending of the stimulus. Each value represents pooled data from three to eight experiments. The vertical bars represent ± SEM. *P < 0.05 (Student’s t-test) when compared with controls (no drugs).

3.2 Monitoring Exocytosis

For the exocytosis (unloading) experiments, we follow the protocol shown in Fig. 3(A). After the loading protocol, we focus on a labeled nerve terminal. Similarly to the loading protocol, fluorescence images are acquired using a 63x objective lens in the real-time mode. Exposure time is adjusted between 150–250 ms (binning is adjusted to 2–3 and gain to 1–2). Regions of interest (ROIs) are manually outlined and the average intensity of the pixels inside this area is calculated by the acquisition software. Three regions may be drawn: the whole nerve terminal (or eventually hotspots), the axon and an outlined region of non-stimulated motor endplate (for background normalization). During nerve stimulation (with 50 Hz intermittent bursts), a rapid destaining pool of vesicles will fuse with the pre-synaptic membrane allowing the release of the FM4-64 dye [Fig. 3(B)]. This causes the fluorescence inside the nerve terminal to decay, but the same does not occur in unspecific labeled regions (axon or background) [Fig. 3(C)]. To facilitate

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comparison between different preparations, absolute fluorescence measurements are converted to a percentage of the maximum fluorescence detected after staining, using the following equation:

%F(t) = 100 x [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) [1, 4, 9, 10]. Under our experimental conditions, nerve-evoked FM4-64 intensity decay reflects synaptic vesicle exocytosis, since it is largely reduced in the absence of extracellular Ca2+ (plus EGTA 1 mM) and in the presence of both tetrodotoxin (1 µM, an action potential generation blocker) and CdCl2 (500 µM, a non-specific voltage sensitive calcium channel blocker) [Fig. 4]. Test drugs depicted in Fig. 4 were added to the superfusion fluid 15 min before electrical nerve stimulation via the automatic perfusion system [Fig. 1].

4. Outcome data - examples

This powerful technique has allowed us to prove previously unpredictable changes in transmitter exocytosis observed in a rat model of Myasthenia Gravis along with the frequency-dependent neuromuscular fatigue due to blockade of the nicotinic receptors at skeletal muscle fibers [4]. Likewise, we recently demonstrated that the myotoxic venom of the Brazilian snake Bothrops jararacussu (bothropstoxin-I, BthTX-I) also decreased acetylcholine exocytosis at the rat neuromuscular junction [4, 11]. These findings are shown here to illustrate the relevance of real-time video microscopy to study exocytosis at the neuromuscular synapse.

4.1 Using real time video microscopy to assess neuromuscular transmission failure

Myasthenia gravis (MG) is a common autoimmune disease which is characterized by fatigable muscle weakness. About 85% of patients with generalized MG have high levels of IgG antibodies against the muscle-type α1-containing nicotinic ACh receptor (nAChR). Antibodies against nAChR reduce the number of effective receptors to nearly one-third of the normal amount using several mechanisms [11]. Antibodies bind to the nAChR to cause receptor internalization and degradation. The antibody-nAChR complex also binds to complement resulting in damage to the post-synaptic membrane, which typically has fewer secondary synaptic folds and a widened synaptic cleft that leads to loss of functional receptors [12, 13, 14, 15], compromising neuromuscular transmission. These features reduce the safety margin of neuromuscular transmission and are particularly evident during high-frequency nerve activity. We, therefore, hypothesized that besides the skeletal muscle block observed in MG these patients could also exhibit changes in the normal transmitter release facilitation triggered by high-frequency nerve stimuli. If this were the case than novel therapeutic targets may be envisaged for the management of neuromuscular transmission deficits in myasthenics.

Fig. 5 Nerve-evoked transmitter exocytosis measured by video microscopy (unloading protocol) using the FM4-64 fluorescent dye in rat phrenic nerve-hemidiaphragm preparations from control and TIMG rats. (A) Time course of the FM4-64 fluorescence decay during electrical stimulation of the phrenic nerve with 50 Hz-Bursts. The vertical dashed lines represent starting and ending of the stimulus. (B) Comparison of FM4-64 fluorescence signal 80 s after starting nerve stimulation in control and TIMG preparations. Fluorescence decay is expressed as a percentage of maximal loading considering that 100% is the fluorescence intensity at zero time.

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Using a rat model of Myasthenia Gravis model (TIMG) generated by repeated injections of the muscle-type nicotinic irreversible antagonist α-bungarotoxin for 3-6 weeks [4], we demonstrated that transmitter exocytosis was significantly attenuated in myasthenic animals as compared to their control littermates. These results were reproduced by measuring the evoked [3H]-acetylcholine outflow following nerve stimulation (50-Hz intermittent bursts of 150 pulses delivered at 20s intervals) in samples collected every 3 min. Fig. 5(A) shows differences in the time course of FM4-64 fluorescence decay during phrenic nerve stimulation between control and TIMG preparations. It is obvious that TIMG motor endplates are unable to sustain transmitter exocytosis during repetitive electrical nerve stimulation as compared with controls, i.e. destaining of FM4-64 fluorescence during phrenic nerve stimulation was significantly lower in TIMG animals as compared with the control group [Fig. 5(A)]. This was evidenced despite no significant differences on FM4-64 fluorescence intensity were observed before stimulus application in both preparations. These changes in transmitter exocytosis were positively correlated with the severity of the neuromuscular blockade (e.g. muscle weakness and breathing impairment) exhibited by TIMG animals when subject to repeated motor nerve activity. Such differences were clearly observed when exocytosis was measured in the time-lapse mode, but they can be partially underestimated if one looks only at the remaining fluorescence after the stimulation period [Fig. 5(B)] or when we used the radiochemical approach that lacks time resolution. Notably, we observed a delay in the FM4-64 fluorescence decay starting 20s after the beginning of phrenic nerve stimulation in myasthenic preparations when compared to controls; the magnitude of this gap increased progressively towards the end of the stimulation period. Clearly, this observation was only possible using a technique with enough time resolution, such as fluorescence video microscopy [4]. Contrary to time-resolved electrophysiology, video microscopy adds spatial resolution allowing nerve terminal imaging, which may be useful to delineate the microdomains most responsible for transmitter exocytosis at a given circumstance and to monitor the coincident morphological changes operated on phrenic motor endplates from control and TIMG animals (see e.g. [4]).

4.2 Pharmacological manipulation of transmitter exocytosis using fluorescence video microscopy

The analysis of FM4-64 fluorescence decay allows monitoring of transmitter exocytosis from nerve terminals in a moment-to-moment basis. One can perceive divergence between different types of fibers and fluorescence fluctuations in discrete microdomains of the nerve terminals throughout stimulus application. These signals can be pharmacologically manipulated and their variations can be followed in the real time mode, i.e. upon activation or inhibition of certain nerve terminal receptors or calcium channels one can detect changes in transmitter exocytosis through modifications of the FM4-64 fluorescence decay (exocytosis). Fig. 6 shows that transmitter release (FM4-64 decay) from stimulated motor nerve terminals is significantly enhanced in the presence of CGS21680C, a selective adenosine A2A receptor agonist. This new finding confirms previous neurochemical studies showing that activation of adenosine A2A receptor on motor nerve terminals may contribute to overcome tetanic depression of the rat neuromuscular transmission during neuronal firing [16, 17]. This situation, which is particularly relevant during high-frequency nerve firing, lead us to propose that A2A receptors activation and, subsequent, Ca2+ influx through Cav1 (L-type) calcium channels play a significant role in re-establishing neuromuscular transmission in myasthenic patients [4, 17, 18]. Real time measurements of transmitter exocytosis using the fluorescence dye, FM4-64 add some information to previously released data by demonstrating that the facilitatory effect of CGS 21680C was only observed 40 s after starting nerve stimulation and increased thereafter [Fig. 6(A)]. This result may be physiologically significant given that transmitter release facilitation triggered by A2A receptors activation requires a coordinated shift in Ca2+ channel dynamics from fast activating “prevalent” Cav2.1 (P-type) to long-lasting “facilitatory” Cav1 (L-type) channels [4, 17, 18].


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Fig. 6 Pharmacological manipulation of nerve-evoked transmitter exocytosis measured by video-microscopy using the FM4-64 fluorescent dye in rat hemidiaphragm preparations. (A) Nerve-evoked exocytosis in the absence (control) and in the presence of a selective adenosine A2A receptor agonist (CGS 21680C, 3 nM, 15 min incubation) (B) Nerve-evoked exocytosis in the absence (control) and in the presence of bothropstoxin-I (BthTX-I, 1 µM, 30 min incubation). Fluorescence decay is expressed as a percentage of maximal loading considering that 100% is the fluorescence intensity at zero time. Phrenic nerve was stimulated with 5 Hz trains of 750 supramaximal intensity pulses. The vertical dashed lines represent starting and ending of the stimulus.

Another example of this powerful technique to detect pathophysiological relevant changes in transmitter exocytosis was applied to study the biological effects of bothropstoxin-I (BthTX-I), a Lys49 phospholipase A2 (PLA2) from Bothrops jararacussu snake venom, on neuromuscular transmission [19]. Understanding the biological activities of snake venoms is paramount for improving the treatment of their life-threatening or highly disabling sequelae and may serve as an inspiration for the development of new drugs of potential clinical use [11]. While BthTX-I induces conspicuous myonecrosis by a catalytically independent mechanism, a series of in vitro studies supported the hypothesis that this toxin could also exert a neuromuscular blocking activity. Thus, direct measurement of acetylcholine release from stimulated motor nerve terminals would be of great help to elucidate the involvement of the presynaptic component in the paradoxical inhibitory effect of BthTX-I on neuromuscular transmission. Our results showed for the first time that neuromuscular blockade produced by in vitro exposure to Bth-TX-I results from the summation of both post- and presynaptic effects. The postsynaptic effect of BthTX-I was characterized by typical histological alterations in the architecture of skeletal muscle fibers (edema, round-shape transformation of polygonal skeletal muscle fibers, which also become blank due to the loss of myoflibrils), leading to a progressive depolarization of the resting membrane potential. Modifications affecting the presynaptic apparatus were revealed by the decrease in the mean size of motor nerve terminals together with the significant reduction of nerve-evoked transmitter release, the last one was demonstrated by radiochemical studies designed to measure [3H]-acetylcholine release and by real-time video microscopy using the FM4-64 fluorescence [11]. Fig. 6(B) shows that exposure of rat hemidiaphragm preparations to BthTX-I (1 µM) for 30 min causes a time-dependent irreversible decrease of transmitter exocytosis by about 50%, which is revealed by a decreased FM4-64 fluorescence decay rate during phrenic nerve stimulation (5 Hz trains of 750 pulses). Real-time video-microscopy also allowed us to record transient myofasciculations 5s after addition of BthTX-I (1 µM) to the organ bath [11]. We correlated these results to skeletal muscle fibers depolarization as a consequence of potassium outflow by membrane cell rupture, since this effect was observed without phrenic nerve stimulation in the presence of the muscle-type nicotinic receptor blocker, α-BTX (4 µM).

5. Conclusions

The use of fluorescence video microscopy to probe biological phenomena is rapidly expanding into all aspects of molecular and cell biology. Fluorescence microscopy strength resides on attributes that are not readily available in other contrast modes with traditional optical microscopy. The application of an array of fluorochromes has made possible identification of cells and sub-microscopic cellular components with a high degree of specificity [20, 21, 22]. Real time fluorescence video microscopy adds spatial resolution to most common techniques used to evaluate cell activity. This

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chapter gives good examples of a prototypical application of this powerful technique. Our aim was to highlight the pros and cons of fluorescence video microscopy to study real-time vesicle exocytosis and endocytosis in living motor nerve terminals using styryl dyes (e.g. FM4-64). This work was designed for non-specialists and covers important practical considerations that should be taken into account while setting up and executing video microscopy experiments with FM dyes. Evidence is provided on how high-frequency stimulation of motor nerve terminals in the presence of FM4-64 evokes hotspots of fluorescence, which co-localize with preferred sites of exocytosis. These dissipate during electrical nerve stimulation, which can be taken as a measure of synaptic vesicle exocytosis. This technique has allowed us to study for the first time transmitter exocytosis deficits occurring in neuromuscular disease conditions, such as Myasthenia Gravis and snake venom intoxications.

Acknowledgements This research was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal, FEDER funding, project REEQ/1168/SAU/2005, REEQ/1264/SAU/2005 and PEst-OE/SAU/UI0215/2014). J.B.N.M. is in receipt of a PhD Fellowship from FCT (SRFH/BD/68584/2010).

References

[1] Betz WJ, Mao F, Bewick GS. Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. Journal of Neuroscience. 1992; 12:363–375.

[2] Betz WJ, Mao F, Smith CB. Imaging exocytosis and endocytosis. Current Opinion in Neurobiology. 1996; 6:365–371. [3] Gaffield MA, Tabares L, Betz WJ. Preferred sites of exocytosis and endocytosis colocalize during high- but not lower

frequency stimulation in mouse motor nerve terminals. Journal of Neuroscience. 2009; 29:15308–15316. [4] Noronha-Matos JB, Morais T, Trigo D, Timóteo MA, Magalhães-Cardoso MT, Oliveira L, Correia-de-Sá P. Tetanic failure due

to decreased endogenous adenosine A2A tonus operating neuronal CaV1 (L-type) influx in Myasthenia gravis. Journal of Neurochemistry. 2011; 117:797–811.

[5] Correia-de-Sá P, Sebastião AM, Ribeiro JA. Inhibitory and excitatory effects of adenosine receptor agonists on evoked transmitter release from phrenic nerve ending of the rat. British Journal of Pharmacology. 1991; 103:1614–1620.

[6] Correia-de-Sá P, Timóteo MA, Ribeiro JA. Presynaptic A1 inhibitory/A2A facilitatory adenosine receptor activation balance depends on motor nerve stimulation paradigm at the rat hemidiaphragm. Journal of Neurophysiology. 1996; 76:3910–3919.

[7] Plomp JJ, van Kempen GT, Molenaar PC. Adaptation of quantal content to decreased postsynaptic sensitivity at single endplates in α-bungarotoxin-treated rats. Journal of Physiology. 1992; 458:487–499.

[8] Faria M, Oliveira L, Timóteo MA, Lobo MG, Correia-de-Sá P. Blockade of neuronal facilitatory nicotinic receptors containing alpha 3 beta 2 subunits contribute to tetanic fade in the rat isolated diaphragm. Synapse. 2003; 49:77–88.

[9] Perissinotti PP, Giugovaz-Tropper B, Uchitel OD. L-Type calcium channels are involved in fast endocytosis at the mouse neuromuscular junction. European Journal of Neuroscience. 2008; 27:1333–1344.

[10] Matsuka Y, Edmonds B, Mitrirattanakul S, Schweizer FE, Spigelman I. Two types of neurotransmitter release patterns in isolectin B4-positive and negative trigeminal ganglion neurons. Neuroscience. 2007; 144:665–674.

[11] Correia-de-Sá P, Noronha-Matos JB, Timóteo MA, Ferreirinha F, Marques P, Soares AM, Carvalho C, Cavalcante WLG, Gallacci M. Bothropstoxin-I reduces evoked acetylcholine release from rat motor nerve terminals: Radiochemical and real-time video-microscopy studies. Toxicon. 2013; 61:16–25.

[12] Lindstrom JM. Acetylcholine receptors and myasthenia. Muscle Nerve. 2000; 23:453–477. [13] Conti-Fine BM, Milani M, Kaminski HJ. Myasthenia gravis: past, present, and future. Journal of Clinical Investigation. 2006;

116:2843–2854. [14] Juel VC, Massey JM. Myasthenia gravis. Orphanet Journal of Rare Diseases. 2007; 2:44. [15] van Kempen GT, Trip SA, Molenaar PC. Acetylcholinesterase activity of skeletal muscle in a non-immunogenic model for

myasthenia gravis in rats. Journal of Neural Transmition. 1999; 106:423–431. [16] Cunha RA, Correia-de-Sá P, Sebastião AM, Ribeiro JA. Preferential activation of excitatory adenosine receptors at rat

hippocampal and neuromuscular synapses by adenosine formed from released adenine nucleotides. British Journal of Pharmacology. 1996; 119:253–260.

[17] Oliveira L, Timóteo MA, Correia-de-Sá P. Tetanic depression is overcome by tonic adenosine A2A receptor facilitation of L-type Ca2+ influx into rat motor nerve terminals. Journal of Physiology. 2004; 560:157–168.

[18] Correia-de-Sá P, Timoteo MA, Ribeiro JA. A2A adenosine receptor facilitation of neuromuscular transmission: influence of stimulus paradigm on calcium mobilization. Journal of Neurochemistry. 2000; 74: 2462-2469.

[19] Valentin E, Lambeau G. Increasing molecular diversity of secreted phospholipases A2 and their receptors and binding proteins. Biochimica et Biophysica Acta. 2000; 1488:59–70.

[20] Lichtman JW, Conchello JA. Fluorescence microscopy. Nature Methods. 2005; 2:910-919. [21] Yuste R. Fluorescence microscopy today. Nature Methods. 2005; 2:902-904. [22] Shaner NC, Steinbach PA, Tsien RY. A guide to choosing fluorescent proteins. Nature Methods. 2005; 2:905-909.


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Real-time video exocytosis in rat neuromuscular … video exocytosis in rat neuromuscular junction: ... this presentation may help setting up experiments ... By cooling the CCD to