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ABSTRACT |
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M), oligomycin (8 g ml-1) or CCCP and oligomycin together.
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INTRODUCTION |
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Mitochondria provide metabolic energy for neuronal function and play a critical role in calcium (Ca2+) homeostasis (Gunter & Gunter, 1994; Babcock & Hille, 1998). Loss of Ca2+ buffering by mitochondria and impairment of energy metabolism are both suggested to play key roles in neuronal death induced by excitotoxic amino acids (Kiedrowski & Costa, 1995; Budd & Nicholls, 1996; Schinder et al. 1996; White & Reynolds, 1996; Castillo et al. 1998; Nicholls & Budd, 1998; Stout et al. 1998). In addition, alteration of mitochondrial function is a critical factor in the aetiology of neurodegenerative diseases (Beal, 1992).
Recently, Calupca et al. (1999) demonstrated that in an elevated extracellular Ca2+ concentration ([Ca2+]o) of 3.6 mM, miniature endplate current (MEPC) frequency declined at snake twitch muscle fibre motor endplates during prolonged K+ stimulation. In contrast, MEPC frequency was maintained at very high levels for many hours in 35 mM potassium propionate with 1 mM [Ca2+]o and 4.2 mM [Mg2+]o. The progressive Ca2+-induced decline in MEPC frequency was correlated with a disruption of the ultrastructure of mitochondria within motor nerve terminals. Furthermore, the addition of glucose to stimulate ATP production by glycolysis reduced the Ca2+-induced decline in MEPC frequency without reversing the alterations in mitochondrial morphology. It was postulated that the inhibition of quantal release, during exposure to elevated K+ with 3.6 mM [Ca2+]o, resulted from a progressive loss of mitochondrial function that led to an interruption of mitochondrial ATP production and progressive decrease in the cytosolic ATP/ADP ratio (Calupca et al. 1999). However, recent direct measurements of Ca2+ accumulation in motor nerve terminals demonstrated that sequestration of Ca2+ by mitochondria contributes significantly to the buffering of the intracellular Ca2+ concentration ([Ca2+]i) during nerve terminal stimulation (David et al. 1998; Peng, 1998; David, 1999; Scotti et al. 1999; David & Barrett, 2000). These observations raise the possibility that interference with [Ca2+]i buffering and consequent sustained elevation of [Ca2+]i could be another key factor contributing to the altered nerve terminal function noted by Calupca et al. (1999). Consequently, in the present study, we have tested whether disruption of mitochondrial function, using pharmacological agents that preferentially disrupt ATP production alone or ATP production and [Ca2+]i buffering, more closely mimicked the alterations in nerve terminal function observed in our prior study.
To test whether inhibition of mitochondrial ATP production was critical, we treated preparations with the ATP synthase (F0/F1-ATPase) inhibitor oligomycin. With oligomycin present, mitochondrial ATP production would be inhibited, but because the mitochondrial proton gradient is not compromised, Ca2+ sequestration should continue (Budd & Nicholls, 1996). To test whether inhibition of [Ca2+]i buffering as well as ATP production by mitochondria was involved, we exposed preparations to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). In CCCP, the mitochondrial proton gradient is dissipated, which disrupts mitochondrial Ca2+ uptake mechanisms (Friel & Tsien, 1994; Werth & Thayer, 1994; Kiedrowski & Costa, 1995; White & Reynolds, 1995; Budd & Nichols, 1996; Herrington et al. 1996; Park et al. 1996; Wang & Thayer, 1996; Babcock et al. 1997; Mostafour et al. 1997; David et al. 1998; David & Barrett, 2000). In separate experiments, we tested the combination of CCCP and oligomycin to ensure that increased consumption of cytoplasmic ATP was not the primary effect of protonophore treatment. We have also tested whether stimulation of glycolysis by the addition of glucose during exposure to elevated K+ prevented the effects noted following exposure to oligomycin, CCCP or oligomycin and CCCP together.
The results of the present study showed that with preparations treated with oligomycin, CCCP or both drugs simultaneously, transmitter release, measured as the MEPC frequency recorded in an elevated K+ solution containing 1 mM [Ca2+]o and 4.2 mM [Mg2+]o, declined over time. In the absence of drug treatment, MEPC frequency remained elevated. The rate of decline in quantal release was more rapid with CCCP or CCCP plus oligomycin than with oligomycin treatment alone. Lastly, following exposure to oligomycin, CCCP or CCCP and oligomycin together, the addition of glucose to stimulate substrate level ATP production by glycolysis attenuated the decline in K+-stimulated MEPC frequency. We propose that the decline in K+-stimulated quantal release in preparations treated with CCCP, oligomycin or CCCP and oligomycin together resulted from a progressive elevation of [Ca2+]i. For oligomycin-treated nerve terminals, a progressive elevation of [Ca2+]i could occur as the cytoplasmic ATP/ADP ratio decreases causing energy-dependent [Ca2+]i buffering mechanisms to fail. In addition, we suggest that the decline in MEPC frequency may occur more rapidly in preparations treated with CCCP or CCCP and oligomycin together because when mitochondrial Ca2+ buffering and ATP production are both inhibited, the sustained elevation of [Ca2+]i happens more rapidly.
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METHODS |
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All experiments were performed on visually identified twitch muscle fibre endplates in the costocutaneous muscle of garter snakes (Thamnophis) at room temperature (19-23 °C). Muscle preparations were removed following killing by rapid decapitation following procedures approved by the University of Vermont Institutional Animal Care and Use Committee. As in prior studies, the control snake solution (CSS) contained (mM): NaCl, 159; KCl, 2.5; CaCl2, 1.0; MgCl2, 4.2; Hepes, 5.0; pH 7.3 (Dionne & Parsons, 1981; Connor et al. 1984, 1997). Nerve-muscle preparations were depolarized by a solution in which 35 mM NaCl was replaced by 35 mM potassium propionate (35 KP). The 35 KP solution contained 1.0 mM Ca2+ and 4.2 mM Mg2+ and 5 mM CsCl. The CsCl was added to facilitate voltage clamping of depolarized muscle fibres to hyperpolarized potentials (Connor et al. 1984, 1997).
Stimulation by K+ depolarization rather than nerve stimulation was chosen for the following reasons. First, with repetitive nerve stimulation, muscle movements made it impossible to obtain voltage clamp recordings for prolonged periods. Second, K+ depolarization ensured that all nerve terminals were consistently stimulated to the same extent over the test period. The concentration of KP (35 mM) was chosen because it would depolarize the nerve terminal sufficiently to stimulate a high frequency of miniature endplate currents (MEPCs). Also, at this concentration of KP, the extent of acidification of the nerve terminal should not affect either exocytosis of transmitter or endocytosis of synaptic vesicle membrane (Lindgren et al. 1997).
Pharmacological agents. Oligomycin (a mixture of oligomycin A, B and C; 8 g ml-1; Sigma Chemical Co.) and CCCP (2 M; Sigma) were diluted each day from frozen aliquots of a DMSO stock solution into either CSS or 35 KP. DMSO was included as a vehicle control in the solutions that did not contain oligomycin or CCCP. A key experimental condition for the present study was that the mitochondria be pharmacologically altered prior to exposing the nerve-muscle preparations to 35 KP. To accomplish this, the muscle preparations were bathed in CSS containing 8 g ml-1 oligomycin and/or 2 M CCCP for 20 min and then exposed to 35 KP, which also contained drug. With this protocol, oligomycin would have inhibited the ATP synthase, and CCCP would have dissipated the mitochondrial membrane potential, prior to the introduction of 35 KP.
In some experiments, glucose (15 mM) was present in the 35 KP solution to stimulate glycolysis in preparations treated with oligomycin, CCCP or CCCP and oligomycin together.
Electrophysiological methods. Twitch fibres were identified using criteria described in previous reports (Dionne & Parsons, 1981; Connor et al. 1984). As in preceding studies (Connor et al. 1997), no distinction was made between twitch fibre types (Wilkinson & Lichtman, 1985; Lichtman & Wilkinson, 1987). Transmitter release, measured as the MEPC frequency, was stimulated by K+-induced depolarization (Connor et al. 1997; Calupca et al. 1999). MEPCs were recorded from twitch fibre endplates bathed in CSS or in 35 KP and voltage clamped to -150 mV using a two-microelectrode voltage clamp system (Connor et al. 1984, 1997). The fibres were voltage clamped to -150 mV in order to increase the signal-to-noise ratio and thus ensure that MEPCs were distinguishable above background noise. Since it was virtually impossible to voltage clamp the fibres for longer than 10 min, no attempt was made to record MEPCs from the same endplate prior to and during exposure to 35 KP.
Current records were stored on a PCM recorder (A. R. Vetter, Rebersburg, PA, USA) or FM tape (Racal Store 4DS, Hythe, Southampton, UK) for subsequent digitization and analysis using the SCAN program (generously provided by Dr John Dempster, University of Strathclyde, Glasgow, Scotland, UK). MEPCs were digitized and their frequencies were determined by visual inspection of computer traces (Connor et al. 1997). Criteria used to select MEPCs for analysis followed those described previously (Dionne & Parsons, 1981; Connor et al. 1984). MEPC frequencies immediately after switching to 35 KP were too high to determine accurately and therefore were not included in the plots of the averaged MEPC frequency versus time.
Averaged data are expressed as means
FM1-43 assay of vesicle recycling. Optical estimates of synaptic vesicle membrane endocytosis were made using the styryl dye FM1-43 (2 M; Molecular Probes, Eugene, OR, USA) (Betz & Bewick, 1992, 1993; Betz et al. 1992) following procedures used in our previous studies (Connor et al. 1997; Calupca et al. 1999; Parsons et al. 1999). The FM1-43 was present for the final 6 min of a 120-180 min exposure to 35 KP. The preparations were then washed for 15 min in CSS that contained rhodamine-conjugated peanut agglutinin (PNA, 33 g ml-1, Sigma), and rinsed with PNA-free CSS. PNA labelled the basal laminae at all motor nerve terminals and thus was used as a marker for neuromuscular junctions (Ko, 1987; Connor et al. 1997). To determine whether nerve terminals destained, muscles were loaded with FM1-43 as described above and were viewed shortly after washout of FM1-43 with dye-free 35 KP to confirm the success of the loading procedure. The preparations were exposed to 35 KP (without FM1-43) for an additional 45 min and then washed with CSS containing PNA.
FM1-43 accumulation and destaining of nerve terminals was monitored with a Zeiss fluorescence photomicroscope equipped with filter sets appropriate for FITC (green emission filter, 520-560 nm) or rhodamine (red emission filter, > 590 nm). A Zeiss 
40 water immersion lens was used to locate individual PNA-labelled nerve terminals. Twitch nerve terminals were examined visually and scored for FM1-43 staining, as in previous studies, as uniformly stained, partially stained, or unstained (Connor et al. 1997). A nerve terminal was considered to be uniformly stained if all boutons within the terminal were stained, and partially stained if some, but not all, boutons appeared stained above background. For each muscle examined, we scored PNA-labelled terminals beginning at one edge of the preparation and continued scoring additional PNA-identifed neuromuscular junctions by following the main nerve trunk until it terminated at the opposite edge of the preparation.
Electron microscopy. Previously described techniques were used for the ultrastructural examination of CCCP-treated twitch nerve terminals (Coniglio et al. 1993; Calupca et al. 1999). Untreated preparations and preparations treated with 2 M CCCP were exposed to 35 KP with drug for 60-90 min, then fixed for 15 min in 2 % glutaraldehyde, washed in fresh Millonig's phosphate buffer, postfixed for 30 min in 1 % osmium tetroxide, and washed again in buffer. The preparations were dehydrated in a graded series of ethanols to 100 % and en bloc stained with 2 % uranyl acetate for 5 min. After en bloc staining, nerve-muscle preparations were returned to 100 % ethanol, then into propylene oxide, and finally embedded in a resin mixture of Embed 812-Araldite 502 (hard) between microscope slides that had been precoated with liquid releasing agent. After polymerization, whole mounts were removed from between the slides and examined on a compound microscope to identify individual endplates. Areas of the whole mount that contained motor endplates were cut away and re-embedded into precast Embed-Araldite blocks. These blocks were then polymerized and thick sectioned (5 m sections). The thick sections were placed on Teflon-coated microscope slides and examined on a compound light microscope to identify thick sections that contained individual endplates. Appropriate thick sections were remounted onto the ends of precast Embed-Araldite blocks. The blocks were polymerized, trimmed, and ultrathin sectioned (78-80 nm). Thin sections were examined and photographed on a JEOL 100CXII, a JEOL 100S or a Philips CM10 transmission electron microscope.
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RESULTS |
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Neither oligomycin nor CCCP affects MEPC amplitude in control snake solution
Experiments were completed to determine whether oligomycin or CCCP treatment affected the amplitude of miniature endplate currents (MEPCs) recorded in CSS. For these experiments, MEPCs recorded from individual snake twitch fibre endplates in CSS were compared to those obtained during a 30 min exposure to CSS containing 8 g ml-1 oligomycin or 2 M CCCP. The mean amplitude of MEPCs recorded at -150 mV from eight control endplates was -6.6 ± 0.4 nA. For four endplates exposed to 8 g ml-1 oligomycin, MEPC amplitude was -7.1 ± 0.3 nA and for six endplates exposed to 2 M CCCP, MEPC amplitude was -6.6 ± 0.3 nA.
Although MEPC amplitudes were not affected by either drug, muscle fibres were depolarized in the presence of CCCP. The resting membrane potential measured at twitch fibre endplates bathed in CSS was -95 ± 0.9 mV (n = 21). In CSS containing 8 g ml-1 oligomycin, the membrane potential measured at unclamped twitch fibre endplates was -97 ± 0.9 mV (n = 9). However, shortly after switching to CSS with 2 M CCCP, individual muscle fibres within many preparations began to twitch repetitively. The duration of fibre twitching lasted for periods ranging from a few minutes to approximately 12 min and then subsided. Muscle fibre membrane potentials, measured after approximately 5 min in CCCP, indicated that muscle fibres were depolarized (average membrane potential -48 ± 2.0 mV, n = 14). Muscle fibres were also depolarized (-40 ± 3.9 mV, n = 6) in the presence of 2 M CCCP in CCS when Ca2+ was omitted and the Mg2+ concentration was elevated to 5.2 mM. This observation indicated that the CCCP-induced depolarization of the muscle fibres was not a secondary effect of release of acetylcholine (ACh) from nerve terminals, which is dependent on external Ca2+.
MEPC frequency also was monitored at different endplates during a 30 min exposure to 2 M CCCP. The mean MEPC frequency at 16 twitch fibre endplates in CSS was 0.2 ± 0.03 s-1. MEPC frequency measured at 13 twitch endplates over the 30 min exposure to CSS containing 2 M CCCP ranged between 0.1 and 0.6 s-1 (mean = 0.3 ± 0.05 s-1). The lack of a consistent change in MEPC frequency suggested that CCCP either did not depolarize the nerve terminal at all or at least did not depolarize it to an extent that stimulated a marked increase in quantal release.
These observations demonstrated that CCCP, but not oligomycin, depolarized snake twitch muscle fibres, an effect we presumed was due to a direct action on the muscle fibres. However, neither drug affected MEPC amplitude during the 30 min exposure in CSS. Thus, we thought it reasonable to treat the preparations with either drug for 20 min prior to stimulation of transmitter release by depolarization in 35 KP. With this protocol, mitochondrial function would have been altered before the K+-induced stimulation of transmitter release.
In preparations depolarized by 35 KP, MEPC frequency declines more rapidly following CCCP treatment than following oligomycin treatment
When oligomycin- or CCCP-treated twitch fibres were exposed to 35 KP, the resting membrane potential was -36 ± 0.6 mV (n = 10) or -37 ± 0.9 mV (n = 20), respectively. These values were similar to that obtained previously for twitch fibres in 35 KP without oligomycin or CCCP treatment (-37 ± 0.7 mV, Connor et al. 1997).
In untreated preparations depolarized by 35 KP, MEPC frequency increased initially from < 1 s-1 to > 350 s-1 and remained elevated during exposure to 35 KP (Fig. 1C; also see Connor et al. 1997; Parsons et al. 1999). At the oligomycin- or CCCP-treated twitch endplates, MEPC frequency also increased to values > 350 s-1 upon initial exposure to 35 KP, but then declined progressively (Fig. 1A and B). For oligomycin-treated preparations, the decline in MEPC frequency occurred slowly such that the averaged frequency remained > 100 s-1 after ~100 min and was ~10 s-1 after 200 min in 35 KP (Fig. 2). In contrast, at CCCP-treated preparations, the MEPC frequency declined to values < 10 s-1 within 80 min (Fig. 2).
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A, MEPC recordings obtained from oligomycin-treated preparations. Traces 1-3 were recorded from oligomycin-treated endplates after 15, 130 and 182 min in 35 KP solution, respectively. B, recordings obtained from CCCP-treated preparations. Traces 1-3 were recorded from CCCP-treated endplates after 5, 29 and 79 min in 35 KP solution, respectively. C, MEPC recording obtained in a control preparation after 200 min in 35 KP.
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Mean MEPC frequency recorded in 35 KP for preparations treated with oligomycin (
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MEPC amplitudes were measured at individual CCCP-treated endplates between 15 and 140 min in 35 KP, a period when the frequency had declined to levels at which individual MEPCs could be reliably resolved. The averaged MEPC amplitude determined for individual CCCP-treated endplates varied widely, i.e. from approximately -3.7 to approximately -8.3 nA. However, the mean MEPC amplitude, determined for 10 endplates in the initial 20-30 min period in 35 KP (-6.5 ± 0.42 nA) was not different from the mean MEPC amplitude determined for five endplates sampled at 115-140 min in 35 KP (-6.7 ± 0.34 nA). Given that the averaged MEPC amplitude did not decline progressively over time in KP, we concluded that the decline in MEPC frequency at CCCP-treated endplates in 35 KP was not due to a progressive loss of smaller amplitude MEPCs into the baseline noise. Rather, the decline in MEPC frequency at CCCP-treated endplates in 35 KP represented a presynaptic alteration in quantal release. Previous studies demonstrated that CCCP treatment can disrupt ACh transport into cholinergic vesicles (Anderson et al. 1982; Parsons et al. 1982). Thus, one might have expected a decrease in MEPC amplitude during exposure to CCCP. It is possible, therefore, that subtle changes in amplitude would have been evident if more sensitive measurement techniques were used (Van der Kloot et al. 2000).
As the mitochondrial proton gradient is dissipated in CCCP, the FO/F1-ATP synthase reverses, consuming cytoplasmic ATP in an attempt to sustain the mitochondrial membrane potential (Budd & Nicholls, 1996). Thus, during exposure to CCCP, the cytoplasmic ATP/ADP ratio can quickly decrease. To test whether a rapid decrease in cytoplasmic ATP/ADP ratio was the primary factor responsible for the CCCP-induced decline of the K+-stimulated MEPC frequency, additional experiments were done with preparations treated with both 8 g ml-1 oligomycin and 2 M CCCP (Budd & Nicholls, 1996). The preparations were treated with both CCCP and oligomycin in CSS for 20 min and then exposed to 35 KP, which also contained both drugs. In these preparations, MEPC frequency increased and then declined progressively following a time course similar to that noted with CCCP alone (Fig. 2). Thus, slowing the decrease in ATP/ADP ratio by the addition of the ATP synthase inhibitor oligomycin did not affect the time course of decline in MEPC frequency.
Addition of glucose during exposure to 35 KP reduces the oligomycin-, CCCP- or CCCP and oligomycin-induced decline in MEPC frequency
In our previous study, we noted that the addition of glucose to stimulate production of ATP by glycolysis partially prevented the decline in MEPC frequency produced following prolonged exposure to 35 KP with a [Ca2+]o of 3.6 mM even though the morphological disruption of mitochondria remained (Calupca et al. 1999). Consequently, we tested whether addition of 15 mM glucose to 35 KP would also reduce the decline in K+-stimulated MEPC frequency that occurred in preparations treated with oligomycin, CCCP or CCCP and oligomycin combined. The results, summarized in Table 1, demonstrate that when glucose was present, the average MEPC frequency at drug-treated endplates after prolonged exposure to KP was similar to that determined for untreated endplates exposed for similar times in 35 KP without glucose.
FM1-43 stains many oligomycin- or CCCP-treated twitch nerve terminals after prolonged stimulation in 35 KP
We also tested whether oligomycin or CCCP affected synaptic vesicle recycling. These experiments were completed using incorporation of the activity-dependent fluorescent dye FM1-43 as an assay of endocytosis of synaptic vesicle membrane (Betz & Bewick, 1992, 1993; Betz et al. 1992). Motor nerve terminals innervating twitch fibre endplates were identified by the pattern of PNA staining (Connor et al. 1997; see Methods). In this series of experiments, for seven control preparations maintained in 35 KP for 120-180 min, every PNA-labelled twitch nerve terminal (> 10 terminals/ preparation) was stained by FM1-43 (Fig. 3A). The intensity of the FM1-43 staining varied among twitch terminals, but, in all cases, the terminals were uniformly stained.
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A, an example of the uniform FM1-43 fluorescence for a control terminal after 120 min in 35 KP. B-D, examples of FM1-43 staining patterns at CCCP- or oligomycin-treated terminals after 120-180 min in 35 KP. B, an example of an oligomycin-treated nerve terminal (180 min in KP) that did not exhibit FM1-43 fluorescence. C, an example of a CCCP-treated terminal (120 min in KP) that exhibited partial FM1-43 staining. D, an example of an oligomycin-treated terminal (180 min in KP) that exhibited uniform FM1-43 staining. E and F, uniformly stained nerve terminals from a CCCP-treated (E) or an oligomycin-treated (F) preparation exposed to 35 KP containing 15 mM glucose. Calibration bar in F, 40
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We then compared FM1-43 staining in CCCP- or oligomycin-treated preparations at times in 35 KP when MEPC frequencies had declined substantially (> 120 min with CCCP or > 180 min with oligomycin). In CCCP- or oligomycin-treated preparations bathed in 35 KP, the pattern of FM1-43 staining differed markedly amongst twitch nerve terminals. For nine CCCP-treated preparations after 120 min in 35 KP, 50 % of the terminals appeared to be uniformly stained, 31 % were partially stained, and 20 % of the twitch terminals were not stained by FM1-43 (Table 2). There was a marked variation in FM1-43 staining among those CCCP-treated terminals identified as uniformly stained: some were intensely fluorescent whereas other terminals exhibited weak fluorescence. In three oligomycin-treated preparations after 180 min in 35 KP, 50 % of the twitch nerve terminals appeared uniformly stained by FM1-43, 14 % appeared partially stained and 36 % were not stained (Table 2). Examples of different FM1-43 staining patterns for either CCCP- or oligomycin-treated twitch terminals after 120 or 180 min in 35 KP, respectively, are shown in Fig. 3B-D.
It has been suggested that the rate of endocytosis of synaptic vesicle membrane is coupled closely to the rate of exocytosis (Betz et al. 1992; Wu & Betz, 1996; Murphy & Stevens, 1998). Using bath solutions containing different K+ concentrations (to elevate MEPC frequency progressively) and a 6 min exposure to FM1-43, Connor et al. (1997) determined the minimum MEPC frequency required to accumulate sufficient dye to identify snake twitch terminals in these nerve muscle preparations as positively stained. It was established that a MEPC frequency of 25-50 s-1 was required before nerve terminals accumulated sufficient dye to be consistently classified as positively stained. In the present study, we tested whether the percentage of nerve terminals stained by FM1-43 in oligomycin-treated or CCCP-treated preparations paralleled the number of endplates with MEPC frequencies greater than 25-50 s-1. For CCCP-treated preparations after 120 min in 35 KP, the MEPC frequency at all endplates was < 1 s-1. We expected that with a MEPC frequency < 1 s-1, no nerve terminals would exhibit FM1-43 fluorescence. Thus, it was unexpected that > 50 % of the CCCP-treated terminals would be stained by FM1-43 at this time (Table 2).
Recordings were made at 20 oligomycin-treated endplates after 180 min in 35 KP. At this time, the MEPC frequency at seven endplates was < 25 s-1, at two other endplates the MEPC frequency ranged between 25 and 50 s-1 and at the 11 remaining endplates, the MEPC frequency was > 50 s-1. Thus, for oligomycin-treated preparations, the FM1-43 staining was consistent with the range of recorded MEPC frequencies.
Destaining experiments were used as an optical assay to test whether synaptic vesicle exocytosis was continuing at a time when MEPCs were essentially absent. Two groups of CCCP-treated muscle preparations (3 preparations in each group) were exposed to FM1-43 during the final 6 min of a 120 min exposure to 35 KP. Two untreated muscles were also exposed to 35 KP with FM1-43 for the final 6 min of a 120 min exposure to 35 KP. In the two control muscles and one group of CCCP-treated muscles, the FM1-43 was washed out with CSS. In the second group of CCCP-treated muscles, the FM1-43 was washed out with 35 KP so that the nerve terminals would remain stimulated to allow destaining to occur. The muscles were viewed immediately to verify dye accumulation in the terminals. The preparations undergoing destaining were then maintained in 35 KP without dye for an additional 45 min before washing in CSS. All muscles were counter stained with PNA prior to the terminals being viewed.
All twitch terminals in both control preparations exhibited FM1-43 staining after 120 min in 35 KP; 56 of 60 were uniformly stained and four appeared partially stained. For the three CCCP-treated preparations that had not been allowed to destain, 40 % of the twitch nerve terminals appeared uniformly stained, 20 % appeared partially stained and 40 % were unstained. For the three CCCP-treated preparations which had been exposed to dye-free 35 KP for an additional 45 min to allow destaining to proceed, 32 % of the twitch terminals appeared uniformly stained, 14 % appeared partially stained and 53 % were unstained.
To ensure that a control preparation would destain under these same conditions, we exposed one of the untreated preparations to 35 KP for 45 min after it had been stained with FM1-43. In this preparation, of 30 PNA-identified terminals, 22 were destained (73 %) and eight were faintly, but uniformly, stained (27 %).
Addition of glucose during exposure to 35 KP enhances FM1-43 accumulation into oligomycin- and CCCP-treated twitch nerve terminals
We determined whether treatment with 15 mM glucose increased the percentage of CCCP- or oligomycin-treated twitch terminals stained by FM1-43 after 120 or 180 min in 35 KP, respectively (Table 2). Essentially all of the PNA-identified twitch terminals in five CCCP-treated preparations exposed to 35 KP with glucose exhibited FM1-43 staining (99 %, 71 out of 72 terminals uniformly stained, 1 unstained) (Fig. 3E). Similarly, all 43 of the PNA-identified terminals in two oligomycin-treated preparations were uniformly stained by FM1-43 when glucose was present (Fig. 3F).
The high percentage of terminals stained by FM1-43 was consistent with the frequency of MEPCs recorded from CCCP-treated (177 ± 32 s-1, n = 12) or oligomycin-treated (228 ± 20 s-1, n = 27) preparations in 35 KP with glucose at 120 or 180 min, respectively.
The ultrastructure of K+-depolarized twitch nerve terminals is not altered by CCCP at times when MEPC frequencies had declined to near zero
Molgo & Pecot-Dechavassine (1988) reported previously that for amphibian motor nerve terminals during prolonged exposure to CCCP, synaptic vesicle numbers declined and mitochondrial ultrastructure was progressively disrupted. Consequently, studies were initiated to determine the morphology of CCCP-treated nerve terminals at times in 35 KP when MEPC activity had declined to < 1 s-1. For twitch nerve terminals in untreated preparations after 120-180 min in 35 KP, all boutons contained numerous synaptic vesicles and mitochondria (Fig. 4A; see also Calupca et al. 1999). Electron micrographs were also obtained of CCCP-treated nerve terminals exposed to 35 KP for 60-90 min. In the CCCP-treated nerve terminals, there was no obvious alteration in the morphology of mitochondria. Furthermore, boutons of twitch terminals exposed to 35 KP and CCCP contained abundant synaptic vesicles (Fig. 4B). Similar results were obtained in electron micrographs obtained from at least three different endplates in each of three separate nerve-muscle preparations.
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A, a terminal from a control preparation after 120 min in 35 KP. B, a terminal from a CCCP-treated preparation maintained in 35 KP for 90 min. Note that the mitochondrial morphology was similar and that synaptic vesicles were abundant in both examples. M, mitochondria; SV, synaptic vesicles.
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DISCUSSION |
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The present study analysed presynaptic function at K+-stimulated motor nerve terminals treated with oligomycin, CCCP, or oligomycin and CCCP combined. For all three treatments, MEPC frequency increased markedly during the initial exposure to 35 KP, but then declined with continued K+ stimulation. The striking difference was that the decline in K+-stimulated MEPC frequency was more rapid in preparations treated with CCCP or with both oligomycin and CCCP than in preparations treated with oligomycin alone.
Numerous synaptic vesicles were present in nerve terminals from CCCP-treated preparations, an indication that synaptic vesicle depletion was not a cause of the decline in quantal release with CCCP treatment. In addition, the ultrastructure of mitochondria in the CCCP-treated, K+-stimulated motor nerve terminals was not altered at times when MEPCs were virtually absent. Thus, the CCCP-induced progressive decline in release of quanta in this study (where [Ca2+]o and [Mg2+]o were 1.0 and 4.2 mM, respectively) was not correlated with an obvious alteration in mitochondrial morphology as noted in the prior study that examined the change in MEPC frequency and nerve terminal morphology after an ~120 min exposure to 35 KP with a raised [Ca2+]o of 3.6 mM (Calupca et al. 1999).
With the ATP synthase inhibited by oligomycin, ATP production by mitochondria would be eliminated. However, during oligomycin treatment, Ca2+ sequestration by mitochondria, at least initially, should not have been impaired (Budd & Nicholls, 1996). Therefore, in the presence of oligomycin and in the absence of added glucose to support glycolysis, a progressive decrease in the cytoplasmic ATP/ADP ratio would have occurred. We suggest that the progressive decline in cytoplasmic ATP/ADP ratio was a factor contributing to the decline in quantal release from oligomycin-treated, K+-stimulated terminals. The time course of the decline in MEPC frequency in oligomycin-treated, K+-stimulated preparations was somewhat similar to that reported previously for the depression of MEPC frequency in preparations bathed in 35 KP with 3.6 mM [Ca2+]o (Connor et al. 1997; Calupca et al. 1999). This similarity in time course provided support for the hypothesis that a decrease in mitochondrial ATP production (as mitochondrial integrity became disrupted) and consequent progressive decline in the cytoplasmic ATP/ADP ratio was a key factor in the Ca2+-induced depression of quantal release (Calupca et al. 1999).
Mitochondria buffer the rise in [Ca2+]i produced by Ca2+ influx through voltage-gated Ca2+ channels (David et al. 1998; Peng, 1998; David, 1999; Scotti et al. 1999; David & Barrett, 2000). In the presence of protonophores, such as CCCP, the mitochondrial proton gradient dissipates, and as a consequence, Ca2+ sequestration by mitochondria is diminished (Budd & Nicholls, 1996). With mitochondrial Ca2+ buffering diminished, the rise in [Ca2+]i during depolarization of nerve terminals is enhanced and prolonged (David et al. 1998; Peng, 1998; David, 1999; David & Barrett, 2000). However, during exposure to CCCP alone, the mitochondrial ATP synthase reverses and metabolizes cytoplasmic ATP in an attempt to restore the proton gradient, causing a faster decrease in cytoplasmic ATP/ADP ratio (Budd & Nicholls, 1996). Consequently, we tested the effect of treatment with oligomycin along with CCCP. Co-treatment with oligomycin and CCCP, a condition that would prevent reversal of the ATP synthase, did not attenuate the decline in K+-stimulated MEPC frequency as would have been predicted if a rapid decrease in the cytoplasmic ATP/ADP ratio had been the primary factor underlying the effect of CCCP. Thus, we argue that the inhibition of [Ca2+]i buffering by mitochondria rather than a more rapid decline in ATP/ADP ratio was responsible for the faster decline in quantal release at K+-stimulated, CCCP-treated terminals than at K+-stimulated, oligomycin-treated terminals.
With glucose present in the KP solution to stimulate ATP production by glycolysis, an elevated MEPC frequency was maintained at K+-stimulated preparations treated with CCCP, oligomycin or CCCP and oligomycin together. Thus, a decline in the cytosolic ATP/ADP ratio must have been a factor that contributed to the decline of K+-stimulated transmitter release in all three drug-treated preparations, and furthermore, ATP production by glycolysis must have been sufficient to support an ATP-dependent process required to maintain K+-stimulated release. We propose that as energy-dependent [Ca2+]i buffering mechanisms gradually failed, a progressive elevation of [Ca2+]i may have resulted. This hypothesized elevation in [Ca2+]i was the primary factor responsible, in all three drug-treated preparations, for the decline in K+-stimulated release. In addition, we suggest that ATP production by glycolysis was sufficient to support Ca2+ homeostasis in these K+-stimulated nerve terminals. For K+-stimulated, oligomycin-treated terminals, mitochondria, along with ATP-dependent Ca2+ extrusion and uptake mechanisms, would contribute to [Ca2+]i buffering until ATP stores were depleted. However, over time [Ca2+]i would rise as the cytoplasmic ATP/ADP ratio decreased progressively, and energy-dependent mechanisms driving Ca2+ extrusion and uptake failed. For preparations treated with CCCP or CCCP and oligomycin together, the sequestration of Ca2+ by mitochondria would be immediately inhibited. In these preparations, the ATP/ADP ratio also would fall progressively and, as a consequence, ATP-dependent [Ca2+]i buffering mechanisms would gradually fail. However, in the absence of Ca2+ sequestration by mitochondria, [Ca2+]i would rise more quickly in terminals treated with CCCP alone or treated with CCCP and oligomycin than in terminals treated only with oligomycin. In all cases, K+-stimulated release would decline as [Ca2+]i became elevated. However, because the suggested change in [Ca2+]i would probably take place more rapidly in the CCCP-treated terminals than in oligomycin-treated terminals, the decline in K+-stimulated MEPC frequency would occur sooner in CCCP-treated preparations. We suggest that a sustained elevation of [Ca2+]i might have disrupted K+-stimulated release by a depression of processes essential to transmitter release (Adams et al. 1985; Hsu et al. 1996). Direct measurement of the effect of prolonged exposure of snake motor nerve terminals to 35 KP on [Ca2+]i will be required to confirm the above hypotheses.
The rate of endocytosis commonly is closely coupled to the rate of exocytosis (Betz et al. 1992; Wu & Betz, 1996; Murphy & Stevens, 1998). Thus, the observation that FM1-43 accumulated in > 50 % of the CCCP-treated nerve terminals, when MEPC frequency was < 1 s-1, suggested that synaptic vesicle exocytosis might be still ongoing in these terminals. CCCP did not affect MEPC amplitude in CSS; thus, we assumed that postjunctional receptor sensitivity to ACh was not affected by CCCP. We propose that if exocytosis continued at some CCCP-treated endplates after > 120 min in 35 KP, then the FM1-43-labelled synaptic vesicles must have been recycled vesicles that had not refilled with ACh. It is plausible that some synaptic vesicles undergoing exocytosis at CCCP-treated, K+-stimulated terminals were empty because CCCP can disrupt ACh transport into cholinergic synaptic vesicles (Anderson et al. 1982; Parsons et al. 1982; Parsons et al. 1993). However, if CCCP inhibited ACh transport into recycled vesicles, then most MEPCs recorded in 35 KP must have been generated by quanta released from either preformed reserve vesicles or vesicles recycled during the initial period of stimulation. This conclusion would be consistent with the observation that averaged MEPC amplitudes were similar when compared at 15-30 min and more prolonged periods in 35 KP. Empty cholinergic vesicles can undergo cycles of exocytosis followed by endocytosis. We showed previously that FM1-43 is accumulated in empty synaptic vesicles undergoing endocytosis at vesamicol-pretreated snake nerve terminals exposed to 35 KP for 120 min (Parsons et al. 1999). Thus, we suggest that a CCCP-induced inhibition of ACh transport into recycling synaptic vesicles could also have contributed to the more rapid decline in MEPC frequency at CCCP-treated than at oligomycin-treated, K+-stimulated preparations.
We initiated destaining experiments to optically demonstrate that exocytosis continued at CCCP-treated, K+-stimulated motor nerve terminals. Minimal destaining of the CCCP-treated motor nerve terminal was observed, whereas terminals in a control muscle were effectively destained using the same method. One interpretation of this result is that although endocytosis continued after prolonged K+ stimulation, exocytosis was not occurring at CCCP-treated terminals. Alternatively, it is possible that exocytosis was ongoing at some terminals, but had shifted to a 'kiss and run' mode (Klingauf et al. 1998). With exocytosis in the 'kiss and run' mode, FM1-43 accumulates in synaptic vesicles, but these vesicles do not destain with subsequent stimulation (Henkel & Betz, 1995). Recently, Ales et al. (1999) demonstrated that high [Ca2+]i shifted exocytosis to a 'kiss and run' mode. Thus, we suggest that an elevation of [Ca2+]i within CCCP-treated, K+-stimulated motor nerve terminals could have shifted exocytosis to the 'kiss and run' mode, thereby reducing the likelihood of FM1-43 destaining.
Snake twitch muscle fibres in CSS were rapidly depolarized in CCCP, an action we attributed to a direct effect on the muscle fibres. A CCCP-induced contraction of lizard muscle fibres was noted by David (1999). This mechanical activity could have been caused by muscle fibre depolarization similar to that noted in the present study. Molgo & Pecot-Dechavassine (1988) did not report any CCCP-induced depolarization of amphibian muscle fibres so this effect of CCCP on reptilian skeletal muscle fibres may not be common to all species.
In conclusion, the results presented in this study clearly demonstrate that motor nerve terminals could not sustain a K+-stimulated elevation of transmitter release when treated with CCCP, oligomycin or CCCP and oligomycin simultaneously. We propose that the decline in K+-stimulated quantal release resulted from a progressive elevation of [Ca2+]i. For oligomycin-treated nerve terminals, the hypothesized progressive elevation of [Ca2+]i occurred as the cytoplasmic ATP/ADP ratio decreased causing energy-dependent [Ca2+]i buffering mechanisms to fail. We also suggest that the decline in MEPC frequency occurred more rapidly in preparations treated with CCCP or CCCP and oligomycin together because with mitochondrial Ca2+ buffering compromised, a sustained elevation of [Ca2+]i could occur more quickly.
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Acknowledgements
We thank Dr Ian G. Marshall (I.G.M.) for helpful discussions during the course of this work and constructive comments on an earlier version of this manuscript. Support for this study was obtained from PHS grant NS 23978 to R.L.P. and a NATO Grant to R.L.P. and I.G.M.
Corresponding author
R. L. Parsons: Department of Anatomy and Neurobiology, University of Vermont College of Medicine, Burlington, VT 05405, USA.
Email: rparsons{at}zoo.uvm.edu
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), CCCP (
) and oligomycin and CCCP together (
). Each data point represents the mean ± S.E.M. from at least 3 endplates.