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Previous Article | Next Article
The Journal of Neuroscience, April 1, 1999, 19(7):2511-2521
Synaptic Vesicle Dynamics in Rat Fast and Slow Motor Nerve Terminals
Brian Reid1, Clarke R. Slater2, and Guy S. Bewick1 1 Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen AB25 2ZD, United Kingdom, and 2 Department of Neurobiology, The Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
We have investigated whether rat motor nerve terminals with different in vivo activity patterns also have different vesicle trafficking characteristics. To do this, we monitored, using combined optical and electrical techniques, the rate of exocytosis (during different frequencies and patterns of activity), the releasable pool size, and the recycle time of synaptic vesicles in terminals on soleus (slow-twitch) and extensor digitorum longus [(EDL); fast-twitch] muscle fibers. EDL terminals had a higher initial quantal content (QC) than soleus, but during tonic or phasic stimulation at 20-80 Hz, EDL QC ran down to a greater extent than soleus QC. By recording loss of fluorescence from exocytosing vesicles labeled with the dye FM1-43, EDL terminals were found to destain faster than those in soleus. Simultaneous intracellular recording of end plate potentials, to count the number of vesicles released, permitted estimation of the total vesicle pool (VP) size and the recycle time by combining the optical and electrophysiological data. Soleus vesicle pool was larger than EDL, but recycle time was not significantly different. These terminals, therefore, are adapted to their in vivo activity patterns by alterations in QC and VP size but not recycle time.
Key words: synaptic vesicles; vesicle recycling; FM1-43; exocytosis; quantal content; neuromuscular junction
INTRODUCTION
The adaptation of neurotransmitter release to a pattern of activity of a neuron in vivo is essential for normal functioning throughout the nervous system. For motoneurons, this suggests that the terminals innervating tonically active postural muscles might be better adapted for sustained transmitter release than those innervating less frequently used phasic muscles (Henneman et al., 1965
; Henneman and Olson, 1965
The EDL is a fast-twitch muscle that is easily fatigued. Terminals in EDL are active in short bursts (~1% of animal's activity time) at a high frequency (80 Hz) (Hennig and Lømo, 1985
Our initial investigations showed that soleus nerve terminals sustained release better than those in EDL during tonic stimulation at 20 Hz. We therefore went on to analyze the cellular processes that might account for these differences. In particular, we wanted to know whether the kinetics of vesicle recycling were different in nerve terminals adapted to such different in vivo activity patterns. We therefore monitored the kinetics of recycling synaptic vesicles by labeling them with the fluorescent styryl pyridinium dye FM1-43 (Betz and Bewick, 1992
Some of this data has appeared in preliminary form (Reid and Bewick, 1996
MATERIALS AND METHODS Many of the techniques used here have been described in detail elsewhere (Betz and Bewick, 1992
Dissection and terminal labeling. Large (>350 gm) male Sprague Dawley rats were used because their EDL and soleus muscles contain an almost homogeneous fiber type. We have confirmed, by myofibrillar ATPase assay (Brooke and Kaiser, 1969
Muscle fiber diameter. Fiber diameters were determined from mature (16-18 weeks) and 6-week-old rats. The midsection of the muscle was cut out with a scalpel blade and wrapped in a strip of ox liver to provide support during freezing. This was done in isopentane cooled to
176°C with liquid nitrogen. Sections 10-µm-thick were made using a Reichert-Jung Cryocut E cryostat and labeled for ATPase activity (Brooke and Kaiser, 1969
Imaging. Stimulation of labeled terminals led to destaining as dye-filled vesicles released neurotransmitter and dye simultaneously during exocytosis. To monitor exocytosis optically, images were acquired at 10 sec intervals from individual terminals during 10 min of activity-dependent destaining. To prevent contraction, muscles were paralyzed by exposure to either 5 µM d-tubocurarine (Sigma, Poole, UK) for 20-30 min to block the binding of acetylcholine to its receptor or 2 µM µ-conotoxin GIIIB (Sigma) for 30-40 min to selectively block muscle voltage-gated sodium channels (Plomp et al., 1992
Intracellular recording. Standard electrophysiological techniques were used to record miniature end plate potentials (MEPPs) and evoked EPPs during destaining using glass microelectrodes filled with 3 M KCl. Muscle fibers were impaled ~50-100 µm from the terminal to be imaged, thus minimizing decay of the synaptic signal (Auerbach and Betz, 1971
Data analysis. Analysis of terminal destaining was performed using NIH Image software (http://rsb.info.nih.gov/nih-image/). Briefly, all fluorescently labeled regions of a terminal that were in focus were selected for measurement of pixel intensity. A background region was also selected, and the data were background-subtracted to give a value of "net intensity" and to account for variability between preparations. The data for all the images of a time series were compiled and analyzed using Excel (Microsoft, Bellevue, WA). To allow comparison of the rates of destain between different terminal types, the net intensity data in pixel intensity units (PIU) were converted into percentage of units of dye loss, such that the dye lost by a terminal at the start of the experiment was 0% and at the end of 10 min stimulation (60 images) was 100%. For example, the percentage of dye lost after n number of images is:
where 0 and 60 are the first and last images, respectively.
![[(<UP>PIU</UP><SUB>0</SUB>−<UP>PIU<SUB>n</SUB></UP>)÷(<UP>PIU</UP><SUB>0</SUB>−<UP>PIU</UP><SUB>60</SUB>)]×100](/content/vol19/issue7/fulltext/2511/img001.gif)
Initial quantal contents (QC) were determined using the direct method, i.e., the mean amplitude of the first EPP of the stimulus train was divided by the mean MEPP amplitude and then corrected for nonlinear summation (Martin, 1955
Percentage dye lost per EPP was calculated as: % dye lost before RT
number of EPPs recorded by same time.
Releasable vesicle pool (VP) size was determined as: (100
In turn, the percentage of the vesicle pool released per initial EPP is: (QC
Statistics. Average data are shown as mean ± SE. The statistical significance of differences between means was tested using Student's t test for samples of equal or unequal variances, as appropriate, and curve fitting was performed via MacCurveFit 1.1.1 (Kevin Raner Software, Mt. Waverley, Victoria, Australia) using the Marquardt-Levenberg algorithm.
RESULTS Quantal release in EDL versus soleus
Rat EDL terminals release more vesicles per impulse (QC) than soleus terminals for isolated stimuli or low-frequency stimulation trains (Gertler and Robbins, 1978
Neither MEPP amplitude (EDL, 0.28 ± 0.01 mV; soleus, 0.28 ± 0.02 mV; n = 10) nor initial MEPP frequency (EDL, 2.7 ± 0.4/sec; soleus, 3.0 ± 0.7/sec; n = 10) were significantly different between the two muscles. These MEPP amplitudes were lower, and their frequencies were higher than those measured previously in 6- to 8-week-old rats (Wood and Slater, 1997
Immediately after 10 min stimulation at 20 Hz, although MEPP frequency was significantly increased (EDL, 8.1 ± 1.3/sec; soleus, 9.9 ± 2.1/sec; n = 10; p < 0.01 for both muscles), the amplitude was unchanged (EDL, 0.26 ± 0.01 mV; soleus, 0.27 ± 0.02 mV; n = 10; p > 0.1 for both), indicating that postsynaptic sensitivity to ACh had not changed and vesicle filling with neurotransmitter was complete, even after prolonged stimulation.
The initial QC of EPPs was significantly higher for EDL than for soleus (101.50 ± 4.43 vs 86.91 ± 4.48; n = 35 and 29, respectively; p < 0.03), confirming previous findings (Table 1) (Gertler and Robbins, 1978
) of 7.2 sec in EDL and 9.7 sec in soleus. The slowest component of EPP depression had a
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Table 1. Synaptic vesicle recycling parameters from terminals stimulated continuously at 20 Hz
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Figure 1. A, Electrophysiological recordings from muscle fibers innervated by motor nerve terminals stimulated at a constant 20 Hz. Spontaneous MEPPs (top) were recorded for 2-3 min before and after stimulation. Evoked EPPs (bottom) were recorded throughout the 10 min stimulation period. There were no significant differences in MEPP amplitude or frequency between EDL and soleus terminals before stimulation. B, Mean QC against time during stimulation at 20 Hz for EDL and soleus terminals (n = 10). Upon stimulation, QC ran down rapidly; EDL QC ran down faster and to a greater extent than soleus. The greater ability of soleus terminals to maintain adequate quantal release during prolonged 20 Hz stimulation is consistent with their in vivo activity pattern.
The apparent adaptation of soleus terminals to tonic stimulation, indicated by these studies, could be caused by several factors: (1) larger vesicle pools and therefore lower fractional release per impulse; (2) faster vesicle recycling; or (3) a combination of 1 and 2.
We therefore went on to use FM1-43 labeling (Betz and Bewick, 1992
Morphology of nerve terminals in EDL and soleus and their destaining
Figure 2 shows various nerve terminals on EDL (above) and soleus (below) muscle fibers after labeling with FM1-43 and washing. The staining highlights apparent morphological differences between these terminal types in adult rat muscle. EDL terminals are approximately circular, with an absence of dye in the very center and the dye distributed in an almost continuous ribbon. In contrast, soleus terminals are significantly elongated along the muscle fiber long axis. Labeling of soleus terminals is usually in a greater number of separate regions, and there is frequently an area free from dye on one side of the terminal, near the point of nerve entry. There is, however, no difference in terminal area (measured as FM1-43 fluorescence) between adult EDL and soleus (370 ± 105.8 vs 388.2 ± 129.8 µm2; n = 100; p > 0.2). These morphological data are the subject of further study and will be reported in detail elsewhere.
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Figure 2. Examples of EDL (top) and soleus (bottom) motor nerve terminals after activity-dependent labeling with the fluorescent dye FM1-43 and washing. The longitudinal axes of the muscle fibers are aligned approximately vertically in these pictures. Bright regions indicate clusters of thousands of labeled vesicles. Morphological differences are apparent; soleus terminals are elongated along the muscle fiber, and their labeling is in smaller but more numerous spots. There is, however, no difference in terminal area or initial labeling intensity between EDL and soleus. Scale bar, 10 µm.
Stimulation of labeled terminals caused dye loss as vesicle exocytosis took place. Figure 3 shows an EDL terminal (above) and a soleus terminal (below) after 0, 5, and 10 min stimulation at 20 Hz. From these images, it appears that, although there is variability in initial intensities, the different terminal regions all destain at similar rates, as shown previously in the frog (Betz and Bewick, 1992
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Figure 3. Nerve stimulation causes labeled terminals to destain as vesicles undergo exocytosis and lose their dye. An EDL (top) and a soleus (bottom) terminal are shown at three different time points during continuous stimulation at 20 Hz. Terminals lose most dye during the first 2-3 min of the experiment. The activity-dependent destaining of terminals is rapid and is independent of the very slow activity-independent dye loss displayed in axonal regions (arrows) (see Fig. 4). Scale bar, 10 µm.
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Figure 4. Analysis of terminal destaining. A, Stained in-focus areas of the terminal were outlined manually, and these regions of interest (terminal ROIs) were analyzed for brightness (pixel intensity). This analysis was repeated for corresponding regions of interest in each image of a time series. The regions of interest are shown here, and a background and axon region of interest have also been drawn. Scale bar, 10 µm. B, Pixel intensity of the regions of interest in A plotted against time over the 10 min stimulation period. The data have been background-subtracted. The circles show the average pixel intensity over the whole terminal, and the squares show the brightness of the axon. Initially, all vesicles in the terminal are labeled with dye, and therefore the terminal fluoresces brightly. Upon stimulation (time 0), large-scale vesicle exocytosis commences, and vesicles release their dye. Initial brightness did not affect the rate of destaining of different parts of the terminal (inset), because the percentage of dye loss for all regions followed very similar time courses. The initial fast rate of destaining slows as the number of labeled vesicles diminish and are replaced by recycled (unlabeled) vesicles, which begin to compete with labeled vesicles for release. The axon myelin sheath is labeled in an activity-independent manner and loses very little dye during the experiment, showing photobleaching does not contribute significantly to the destaining.
Activity-dependent nerve terminal destaining appeared to consist of two components. The first, most evident over the initial 3-4 min, was characterized by a high rate of dye loss. This gave way to a second component of slower dye loss as unlabeled vesicles reentered the vesicle pool and competed with labeled vesicles for release (Betz and Bewick, 1993
During the initial component, all regions of the nerve terminal appeared to destain at the same rate. In the soleus terminal in Figure 4, the mean destaining rate of all boutons was 0.23 ± 0.01 PIU/sec (n = 14) over the first 3 min, regardless of their initial intensity. Thus, for all subsequent experiments, the rate of destaining of a terminal was expressed as the average over all in-focus regions. Toward the end of the period of stimulation, the rate of dye loss from terminals was very low and not different between EDL and soleus (EDL, 0.013 ± 0.001 PIU/sec; soleus, 0.017 ± 0.002 PIU/sec; n = 20; p > 0.5).
The activity-dependent destaining appeared to be superimposed on a level of activity-independent labeling that varied considerably between the different regions of the terminal. Subtraction of this component from each of the individual regional destaining curves revealed that destaining was essentially identical across the entire terminal (Fig. 4B, inset). The similarity in the intensity data after subtraction of the slow component also shows that vesicle densities are probably very similar throughout the terminal. Thus, variations in initial intensities are probably caused by differences in the underlying background level of staining. It is not clear what this variation in background between different regions of the same terminal is attributable to, but dye accumulation in membrane of terminal Schwann cells or postsynaptic folds are obvious candidates.
In contrast to terminals, axons (in EDL and soleus), which take up FM1-43 in an activity-independent manner because of the large amount of membrane in the myelin sheath (Betz et al., 1992
The rate of terminal destaining, even at the end of the period of stimulation, was significantly higher (p < 0.01) than that in axonal regions (Figs. 3, 4B). This is probably because of a small number of labeled vesicles still being lost from the terminals. In addition, the highly lipophilic myelin sheath may be slower than terminals to lose its dye to washout and/or photobleaching.
Kinetics of destaining in EDL versus soleus
Ribchester et al. (1994)
The first 5 min of destaining of EDL and soleus terminals, expressed as percentage of dye lost over the full 10 min experiment, is compared in Figure 5A. During the first minute of stimulation, EDL terminals lost on average 0.70 ± 0.05% of dye per second compared with 0.43 ± 0.03 in soleus (n = 10; p < 0.0005). Consequently, EDL terminals took only 75 sec to lose 50% of their initial fluorescence compared with 140 sec for soleus (Fig. 5A). These data were acquired from muscles that had been paralyzed with d-tubocurarine, which blocks the postsynaptic acetylcholine receptors. This compound, however, reportedly increases the rate of rundown of QC in rat terminals during high-frequency stimulation (Glavinovic 1979b
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Figure 5. Comparison of dye release from EDL and soleus terminals. A, Destaining of EDL and soleus terminals during the first 5 min of stimulation at 20 Hz expressed as cumulative percentage of dye lost. The EDL terminals have a higher initial rate of dye release: 0.70 versus 0.43 PIU/sec in soleus (n = 10). B, Comparison of the percentage of dye lost by EDL and soleus terminals during the first 2 min of stimulation at 20 Hz in the presence of 5 µM d-tubocurarine (n = 10) or 2 µM µ-conotoxin GIIIB (n = 5) to block muscle contraction. EDL terminals show a higher rate of dye loss in both treatments (EDL vs soleus; d-tubocurarine, p < 0.001; µ-conotoxin GIIIB, p < 0.003). There is no significant difference in the rate of dye loss between the two treatments (d-tubocurarine versus µ-conotoxin GIIIB; EDL, p > 0.9; soleus, p > 0.5).
Basis for different rates of destaining
The higher initial rate of dye release per unit time, and hence per nerve impulse, in EDL nerve terminals means a greater fraction of their pool of labeled vesicles (VP) undergo exocytosis during prolonged continuous stimulation than in soleus terminals. Although the number of vesicles lost per impulse (QC) from the terminals is initially higher in EDL than in soleus, this changes with prolonged stimulation. The higher rate of destain with prolonged stimulation could be because of either of two options: (1) the mean QC is higher in EDL, but the VP is the same in the two terminal types, or (2) both the mean number of vesicles lost per impulse and VP differ.
To determine which scenario is correct, we therefore estimated VP in EDL and soleus nerve terminals and then compared it with the electrophysiological data on QC.
We estimated VP size by correlating the fraction of dye lost (measured optically) with the actual number of vesicles lost (measured electrophysiologically). This is a novel extension of the correlation of styryl dye and transmitter release first used by Betz and Bewick (1992
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Figure 6. Correlation of cumulative transmitter release (summed QC) and dye loss during the first 100 sec of destaining. Transmitter release is proportional to dye loss during this time because in fully loaded terminals, every vesicle that releases transmitter also loses dye (n = 10 terminals). These data were aligned manually by adjusting the dye loss scale so that the lines correspond for as long as possible during stimulation.
From the dye loss and electrophysiological data, the estimation of the initial VP size was as follows, illustrated using the data in Figure 6. In this case, for EDL, 30% of dye loss corresponds to a loss of 5 × 104 vesicles. Thus, the total number of vesicles in the terminal initially (VP) would be:
The results of this procedure for all 10 terminals of each type are shown in Table 1. Our results correlate well with many other estimates from similar preparations using different techniques. Elmqvist and Quastel (1965)
It is clear from the data in Table 1 that the VP in EDL terminals is only ~70% of that in soleus terminals. Thus, EDL terminals destain faster not only because they have a larger quantal vesicle release per impulse but also because of a smaller pool of vesicles from which to lose those vesicles. These data also allow us to compare the fraction of VP released over 2 min with the fraction of dye lost over the same time frame (Table 1). EDL terminals release a greater proportion of their vesicle pool in 2 min than soleus terminals under these conditions, there being no significant difference between the percentage of dye lost and percentage of VP lost in 2 min in EDL (p > 0.7) or soleus (p > 0.3).
As mentioned above, our studies of quantal release suggest that part of the VP appears to be more readily releasable than the rest. We estimate that the sum of vesicles contained in the two fastest components of release (
Vesicle recycling
Given the higher QC and the smaller VP size in the EDL terminals, it was of great interest to know whether the rate at which vesicles are recycled after exocytosis differs between the two muscles. It might be expected that EDL terminals, losing a higher proportion of their vesicles per impulse, would require the vesicles to be returned to the pool more rapidly to maintain transmitter release. We therefore went on to estimate the vesicle recycle time for the two different types of terminal.
During prolonged stimulation of frog motor nerve terminals, dye loss and summed QC curves deviate at later times, the rate of dye loss eventually falling below the rate of quantal release (Betz and Bewick, 1992
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Figure 7. During prolonged continuous stimulation, dye loss begins to lag behind transmitter release (summed QC) after a period of ~110 sec (n = 10 terminals). Dye loss falls below transmitter release when vesicles that have previously lost their dye reenter the vesicle pool and again undergo exocytosis but this time release transmitter but not dye. The time of first deviation between the two lines was determined by eye. This "vesicle recycle time" shows no significant difference between EDL and soleus (Table 1). The inset shows the trace from a single terminal; the point of deviation is indicated with an arrow.
Effect of stimulation frequency on vesicle recycling
Studies investigating the effect of changing activity on the total recycle time and the rate of endocytosis (the first step in the recycling of exocytosed vesicles) in frog motor nerve terminals (Betz and Bewick, 1993
Initially, to obtain as direct a comparison as possible, experiments were performed at continuous 80 Hz stimulation, the same rate as the in vivo EDL firing pattern but much longer duration (median in vivo burst duration, 1.6 sec). Our measurements of VP size and QC above would predict that most preparations would be depleted of vesicles before any vesicles were completely recycled ready for release again. In good agreement with this prediction, very few preparations were able to maintain transmitter release long enough for deviations between dye loss and transmitter release to be seen (~3 min). Further, again as predicted, soleus terminals consistently lasted longer than those in EDL. Unsuccessful experiments were terminated when EPPs had run down to undetectable levels or when evidence of axonal block (Krnjevic and Miledi, 1958
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Table 2. Synaptic vesicle recycling parameters in soleus terminals stimulated at continuous 60 and 80 Hz Effect of activity pattern on vesicle recycling
To approximate more closely the pattern of activity of the EDL in vivo, we next investigated the effect of different stimulation patterns on recycle time. The same number of stimuli were applied as in the 20 Hz continuous stimulation experiments but in a pattern approximating that in the EDL in vivo. Thus, we stimulated with short bursts at 80 Hz (2.5 sec) interspersed with 7.5 sec of rest, repeated each 10 sec for 10 min. The data for three EDLs and two soleus terminals are shown in Table 3. As can be seen, there was again no detectable effect of changes in activity frequency or pattern on total recycle time or fraction of dye lost per EPP. Further, the destaining kinetics of soleus terminals stimulated intermittently at 80 Hz were not significantly different from those stimulated continuously at 20 Hz (e.g., R2 = 0.97; fast
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Table 3. Synaptic vesicle recycling parameters from terminals stimulated phasically at 80 Hz.
DISCUSSION This study reveals significant differences in synaptic vesicle trafficking in motor nerve terminals, which apparently adapt them to their distinctive in vivo patterns of impulse activity. In particular, soleus terminals, which fire in vivo for long periods of time at ~20 Hz, support sustained quantal transmitter release much better than the terminals in EDL, which fire in short bursts at 60-80 Hz.
Soleus terminals have a lower QC but maintain release better
As in previous studies of these rodent muscles (mouse, Tonge, 1974
Another marked difference in vesicle release characteristics between EDL and soleus terminals was revealed during tonic 20 Hz stimulation. Although triphasic in both muscles, QC rundown was much greater in EDL at all frequencies (Fig. 1), suggesting soleus terminals are much better adapted to tonic transmitter release. There are few relevant previous studies available for direct comparison. In the garter snake, terminals on tonic muscle fibers maintained spontaneous transmitter release much better than terminals on twitch fibers when depolarized with high-potassium solutions for several hours (Morgan and Proske, 1984
Thus, one or more aspects of synaptic vesicle trafficking properties appears better adapted to maintaining transmitter output during tonic activity in soleus terminals. We therefore went on to use the FM 1-43 technique to investigate which aspects of vesicle trafficking might underlie this adaptation, beginning with the synaptic vesicle distribution and terminal destaining characteristics.
Are all vesicles labeled by the labeling regimen?
An important consideration for these data are whether the loading regimen labels all releasable vesicles in the terminals. Three lines of evidence suggest that this is so. First, our labeling stimulation regimen recycled ~378,000 vesicles per terminal (15 min, alternating trains of 5 sec at 1 Hz and 10 sec at 10 Hz = 6300 stimuli × estimated mean QC of 60; probably an underestimate under these conditions), which comfortably exceeds the largest VP measured (~280,000), although some vesicles undoubtedly recycle more than once. Second, increasing the stimulation time did not produce brighter labeling (20 min; data not shown). Finally, a similar regimen labeled all vesicles within frog motor nerve terminals (Henkel et al., 1996a
Terminal morphology, QC per unit area, and regional terminal destaining properties
FM1-43 labeling revealed morphological differences between terminals in EDL and soleus. A detailed quantification of these differences is the subject of another study in this laboratory. We report here, however, that terminals in the two muscles have similar areas. Thus, at least initially, the QC per unit area is higher in EDL than soleus terminals: 0.27 ± 0.01 versus 0.22 ± 0.01 vesicles/µm2 (n = 10; p < 0.03), consistent with previous findings in these muscles by Wood and Slater (1997)
We found considerable variability in fluorescence intensities between different regions of the same terminal, as reported previously for terminals in mouse triangularis sterni muscles (Ribchester et al., 1994
Although all terminals destained after stimulation, the rate of dye loss was consistently greater in EDL terminals at each frequency and activity pattern tested, irrespective of neuromuscular blocking agent. Both the optical and electrophysiological data, therefore, indicate real differences in the vesicle trafficking characteristics between terminals in the two muscles.
Quantification of vesicle trafficking characteristics in EDL and soleus
Correlation of dye loss with cumulative quantal release allowed us to determine several vesicle trafficking parameters, including total vesicle recycle time (Betz and Bewick, 1992
The VP size estimates revealed significant differences between EDL and soleus terminals. The ratio of vesicle density (VP/area) in soleus and EDL terminals (651 ± 55 vs 481 ± 37 vesicles/µm2, respectively; n = 10; ratio, 1.4:1) is very similar to the ratio of their intensities per unit area (0.14 ± 0.02 vs 0.11 ± 0.01 PIU/µm2, soleus and EDL, respectively; n = 25 and 23, respectively; ratio, 1.3:1). Thus, the larger VP in soleus terminals is primarily attributable to a significantly (p < 0.02) higher vesicle density, although what determines terminal vesicle density is not known.
The ratio between VP size and initial QC is therefore quite different in EDL and soleus terminals (Table 1). The smaller VP and higher initial QC in EDL is entirely consistent with the observations that EDL terminals destained faster, exhibited faster and more extensive EPP rundown, and failed earlier during prolonged high-frequency stimulation. Thus, VP size is not proportional to the QC of a terminal. So what determines VP size in a terminal? The demands of the in vivo activity pattern are presumably an important determinant, although not necessarily in the way some studies have predicted (Brodin et al., 1997
The balance of VP size and QC seem crucial for maintaining transmitter release, particularly because vesicle recycle time is similar in the two muscles and is constant. Interestingly, the vesicle recycle times reported here are similar to previous estimates from other methods and neuronal preparations [frog motor nerve terminals, ~94 sec, Betz and Bewick, 1993
In conclusion, motor nerve terminals appear to be adapted to their particular functional demands, maintaining vesicular output by adapting VP size and QC but not vesicle recycle time. If this is a general principle, it might place important constraints on the plasticity of terminal function, e.g., in learning and memory. How the consistent vesicle recycle times reported to date relate to recent data showing activity-dependent modulation of endocytosis (von Gersdorff and Matthews, 1994a
FOOTNOTES Received July 30, 1998; revised Jan. 19, 1999; accepted Jan. 22, 1999.
This work was supported by The Wellcome Trust, The Physiological Society, and The Royal Society. We thank Dr. Sarah J. Wood and Dr. John Pulham for helpful comments and discussion about this work.
Correspondence should be addressed to Dr. Guy S. Bewick, Department of Biomedical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, AB25 2ZD, United Kingdom.
REFERENCES
- Auerbach A, Betz W (1971) Does curare affect transmitter release? J Physiol (Lond) 213:681-705.
- Bennett MK, Scheller RH (1994) A molecular description of synaptic vesicle membrane trafficking. Annu Rev Biochem 63:63-100[Web of Science][Medline].
- Betz WJ, Bewick GS (1992) Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255:200-203
[Abstract/Free Full Text] .- Betz WJ, Bewick GS (1993) Optical monitoring of transmitter release and synaptic vesicle recycling at the frog neuromuscular junction. J Physiol (Lond) 460:287-309
[Abstract/Free Full Text] .- Betz WJ, Mao F, Bewick GS (1992) Activity-dependent fluorescent staining and destaining of living vertebrate motor nerve terminals. J Neurosci 12:363-375[Abstract].
- Betz WJ, Ridge RMAP, Bewick GS (1993) Comparison of FM1-43 staining patterns and electrophysiological measures of transmitter release at the frog neuromuscular junction. J Physiol (Paris) 87:193-202[Web of Science][Medline].
- Bewick GS, Tonge DA (1991) Characteristics of endplates formed in mouse skeletal muscles reinnervated by their own or by foreign nerves. Anat Rec 230:273-282[Medline].
- Braga MF, Anderson AJ, Harvey AL, Rowan EG (1992) Apparent block of K+ currents in mouse motor nerve terminals by tetrodotoxin, µ-conotoxin and reduced external sodium. Br J Pharmacol 106:91-94[Web of Science][Medline].
- Brodin L, Low P, Gad H, Gustafsson J, Pieribone VA, Shupliakov O (1997) Sustained neurotransmitter release: new molecular clues. Eur J Neurosci 7:2503-2511.
- Brooke MH, Kaiser KK (1969) Some comments on the histochemical characterization of muscle adenosine triphosphatase. J Histochem Cytochem 17:431-432[Web of Science][Medline].
- Coniglio LM, Hardwick JC, Parsons RL (1993) Quantal transmitter release at snake twitch and tonic muscle fibres during prolonged nerve terminal depolarization. J Physiol (Lond) 466:383-403
[Abstract/Free Full Text] .- Connor EA, Dunaevsky A, Griffiths DJG, Hardwick JC, Parsons RL (1997) Transmitter release differs at snake twitch and tonic endplates during potassium-induced deploarization. J Neurophysiol 77:749-760
[Abstract/Free Full Text] .- Elmqvist D, Quastel DMJ (1965) Presynaptic action of hemicholinium at the neuromuscular junction. J Physiol (Lond) 177:463-482.
- Ferry CB, Kelly SS (1988) The nature of the presynaptic effects of (+)-tubocurarine at the mouse neuromuscular junction. J Physiol (Lond) 403:425-437
[Abstract/Free Full Text] .- Gertler RA, Robbins N (1978) Differences in neuromuscular transmission in red and white muscles. Brain Res 142:160-164[Web of Science][Medline].
- Glavinovic MI (1979a) Change of statistical parameters of transmitter release during various kinetic tests in unparalysed voltage-clamped rat diaphragm. J Physiol (Lond) 290:481-497
[Abstract/Free Full Text] .- Glavinovic MI (1979b) Presynaptic action of curare. J Physiol (Lond) 290:499-506
[Abstract/Free Full Text] .- Henkel AW, Lübke J, Betz WJ (1996a) FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals. Proc Nat Acad Sci USA 93:1918-1923
[Abstract/Free Full Text] .- Henkel AW, Simpson LL, Ridge RMAP, Betz WJ (1996b) Synaptic vesicle movements monitored by fluorescent recovery after photobleaching in nerve terminals stained with FM1-43. J Neurosci 16:3960-3967
[Abstract/Free Full Text] .- Henneman E, Olson CB (1965) Relations between structure and function in the design of skeletal muscles. J Neurophysiol 28:581-598
[Free Full Text] .- Henneman E, Somjen G, Carpenter DO (1965) Functional significance of cell size in spinal motoneurons. J Neurophysiol 28:560-580
[Free Full Text] .- Hennig R, Lømo T (1985) Firing patterns of motor units in normal rats. Nature 314:164-166[Medline].
- Ireland DR, Davies PJ, McLachlan EM (1999) Calcium channel subtypes differ at two types of cholinergic synapse in lumbar sympathetic neurones of guinea pigs. J Physiol (Lond) 514:59-69
[Abstract/Free Full Text] .- Kamenskaya MA, Elmqvist D, Thesleff S (1975) Guanidine and neuromuscular transmission. II. Effect on transmitter release in response to repetitive nerve stimulation. Arch Neurol 32:510-518
[Abstract/Free Full Text] .- Klingauf J, Kavalali ET, Tsien RW (1998) Kinetics and regulation of fast endocytosis at hippocampal synapses. Nature 394:581-585[Medline].
- Krnjevic K, Miledi R (1958) Some effects produced by adrenaline upon neuromuscular propagation in rats. J Physiol (Lond) 141:291-304[Web of Science][Medline].
- Lagnado LL, Gomis A, Job C (1996) Continuous vesicle cycling in the synaptic terminal of retinal bipolar cells. Neuron 17:957-967[Web of Science][Medline].
- Liley AW (1956) An investigation of spontaneous activity at the neuromuscular junction of the rat. J Physiol (Lond) 132:650-666.
- Martin AR (1955) A further study of the statistical composition of the endplate potential. J Physiol (Lond) 130:114-122.
- McLachlan EM (1978) The statistics of transmitter release at chemical synapses. Int Rev Physiol 17:49-117[Medline].
- Morgan DL, Proske U (1984) Vertebrate slow muscle: its structure, pattern of innervation, and mechanical properties. Physiol Rev 64:103-169
[Free Full Text] .- Plomp JJ, van Kempen GTH, Molenaar PC (1992) Adaptation of quantal content to decreased postsynaptic sensitivity at single endplates in
-bungarotoxin-treated rats. J Physiol (Lond) 458:487-499
[Abstract/Free Full Text] .- Reid B, Bewick GS (1996) Synaptic vesicle trafficking in motor nerve terminals of rat fast- and slow-twitch muscles. J Physiol (Lond) 495:59P.
- Reid B, Bewick GS (1997) Synaptic vesicle recycle time and releasable pool size in motor nerve terminals of rat fast- and slow-twitch muscles. J Physiol (Lond) 499:28P.
- Ribchester RR, Mao F, Betz WJ (1994) Optical measurements of activity-dependent membrane recycling in motor nerve terminals of mammalian skeletal muscle. Proc R Soc Lond B Biol Sci 255:61-66[Medline].
- Ryan TA, Reuter H, Wendland B, Schweizer FE, Tsien RW, Smith SJ (1993) The kinetics of synaptic vesicle recycling measured at single presynaptic boutons. Neuron 11:713-724[Web of Science][Medline].
- Schofield GG, Marshall IG (1980) Neuromuscular transmission in the athymic nude mouse. J Neurol Sci 48:21-34[Web of Science][Medline].
- Searl T, Prior C, Marshall IG (1990) The effects of L-vesamicol, an inhibitor of vesicular acetylcholine uptake, on two populations of miniature endplate currents at the snake neuromuscular junction. Neuroscience 35:145-156[Web of Science][Medline].
- Searl T, Prior C, Marshall IG (1991) Acetylcholine recycling and release at rat motor nerve terminals studied using (
[Abstract/Free Full Text] .- Slater CR, Lyons PR, Wallis TJ, Fawcett PRW, Young C (1992) Structure and function of neuromuscular junctions in the vastus lateralis of man: a motor point biopsy study of two groups of patients. Brain 11:451-478.
- Tonge DA (1974) Physiological characteristics of re-innervation of skeletal muscle in the mouse. J Physiol (Lond) 241:141-153
[Abstract/Free Full Text] .- von Gersdorff H, Matthews G (1994a) Dynamics of synaptic vesicle fusion and membrane retrieval in synaptic terminals. Nature 367:735-773[Medline].
- von Gersdorff H, Matthews G (1994b) Inhibition of endocytosis by elevated internal calcium in a synaptic terminal. Nature 370:652-655[Medline].
- Walrond JP, Reese TS (1985) Structure of axon terminals and active zones at synapses on lizard twitch and tonic muscle fibers. J Neurosci 5:1118-1131[Abstract].
- Wood S, Slater C (1997) The contribution of postsynaptic folds to the safety factor for neuromuscular transmission in rat fast- and slow-twitch muscles. J Physiol (Lond) 500:165-176
[Abstract/Free Full Text] .- Wu LG, Betz WJ (1996) Nerve activity but not intracellular calcium determines the time course of endocytosis at the frog neuromuscular junction. Neuron 17:769-779[Web of Science][Medline].
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