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. 2005 Aug 25;568(Pt 2):413–421. doi: 10.1113/jphysiol.2005.096131

Vesicles in snake motor terminals comprise one functional pool and utilize a single recycling strategy at all stimulus frequencies

Michael Y Lin 1, Haibing Teng 1, Robert S Wilkinson 1
PMCID: PMC1474750  PMID: 16123101

Abstract

At a variety of fast chemical synapses, spent synaptic vesicles are recycled via a large ‘reserve’ vesicle pool at high stimulus frequencies, and via fast ‘local cycling’ near release sites (e.g. ‘kiss and run’ transmitter release) at low stimulus frequencies. We have investigated recycling at the snake neuromuscular junction (NMJ), specifically seeking evidence for local cycling. Activity-dependent staining and destaining of the endocytic probe FM1-43 were directly compared to transmitter release over a range of stimulus frequencies. We found a fixed proportionality between staining/destaining and summed endplate potentials (EPPs) representing total transmitter release. There was no direct dependence of staining or destaining on stimulus frequency, as would be expected if local cycling (and consequent altered FM1-43 retention) were more prevalent at one frequency than another. In other experiments the drug vesamicol was used to abolish refilling of vesicles with transmitter, thereby blocking EPPs contributed by recycled vesicles. Control and vesamicol-treated NMJs had identical quantal content for the first 10 min of 1 Hz stimulation. Afterwards EPP amplitudes at vesamicol-treated NMJs declined at a rate consistent with use of a large pool containing ∼130 000 vesicles. Finally, calibrated paired stimulations show that regenerated vesicles have poorer than random probability of re-release. Our findings are inconsistent with local cycling and suggest that the snake motor terminal utilizes exclusively a single large vesicle pool.

The efficacy of a nerve terminal depends both on its ability to sustain transmitter release and on its ability to recycle spent vesicles for reuse. Two principal recycling strategies have been described. In one, a single vesicular pool supplies the exocytic machinery with preformed vesicles, and is replenished via clathrin-mediated endocytosis (CME; Heuser & Reese, 1973). In the other, subsets of vesicles recycle at or near individual active zones (AZs; Ceccarelli et al. 1973). These ‘local pool’ vesicles are preferentially reused without entering the larger ‘reserve pool.’ An attractive hypothesis is that both strategies are available to nerve terminals, and are differentially recruited in some use-dependent manner. Supporting evidence has come from studies of the stimulus frequency dependence of endocytosis in frogs (Richards et al. 2003), Drosophila shibire (Kuromi & Kidokoro, 2000), rodent hippocampal neurones (Klingauf et al. 1998; Pyle et al. 2000), the rat calyx of Held (Sun et al. 2002; de Lange et al. 2003) and other preparations. With some exceptions, there is consensus that local cycling prevails whenever use is minimal (low frequency stimulation) while recycling through the reserve pool (often involving budding from macroendosomes internalized by bulk membrane invagination; Richards et al. 2000; Teng & Wilkinson, 2000) prevails as level of use increases (high frequency stimulation; reviewed by Kuromi & Kidokoro, 2005; Rizzoli & Betz, 2005). In snake motor terminals, CME occurs preferentially near AZs (Teng & Wilkinson, 2000), placing recently internalized vesicles in a seemingly ideal position to undergo local cycling and re-release. We therefore undertook a study of vesicle recycling at the snake NMJ, seeking evidence for a systematic change in recycling strategy as we altered stimulus frequency.

To do so we directly compared transmitter release, assessed by postsynaptic electrophysiological recording, with both uptake and release (‘destaining’) of the activity-dependent dye FM1-43. We also functionally blocked vesicle recycling with vesamicol, an inhibitor of the ACh–proton exchanger found on cholinergic vesicles. We observed the same direct proportionality between staining/destaining and transmitter release at all stimulus frequencies. There was no evidence suggesting an influence of stimulus frequency on recycling strategy. Moreover, a recently recycled vesicle had a poorer than random chance of being re-released. Our results support a ‘first in, first out’ model of recycling wherein vesicles return to the back of a single large pool and are re-released after considerable delay.

Methods

Garter snakes (Thamnophis sirtalis) were anaesthetized with pentobarbital sodium (80 mg kg−1, i.p.) and killed by rapid decapitation. Several contiguous segments of the single-fibre-thick transvs. abdominis muscle were dissected from the animal, placed in reptilian saline solution and divided to provide 4–12 individual nerve–muscle preparations. Each preparation contained three segmental muscles. Details of the muscle's anatomy and of the composition of reptilian saline are given elsewhere (Wilkinson & Lichtman, 1985). All procedures followed the Washington University Guidelines for Animal Studies.

Electrical stimulation and intracellular recording

Preparations were mounted in a chamber containing reptilian saline. The cut end of the central muscle's nerve was drawn into a suction electrode for stimulation with 200 µs negative-going rectangular pulses of 2–7 V. Intracellular recording of endplate potentials (EPPs) and miniature endplate potentials (mEPPs) and subsequent analyses were performed using methods described elsewhere (Wilkinson et al. 1992). Under these conditions, change in EPP amplitude reflects alteration in quantal content (Wilkinson et al. 1996), not change in endplate sensitivity. Recording of mEPPs immediately after tetanic stimulation in separate experiments indicated that asynchronous transmitter release (which was not detected in curarized preparations and therefore ignored) accounted for 0.7% of total release, or less, in all experiments. Simultaneous imaging of FM1-43 and recording of EPPs was possible in destaining (dye-free) experiments only. The muscle was damaged (low resting potential) when placed in a dye-containing bath and repeatedly exposed to light, as would be necessary for staining experiments.

Activity-dependent staining and destaining

For staining, FM1-43 (12 μm) or FM2-10 (48 μm; Molecular Probes, Eugene, OR, USA) was dissolved in curarized (30 μm) reptilian saline and applied 2 min prior to electrical stimulation. After stimulation, preparations were vigorously washed at the times indicated in Results. We used an inverted microscope equipped with an Intelligent Imaging Innovations (Denver, CO, USA) digital imaging system (12 bit, Cooke SensiCam camera) to quantify brightness of internalized FM1-43. Light levels were kept as low as possible to prevent photobleaching. Regions of interest (individual boutons) were manually outlined and their pixel values determined. Background fluorescence was estimated from an outlined region of the muscle fibre surrounding the nerve terminal boutons or from fluorescence of unstimulated boutons and subtracted from the measured average bouton fluorescence (see Fig. 2A). Fluorescence is reported as arbitrary brightness units (ABUs; 1–4096).

Figure 2. Destaining of nerve terminals reveals a constant ratio between dye loss and transmitter release.

Figure 2

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Nerve–muscle preparations were loaded with FM1-43, rested overnight (see Methods) and destained by stimulating in a curarized dye-free bath. EPP amplitudes and fluorescence loss were recorded simultaneously. A, representative image of FM1-43 loaded nerve terminal prior to destaining. Manually outlined regions of interest (boutons) are shown in red and the selected background region is shown in yellow. B, typical dye loss plotted versus stimulus number. Each curve is the loss from one nerve terminal destained at the frequency indicated. Dye loss per stimulus was greatest at 1 Hz. C, typical EPPs recorded during the stimulus train (from the 3 terminals in B), plotted versus stimulus number. EPPs were largest at 1 Hz. D, EPP amplitudes (in C) were added successively to plot summed EPP amplitude versus stimulus number. E, fluorescence loss (B) and corresponding summed EPPs (D) from all experiments are shown plotted against each other. Dye loss versus transmitter release was linear at each frequency. All data begin at 100% fluorescence; for clarity the 10 and 30 Hz data are offset −10% and −20%, respectively. F, linear regressions (from E) showed identical dye–transmitter proportionality regardless of frequency.

For destaining experiments, nerve terminals were maximally stained (100%; 30 min, 1 Hz) or partially stained (∼40% or ∼20%; see Results). Maximal staining at each frequency was determined by subjecting nerve terminals to various durations of prolonged stimulation in the dye bath. Maximally stained preparations were rested overnight (4°C) to ensure mixing of vesicle pools (Richards et al. 2000). Spontaneous transmitter release during this period (< 1200 mEPPs/12 HR) was small compared to release during experiments (∼40 000 quanta), and in separate experiments we compared brightness of terminals before and after the rest period and detected no destaining. Partially stained preparations were rested for at least 5 min (40% stained) or 40 s (20% stained) prior to destaining, times sufficient to allow recovery from depression as determined in separate electrophysiological experiments. Destaining was performed in a curarized bath. Residual fluorescence (that remaining after ∼2 h of 1 Hz destaining; less than 5–8%) was subtracted from all data prior to analyses.

Curare washout and estimation of released quanta

Several amino acid substitutions (Samson et al. 2002) impart to the snake ACh receptor resistance to bungarotoxin and relatively weak affinity for curare. The latter permits rapid and complete removal of curare with three to four solution changes, which we confirmed by comparing mEPPs prior and subsequent to incubation (data not shown).

The number of quanta released per stimulus was taken as the quotient of the ‘theoretical endplate response’ (see Results) and the mean mEPP amplitude (i.e. 90 mV/0.6 mV =∼150 quanta).

Results

FM1-43 uptake is proportional to transmitter release and independent of stimulus frequency

To investigate the effect of increasing stimulus frequency on transmitter output and dye uptake, we delivered trains of exactly 1800 stimuli at 1, 2, 5, 10 and 30 Hz to paired preparations from the same snake. This number of stimuli was below that required to saturate dye uptake at all frequencies (Methods). One preparation was stimulated in a curarized bath containing FM1-43. Prior to washout and image acquisition, it remained in the dye-containing bath for 10 min to ensure that all endocytosis was complete (Teng & Wilkinson, 2003). Shown in Fig. 1A are representative images of living nerve terminals stimulated at the frequencies indicated. Increasing stimulus frequency monotonically diminished dye uptake. On average, nerve terminals stimulated at 1 Hz were ∼2.5 times as bright as those stimulated at 30 Hz (797 ± 114 versus 323 ± 35; ABUs ± s.e.m.; n = 8–16 each frequency).

Figure 1. Activity-dependent staining depends on total transmitter output and not stimulus frequency.

Figure 1

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A, FM1-43 uptake in response to 1800 stimuli decreased with increasing stimulus frequency. Images are of living nerve terminals, shown brightness-enhanced ∼5-fold. B, first and last EPPs recorded from preparations receiving identical stimulus trains as in A, showing depression. C, mean EPP amplitudes evoked at 1, 2, 5, 10 and 30 Hz are plotted against stimulus number. Depression with continued stimulation was more pronounced at higher frequencies. D, mean EPP amplitudes (in C) were added successively to plot summed EPP amplitude versus stimulus number. The end value of each curve reflects total transmitter output during the stimulus train. E, increasing stimulus frequency depressed total dye-uptake and total transmitter output similarly. Average nerve terminal fluorescence and summed EPP amplitude (end values from D) are compared. Error bars are s.e.m. F, plot of FM1-43 fluorescence versus total transmitter output, showing approximate proportional relationship (continuous regression line forced through origin). Best regression fit (dashed line) has 0.77 slope on double-logarithmic axes.

The second preparation from each pair was stimulated in a bath containing curare but not FM1-43 to facilitate the recording of EPPs (Methods). Increasing the stimulus frequency increased synaptic depression during the electrical stimulation, as is shown by the representative first and last EPPs in Fig. 1B. The average EPP amplitudes during the stimulus train are shown in Fig. 1C (n = 4 each frequency; 4 snakes; all s.e.m.s < 8%, error bars omitted for clarity). All stimulus frequencies evoked initial EPP amplitudes of approximately 7 mV, and all EPPs exhibited sharp declines in amplitude during the first 50 stimuli. Following this initial rapid decay, EPP amplitudes decreased by various extents. By the end of the stimulus train, the slower rates (1, 2, and 5 Hz) evoked EPPs of 2.5, 2 and 1 mV, respectively; EPP amplitudes at 10 Hz and 30 Hz had decreased to 0.5 mV. The average EPP amplitudes (Fig. 1C) were summed successively to reveal the cumulative transmitter output during the stimulus train (Fig. 1D). The total transmitter output, taken as the end value of each cumulative EPP curve, decreased in response to increasing stimulus frequency. Stimuli delivered at 1 Hz evoked ∼3 times the transmitter output (summed EPPs, 6 V) as stimuli delivered at 30 Hz (2 V).

The measured FM1-43 fluorescence and total transmitter output after 1800 stimuli are presented in Fig. 1E as functions of stimulus frequency. The same data are compared directly in Fig. 1F, showing a direct proportionality. Thus depression in transmitter release at higher stimulus rates was matched by decrease in compensatory endocytosis. Stimulus rate did not independently alter dye uptake, as would occur if recycling were more prominent at one stimulus frequency than another.

Destaining of FM1-43 is proportional to transmitter release and independent of stimulus frequency

We stimulated maximally labelled and equilibrated nerve terminals (Fig. 2A; Methods) in a dye-free curarized bath and simultaneously monitored transmitter output and dye loss. We expected preformed vesicles to release both transmitter and previously loaded dye, and vesicles recycled during the stimulus train to release only transmitter. At high, medium and low stimulus frequencies (30 Hz, 10 Hz and 1 Hz), we acquired ∼25 images over periods sufficiently long to characterize the time course of destaining. Figure 2B shows the effect of stimulus frequency on destaining for three representative terminals. On average (n = 3 at 1 Hz, 3 at 10 Hz, 2 at 30 Hz), a 20% dye loss required ∼400 stimuli at 1 Hz, ∼800 stimuli at 10 Hz, and ∼1100 stimuli at 30 Hz. Figure 2C shows the EPP amplitudes recorded from the endplates of Fig. 2B. Depression increased with frequency as in staining experiments. The EPPs from Fig. 2C were summed successively to indicate cumulative transmitter release (Fig. 2D). The fluorescence loss and transmitter release from the terminals of Fig. 2B–D, plus all other terminals studied, are compared directly in Fig. 2E (plots offset vertically for clarity; see legend). Destaining and transmitter output were proportional at each frequency. Linear regression fits (Fig. 2F) overlapped and their slopes were statistically identical (1 Hz, m = −11.7% V−1; 10 Hz, m = −12.6% V−1; 30 Hz, m = −12.7% V−1; P > 0.14).

We repeated the experiments of Fig. 2 at 1 Hz (n = 5) and 30 Hz (n = 5) using the dye FM2-10 (a less hydrophobic version of FM1-43), which behaves differently from FM1-43 in frog (Richards et al. 2000). Results were similar to those using FM1-43: the relationships between destaining and summed EPPs (Fig. 2D) were linear at both frequencies with nearly identical regression slopes (1 Hz, m = −17.9% V−1; 30 Hz, m = −18.5% V−1).

Thus, comparable to the results from staining experiments, there was no detectable change in the proportionality between transmitter release and FM1-43 (or FM2-10) destaining, either as a function of stimulus frequency (which would yield a different slope at each frequency) or as a function of time or stimulus number (which would cause a change in slope with increasing summed EPP amplitude at a single stimulus frequency, resulting in a non-linear relation).

Contribution of recycled vesicles during sustained stimulation

The experiments of Figs 1 and 2 showed that the ratio of recycled vesicles to preformed vesicles released was the same at all frequencies, but did not address the actual extent of recycling. We therefore utilized vesamicol (ICN Biomedicals Inc., Aurora, OH, USA), an inhibitor of the ACh/proton exchanger of cholinergic vesicles, to determine the contribution of recycled vesicles during sustained transmitter release. At 5 µm bath concentration, vesamicol has no detectable postsynaptic effect (Enomoto, 1988), does not alter the quantal size of preformed vesicles (reviewed by Van der Kloot, 2003), does not disturb synaptic vesicle recycling (Parsons et al. 1999), but completely abolishes transmitter refilling (Bahr & Parsons, 1986). After vesamicol treatment, cholinergic terminals release normal previously formed vesicles, but recycled vesicles are transmitter-free and therefore postsynaptically silent (Parsons et al. 1999). Thus decrease in EPP amplitude at a vesamicol-treated terminal, as compared to an identically stimulated control terminal, indicates reuse of vesicles during the stimulus train.

Figure 3 shows the effect of vesamicol on EPP amplitudes recorded during prolonged 1 Hz stimulation. Plotted in Fig. 3A are the averaged EPP amplitudes from curarized control (n = 4) and curarized vesamicol-treated terminals (n = 4). The two plots superimpose for ∼600 s. Subsequently, EPPs from vesamicol treated terminals diminished gradually in size, becoming undetectable in the curarized bath after ∼1800 s, or 30 min. In contrast, EPPs from control preparations reached a steady level of approximately 3 mV. The initial superimposition of control and vesamicol data suggests that nerve terminals utilized preformed vesicles predominantly for ∼10 min before they began using recycled vesicles at detectable levels. Estimating from Fig. 3A, recycled vesicles comprised ∼30% ((EPPCntrl− EPPVes)/EPPCntrl) of released vesicles after 20 min of stimulation and ∼80% after 30 min.

Figure 3. Characterizing recycling with vesamicol.

Figure 3

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EPPs in vesamicol are from preformed vesicles; control EPPs are from both preformed and recycled vesicles. Error bars are s.e.m.A, terminals used preformed vesicles before detectable onset of recycling. Average EPPs from curarized and control preparations superimposed for ∼600 stimuli at 1 Hz (10 min). Vesamicol EPPs then approached 0 mV as the preformed vesicle supply became exhausted. Control EPPs remained at ∼3 mV for at least 100 min, utilizing recycled vesicles. At ∼1800 s, curare was removed from the vesamicol preparations and EPP recording resumed in normal reptilian saline (▵). B, the functional releasable pool comprises ∼130 000 vesicles. Data (in A) were corrected for the effect of curare and plotted as quantal content/stimulus. Averaged results from vesamicol preparations were fit by a single exponential (note semilog axes) from which the pool size was estimated (see text).

Estimation of the functional releasable pool size

Without interrupting the prolonged 1 Hz stimulus train, we rapidly removed curare from each vesamicol-treated preparation after 1800 stimuli and resumed recording of previously undetectable EPPs in normal saline (Fig. 3A; Methods). By comparing EPPs just before and just after removal of curare, we corrected for the effect of curare in both vesamicol and control preparations, yielding theoretical EPP amplitudes (i.e. without non-linear summation; ∼90 mV for the first stimulus) that were proportional to quantal content, m. These normalized data from Fig. 3A are plotted in Fig. 3B as quantal content (m= EPP/mEPP), with mean mEPP amplitude taken as 0.6 mV from prior experiments (Methods). On average, ∼150 quanta were released initially from both normal and vesamicol-treated terminals, consistent with previous estimates (m = 1.4 quanta/bouton, 58 boutons/terminal; Wilkinson et al. 1996). Removal of curare permitted the releasable pool's depletion time course to be assessed. The vesamicol curve in Fig. 3B (4 terminals, 3 snakes) was fitted by the single exponential m = 150 e−0.0011S where m is the number of vesicles (quanta) released per stimulus and S is the stimulus number. By integrating under this curve, we estimate that the number of releasable preformed vesicles (the functional releasable pool size) is 130 000 vesicles per terminal, or roughly 2000 vesicles per bouton (see also Parsons et al. 1999).

Recycled vesicles have a poorer than random chance of re-release

We asked whether newly regenerated vesicles have greater probability, lesser probability, or the same probability of re-release as a vesicle selected randomly from the releasable pool, and whether this probability changes with time. In the experiments of Fig. 4, two stimulus trains, both calibrated to release ∼50 000 vesicles (40% of the ∼130 000 releasable vesicles determined from vesamicol data of Fig. 3), were delivered sequentially to the same nerve terminal. The first (loading stimulus; either 1 Hz, 7.5 min or 30 Hz, 1 min) was in a dye-containing bath, thereby labelling ∼40% of releasable vesicles. The second (destaining stimulus, 30 Hz, 1 min) was in a dye-free bath. We imaged the nerve terminal prior to and subsequent to the destaining stimulus train and determined the percentage dye loss. This indicated the recycled vesicles' re-release probability according to the following logic. If newly formed vesicles (those labelled during the first train) have a 100% probability of re-release, the second, destaining stimulus train should exocytose 100% regenerated vesicles, resulting in 100% dye loss. Conversely, if regenerated vesicles have, say, a 20% probability of re-release, a 20% dye loss should occur. Finally, a 40% dye loss is expected if the regenerated vesicles are randomly accessible, as should be the case after times sufficient for equilibration of vesicles in different pools. Therefore, > 40% dye loss reflects greater-than-random probability of re-release, while < 40% dye loss reflects less-than-random probability of re-release.

Figure 4. Recycled vesicles have a poorer than random chance of re-release for 15–20 min.

Figure 4

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Shown above each plot is the experimental protocol. Preparations were partially stained to label 40% of the ∼130 000 releasable vesicles. After variable delays, they were re-stimulated in a dye-free bath to again release 40% of vesicles. The percentage dye loss from images taken prior to and subsequent to the second stimulus indicates re-release probability of stained vesicles (see text). Error bars are s.e.m.A, results from nerve terminals stained at 30 Hz. Only ∼17% of FM1-43 (•) taken up was released after 5 min delay. This percentage increased slowly over the next 15–20 min to a maximum approaching ∼40%, the amount expected for random selection from a pool in which labelled preformed vesicles were uniformly distributed. The dye FM2-10 (○) behaved similarly. B, results from nerve terminals stained at 1 Hz were the same (P= 0.50).

Whether the re-release probability changes over time was investigated by systematically varying the time elapsed between the loading and the destaining stimuli. Figure 4A shows results from nerve terminals loaded at 30 Hz. Approximately 17% dye loss accompanied a destaining stimulus train 5 min after loading (the shortest delay possible to allow recovery from depression after 40% loading; Methods; n = 5), a poorer than random probability of re-release. This low probability persisted for nearly 15 min, then increased to a plateau value of ∼40% after 30 min. We repeated the experiment using FM2-10, and obtained similar results (Fig. 4A). There was also no evidence for an influence of loading frequency, as similar results were also obtained from nerve terminals loaded at 1 Hz (Fig. 4B; FM1-43 only): the probability of re-release was ∼17% initially, rising to ∼40% after ∼30 min (n = 5). Finally, we tested whether decreased (∼20%) initial dye loading, at 1 Hz (173 s, n = 16) or 30 Hz (18 s, n = 14), made vesicles more available for re-release, and found that it did not. We destained by repeating the staining stimulations at both frequencies, after either 40 s (minimum time for recovery from depression) or 30 min. There was no significant destaining after 40 s (n = 16, P > 0.12 at 1 Hz; n = 13, P > 0.17 at 30 Hz). After 30 min, destaining was 30% at 1 Hz (n = 16, P < 0.001) and 20% at 30 Hz (n = 14, P < 0.03).

Discussion

We conclude that re-formed vesicles enter a single large (reserve) pool and remain there for some time before they are mobilized to AZs for reuse. We found no evidence for use of smaller (local) pools over the 1 Hz to 30 Hz range of stimulus frequencies. The delay before reuse (Fig. 4) is consistent with a ‘first in, first out’ storage mechanism similar to that proposed by Heuser and colleagues (Heuser & Reese, 1973; Miller & Heuser, 1984). In that model, endo-cytosis occurs at sites distant from the AZ. Because of this spatial separation, the model predicts a less-than-random probability, compared to the remainder of the pool, that a newly formed vesicle would be among the first arriving at the AZ for re-release. In contrast, local or fast recycling near AZs implies a greater-than-random probability of re-release. In the snake, CME occurs near AZs (Teng & Wilkinson, 2000), suggesting that delayed reuse must be due to some mechanism other than spatial separation. One possibility is that other vesicles – those budded from internalized membrane (macroendosomes) – are initially unavailable and contribute substantially to the delay (see Richards et al. 2000). Consistent with this, macroendosomes form at all stimulus frequencies in snake and require several minutes to dissipate via CME (Teng et al. 2005). A second possibility is restraint of newly formed vesicles, perhaps via an interaction of actin with the vesicle membrane protein synapsin (Hilfiker et al. 2005), by regulation of ATP-dependent myosin motors (Verstreken et al. 2005), or both. These mechanisms might ensure the orderly mobilization of vesicles to AZs while allowing ample time for refilling with transmitter.

We were surprised to observe only a single recycling strategy in snake. The Drosophila NMJ contains two anatomically distinct vesicle pools, one reserve and one local cycling near AZs, and utilizes the latter at low stimulus frequencies (reviewed by Kuromi & Kidokoro, 2005; but see also Koenig & Ikeda, 2005). The frog NMJ also employs local cycling under low use conditions (Richards et al. 2003; reviewed by Rizzoli & Betz, 2005). Curiously, the frog's cycling pool is not anatomically distinct, but appears scattered throughout the reserve pool (Rizzoli & Betz, 2004). Local cycling, probably via kiss-and-run transmitter release, occurs in rodent CNS terminals as well (Klingauf et al. 1998; Pyle et al. 2000; Aravanis et al. 2003). However, there is some dispute with this preparation, as recent studies using the pH-sensitive protein synaptopHlorin provide evidence for a single recycling strategy (Fernandez-Alfonso & Ryan, 2004; Li et al. 2005; see Zenisek, 2005), in agreement with results presented here.

Acknowledgments

We thank J. Heuser and S. Misler for helpful discussions. This work was supported by U.S. Public Health Service grant NS-24752.

Supplemental material

The online version of this paper can be accessed at: 10.1113/jphysiol.2005.096131 jp.physoc.org/cgi/content/full/jphysiol.2005.096131/DC1 and contains supplemental material consisting of two figures:

Supplemental Figure A. Destaining of FM 2-10 labelled nerve terminals reveals a constant ratio between dye loss and transmitter release

Supplemental Figure B. Preformed, not recycled, vesicles are released with a brief second stimulation after a 40 s delay

This material can also be found as part of the full-text HTML version available from www.blackwell-synergy.com

Supplemental Data
jphysiol_2005.096131_index.html (871B, html)

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