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Previous Article | Next Article
The Journal of Neuroscience, March 1, 1999, 19(5):1557-1565
The Optically Determined Size of Exo/Endo Cycling Vesicle Pool Correlates with the Quantal Content at the Neuromuscular Junction of Drosophila Larvae
Hiroshi Kuromi and Yoshiaki Kidokoro Institute for Behavioral Sciences, Gunma University School of Medicine, 3-39-22, Showa-machi, Maebashi, 371-8511, Japan
ABSTRACT
Top
Abstract
Introduction
According to the current theory of synaptic transmission, the amplitude of evoked synaptic potentials correlates with the number of synaptic vesicles released at the presynaptic terminals. Synaptic vesicles in presynaptic boutons constitute two distinct pools, namely, exo/endo cycling and reserve pools (Kuromi and Kidokoro, 1998a
). We defined the vesicles that were endocytosed and exocytosed during high K+ stimulation as the exo/endo cycling vesicle pool. To determine the role of exo/endo cycling vesicle pool in synaptic transmission, we estimated the quantal content electrophysiologically, whereas the pool size was determined optically using fluorescent dye FM1-43. We then manipulated the size of the pool with following treatments. First, to change the state of boutons of nerve terminals, motoneuronal axons were severed. With this treatment, the size of exo/endo cycling vesicle pool decreased together with the quantal content. Second, we promoted the FM1-43 uptake using cyclosporin A, which inhibits calcineurin activities and enhances endocytosis. Cyclosporin A increased the total uptake of FM1-43, but neither the size of exo/endo cycling vesicle pool nor the quantal content changed. Third, we increased the size of exo/endo cycling vesicle pool by forskolin, which enhances synaptic transmission. The forskolin treatment increased both the size of exo/endo cycling vesicle pool and the quantal content. Thus, we found that the quantal content was closely correlated with the size of exo/endo cycling vesicle pool but not necessarily with the total uptake of FM1-43 fluorescence by boutons. The results suggest that vesicles in the exo/endo cycling pool primarily participate in evoked exocytosis of vesicles.
Key words: synaptic vesicle pools; FM1-43; quantal con tent; neuromuscular junction; Drosophila larva; forskolin; cyclosporin A
INTRODUCTION
Signal transmission at the synapse is mediated by the neurotransmitter packaged in synaptic vesicles at the nerve terminal. Transmitter release occurs in discrete quantal units as the result of exocytosis of these vesicles. According to the current theory of neurotransmission, the magnitude of evoked release depends on the number of quanta released after arrival of an action potential. To test this prediction, Koenig et al. (1989)
When the shits1 preparation was treated with cytochalasin D, the reserve pool size was greatly reduced, whereas the exo/endo cycling pool was unchanged. In this preparation, the amplitude of synaptic potentials did not change at 0.3 Hz stimulation but decreased rapidly at 10 Hz (Kuromi and Kidokoro, 1998a
In this study we used the following three treatments to manipulate the size of the vesicle pool, and we examined the effects on the quantal content of synaptic transmission: (1) the axon severance, which might affect the state of nerve terminals and change the pool size, (2) the treatment with cyclosporin A, which enhances the endocytosis of synaptic vesicles by inhibiting calcineurin activities (Kuromi et al., 1997
Here we report that the optically determined size of exo/endo cycling vesicle pool is closely correlated with the quantal content of synaptic potentials at the neuromuscular junction of Drosophila larvae.
A preliminary report of these results has appeared elsewhere (Kuromi and Kidokoro, 1998b
MATERIALS AND METHODS Fly stocks. A wild-type strain of Drosophila melanogaster, Canton S, and a single-gene mutant of Drosophila, shits1, were used in this study. The stock flies were bred at 24°C on the standard cornmeal medium supplemented with yeast under a 12 hr light/dark cycle.
Preparations. Larval body wall neuromuscular preparations were made from third instar larvae. For dissection, a larva was placed prone on a thin layer of sylgard resin (Dow Corning, Tokyo, Japan) in a plastic dish with the HL3 medium (Stewart et al., 1994
Solutions. Preparations with intact or severed axons were incubated for 1-3 hr in HL3 medium, which was suitable for maintaining preparations for a long period (Stewart et al., 1994
FM1-43 loading and unloading. For study of FM1-43 uptake into vesicles at nerve terminals, we replaced the Drosophila medium with high K+ saline containing 10 µM FM1-43. After incubation in high K+ saline containing FM1-43 and Ca2+ for 5 min, the preparations were washed three times with Ca2+-free saline and then incubated in Ca2+-free saline for additional 15 min to remove surface-bound FM1-43. The fluorescence intensity measured in this condition represents the total uptake of FM1-43 and is indicated by the height of each column in Figures 2-4.
To release synaptic vesicles, the synapses loaded with FM1-43 were stimulated at 22°C (except described otherwise) with high K+ saline in the presence of external Ca2+ and in the absence of FM1-43 for 5 min and then viewed under a fluorescence microscope in Ca2+-free saline. The fluorescence intensity measured in this condition represents the unreleasable portion of fluorescence and is indicated by the dotted or filled portion of each column in Figures 2-4. Thus, the blank portion of each column represents the amounts of vesicles that are incorporated into boutons by endocytosis and released by exocytosis during high K+ stimulation. We referred to these vesicles in wild-type larvae as the exo/endo cycling vesicle pool. In the previous study using shi, we defined the fraction of vesicles that were endocytosed and exocytosed during high K+ stimulation at permissive temperature (22°C) as the exo/endo cycling vesicle pool (Kuromi and Kidokoro, 1998a
Fluorescence microscopy and image processing. The preparations were viewed with an upright microscope (BX50WI; Olympus, Tokyo, Japan) equipped with DIC and epifluorescence optics. We used a 40×, 0.80 NA water immersion objective lens (Lumplane FI; Olympus) or a 63×, 0.90 NA Zeiss Achroplan water immersion objective for observation. The preparations were excited by light with 488 nm wavelength (Polychrome II; TILL Photonics GmbH, Planegy, Germany), and light emitted at >530 nm was collected.
Images were captured with a CCD camera (usually 0.27 sec/frame, C4880-81S; Hamamatsu Photonics, Hamamatsu, Japan). Images were acquired, stored, and processed with a digital Celebris computer (GLST 5133; Nippon Digital Equipment, Tokyo, Japan) by an intracellular Ca2+ analysis system (Argus-HiSCA; Hamamatsu Photonics) and printed with an Epson printer (MJ-930C). Twelve bit images were acquired with the CCD camera, and pixel values were compressed identically to 8 bits. The average light intensity emitted from five to six brightly stained boutons with diameters longer than 3 µm, which were selected from one field (86 times 64 µm), was measured before and after the high K+ stimulation. The background intensities were subtracted from the intensities in the bouton.
Electrophysiology. Membrane potentials were recorded from muscle 6 in the abdominal segments A-2 and A-3 using glass microelectrodes filled with 4 M potassium acetate. Electrode resistances were usually between 20 and 40 M
. To evoke excitatory junctional potentials (EJPs), pulses (1 msec in duration and 2× threshold voltage in intensity) were delivered to an appropriate segmental nerve via a glass suction electrode (20-50 µm inside diameter) at a frequency of 0.1 Hz. Spontaneous miniature excitatory junctional potentials (MEJPs) were recorded for 1 min, and the mean amplitude was calculated.
Chemicals. Chemicals used in these experiments and their sources are as follows: FM1-43 (Molecular Probes, Eugene, OR), cytochalasin B and cytochalasin D (Sigma, St. Louis, MO), cyclosporin A, forskolin, and dibutyryl cAMP sodium salt (db-cAMP) (Wako Chemicals, Tokyo, Japan).
Statistical analysis. Statistical analysis was performed by using Student's t test.
RESULTS Effects of axon severance on FM1-43 loading and unloading in the nerve terminals
In an attempt to affect the state of presynaptic boutons of nerve terminals, we severed the motoneuronal axons and examined the FM1-43 fluorescence uptake.
Acute effects of axon severance
We loaded and unloaded nerve terminals with FM1-43 in freshly isolated preparations with the intact CNS or after sectioning nerves near their entry to the ganglion. The staining patterns of boutons with FM1-43 were the same in both preparations, with staining predominantly in the periphery of boutons (Fig. 1A, top panel). Between these fresh preparations, there were no differences in FM1-43 fluorescence intensities before and after high K+ stimulation (Fig. 2A,B; marked with "0 h" at the bottom, the height of each column represents the total fluorescence intensity after loading, and that of the dotted portion indicates the fluorescence intensity after unloading. Thus, the blank portion of each column depicts the fraction of vesicles that are endocytosed and exocytosed in the normal condition and is called the exo/endo cycling vesicle pool). No effect of axon severance was found in the vesicle pool. However, it is possible that the effect of axon severance could become apparent after a certain period of time.
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Figure 1. FM1-43 staining and destaining of Drosophila nerve terminals in variously treated preparations. Preparations that were freshly isolated (A), pretreated for 7 min with 20 µM forskolin (B), with severed axons incubated for 2 hr (C), and pretreated for 20 min with 10 µM cyclosporin A (D), were incubated for 5 min in high K+ saline containing 10 µM FM1-43, then washed with Ca2+-free saline for 15 min and viewed (top picture in each preparation). The stained preparations were then stimulated at 22°C for 5 min by high K+ saline (bottom picture in each preparation). For details, see Results. Scale bar, 10 µm.
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Figure 2. FM1-43 fluorescence intensities in boutons of preparations that were incubated from various lengths of time in different media before (A, B) or after (C, D) loading with FM1-43. Preparations with intact axons (A, C) and with severed axons (B, D) were incubated in HL3 medium (not labeled) or in Ca2+-free saline (labeled with " Ca2+"). Some preparations were pretreated for 20 min with cytochalasin B (10 µM, CB) or cytochalasin D (10 µM, CD), which were also present in the medium during incubation. The ordinates show FM1-43 fluorescence intensities (pixel values) in boutons before (the heights of columns) and after (top ends of dotted portion of columns) high K+ stimulation. For measurements of the intensities of fluorescence of boutons, 12-bit images were acquired with the CCD camera, and pixel values were compressed to 8 bits. Average intensities were computed for each bouton. Five to six boutons were examined in each preparation. Mean fluorescence values of boutons were determined in each preparation and summarized (mean and SEM). The numbers in columns are the number of preparations examined. Vertical bar of each column is SEM. The time below columns shows the incubation time.
Effects of axonal severance after 1-3 hr of incubation
When preparations with intact axons were incubated for 2 hr in HL3 medium and then loaded with FM1-43 in high K+ saline, the FM1-43 staining pattern of boutons and the fluorescence intensity did not differ from those in freshly isolated preparations (Fig. 2A, column marked with "2 h" at the bottom). The similar results were obtained when preparations were incubated in Ca2+-free saline to prevent spontaneous activities evoked by action potentials generated in the CNS (Fig. 2A, column marked with "
When preparations with severed axons were loaded with FM1-43 at 1, 2, and 3 hr after incubation in HL3 medium, the boutons showed staining patterns and the total fluorescence intensities similar to those in freshly isolated preparations (Fig. 1C, top panel; Fig. 2B, columns marked with "1 h", "2 h", and "3 h"). Similar results were obtained when preparations were incubated in Ca2+-free saline (Fig. 2B, column marked with "
These results suggest either that a part of previously releasable fraction became unreleasable or that a new unreleasable fraction appeared during the incubation period. To distinguish these alternatives, the following experiment was performed in which synaptic vesicles were first loaded with FM1-43 and then incubated for 2 hr.
FM1-43 was loaded in freshly isolated preparations with intact or severed axons, and then the preparations were incubated for 2 hr in Ca2+-free saline. The fluorescence intensity was measured before and after high K+ stimulation to assess a releasable portion of the fluorescence intensity. The total FM1-43 fluorescence intensities in boutons were slightly decreased after 2 hr incubation compared with those observed in freshly isolated preparations, but the difference was not significant (Fig. 2C,D, compare the height of columns marked with "0 h" and "2 h"). In preparations with intact axons, ~85% of FM1-43 fluorescence in boutons was destained by high K+ stimulation (Fig. 2C, the blank portion of column marked with "
Effects of cytochalasin B and D on the FM1-43 fluorescence fractions
It is plausible that synaptic vesicles became unreleasable as the result of binding to actin filaments (Hirokawa et al., 1989
To further confirm the effect of actin polymerization disrupting agents, preparations with severed axons were first loaded with FM1-43 and then incubated for 2 hr in Ca2+-free saline containing cytochalasin D (10 µM). The same effects of the drug were observed as described above (Fig. 2D, the blank portion of column marked with "
High K+ stimulation at 22 and 34°C in shits1 mutants
Previously we have shown in shits1 mutants that vesicles in the reserve pool are not releasable with high K+ stimulation at room temperature but can be released at nonpermissive temperature (Kuromi and Kidokoro, 1998a
As was observed in wild-type larvae, 60% of FM1-43 fluorescence remained after high K+ stimulation at 22°C in shits1 terminals of axons severed and incubated in HL3 medium for 2 hr (Fig. 3A, preparations incubated and then loaded with FM1-43; Fig. 3B, preparations loaded with FM 1-43 and then incubated, the dotted area of column marked with "22°C" in shibire). On the other hand, after high K+ stimulation at 34°C, only ~10% of FM1-43 fluorescence remained in shits1 (Fig. 3A,B, the filled area of columns marked with "34°C" in shibire). These results suggest that the FM1-43 fluorescence that remained after high K+ stimulation at 22°C in preparations with severed axons and incubated for 2 hr was not caused by dye taken up nonspecifically in the boutons, but rather represented dye in the releasable vesicles.
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Figure 3. FM1-43 fluorescence intensities in boutons of wild-type and shibire (shits1) preparations with axons severed and incubated in the medium for 2 hr. The ordinates show FM1-43 fluorescence intensities (pixel values) in boutons before (heights of columns) and after (top ends of dotted and filled portions) high K+ stimulation at 22°C (dotted) or 34°C (filled). Wild-type and shits1 preparations with severed axons were incubated in HL-3 medium for 2 hr and then loaded with FM1-43 (A), or first loaded with FM1-43 and then incubated in Ca2+-free saline for 2 hr (B). For details, see the legend to Figure 2.
These results were not simply the effect of temperature, because 60% of FM1-43 fluorescence of wild-type larvae with axons severed and incubated in HL3 medium for 2 hr remained after high K+ stimulation both at 22°C and at 34°C (Fig. 3A,B, dotted and filled areas of columns marked with "22°C" and "34°C" in wild-type).
Effects of cyclosporin A on FM1-43 loading and unloading in the nerve terminals
Previously we have shown that pretreatment with cyclosporin A increases the number of nerve terminals visibly stained with FM1-43 by high K+ stimulation in Drosophila larvae and suggested that cyclosporin A enhances endocytosis of synaptic vesicles in the nerve terminals (Kuromi et al., 1997
Preparations with intact axons were incubated for 20 min in Drosophila medium containing 10 µM cyclosporin A and then loaded with FM1-43 by high K+ stimulation. In these cyclosporin A-pretreated preparations, entire boutons were stained, as shown in Figure 1D (top panel). The FM1-43 fluorescence intensity in boutons of cyclosporin A-pretreated preparations was significantly higher than that in nontreated ones (Fig. 4, compare the height of column marked with "10 µM, cyclosporin A" with that of column marked with "control"). In cyclosporin A-pretreated preparations some amounts of FM1-43 fluorescence remained in the central part of boutons after high K+ stimulation (Fig. 1D, bottom panel; Fig. 4, the dotted portion of column marked with "10 µM cyclosporin A").
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Figure 4. Effects of drugs on FM1-43 fluorescence fractions in boutons of preparations with intact axons. The heights of columns are the fluorescence intensities of boutons before high K+ stimulation, and dotted portions of columns represent fluorescence intensities remaining after high K+ stimulation. Preparations were treated with the drug shown: cyclosporin A (20 min), forskolin (7 min), or db-cAMP (15 min). Some preparations were first loaded with FM1-43 by high K+ stimulation, and these stained preparations were then treated with cyclosporin A (10 µM) for 20 min (loaded with FM1-43 and then treated with cyclosporin A). *p ± 0.05; **p ± 0.01, compared with corresponding fluorescence intensities in control preparations (Drosophila medium).
When the preparations with intact axons were first loaded with FM1-43 and then treated with cyclosporin A, no change was observed in the total intensity of FM1-43 fluorescence and in its sensitivity to high K+ stimulation (Fig. 4, the column marked with "loaded with FM1-43 and then treated with cyclosporin A"), indicating that cyclosporin A had no effect on the vesicles already incorporated in the nerve terminals.
Cyclosporin A enhances endocytosis of synaptic vesicles at nerve terminals. The endocytosed vesicles are incorporated into two distinct vesicle fractions; one is high K+-releasable and located in periphery of boutons, and the other is high K+-unreleasable and located in the center of boutons.
Effects of forskolin and db-cAMP on FM1-43 loading and unloading in the nerve terminals
An elevation of cAMP in the presynaptic terminal has been shown to enhance synaptic transmission (Dixon and Atwood, 1989
Pretreatment with db-cAMP (150 µM for 15 min) had essentially the same effects on FM1-43 loading and unloading as pretreatment with forskolin (Fig. 4, the column labeled with "150 µM db-cAMP")
Relationships between the FM1-43 loading time and the size of exo/endo cycling vesicle pool in variously treated preparations
As shown above, in forskolin-treated preparations, the size of exo/endo cycling vesicle pool increased, whereas it decreased in preparations with axons severed and incubated for 2 hr. Because we loaded boutons with FM1-43 for 5 min in experiments so far described, the possibility remains that the 5 min loading time might not be long enough to estimate the size of the vesicle pool.
To test this possibility, we varied the loading time of FM1-43 and measured the amounts of FM1-43 fluorescence released by high K+ stimulation. The amount of the fluorescence increased as the loading time was prolonged, and the steady state was achieved within 1 min in nontreated preparations (Fig. 5, open circles). As shown in Figure 5, although the loading rate of the exo/endo cycling vesicle pool was similar, the magnitude of the steady-state levels was significantly enhanced in forskolin-pretreated preparations (Fig. 5, filled circles) and significantly reduced in preparations with axons severed and incubated for 2 hr (Fig. 5, open triangles), compared with nontreated preparations. There were no significant differences in the loading rate of the exo/endo cycling vesicle pool and the magnitude of steady-state levels between cyclosporin A-pretreated and nontreated preparations (Fig. 5, filled triangles, open circles)
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Figure 5. Time courses of FM1-43 incorporation into exo/endo cycling vesicle pool in boutons of variously treated preparations. Preparations were pre-exposed to 10 µM FM1-43 in the normal medium for 5 min and then exposed for various times to high K+ saline in the presence of 10 µM FM1-43 (loading time). These preparations were washed three times with Ca2+-free saline and incubated in Ca2+-free saline for additional 15 min to remove surface-bound dye. To measure the size of exo/endo cycling vesicle pool, the preparation was then stimulated with high K+ saline for 5 min in the absence of FM1-43. The ordinate shows difference in the fluorescence of boutons before and after high K+ stimulation. The abscissa shows time of exposure to high K+ saline containing 10 µM FM1-43. Forskolin-treated (20 µM, filled circles), cyclosporin A-treated (10 µM, filled triangles), or nontreated (open circles) preparations and preparations with axons severed and incubated for 2 hr in HL3 medium (open triangles). Numbers in parentheses are the number of preparations examined. Asterisks indicate significant difference from corresponding values of nontreated preparations at p < 0.001.
Synaptic transmission at the neuromuscular junction in variously treated preparations
Despite variations in the EJP amplitude in variously treated preparations, no change was detected in the MEJP amplitude (Table 1). The amplitude of the EJP was significantly smaller in preparations with axons severed and incubated for 2 hr (Fig. 6D, Table 1), but was not different in preparations with axons severed and incubated in the presence of cytochalasin D (Fig. 6E, Table 1), compared with that in freshly isolated nontreated preparations with intact axons. The ratio of mean EJP amplitude to mean MEJP amplitude was significantly enhanced in forskolin-pretreated (20 µM) preparations and significantly reduced in preparations with axons severed and incubated for 2 hr, compared with that in freshly isolated nontreated preparations with intact axons (Table 1). There were no significant differences in the parameters examined in the present study and also in the frequency of MEJPs (data not shown) between cyclosporin A-pretreated (10 µM) and nontreated preparations (Fig. 6B, Table 1), in accord with the result reported in our previous paper (Kuromi et al., 1997
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Table 1. Membrane potentials and quantal contents of synaptic potentials in variously treated preparations
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Figure 6. EJPs and MEJPs in variously treated preparations (A-E) and Ca2+ dependence of evoked release (F). A-E, Representative traces of synaptic potential in freshly isolated preparation (A), cyclosporin A-treated (10 µM for 20 min) preparation (B), forskolin-treated (20 µM for 7 min) preparation (C), preparation with axons severed and incubated for 2 hr in HL3 medium (D), and preparation with axons severed and incubated for 2 hr in the presence of cytochalasin D (E, 10 µM). The topmost record in each panel shows evoked junctional potentials, and the bottom four traces show spontaneous events recorded in the same muscle fibers. MEJP records were selected to show representative amplitudes. Calibration: 8 mV, 0.1 sec (for EJPs); 1 mV, 0.2 sec (for MEJP). F, Quantal content is plotted as a function of extracellular Ca2+ concentration for forskolin-treated (20 µM, filled circles), nontreated (open circles), and cyclosporin A-treated (10 µM, filled triangles) preparations. The Ca2+ concentration of the recording solution was adjusted to 0.1, 0.2, 0.4, and 0.8 mM with fixed 5 mM MgCl2.
Quantal contents in forskolin- and cyclosporin A-pretreated preparations
In saline containing 2 mM Ca2+, the quantal content was larger in the forskolin-treated preparations and unchanged in the cyclosporin A-treated preparations. To assess the Ca2+ dependence of these effects, we measured EJPs and MEJPs at different extracellular Ca2+ concentrations. The ratio of mean EJP amplitude to mean MEJP amplitude [corrected for nonlinear summation, (Martin, 1955
DISCUSSION To study the functional role of the exo/endo cycling vesicle pool in synaptic transmission, the size of vesicle pool in single boutons was optically determined using fluorescent dye FM1-43, and the quantal content was estimated by the ratio of the EJP amplitude to that of MEJP at the neuromuscular junction of Drosophila larvae. Among the FM1-43 fractions taken up in boutons in high K+ saline, the fraction that was released by high K+ stimulation in the absence of the dye is considered the exo/endo cycling vesicle pool because these vesicles are involved in both endocytosis and exocytosis processes. In the following three types of preparations, the size of the exo/endo cycling vesicle pool and the quantal content of synaptic transmission were found to change in parallel. (1) In preparations with axons severed and incubated for 2-3 hr, the total amount of FM1-43 fluorescence taken up by these boutons was in the normal range, but the fraction released by high K+ stimulation was sharply decreased, compared with that in freshly isolated control preparations. In these preparations, the quantal content was significantly smaller than controls. Furthermore, the treatment of these preparations with cytochalasin B or D during the incubation period prevented the decrease in the FM1-43 fraction released by high K+ stimulation and, at the same time, blocked the reduction in the quantal content. (2) The cyclosporin A treatment increased the total amount of FM1-43 fluorescence taken up by boutons, but the fraction releasable by high K+ stimulation did not change. The quantal content in these preparations was not different from controls. (3) Forskolin increased the total amount of FM1-43 fluorescence taken up by boutons, and the exo/endo cycling vesicle pool was enlarged. The quantal content in these preparations was also increased. Thus, the present study showed that changes in the exo/endo cycling vesicle pool paralleled those in the quantal content and that there is not necessarily a tight relationship between the total FM1-43 fluorescence taken up by boutons and the quantal content. These results suggest that synaptic vesicles in the exo/endo cycling vesicle pool participate primarily in impulse-evoked release of synaptic vesicles from nerve terminals.
In preparations with axons severed and incubated and also in those pretreated with cyclosporin A, there was a difference between FM1-43 loading and unloading efficiency. If exocytosis of vesicles caused the same extent of endocytosis of vesicles, we cannot account for this discrepancy. It appears that although endocytosis is closely coupled to exocytosis, two processes are differentially controlled. For example, exocytosis of vesicles occurs at the active sites of nerve terminals, whereas endocytosis is also observed in other sites (Miller and Heuser, 1984
It has been hypothesized that among synaptic vesicles in nerve terminals, only few vesicles at active sites of synapse participate in the immediate release of transmitter (Von Gersdorff et al., 1996
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Figure 7. Relationship between the size of exo/endo cycling vesicle pool and the quantal content. The size of exo/endo cycling vesicle pool (abscissa) and the quantal content (ordinate) were determined in various preparations in the medium containing 2 mM Ca2+ as described in Results. Vertical and horizontal bars of each circle are SEM. FSK, Forskolin-treated preparations; Cyc.A., cyclosporin A-treated preparations; Fresh, freshly isolated nontreated preparations; Severed, Cyt.D. 2 h, preparations with axons severed and incubated for 2 hr in HL3 medium containing cytochalasin D; Intact, 2 h, preparations with intact axons incubated for 2 hr in HL3 medium; Severed, 2 h, preparations with axons severed and incubated for 2 hr in HL3 medium.
When axons were severed and incubated for 2-3 hr, FM1-43 staining patterns of boutons were essentially the same as those observed in the freshly isolated preparation with severed axons. Nevertheless, some of the fluorescence was not released by high K+ stimulation. Furthermore, when nerve terminals with axons severed were first loaded with FM1-43 and then incubated, the fluorescence was only partially released by high K+ stimulation. These results suggest that some changes occurred in the exo/endo cycling vesicle pool during incubation in nerve terminals disconnected from the cell bodies. When cytochalasin B or D was present during the incubation period, however, the appearance of an FM1-43 fraction unreleased by high K+ stimulation was prevented. Cytochalasin B and D are known to disrupt actin polymerization (Cooper, 1987
Previously using the shits1 mutant, we have demonstrated that the exo/endo cycling pool of synaptic vesicles is localized in the periphery of boutons and is released after high K+ stimulation at permissive temperature (Kuromi and Kidokoro, 1998a
Both forskolin, a specific activator of adenylyl cyclase (Seamon and Daly, 1983
In conclusion, previous (Kuromi and Kidokoro, 1998a
FOOTNOTES Received Oct. 5, 1998; revised Dec. 7, 1998; accepted Dec. 9, 1998.
This work was supported by grant-in-aids from the Ministry of Education, Science, Sports, and Culture of Japan to H.K. and Y.K. We thank Dr. A. D. Grinnell for his critical reading of this manuscript, important suggestions, and improvement of English.
Correspondence should be addressed to Dr. Hiroshi Kuromi, Institute for Behavioral Sciences, Gunma University School of Medicine, 3-39-22 Showa-machi, Maebashi 371-8511, Japan.
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