Contribution of Active Zone Subpopulation of Vesicles to Evoked and Spontaneous Release

J. H. Koenig and Kazuo Ikeda

Division of Neurosciences, Beckman Research Institute of the City of Hope, Duarte, California 91010


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Koenig, J. H. and Kazuo Ikeda. Contribution of active zone subpopulation of vesicles to evoked and spontaneous release. Our previous work on Drosophila synapses has suggested that two vesicle populations possessing different recycling pathways, a fast pathway emanating from the active zone and a slower pathway emanating from sites away from the active zone, exist in the terminal. The difference in recycling time between these two pathways has allowed us to create a synapse that possesses the small, active zone subpopulation without the larger, nonactive zone population. Synapses were depleted using the temperature-sensitive endocytosis mutant, shibire, which reversibly blocks vesicle recycling at the restrictive temperature. In the depleted state, both the excitatory junction potential (EJP) and spontaneous release are abolished. After shibire-induced depletion, the active zone population begins to reform within 30 s at the permissive temperature, whereas the nonactive zone population does not begin to reform until ~10-15 min later. Evoked release recovered at approximately the same time as the active zone population. During the time when the active zone population existed in the terminal without the nonactive zone population, enough transmitter release was available to sustain a normal evoked response for many minutes at frequencies above those produced during normal activity (flight) by this motor neuron. When only the active zone population existed in the terminal, the frequency of spontaneous release was greatly attenuated and possessed abnormal release characteristics. Spontaneous release recovered its predepletion frequency and release characteristics only after the nonactive zone population was reformed.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Two distinct recycling pathways have been proposed to exist in a synaptic terminal: 1) the "classical" recycling pathway, which emanates from presynaptic membrane away from the active zone and involves intermediate structures such as coated vesicles and endosomes (Heuser and Reese 1973; for review see Heuser 1989), and 2) the active zone pathway, which emanates from the presynaptic membrane adjacent to the dense body at the active zone and operates by direct pinch-off of the presynaptic membrane into vesicles without the involvement of endosomal intermediates (Ceccarelli et al. 1973; for review see Fesce et al. 1994). Our data have suggested that both of these pathways exist in a terminal and that they may replenish two distinct vesicle pools (Koenig and Ikeda l996). In Drosophila, the active zone pathway appears to replenish a small subpopulation of vesicles that remains within 150-200 nm of the dense body, attached to it by thin filaments, whereas the classical, or nonactive zone, pathway replenishes a much larger vesicle population that is dispersed throughout the terminal cytoplasm. The active zone pathway, operating by direct pinch-off, begins to reform new vesicles within ~30 s and appears to replenish its entire population within ~1 min, whereas the nonactive zone pathway, which involves various intermediate steps, begins to reform vesicles after ~10 min and completely replenishes its population within ~25 min. This difference in time courses allowed us to investigate the relative contributions of the two vesicle populations to transmitter release by depleting the terminal of vesicles and then correlating the reformation of the two populations with the recovery of the excitatory junction potential (EJP) and spontaneous release.

To deplete the terminal, the temperature-sensitive endocytosis mutant, shibire (shi), which blocks recycling at the restrictive temperature, was used. After 10 min at 29°C, the terminal becomes completely depleted, resulting in the loss of the EJP and spontaneous release (Koenig et al. l989). When the temperature is lowered, vesicle recycling again proceeds normally, resulting in the reformation of the vesicle populations as well as the return of evoked and spontaneous release. The reformation of the active zone and nonactive zone vesicle populations after inducing complete vesicle depletion was correlated with the recovery of the EJP and spontaneous release. It was observed that the recovery of the full evoked response correlates well with the reformation of the active zone vesicle population, which is at a time when vesicles of the nonactive zone population have not yet begun to reform. A normal response at and above physiological frequencies could be sustained at this time. On the other hand, spontaneous release remained greatly attenuated in frequency and exhibited abnormal release characteristics even after the evoked response had fully recovered. The recovery of the predepletion frequency and release characteristics of spontaneous release took ~10-15 min, which was similar to the time course of the recovery of the nonactive zone population of vesicles.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

For these experiments, 4-day-old female Drosophila melanogaster of the wild-type strain, Oregon-R, and the temperature-sensitive mutant, shibire ts1, were used. The shi gene encodes the protein, dynamin (Chen et al. 1991; van der Bliek and Meyerowitz 1991), which is involved in the process whereby invaginations of the plasma membrane pinch off to form vesicles or cisternae (Damke et al. 1994; Hinshaw and Schmid 1995; Kosaka and Ikeda 1983a,b; Takei et al. 1995). In shi flies the dynamin molecule functions normally at 19°C (permissive temperature), but becomes dysfunctional above 27°C (restrictive temperature). In the shi nerve terminal, as exocytosis proceeds normally at the restrictive temperature while vesicle recycling is blocked, vesicle depletion occurs. When the depleted terminal is exposed to the permissive temperature, vesicle recycling begins and the vesicle populations reform.

The terminals of the dorsal longitudinal flight muscle (DLM) were used because of the ease of recording intracellularly from the DLM fibers. Similar results were observed in the coxal muscle (unpublished). The DLM is composed of six fibers that attach anteriorly to the dorsal thoracic cuticle and posteriorly to the posterior phragma. Each fiber is multiterminally innervated by a single excitatory motor neuron that sends its axon through the posterior dorsal mesothoracic nerve (PDMN) (Ikeda et al. 1980).

For details on both intracellular recording and electron microscopic techniques, see Koenig et al. (1983) and Ikeda and Koenig (1988). Briefly, the fly was dissected in saline containing (in mM) 128 NaCl, 4.7 KCl, 1.8 CaCl2, and 5 Tris aminomethane HCl (pH 7.4) to expose the lateral surface of the DLM fibers and the thoracic ganglion. A glass micropipette was inserted into one of the muscle fibers for intracellular recording. To elicit the evoked response the PDMN was cut where it leaves the thoracic ganglion and sucked into a suction electrode for stimulation. Both the evoked response and spontaneous release were monitored in each recording. The temperature was raised at a rate of 1°C/15 s with a Peltier heating-cooling device. It should be noted that in some of our previous experiments on the DLM the temperature was raised and lowered instantly by replacing the 19°C saline with 29°C saline and vice versa (Ikeda and Koenig 1988; Koenig et al. 1989) or was raised and lowered more slowly than was done here (Koenig et al. 1983). Thus in these aforementioned papers the number of minutes at 29°C necessary to cause complete depletion or the number of minutes necessary for complete recovery is different from that in this paper. Also, in the paper of Koenig et al. (1983), the recovery of spontaneous release was observed at 25°C, a temperature that enhances spontaneous release.

The shi flies were exposed to 29°C for various periods of time depending on the experiment, before lowering the temperature. Both the evoked response and spontaneous release were monitored during each recording. Only those muscle fibers that maintained a resting potential of -90 mV or more throughout the experiment were used for these results. After the experiment the saline was instantly replaced by fixative (2% paraformaldehyde, 2% gluteraldehyde in 0.1 M phosphate buffer, pH 7.4, for 30 min, followed by 4% gluteraldehyde in 0.1 M phosphate buffer, pH 7.4, for 2 h). The recording electrode remained in place at this time. With the nerve cut no response was observed as a result of application of fixative. Because of the very small size of the terminals ( ~1 µm diam) and the small size of the muscle fibers (~50 µm diam), fixation of the terminals occurs within milliseconds. It is unlikely that the muscle fixes before the terminals because spontaneous release ceases completely before the muscle begins to depolarize (Koenig, unpublished observations).

For measurement of the positioning of vesicles relative to the dense body at the active zone, electron micrographs of many synapses were measured by hand. Only those synapses that had clearly cross-sectioned dense bodies were used. A synaptic vesicle was defined as a circular structure of 45 to 55 nm diam. Other structures in the terminal cytoplasm, such as microtubules or recycling intermediates, were easily distinguishable from vesicles by differences in their sizes and shapes.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

As mentioned in the INTRODUCTION, evidence has been presented suggesting that two distinct recycling pathways exist in the terminal, which appear to replenish two distinct vesicle populations. The fast pathway, which emanates from active zone membrane adjacent to the dense body, appears to replenish a small subpopulation of vesicles that remains close to the active zone, attached to the dense body by filaments. A typical active zone (specialized release site) of Drosophila neuromuscular junctions is composed of a dense body attached by short, electron-dense strands to a small area of presynaptic membrane that is closely associated with specialized postsynaptic membrane. The dense body is composed of an electron-dense base over which lies a meshwork of filaments. The base is shown diagramatically in Fig. 1A, cross sectioned at three different locations. Depending on the plane of sectioning, this structure can have a variety of shapes, including round, oblong, and double lobed, as demonstrated in the figure. The meshwork of filaments that caps the base, called the dense body plate, appears to originate from two sites on the plasma membrane to either side of the base. The filaments pass over the base and radiate into the cytoplasm above the dense body. Attached to these filaments is a small subpopulation of vesicles. In cross section through the dense body this meshwork of filaments appears as an electron dense bar-like structure running parallel to the plasma membrane above the dense body base. A cross section of the dense body base capped by filaments is expressed diagramatically in Fig. 1B. An electron micrograph of the dense body base sectioned horizontally is shown in Fig. 1C (compare with Fig. 1A). An electron micrograph of an active zone in cross section is shown in Fig. 1D (compare with Fig. 1B). The filamentous cap can be seen to be attached to the plasma membrane on either side of the base with this particular plane of sectioning. Note the specialized postsynaptic membrane that is thickened and more electron dense than adjacent membrane and possesses a brush-like substance extending from it into the synaptic cleft. This is the only area where the pre- and postsynaptic membranes become closely aligned. In this paper, the presynaptic membrane that is closely aligned with this specialized postsynaptic membrane will be referred to as active zone membrane, i.e., the membrane from which release occurs. A tangential section of the active zone is shown in Fig. 1E. With this plane of sectioning, it can be seen that the filaments of the dense body plate radiate out into the cytoplasm and attach to a small subpopulation of vesicles located close to the dense body. (For additional examples of the dense body structure see Koenig et al. 1998; Koenig et al. l993).



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Fig. 1. Examples of the dense body at the active zone of Drosophila neuromuscular junctions. A: diagrammatic representation of dense body base sectioned at 3 different cross-sectional planes. It is presented as viewed from above, lying on the plasma membrane. B: diagrammatic representation of cross section of dense body base (large arrow) attached to presynaptic membrane by thin fibrils. Synaptic vesicles are attached to the filamentous plate (small arrows) overlying the base. Note specialized postsynaptic membrane (arrowheads). C: dense body base sectioned longitudinally. Compare with A. D: cross-sectioned dense body. Compare with B. E: dense body sectioned tangentially. Large arrow points to base. Note filamentous plate (small arrows) and population of vesicles tethered to it. Scale bar in A, 100 nm (×135,000) for B also; in C, 100 nm (×90,000).

During recovery from complete depletion induced by the shi mutant, the active zone subpopulation reforms much faster than the nonactive zone population. This is demonstrated in Fig. 2. Figure 2A shows a typical shi terminal (neuromuscular junction) at 19°C, which is indistinguishable from a wild-type terminal. As can be seen, the terminal possesses many vesicles dispersed throughout the cytoplasm. In Fig. 2B, a shi terminal that was exposed to 29°C while stimulating to cause complete vesicle depletion, followed by 2 min at the permissive temperature, 19°C, is shown. In this terminal, a small cluster of vesicles has formed at the active zone, and the rest of the terminal remains depleted and possesses invaginations ("collared pits") and cisternae, which have been shown to represent intermediate steps in the nonactive zone recycling pathway (Koenig and Ikeda l989) (For additional examples of the fast recovery of the active zone subpopulation see Koenig and Ikeda l996). Thus it is possible to create a terminal in which the active zone population has reformed and the nonactive zone population has not. This allows investigation of the release characteristics of the active zone population without the contribution of the nonactive zone population.



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Fig. 2. Typical shibire (shi) terminals (coxal neuromuscular junction) at 19°C before heating (A) and 19°C for 2 min after exposure to 29°C for 10 min while stimulating at 0.5 Hz (B). Many vesicles are dispersed in the cytoplasm in A, but only a few vesicles are clustered around the dense body at the active zone in B. (Active zones denoted by large arrows.) Also note collared pits (arrowheads) along presynaptic membrane and cisternae in cytoplasm (small arrows), which are intermediate steps in nonactive zone recycling pathway. Scale bar in A and B, 0.5 µm (×47,500).

The current work was performed on fibers 5 and 6 of the DLM, which are innervated by a single motor neuron located at the contralateral midline of the thoracic ganglion (Ikeda and Koenig l988). The motor neuron makes many hundreds of en passant-type synapses on these fibers. A longitudinal section of a typical DLM axon of a shi fly at the permissive temperature is shown in Fig. 3A. At this temperature the synapses are indistinguishable from wild-type DLM synapses, possessing synaptic vesicles, mitochondria, and a presynaptic dense body, which identifies the release site or active zone. It has been demonstrated that if shi DLM synapses are exposed to 29°C and stimulated the population of synaptic vesicles gradually diminishes until complete vesicle depletion is observed in most of the terminals (Koenig et al. l989). An example of a depleted shi DLM terminal is shown in Fig. 3B. As can be seen in this figure, immediately after complete depletion occurred (1 min at 29°C), no membrane compartment appears in the form of swelling or invagination of the plasma membrane, coated vesicles, or cisternae, which would suggest that the lost vesicle membrane compartment had been inserted into the plasma membrane. This apparent loss of the vesicle membrane compartment without a reciprocal increase in the plasma membrane compartment is typical of all shi synapses we have observed (Koenig and Ikeda 1989, 1996) and has been observed in other terminals as well (Fox and Kriebel 1994). With longer exposure to 29°C, invaginations of the plasma membrane develop within the terminal. These invaginations were termed collared pits because of the electron-dense substance (dynamin) that encircles their neck portions. Even at 29°C, these pits begin to elongate, which has been shown to represent the accumulation of recycling membrane that is prevented from pinching off at the nonpermissive temperature in shi. At a slightly lowered temperature (28 or 27°C), these elongations become even more pronounced. The accumulations of membrane from both the active zone and the nonactive zone recycling pathways that develop at the restrictive temperature in a shi DLM synapse are demonstrated in Fig. 3C. In Fig. 3D, another example of active zone recycling is shown at higher magnification. A large cisterna-like invagination emanating from the active zone membrane can be seen to the right of the dense body. Also, a smaller cisterna-like structure is located adjacent to the dense body under the filamentous cap on the right side. The connection of this cisterna with the extracellular space is out of the plane of sectioning. On the left side of the dense body base, another large cisterna is seen, but its connection with the extracellular space is also out of the plane of sectioning. Thus the vesicle depletion that occurs at 29°C in shi terminals is due to a blockage of vesicle recycling resulting from the blockage of pinch-off of recycling membrane from the plasma membrane (Kosaka and Ikeda l983a,b). With time, this blockage causes recycling membrane to accumulate, forming cisterna-like invaginations. No vesicle depletion or appearance of cisterna-like invaginations occurs in wild-type synapses at 29°C.



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Fig. 3. Terminals on the dorsal longitudinal flight muscle (DLM) . A: typical shi terminal at 19°C. (Arrow denotes active zone in A-D). Note many synaptic vesicles dispersed in cytoplasm. B: typical shi terminal after 1 min at 29°C. The temperature was raised at a rate of 1°/15 s while stimulating at 0.5 Hz. Note vesicle depletion. C: typical shi terminal at the restrictive temperature demonstrating accumulation of 2 types of recycling membrane: active zone (small arrows) and nonactive zone (arrowheads). Active zone recycling is defined as emanating from the presynaptic membrane that is aligned with the specialized postsynaptic membrane, as shown in Fig. 1. Nonactive zone recycling is defined as that which emanates from presynaptic membrane not aligned with specialized postsynaptic membrane. D: example of accumulations of active zone recycling membrane (small arrows). Cisterna on right opens to extracellular space at arrowhead. m, mitochondria. Scale bar in A-C, 0.5 µm (×40,500); in D, 0.5 µm (×66,500).

The gradual loss of synaptic vesicles that occurs in shi synapses when the temperature is raised has been correlated with a gradual reduction in the amplitude of the EJP and a gradual decrease in the frequency of spontaneous release. On the other hand, the amplitude of the wild-type evoked response does not change as the temperature is raised, whereas the frequency of spontaneous release greatly increases with an increase in temperature (Koenig et al. l983). It is the purpose of this paper to correlate the reformation after shi-induced depletion of the two distinct synaptic vesicle populations (active zone and nonactive zone) with the recovery of the EJP and spontaneous release as normal recycling is allowed to proceed by lowering the temperature. Because the active zone recycling pathway has a much faster time course than the nonactive zone pathway, it is possible in this way to observe the contribution of the active zone population to release before the nonactive zone population has reformed. The recovery of these processes is described separately, followed by a comparative analysis.

Recovery of the EJP

Continuous intracellular recordings were made from DLM fibers of shi flies while raising the temperature at a rate of ~1°C/15 s and stimulating at a rate of 0.5 Hz. (It should be noted that the conditions eliciting depletion and recovery used here are different from those used in our previous papers so that the depletion and recovery times are different; see METHODS for details). The evoked response gradually decreased in amplitude as shown in Fig. 4A. After exposing the terminal to 29°C for 1 min, the amplitude of the EJP was reduced to ~2 mV, which represents the release of approximately four quanta. The temperature was then lowered at the same rate while stimulating. It was observed that the EJP remained at essentially the same depressed amplitude between 29 and 27°C. In 32 flies tested, the EJP amplitude began to increase between 26 and 25°C and followed a very similar time course, although the exact temperature at which the amplitude began to increase was slightly variable from fly to fly. An example of the recovery of the EJP as the temperature was lowered is demonstrated in Fig. 4B. The recovery of the EJP amplitude from ~2 mV at 26°C to threshold level for the electrogenic response (~30 mV) at 25°C took ~10 s. As the temperature was lowered further, the action potential (combination of EJP and electrogenic response) continued to increase in amplitude until at 22°C it reached its original level before raising the temperature. This suggests that the EJP amplitude continued to increase as the temperature was lowered from 25 and 22°C. As can be seen, the recovery of the full amplitude of the evoked response took ~1 min.



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Fig. 4. Effect of temperature on excitatory junction potential (EJP) in shi fly. A: excerpts from a typical continuous intracellular recording from a shi DLM fiber as the temperature is raised from 22 to 29°C. Evoked response remains undiminished until ~27°C, after which the EJP amplitude becomes reduced to the point where the electrogenic response disappears. (28°C). By 29°C, the EJP amplitude has been reduced to ~2 mV. B: excerpts from recording of A, as the temperature is lowered from 29 to 22°C. The amplitude of the EJP begins to increase dramatically at 25°C. Traces 25a-d are 4 consecutive traces, each 15 s apart. Note that within this 60-s period the EJP increases in amplitude from ~2 to ~40 mV, which elicits the graded electrogenic response. Scale bars: 80 mV, 10 ms

After the amplitude of the EJP had fully recovered, no abnormality in release characteristics was observed. The terminals were capable of sustaining firing frequencies of up to 20 Hz for 15 min, the period of time when only the active zone cluster of vesicles was reformed. The normal firing frequency for this muscle during flight is between 5 and 10 Hz (Koenig and Ikeda l980). Stimulation at higher frequencies (up to 50 Hz) was performed, but above 20 Hz this muscle exhibits an intermittent, abnormal, 5- to 10-s depolarizing response, which damages the muscle. This occurred in shi as well as in wild type. Thus no difference in evoked response could be detected in the newly recovered terminals from that of wild type.

Recovery of spontaneous release

As mentioned previously, the frequency of spontaneous release onto a DLM fiber in a wild-type fly increases as the temperature is elevated and decreases as the temperature is subsequently lowered. This is demonstrated by excerpts from a continuous recording in Fig. 5A, left column. As can be seen, many small (0.5 mV) miniature EJPs (MEJPs), as well as a few larger ones, are occurring at 19°C, which gives the impression of a noisy baseline. Although the overlapping of individual events makes it impossible to determine the MEJP frequency precisely, a count of distinguishable bumps suggests that the frequency must be at least 40/s. As the temperature increases, a great deal of summation occurs, causing larger fluctuations of the baseline. Although the frequency could not be determined at this time, it is possible that literally hundreds of events may be occurring per second at 29°C, considering the amplitude of the large fluctuations. The high frequency of MEJPs, even at 19°C, is a consequence of the fact that many hundreds of active zones exist on this muscle fiber, which is isopotential.



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Fig. 5. Effect of temperature on miniature EJPs (MEJPs). A: excerpts from continuous intracellular recordings of wild-type (left column) and shi (right column) DLM fibers as the temperature is raised and lowered. Note how the frequency of spontaneous release is greatly diminished at 29°C in the shi fly, exposing individual MEJPs (arrowheads); 19°C a-c represent excerpts after 1, 10, and 20 min at 19°C, respectively. The arrowheads point to a single MEJP (left) and a multiquantal MEJP (right). B: example of a greatly diminished evoked response (EJP) at 29°C in a shi DLM terminal followed by a multiquantal MEJP of similar amplitude (arrowhead). Note faster time course of evoked response. C: examples of clustering of MEJPs (underlined) in shi terminal after exposure to 29°C. Scale bars: 1 mV, 1 s

As shown by excerpts from a continuous recording in Fig. 5A, right column, the frequency of spontaneous release onto a DLM fiber in a shi fly increases as the temperature is raised from 19 to ~26°C, after which it gradually decreases to almost zero as the temperature is raised to 29°C. (For other examples of this phenomenon see Ikeda and Koenig 1988; Koenig et al. 1983). At 29°C, with the loss of almost all of the MEJPs, the true baseline now becomes apparent.

When the temperature was lowered after exposure to 29°C, the frequency increased to some extent between 29 and 25°C. Thus the average frequency (32 flies) increased from 0.42 events/s at 29°C to 1.98 events/s at 27°C to 5.77 events/s at 25°C. The difference in frequency within a single shi fly at 25°C before and after depletion was dramatic and consistent in 32 flies tested. Thus in every fly the frequency was far beyond the level where individual events could be recognized at 25°C before heating, whereas it averaged 5.77 events/s at 25°C after exposure to 29°C. At this time, the EJP recovered to the point where the electrogenic response was elicited. As the temperature was lowered to l9°C, the frequency increased somewhat, averaging 9.1 events/s after 1 min at 19°C, but continued to be depressed in comparison with the predepletion frequency at 19°C. (Fig. 5A, 19°a) Thus, although the EJP had recovered fully at this point, the MEJP frequency had not recovered. The frequency of spontaneous release remained attenuated for 10-15 min after lowering the temperature to 19°C (Fig. 5A, 19°b), after which it increased to the point where individual small events overlapped, as shown in Fig. 5A, 19°c. The exact timing at which the increase in spontaneous release occurred varied somewhat from fly to fly. Furthermore, in ~20% of the recordings, the frequency remained attenuated for 30 min or more, until the muscle began to depolarize. Possibly, the dissection damaged these preparations so that they did not recover. However, in these preparations the EJP recovered completely. A graph of the changes in spontaneous release frequency with temperature over time taken from a continuous recording from a shi fly is shown in Fig. 6. The recovery of the EJP for this particular fly is also included. As mentioned previously, the exact time course of recovery varied from fly to fly so that this graph is presented only to demonstrate that the return to the predepletion frequency took ~15 min at 19°C, although the EJP was fully recovered in 2 min as the temperature was lowered from 29 to 21°C.



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Fig. 6. Graph of recovery of the amplitude of the evoked response and MEJP frequency over time as the temperature is lowered from 29 to 19°C and maintained at that level. The data are from a single shi fly. The temperature was lowered at a rate of 4°C/min. The amplitude of the evoked response recovered fully (~100 mV) within 1 min between the temperatures of 25 and 21°C, whereas the MEJP frequency did not recover fully (~40 events/s) until ~15 min later.

In addition to a dramatic and prolonged decrease in the frequency of spontaneous release, the release characteristics after shi-induced depletion were also changed. It has been published previously that during recovery from depletion in shi terminals, the probability that a given MEJP will fire either simultaneously with or soon after another MEJP is much higher than would occur by chance (Ikeda and Koenig 1988). This phenomenon results in two release characteristics that are apparent in the recordings shown in Fig. 5, clusters of MEJPs and a preponderance of larger-sized MEJPs made up of two or more synchronously released quanta. The larger-sized MEJPs give the impression that the MEJPs after depletion are larger than they were before heating, as can be seen in Fig. 5A (compare recordings at 19°C before and after raising the temperature). However, MEJP amplitude histograms demonstrate that the amplitudes of the larger-sized events are roughly integral multiples of the events observed at 19°C before raising the temperature, suggesting they are made up of two or more synchronously released single quanta (Ikeda and Koenig 1988). In Fig. 5B, it can be seen that these multiquantal MEJPs have slower rise and fall times than an attenuated EJP of approximately the same amplitude. This demonstrates that the quanta are not as highly synchronized in the multiquantal MEJP as they are in the EJP. Figure 5C demonstrates clusters of MEJPs, which represent even less synchronized spontaneous release than the spontaneous multiquantal events. This tendency toward clustered release begins to occur in shi at 29°C, although very few events are observed at this temperature. As the frequency of spontaneous release increases as the temperature is lowered, this effect becomes more obvious.

The reduced frequency and abnormal release characteristics described above persisted in the terminal for ~10-15 min after the temperature was lowered to 19°C. After this time the frequency of release increased to the point where individual events could not be distinguished, so that the release characteristics could not be determined. Thus it could not be determined by statistical analysis if the nonrandom release characteristics observed earlier were simply masked by the increase in frequency or if the release characteristics became random. In both wild-type and 19°C shi, some larger-sized MEJPs were observed in addition to the many, very small, single quantal events that characterize spontaneous release on this muscle. Possibly these larger MEJPs represent the multiquantal events observed while only the active zone vesicle population was reformed.

Synaptic vesicle recovery

To correlate the recovery of the EJP and MEJPs with the reformation of synaptic vesicles by recycling, preparations were recorded intracellularly and then fixed for electron microscopy at various stages of recovery after the initial exposure to 29°C. The amplitude of the EJP and the frequency of spontaneous release at the time of fixation were then correlated with the number of vesicles per synapse per plane of sectioning for many DLM synapses for each preparation.

The number of vesicles observed in a single section through a dense body at the active zone varies from synapse to synapse, depending on the plane of sectioning and the positioning of vesicles relative to the dense body at each particular synapse. This variability is demonstrated by the histogram in Fig. 7A. Sixty-two different synapses on a single fiber were observed for this histogram. As can be seen, the number of vesicles that is observed in a single section through an active zone of a shi synapse at l9°C varies from 0 to ~30, depending on the section (for additional histograms of vesicle distribution see Koenig et al. l989). Because a dense body covers approximately six thin sections, the actual number of vesicles per synapse is much higher than the numbers presented here for vesicles per synapse per section. Although the number of vesicles observed in different sections through different active zones synapsing on a single fiber varies considerably, the average number of vesicles per section for many synapses is quite consistent from fly to fly (Koenig et al. 1989). In the case of the histogram in Fig. 7A, the average number of vesicles per section is 10.6. For 8 flies (4 wild-type and 4 shi at 19°C), the averages ranged between 9.5 and 11.3. Again it should be remembered that this average only represents the average number of vesicles observed in a single plane of sectioning through a synapse and does not represent the average number of vesicles per synapse.



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Fig. 7. Histograms of number of vesicles/single plane of sectioning through active zones. A: histogram of the number of vesicles observed in single sections through 62 different synapses from a single DLM fiber. Ordinate: number of synapses; abscissa: synapses containing x number of vesicles. Note the variability in the number of vesicles observed in a single section. B: histograms of the average number of vesicles/zone/plane of sectioning observed at different temperatures in shi DLM synapses. Averages are of data similar to those presented in A. Figure should be read from left to right. Zone 1: area adjacent to the dense body base and under dense body filamentous plate; zone 2: area within 150 nm of dense body base; zone 3: area further than 150 nm from the dense body; 19° before heating (far left): 292 synapses, 4 flies; 25° before heating: 156 synapses, 2 flies; 29°: 136 synapses, 2 flies; 25° after depletion: 226 synapses, 3 flies; 19° for 1 min after depletion: 197 synapses, 3 flies; 19° for 30 after depletion: 284 synapses, 4 flies. Ordinate: average number of vesicles/section; abscissa: zones 1, 2, or 3.

Because the relative contributions of vesicles formed via the active zone and nonactive zone pathways were in question, the synapse was divided into three zones. 1) The area under the dense body plate adjacent to the dense body base. Vesicles in this area are attached to the plasma membrane (docked) and may represent the readily releasable population. This also represents the area from which active zone vesicle recycling has been shown to emanate, so that these might represent newly formed vesicles as well. 2) The area within 150 nm of the dense body base. Vesicles in this area are attached to the dense body by filaments. 3) The area further than 150 nm from the dense body. This represents the approximate area where the nonactive zone population, which is dispersed throughout the cytoplasm, would be located. Some vesicles tethered to the dense body may be included in this group because it is not possible with most planes of sectioning to determine precisely how far from the dense body these vesicles might be located.

The data are presented in Fig. 7B as the average number of vesicles per section for each of the three zones mentioned above. Each group of histograms (zones 1-3) represents an average of data from several different flies. Figure 7B demonstrates that at 19°C before raising the temperature, at a time when the EJP and spontaneous release were normal, virtually all of the vesicles were located in zones 2 and 3. This means that the vast majority of vesicles were not located in a docked position at the active zone presynaptic membrane, so that the probability of observing one in this position was extremely low. Rather, most vesicles were either tethered to the dense body on filaments (zone 2) or were dispersed throughout the cytoplasm, presumably attached to the cytonet (zone 3). This is the typical positioning of vesicles during both rest and activity in this as well as other types of Drosophila synapses (Koenig et al. l993). A similar distribution of vesicles was observed as the temperature was raised to 25°C.

After raising the temperature to 29°C and allowing it to remain there until the EJP amplitude was ~1 mV, almost all of the few remaining vesicles were located in zone 3. The averages of less than one reflect the fact that most of the terminals possessed no vesicles. When the temperature was lowered to 25°C, at a time when the EJP had recovered to the point where the electrogenic response was elicited, but the frequency of spontaneous release only increased slightly and displayed nonrandom release characteristics, many of the synapses possessed no vesicles, but some possessed one or two vesicles in a docked position under the dense body plate (zone 1). This represents a substantial increase in the number of docked vesicles over that which was observed before depletion at 19 or 25°C at a time when the EJP was of normal size. The number of vesicles in zones 2 and 3 remained very low, essentially the same as at 29°C. When the temperature was lowered to 19°C, at a time when the action potential reached its full predepletion amplitude but spontaneous release continued to be depressed and nonrandom (1 min at 19°C), most synapses possessed several vesicles, mainly located in zones 1 and 2, but some continued to be depleted. The number of vesicles in zone 3 remained very reduced, essentially the same as it was at 29°C. If the data taken after 1 min at 19°C are compared with the data taken at 19°C before the temperature was raised, it can be seen that the average number of vesicles in the postdepletion terminals is reduced, and furthermore, the distribution of vesicles in the terminal is altered considerably. In particular, at 19°C before raising the temperature, essentially no vesicles are observed in zone 1, i.e., under the dense body plate adjacent to the dense body base, whereas many vesicles are observed in zone 1 in terminals recovering from shi-induced depletion. After ~10 min at 19°C, vesicles begin to appear in zone 3, away from the active zone. By 30 min at 19°C, many vesicles are observed in zone 3, and the number of vesicles in zone 1 has decreased to almost zero, whereas the number of vesicles in zone 2 has increased considerably. The average number of vesicles in postdepletion terminals after 30 min at 19°C is reduced in comparison to either 19 or 25°C before depletion. This is because two of the four flies observed possessed vesicle averages identical to the predepletion averages, whereas the other two possessed somewhat lower averages. Possibly, recovery was simply slower in the two flies with fewer vesicles. At any rate, full recovery was observed in some flies. Electron micrographs of active zones during early recovery at 19°C after exposure to 29°C are shown in Fig. 8, A-C. In Fig. 8A (1 min at 19°C), it can be seen that several vesicles are located on either side of the dense body base under the filamentous plate. A cluster of vesicles is also observed above the plate. Many terminals possessed several vesicles above and below the plate, but other terminals continued to be depleted. Thus vesicle averages in the histograms of Fig. 7B are lower than would be expected by observing these terminals. In Fig. 8B (2 min at 19°C), in addition to a cluster of vesicles at the dense body, recycling membrane can be seen in these terminals. This recycling membrane represents the nonactive zone recycling pathway, which involves cisternae from which vesicles are assumed to bud. The various steps of the nonactive zone pathway were described in detail elsewhere (Koenig and Ikeda 1989). After 20 min at 19°C, some terminals continued to possess cisternae in addition to a normal compliment of vesicles (Fig. 8D). Also, whereas most terminals appeared to possess a normal compliment of vesicles, a few became densely packed with vesicles (Fig. 8E). These terminals were not included in the data of Fig. 7 because they are clearly abnormal. However, flies that possessed some densely packed terminals exhibited normal evoked and spontaneous release.



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Fig. 8. Electron micrographs of shi synapses at 19°C after depletion. A: 1 min at 19°C. B and C: 2 min at 19°C. Examples of cluster of vesicles around the dense body at the active zone (large arrow). Note many "docked" vesicles (arrowheads) in zone 1. Note recycling cisternae of nonactive zone pathway (small arrows) in B. D: note many vesicles dispersed throughout cytoplasm, lack of docked vesicles at active zone (large arrow), and recycling cisternae (small arrow). E: note densely packed vesicles throughout cytoplasm. Large arrow points to dense body at active zone. Scale bars: in A-C, 250 nm (×80,000); in D, 0.5 µm (×60,000); in E, 0.5 µm (×50,000).

Correlation between reformation of vesicle populations and recovery of EJP and spontaneous release

The data demonstrate the following. 1) At 29°C, the vesicle population is almost completely depleted, the amplitude of the EJP is reduced to approximately four quanta, and the frequency of spontaneous release is reduced from possibly hundreds/s to 0.5/s. 2) When the temperature is lowered to ~25°C, the EJP begins to recover very rapidly (from 2 to 30 mV in ~10 s). When the EJP has recovered to ~30 mV in amplitude (threshold for the electrogenic response), the frequency of spontaneous release has increased slightly to ~5/s, which is greatly reduced from what it was at 25°C before raising the temperature. Also, the vesicle population has only increased slightly from what it was at 29°C, and the distribution of these few vesicles relative to the active zone is quite different from what it was before raising the temperature. In particular, a large percentage of the vesicles is now located under the dense body in a docked position, which was never seen before heating. A few are located in the tethered position, and the remainder of the vesicles are located away from the active zone. The number of vesicles located away from the active zone represents about the same number of vesicles that was in this location at 29°C. 3) After 1 min at 19°C following exposure to 29°C, the evoked response has recovered fully, whereas the frequency of spontaneous release, although slightly higher than it was at 25°C, is still greatly attenuated relative to what it was at 19°C before heating. Also, the number of vesicles, although increased to some extent, continues to be greatly reduced, and furthermore, the vast majority of these vesicles is located under the dense body plate or tethered to the dense body, a distribution never observed before heating. The number of vesicles located away from the active zone continues to be approximately the same as it was at 29°C. 4) After 30 min at 19°C, the evoked response, frequency of spontaneous release, and number and distribution of the vesicles are similar to what was observed before raising the temperature.

In summary, the data demonstrate that the EJP is fully recovered at a time when the terminal is almost completely depleted of vesicles except for a small population located at the active zone. Many of these vesicles are located adjacent to the dense body base at the plasma membrane, in a seemingly docked position, a location where vesicles are almost never seen under predepletion conditions, either at rest or stimulated. Although the EJP has recovered fully, the frequency of spontaneous release remains greatly reduced, and the release characteristics are nonrandom. Only when the normal compliment of vesicles is reformed does the frequency of spontaneous release return to its predepletion level.


    DISCUSSION
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These data demonstrate that when a small population of vesicles at the active zone exists without the larger population of vesicles dispersed throughout the cytoplasm, the evoked response is normal, but the frequency of spontaneous release is greatly attenuated and possesses abnormal release characteristics. The first question to be addressed is the origin of this small population. Is it really a small subpopulation of vesicles newly formed by a special, active zone recycling pathway as suggested here, or does it represent a group of vesicles from the predepletion pool that have migrated to the active zone when the temperature is lowered? Our reasons for believing in the former possibility are as follows. 1) An active zone recycling pathway is suggested at this synapse by the fact that incubation at 27°C causes an accumulation of recycling membrane that emanates from the active zone. The existence of an active zone pathway has been described in detail in other shi synapses (Koenig and Ikeda 1996) and also in wild-type synapses (Koenig et al. 1998). 2) A clump of vesicles that was not there at 29°C appears at the active zone as soon as the temperature is lowered below 27°C. Because lowering the temperature is known to release the block on vesicle recycling at shi synapses, it seems likely that these vesicles represent newly recycled vesicles. Furthermore, the alternative possibility---that vesicle translocation to the active zone is blocked at 29°C, and lowering the temperature releases vesicles to translocate to the active zone and accumulate there---does not seem likely considering the fact that the exocytotic mechanism (including the process of vesicle translocation to the active zone) is normal at the restrictive temperature, as is evidenced by the depletion of the entire terminal by exocytosis at 29°C. 3) Many of the active zone vesicles that appear on lowering the temperature are located next to the dense body, the exact location from which recycling membrane accumulates above 27°C. No vesicles are observed to appear away from the active zone at this time. 4) The various stages of the nonactive zone pathway (described by Koenig and Ikeda 1989, 1996) can be observed to be taking place during the time when this clump of vesicles appears and changes locations. After ~10 min at 19°C, vesicles begin to appear in the cytoplasm away from the active zone, associated with recycling cisternae. There seems to be no overlap in time between these two waves of vesicle reformation. Although all of the previous observations represent circumstantial evidence, taken together these authors feel they strongly suggest that the small subpopulation shown here is newly formed at the active zone.

Regardless of how or where the vesicles observed at 19°C after heating are formed, the relationship among the number of docked vesicles, the amplitude of the EJP, and the frequency of spontaneous release is unexpected. Under normal conditions, either in wild type at any temperature or shi at 19°C, docked vesicles, i.e., those located adjacent to the dense body attached to the plasma membrane, are rarely observed at the DLM terminals. This can be explained by the fact that there are literally thousands of active zones located on each DLM muscle fiber, and only a small percentage of them would need to have even one vesicle docked to produce a normal-sized EJP. Thus the probability of sectioning through an active zone possessing a docked vesicle would be low. At 25°C, at a time when the EJP has only recovered partially, vesicles adjacent to the dense body at the plasma membrane are observed in ~30% of the sections through active zones. Because a single active zone spans approximately six sections, the percentage of active zones with vesicles in this location is even higher. This represents an enormous increase over what is observed in wild type or shi at 19°C. It is currently accepted that vesicles attached to the active zone presynaptic membrane represent a readily releasable subpopulation. However, if the vesicles observed here were readily releasable, the EJP should be at least of normal amplitude, and the frequency of spontaneous release should not be greatly attenuated. This suggests that the majority of these seemingly docked vesicles are not in readily releasable form. One possibility is that they are newly formed and not yet ready for release. This possibility is also suggested by recent observations on the active zone recycling pathway in wild-type flies, which suggest that the dense body structure may be involved in vesicle recycling rather than release and that docked vesicles and omega-shaped images observed adjacent to this structure represent newly forming vesicles rather than vesicles in the process of release (Koenig et al. 1998). It is interesting that the number of docked vesicles decreases at a time when the frequency of spontaneous release is increasing dramatically. This observation could be explained if the docked vesicles are actually newly forming vesicles that subsequently disperse into the cytoplasm and mature for release.

When the temperature is lowered to 19°C, most synapses possess an accumulation of docked vesicles, although the vesicle population located away from the active zone remains as depleted as at 29°C. At this time the evoked response is completely normal. However, spontaneous release remains greatly attenuated and abnormal in release characteristics. It has been demonstrated statistically that in synapses newly recovered from shi-induced depletion, there is a much higher probability that one MEJP will occur simultaneously with or immediately after another than the probability observed in wild type or shi before heating (Koenig and Ikeda l989). One question that arises is whether the nonrandom spontaneous release characteristics exhibited at a time when only the active zone population has been reformed represent normal release characteristics for this small subpopulation of vesicles. It has been reported that clustering of spontaneous release occurs under normal conditions in various synapses (Bornstein 1978; Fatt and Katz 1952; Kriebel and Bridy 1996; Kriebel and Stolper 1975c,e). Thus it is possible that the nonrandom, low-frequency release observed when only the active zone vesicle population has been reformed might represent normal release for this population that is usually masked by a more randomly distributed, higher-frequency release contributed by the nonactive zone population of vesicles. Another possibility is suggested, however, by the observation that multiquantal MEJPs and clustering of MEJPs have been shown to result from exposure of a terminal to high Ca2+ saline (Bornstein l978; Dennis et al. 1971; Rotshenker and Rahamimoff l970). In wild-type Drosophila terminals, both multiquantal and clustered release also have been demonstrated to increase dramatically in high-Ca2+ saline, although the frequency is not reduced as in shi terminals (Koenig et al. l993). This suggests the possibility that postdepletion shi terminals may have elevated cytosolic Ca2+ levels. This possibility is further suggested by the observation that exposure of wild-type Drosophila terminals to high-Ca2+ saline also causes an accumulation of vesicles in a docked position under the dense body plate (Koenig et al. l993), the same effect that is reported here for synapses recovering from shi-induced depletion. Thus both the unusual release characteristics and the unusual accumulation of vesicles under the dense body plate described here during early recovery from shi-induced depletion can be induced in wild-type terminals by exposing them to high Ca2+ saline (3.6-18 mM). The similarity in both the morphology of the active zone and the spontaneous release characteristics between these two seemingly unrelated conditions (high Ca2+ and recovery from depletion) is truly striking (for examples of spontaneous release characteristics and vesicle positioning at the active zone in high Ca2+ see Ikeda and Koenig l988; Koenig et al. l993). Certainly the possibility must be considered that the recovering shi terminals may possess a higher-than-normal cytoplasmic concentration of Ca2+. How this condition might be brought about by shi-induced depletion is in the realm of speculation, but one possibility is suggested by observations demonstrating that vesicles have the ability to sequester Ca2+ (Israel et al. 1980; Michaelson et al. 1980) and that stimulation transiently increases Ca2+ in synaptic vesicles (Parducz and Dunant 1993). If Ca2+ sequestration were one function of vesicles as has been suggested previously (Tauc 1979), it would be expected that the levels of Ca2+ in the cytoplasm would rise in a depleted terminal as Ca2+ enters with stimulation. A high cytosolic Ca2+ level in depleted shi terminals could explain the observation that stimulus-induced Ca2+ influx is attenuated in shi terminals at 32°C (Umbach et al. 1998), because the concentration gradient driving Ca2+ entry would be reduced.

After ~10 min at 19°C, vesicles begin to appear in the cytoplasm away from the active zone (zone 3). By this time the accumulation of docked vesicles has disappeared for the most part, and a population of tethered vesicles is observed (zone 2). One interpretation of this change in vesicle distribution is that the docked vesicles have detached from the presynaptic membrane but remain tethered to the dense body, whereas the nonactive zone population is newly formed by the slower pathway that emanates away from the active zone. Accompanying the second wave of vesicle reappearance, the frequency of spontaneous release increases dramatically to its predepletion level. This correspondence between the recovery of the nonactive zone population and the increase in frequency of spontaneous release suggests that this population is the main contributor to spontaneous release. This may simply mean that the more vesicles there are in the terminal the more spontaneous release occurs.

In conclusion, these data suggest that there may be a small population of quickly recycling vesicles at the active zone that is capable of producing enough transmitter release to sustain a normal EJP at physiological frequencies. Although the idea that there are two populations of vesicles, one more releasable than the other, is not new (e.g., Àgoston et al. 1985; Birks and MacIntosh 1961; Kristensen et al. 1994; Pieribone et al. 1995; Prado et al. 1992; Zimmerman and Denston 1977), it has not been demonstrated that the more releasable population is capable of sustaining normal release without the other population. This is the first direct demonstration that a synapse devoid of its larger population of vesicles can indeed sustain normal release. Of course, these observations do not preclude the possibility that the nonactive zone vesicle population can supplement the active zone population during times of intense stimulation. Kuromi and Kidokoro (1998) have shown that disruption of the more centrally located vesicle pool (nonactive zone pool) by cytochalasin D causes a reduction in transmitter release with high-frequency stimulation. However, it should be kept in mind that the high-intensity release required to access the larger population may not ever occur under normal physiological conditions. This would suggest that the larger population might have a separate function from evoked release. If this were so, the function of this population would most likely involve spontaneous release. It is interesting to note that staurosporine, a protein kinase inhibitor, has been shown to block the stimulus-induced unloading of the fluorescent dye FM1-43 from synaptic vesicles, although the evoked response is unaffected. However, staurosporine did depress the MEEP frequency, particularly that induced by tetanic nerve stimulation (Henkel and Betz 1995). This suggests that loading and unloading of FM1-43 might be accomplished by spontaneous release rather than evoked release, which fits with the fact that it is the larger nonactive zone vesicle population that is visualized by this dye, the active zone subpopulation being too small to observe at the light microscopic level. Thus the possibility that evoked release may be accomplished by the small, active zone vesicle population, whereas the majority of spontaneous release may be accomplished by the larger, nonactive zone vesicle population, should be considered when correlating the evoked response with effects on the larger, nonactive zone vesicle population.


    ACKNOWLEDGMENTS

This work was supported by National Institute of Neurological Disorders and Stroke Grant NS-18856 and National Science Foundation Grant BNS 8415920.


    FOOTNOTES

Address for reprint requests: J. H. Koenig, Division of Neurosciences, Beckman Research Institute of the City of Hope, 1450 East Duarte Rd., Duarte, CA 91010.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 29 September 1998; accepted in final form 15 December 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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