1Department of Anatomy and Neurobiology, University of Vermont, College of Medicine, Burlington, Vermont 05405-0160; and 2Department of Physiology and Pharmacology, University of Strathclyde, Strathclyde Institute for Biomedical Sciences, Glasgow G4 0NR, Scotland
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ABSTRACT |
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Parsons, Rodney L.,
Michelle A. Calupca,
Laura A. Merriam, and
Chris Prior.
Empty synaptic vesicles recycle and undergo exocytosis at
vesamicol-treated motor nerve terminals. We investigated
whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were
treated with 5 µM vesamicol to inhibit the vesicular ACh transporter
and then were exposed to an elevated potassium solution, 35 mM
potassium propionate (35 KP), to release all preformed quanta of ACh.
At vesamicol-treated endplates, miniature endplate current (MEPC)
frequency increased initially from 0.4 to >300 s1 in 35 KP but then declined to <1 s
1 by 90 min. The decrease in
frequency was not accompanied by a decrease in MEPC average amplitude.
Nerve terminals accumulated the activity-dependent dye FM1-43 when
exposed to the dye for the final 6 min of a 120-min exposure to 35 KP.
Thus synaptic membrane endocytosis continued at a high rate, although
MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results
demonstrate that recycled cholinergic synaptic vesicles that were not
refilled with ACh continued to dock and undergo exocytosis after
membrane retrieval. Thus transport of ACh into recycled cholinergic
vesicles is not a requirement for repeated cycles of exocytosis and
retrieval of synaptic vesicle membrane during prolonged stimulation of
motor nerve terminals.
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INTRODUCTION |
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Neuromuscular transmission occurs when ACh, which
is released from motor nerve terminals through exocytosis of synaptic
vesicles, crosses the synaptic cleft and activates postsynaptic
nicotinic receptors. Each synaptic vesicle normally contains and
releases a discrete amount of ACh, called a quantum. The postsynaptic
current caused by one quantum of ACh is the miniature endplate current (MEPC). Normally, sufficient ACh is released so that the postsynaptic response, the endplate potential, remains suprathreshold even with
continued repetitive stimulation, thereby ensuring efficient neuromuscular transmission. To sustain neuromuscular transmission, appropriate numbers of synaptic vesicles must be docked and primed for
release at the active zones at the inner side of the presynaptic terminal (reviewed in van der Kloot and Molgo 1994). Two
pools of synaptic vesicles are proposed, the readily releasable pool positioned near the active zones and a larger, more distant main store
(reviewed in Parsons et al. 1993
; van der Kloot
and Molgo 1994
). Two mechanisms maintain the needed supply of
synaptic vesicles in the readily releasable pool. First, synaptic
vesicles are mobilized from the main store into the releasable pool
during continued stimulation. Second, after exocytosis, synaptic
membrane must be retrieved through endocytosis quite rapidly. The
retrieved vesicular membrane must be transformed into vesicles,
refilled with ACh, and then returned to the readily releasable pool
near active zones. Recent studies at the neuromuscular junction and at
synapses between cultured hippocampal neurons suggest that exocytosis
and endocytotic retrieval of synaptic vesicle membrane are closely
coupled (Betz et al. 1992b
; Murphy and Stevens
1998
). Thus the rate of synaptic vesicle recycling is
determined by the rate of exocytosis (Betz et al. 1992b
;
Wu and Betz 1996
). In addition, Betz and colleagues
(Betz and Bewick 1992
, 1993
; Betz et al.
1992a
) have shown that recycled, refilled vesicles mix readily
and randomly within the vesicular pool so that the recycled, refilled
synaptic vesicles are indistinguishable from their nonrecycled neighbors.
It might be expected that some mechanism would exist to ensure that
vesicles are refilled, at least partially, with transmitter before
entering the readily releasable pool. Previous electrophysiological measurements indicate that partially filled vesicles undergo exocytosis (Elmqvist and Quastel 1965; Searl et al.
1990
, 1991
). However, it is not established
whether exocytosis continues or stops once the recycled vesicles no
longer contain any transmitter. Thus it is unknown whether refilling of
synaptic vesicles is a prerequisite for recycling synaptic vesicles to
merge with the readily releasable pool of vesicles and continue to
undergo exocytosis (reviewed in van der Kloot and Molgo
1994
).
Recently, Cousin and Nicholls (1997) presented evidence that recycled,
unfilled glutamatergic vesicles re-enter the available pool of vesicles
and undergo exocytosis in cerebellar granule cells. However, key
characteristics of synaptic transmission differ between central
neuronal and peripheral neuromuscular synapses, such as vesicle pool
size, probability of synaptic vesicle release, mechanism of recycling,
and need for sustained suprathreshold transmission. Thus it is
reasonable to propose that mechanisms regulating the replenishment of
recycled vesicles might differ between central nerve terminals and
motor nerve terminals.
Consequently, we asked the basic question do empty, recycled cholinergic synaptic vesicles at continually stimulated motor nerve terminals re-enter the releasable vesicle pool near active zones, dock, and undergo exocytosis? To test this question, we inhibited the ACh transporter with vesamicol and then stimulated the nerve terminal until all preformed stores of quanta were depleted. Once preformed transmitter stores were depleted and postsynaptic currents were eliminated, we then tested whether endocytosis and exocytosis continued to occur at the motor nerve terminal by using the activity-dependent dye FM1-43.
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METHODS |
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Experiments were performed on twitch muscle fiber neuromuscular
junctions in costocutaneous muscles of garter snakes
(Thamnophis) at room temperature (21-23°C). Nerve-muscle
preparations were initially bathed in a control physiological solution
containing (in mM) 159 NaCl, 2.5 KCl, 1.0 CaCl2, 4.2 MgCl2, and 5.0 HEPES, pH 7.3 (Connor et al. 1984,
1997
). Depolarization-stimulated transmitter release was
induced by exposure to an elevated potassium solution (35 mM potassium
propionate, 35 KP) (Connor et al. 1997
). CsCl (5 mM) was
included in the 35 KP solution to facilitate voltage clamping of
depolarized muscle fibers to hyperpolarized potentials (Connor
et al. 1984
, 1997
). Stimulation by K+
depolarization rather than nerve stimulation was chosen because with
repetitive neural stimulation muscle movement made it impossible to
maintain stable intracellular impalements. In addition, vesamicol can
have local anesthetic-like effects at high concentrations (Pemberton et al. 1992
). Thus during repetitive neural
stimulation action potential propagation into vesamicol-treated nerve
terminals might become depressed, causing an intermittent interruption
of neuromuscular transmission.
Vesamicol was dissolved in distilled water as a 10-mM stock solution each day and diluted to 5 µM in either control physiological solution or 35 KP. Muscle preparations were pretreated with vesamicol in the control physiological solution for 20 min before exposure to 35 KP that also contained vesamicol.
Electrophysiology
Twitch muscle fibers were identified with criteria described
previously (Connor et al. 1984, 1997
; Dionne and
Parsons 1981
). MEPCs were recorded at
150 mV with a
two-microelectrode voltage-clamp system and were stored on a PCM
recorder (A. R. Vetter; Rebersburg, PA) for subsequent
digitization and analysis (Connor et al. 1997
). The
integral of a single-exponential function fitting the decline in MEPC
frequency at vesamicol-treated endplates was calculated with a graphic
plotting program (Microcal Origin 4.1, Microcal Software; Northhampton,
MA). Amplitude measurements were made on individual digitized MEPCs
that exhibited a fast rise from baseline to peak with the use of the
SCAN program (generously provided by Dr. John Dempster, Univ. of
Strathclyde, Glasgow, Scotland).
FM1-43 assay of vesicle membrane endocytosis
Neuromuscular junctions were identified with
rhodamine-conjugated peanut agglutinin (PNA, Sigma; St. Louis, MO),
which marks terminals and synaptic Schwann cell basal laminae
(Connor et al. 1997; Ko 1987
).
Accumulation of the fluorophore FM1-43 (2 µM, Molecular Probes;
Eugene, OR) into nerve terminals was used to demonstrate endocytotic
retrieval of synaptic vesicle membrane (Betz and Bewick 1992
,
1993
; Betz et al. 1992a
,b
). Nerve-muscle preparations were exposed to FM1-43 during the final 6 min of a
120-min exposure to 35 KP, washed for 15 min in control physiological solution containing PNA (33 µg/ml), rinsed with PNA-free control physiological solution, and viewed. To demonstrate that the terminals destained, other muscles were loaded with FM1-43 as described previously and were viewed shortly after washout of FM1-43 to confirm
the success of the loading procedure. While still in 35 KP, nerve
terminals were then examined over time to determine whether the FM1-43
staining diminished. FM1-43 commonly stained the myelinated
preterminal axon (Fig. 3). This nonspecific staining served as a
control for the staining conditions and imaging. FM1-43 accumulation
and destaining of nerve terminals was examined with a Zeiss
fluorescence photomicroscope equipped with filter sets appropriate for
FM1-43 (green emission filter, 520-560 nm) or rhodamine (red emission
filter, >590 nm). A ×40 water immersion lens was used to locate
individual nerve terminals.
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RESULTS |
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MEPC frequency declines at vesamicol-treated endplates exposed to 35 KP
MEPCs were recorded at control snake twitch fiber endplates and at
endplates that had been treated with vesamicol (5 µM), a drug that
potently inhibits the cholinergic vesicular transporter (IC50 of ~40 nM) (Anderson et al. 1983;
Marshall 1970
). Transmitter release was stimulated by
depolarization in an elevated potassium solution (35 KP). In both
vesamicol-treated and control preparations, MEPC frequency increased
from ~0.4 s
1 to >300 s
1 on exposure to
35 KP (Fig. 1, trace 1). At
control endplates, MEPC frequency remained elevated during a 120-min
exposure to 35 KP (Fig. 1, trace 4) (see also Connor
et al. 1997
). In contrast, at vesamicol-treated endplates, MEPC
frequency declined progressively in 35 KP so that by 90 min MEPCs were
either not recorded or were recorded at a very low frequency (Fig. 1,
trace 3).
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The decrease in MEPC frequency was not accompanied by a decrease in MEPC amplitude at vesamicol-treated endplates (Fig. 2, A and B). In addition, although mean MEPC amplitude varied among individual endplates, the MEPC amplitude distribution histograms remained unimodal with no evidence of a population of smaller-amplitude MEPCs (Fig. 2C). Thus no ACh was released from partially filled vesicles that contributed to MEPC generation.
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We then used the MEPC frequency-versus-time relationship to approximate
the number of preformed quanta released at vesamicol-treated endplates.
The time-dependent decline in MEPC frequency (Fig. 2A) was
fitted to a single exponential described by
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FM1-43 accumulates in vesamicol-treated nerve terminals when the MEPC frequency is low
We next compared the accumulation of FM1-43 into control and
vesamicol-treated nerve terminals maintained in 35 KP for 120 min. All
nerve terminals in both control and vesamicol-treated preparations
incorporated FM1-43 during the final 6 min of a 120-min exposure to 35 KP (10-15 PNA-identified endplates/muscle; 6 control muscle
preparations from 4 different snakes and 9 vesamicol-pretreated muscle
preparations from the same 4 snakes). Representative examples in Fig.
3, C and D,
illustrate that the FM1-43 fluorescence is comparable for nerve
terminals innervating control and vesamicol-treated endplates. Also we
confirmed that nerve terminals that were not stimulated (i.e., kept in
control physiological solution for 120 min) only exhibited background
FM1-43 staining (Fig. 3B). The lack of FM1-43 accumulation
in these preparations was consistent with the low resting MEPC
frequency (~0.4 s1) recorded at endplates in control
physiological solution (Connor et al. 1997
).
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FM1-43 destains with continued exposure to 35 KP
FM1-43 staining of vesamicol-treated nerve terminals indicated
that endocytosis of synaptic vesicle membrane was continuing at a high
rate after 120 min in 35 KP, although MEPC frequency was very low.
FM1-43 destaining experiments were completed to directly demonstrate
that exocytosis also continued at vesamicol-treated terminals after 120 min in 35 KP. For the destaining experiments, two groups of
vesamicol-treated nerve-muscle preparations were exposed to FM1-43
for the final 6 min of a 120-min exposure to 35 KP. In one group of
muscles, bath solution containing FM1-43 was exchanged by dye-free 35 KP, and the muscles were viewed immediately to verify FM1-43
accumulation. These muscles were then maintained in 35 KP to allow
destaining to proceed. A second group of muscles was similarly exposed
to FM1-43 and then washed in control physiological solution. These
muscles were viewed after >50 min to verify that twitch terminals,
which were not stimulated continually after being allowed to accumulate
FM1-43, still exhibited FM1-43 fluorescence. In the preparations
maintained in 35 KP, all twitch terminals exhibited FM1-43
fluorescence shortly after switching to dye-free 35 KP. However, after
a continued stimulation with 35 KP for >15 min, the majority of
terminals only exhibited background FM1-43 fluorescence (18 unstained,
3 weakly stained in 2 muscle preparations; Fig.
4B). In contrast, 25 of 28 twitch terminals from the other 2 muscle preparations that had been
stained with FM1-43 and then washed and maintained in control
physiological solution retained bright FM1-43 fluorescence.
Photographs of these terminals were taken over a period of 60-120 min
(Fig. 4D). These observations demonstrated that the
vesamicol-treated, FM1-43-stained terminals destained when kept in 35 KP, a demonstration that exocytosis of synaptic vesicle membrane
continued at a high rate. These results also indicated that the FM1-43
fluorescence was due to accumulation into recycling synaptic vesicles
and not nonspecific staining of the nerve terminal (Betz et al.
1992a).
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DISCUSSION |
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At a concentration of 5 µM, vesamicol should have
effectively inhibited the vesicular ACh transporter so that all of the
quanta of ACh released by K+ stimulation must have come
from preformed stores. The observation that the MEPC amplitude
histograms were unimodal confirmed that no partially filled vesicles
contributed to MEPC generation (Searl et al. 1990). In
addition, the MEPC average amplitude did not decrease in
vesamicol-treated preparations during the 120-min exposure to 35 KP.
Thus it appears that preloaded stores of ACh remained within the
vesicles under the conditions of these experiments. We concluded
therefore that the decline in MEPC frequency was the result of a
depletion of the preformed quantal stores. We estimated that 490,000 preformed quanta were released during the 120 min in 35 KP, a value
consistent with other published values for ACh stores (reviewed in
van der Kloot and Molgo 1994
). However, our value is
somewhat larger than that reported previously (290,000) for snake
twitch motor nerve terminals (Searl et al. 1990
). This difference may reflect differences in experimental approach. We stimulated release by elevating K+, whereas Searl et al.
(1990)
estimated numbers of quanta released when nerve stimulation was
used to exhaust vesamicol-treated (5 µM) motor nerve terminals.
Our results show that vesamicol-treated snake twitch nerve terminals
accumulated FM1-43 after 120 min in 35 KP, a time when the MEPC
frequency was <1 s1. This frequency of MEPCs is well
below the frequency normally required (25-50 MEPCs s
1)
to enable twitch nerve terminals to accumulate enough FM1-43 (with the
6-min exposure) to exhibit noticeable fluorescence with our optical
system (Connor et al. 1997
). Thus, although the
frequency of synaptic currents was very low, endocytosis of synaptic
vesicle membrane must have continued at a high rate.
Betz and coworkers (Betz and Bewick 1992, 1993
;
Betz et al. 1992a
,b
) demonstrated that FM1-43
accumulates in synaptic vesicles that have undergone recycling via
endocytosis. It also was concluded that the extent of endocytosis is
dependent on the amount of preceding exocytosis. Thus the rate of
endocytosis appears to be closely coupled to the rate of exocytosis
(Betz et al. 1992b
; Wu and Betz 1996
).
The accumulation of FM1-43 at vesamicol-treated terminals after 120 min in 35 KP provided strong, albeit indirect, evidence that exocytosis
continued a high rate, although very few MEPCs were recorded.
Destaining experiments provided direct evidence that exocytosis was
continuing at a high rate. The nerve terminals were destained with
continued exposure to 35 KP. In contrast, FM1-43 fluorescence remained
in terminals that were washed in control physiological solution and not
continually stimulated by exposure to 35 KP. We have not quantitated
the time course of FM1-43 destaining in the current studies. However,
the extent of destaining we observed after 15 min is very similar to
that reported previously by Lindgren et al. (1997)
in studies of
FM1-43 staining and destaining induced by exposure to elevated
potassium at lizard nerve terminals in the absence of vesamicol.
The ability to destain at a time when recorded MEPC frequency was
consistently <1 s1 indicated that exocytosis continued
but the vesicles did not contain ACh. We conclude that at
vesamicol-treated terminals exocytosis continued at an elevated rate
throughout the 120-min exposure to 35 KP, just as that which occurred
with preparations not exposed to vesamicol but kept in 35 KP. However,
in vesamicol-treated preparations, more and more of the docked vesicles
were unfilled, recycled vesicles. Consequently, as the percentage of
the empty, docked vesicles undergoing exocytosis increased, the number
of recorded postsynaptic currents progressively declined. We conclude therefore that recycled cholinergic synaptic vesicles that were not
refilled with ACh continued to undergo exocytosis and endocytosis during prolonged K+ stimulation. Thus, like glutamatergic
terminals in cerebellar granule cells (Cousin and Nicholls
1997
), transport of transmitter, in this case ACh, into
recycled cholinergic vesicles is not a requirement for repeated cycles
of exocytosis and retrieval of synaptic vesicle membrane during
prolonged stimulation of motor nerve terminals.
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ACKNOWLEDGMENTS |
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We thank Dr. Ian G. Marshall for the gift of vesamicol and Dr. John Dempster for providing the program SCAN. We also acknowledge the helpful comments made by Dr. Marshall on an earlier version of this manuscript.
This work was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-23978 to R. L. Parsons and by a North Atlantic Treaty Organization grant to R. L. Parsons and Dr. Marshall.
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FOOTNOTES |
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Address for reprint requests: R. L. Parsons, Dept. of Anatomy and Neurobiology, University of Vermont, College of Medicine, C 427 Given Building, Burlington, VT 05405-0160.
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 23 November 1998; accepted in final form 11 February 1999.
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REFERENCES |
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