1Department of Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, Vermont 05405; and 2Department of Biology, Ithaca College, Ithaca, New York 14850
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ABSTRACT |
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Calupca, Michelle A.,
Gregory M. Hendricks,
Jean C. Hardwick, and
Rodney L. Parsons.
Role of mitochondrial dysfunction in the Ca2+-induced
decline of transmitter release at K+-depolarized motor
neuron terminals. The present study tested whether a
Ca2+-induced disruption of mitochondrial function was
responsible for the decline in miniature endplate current (MEPC)
frequency that occurs with nerve-muscle preparations maintained in a 35 mM potassium propionate (35 mM KP) solution containing elevated calcium. When the 35 mM KP contained control Ca2+
(1 mM), the MEPC frequency increased and remained elevated for many
hours, and the mitochondria within twitch motor neuron terminals were
similar in appearance to those in unstimulated terminals. All nerve
terminals accumulated FM1-43 when the dye was present for the final 6 min of a 300-min exposure to 35 mM KP with control Ca2+. In
contrast, when Ca2+ was increased to 3.6 mM in the 35 mM KP
solution, the MEPC frequency initially reached frequencies >350
s1 but then gradually fell approaching
frequencies <50 s
1. A progressive swelling and eventual
distortion of mitochondria within the twitch motor neuron terminals
occurred during prolonged exposure to 35 mM KP with elevated
Ca2+. After ~300 min in 35 mM KP with elevated
Ca2+, only 58% of the twitch terminals accumulated
FM1-43. The decline in MEPC frequency in 35 mM KP with elevated
Ca2+ was less when 15 mM glucose was present or when
preparations were pretreated with 10 µM oligomycin and then bathed in
the 35 mM KP with glucose. When glucose was present, with or without oligomycin pretreatment, a greater percentage of twitch terminals accumulated FM1-43. However, the mitochondria in these preparations were still greatly swollen and distorted. We propose that prolonged depolarization of twitch motor neuron terminals by 35 mM KP with elevated Ca2+ produced a Ca2+-induced decrease
in mitochondrial ATP production. Under these conditions, the cytosolic
ATP/ADP ratio was decreased thereby compromising both transmitter
release and refilling of recycled synaptic vesicles. The addition of
glucose stimulated glycolysis which contributed to the maintenance of
required ATP levels.
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INTRODUCTION |
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Previous studies from our laboratory have
demonstrated a calcium (Ca2+)-dependent decline in
transmitter release at potassium (K+)-stimulated snake
twitch fiber endplates (Connor et al. 1997). With
control Ca2+ (1 mM) in the K+-depolarizing
solution (35 mM potassium propionate, KP), miniature endplate current
(MEPC) frequency increased and remained elevated for many hours
(Connor et al. 1997
). In contrast, with the
Ca2+ concentration elevated in the 35 mM KP solution to 3.6 mM, the MEPC frequency increased initially to many hundreds per second but then declined progressively to much lower frequencies, although MEPC amplitudes remained unchanged. On the basis of these observations, it was hypothesized that the progressive decline in transmitter release
was related to an intraterminal accumulation of Ca2+,
which, in turn, either depressed some step in the release process or
inhibited endocytosis leading to depletion of synaptic vesicles (Connor et al. 1997
).
In an earlier study, Coniglio et al. (1993) had
demonstrated that during exposure to isotonic KP with both control and
elevated Ca2+, the number of synaptic vesicles present in
the chronically depolarized nerve terminals was consistently less than
that in resting terminals. More recently, Lindgren et al.
(1997)
demonstrated that exposure to solutions that contained
organic anions such as propionate in place of chloride resulted in
acidification of the nerve terminal cytosol. Acidification of the
terminal produced by concentrations of K propinate >100 mM reversibly
inhibited endocytotic retrieval of synaptic vesicle membrane
(Lindgren et al. 1997
). Thus the progressive decline in
transmitter release observed in the study of Coniglio et al.
(1993)
very likely resulted, at least in part, from a low
pH-induced inhibition of endocytosis and depletion of synaptic
vesicles. In contrast, with 35 mM KP, the extent of acidification of
the nerve terminal cytosol did not affect endocytosis (Lindgren
et al. 1997
). Thus the Ca2+-induced decrease in
transmitter release produced during continued exposure to 35 mM KP very
likely was due to other mechanisms (Connor et al. 1997
).
von Gersdorff and Matthews (1994) reported that an
elevation of intracellular Ca2+ directly depressed
endocytosis in goldfish bipolar cells. Thus with prolonged
depolarization, one mechanism considered previously by Connor et
al. (1997)
was that endocytosis was gradually inhibited as
Ca2+ accumulated in the nerve terminal. However, recent
results of Reuter and Prozig (1995)
and Wu and
Betz (1996)
suggested that the rate of endocytosis was
not correlated with the intracellular Ca2+ concentration
for cultured hippocampal neurons and frog motor neuron terminals. Thus
direct inhibition of endocytosis by sustained elevation of
intraterminal Ca2+ might not be the underlying mechanism.
Coniglio et al. (1993) also noted that with continued
exposure to isotonic KP (with either control or elevated
Ca2+), the mitochondria within the chronically depolarized
nerve terminals progressively became swollen and distorted.
Mitochondria take up Ca2+ when intracellular
Ca2+ concentrations rise to levels >500 nM, a mechanism
that is now recognized as an important aspect of cytosolic
Ca2+ homeostasis (Gunter and Gunter 1994
;
Gunter et al. 1994
; Herrington et al.
1996
; Park et al. 1996
; Werth and Thayer
1994
). However, when the intracellular Ca2+
concentration remains elevated for prolonged periods causing mitochondria to accumulate excessive Ca2+, the
mitochondrial membrane potential begins to diminish (Isaev et
al. 1996
; Schinder et al. 1996
;
White and Reynolds 1996
). Excessive accumulation
of Ca2+ coupled with the loss of the potential gradient can
activate the mitochondrial transition pore, which leads to
mitochondrial swelling and interruption of ATP production (Isaev
et al. 1996
; Kristal and Dubinski 1997
;
Zamzami et al. 1997
). Given that mitochondria produce
the ATP required to support energy-dependent processes in motor neuron
terminals, a progressive loss of ATP production potentially could
compromise nerve terminal function.
We considered that an alteration in mitochondrial function might be involved in the Ca2+-induced depression of transmitter release at nerve terminals exposed to 35 mM KP. Consequently, we hypothesized that in 35 mM KP with elevated Ca2+, but not control Ca2+, the depolarization-induced influx of Ca2+ into twitch motor neuron terminals might elevate intraterminal Ca2+ to levels high enough to promote excessive accumulation of Ca2+ within mitochondria. Furthermore we proposed that as the mitochondria continued to accumulate Ca2+, mitochondrial ATP production gradually became depressed, which led to a progressive decline in nerve terminal ATP levels and depression of ATP-dependent mechanisms required to sustain transmitter release.
In the present study, we have tested whether a Ca2+-induced
alteration of mitochondrial morphology and depression of ATP production contributed to the Ca2+-induced decline in transmitter
release at snake twitch motor neuron terminals maintained in 35 mM KP.
The study combines voltage-clamp recordings of MEPCs, optical assay of
vesicle membrane recycling using the activity-dependent dye FM1-43,
and electron microscopic examination of mitochondrial morphology.
Ultrastructural studies were needed because change in intraterminal
organelle morphology was not examined by Connor et al.
(1997) in their study of the decline in MEPC frequency during
exposure to 35 mM KP with elevated Ca2+.
The results of this study demonstrated that in twitch nerve terminals exposed to 35 mM KP with elevated Ca2+, but not with control Ca2+, the mitochondria progressively became swollen and distorted. Furthermore the alteration in mitochondrial morphology occurred when MEPC frequency began to decline. In addition, exposure to glucose to stimulate nerve terminal glycolysis, and thus increase ATP production, reduced the decline in transmitter release.
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METHODS |
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Experiments were performed on visually identified twitch muscle
fiber endplates in the costocutaneous muscle of garter snakes (Thamnophis) at room temperature (21-23°C). Snakes were
killed by rapid decapitation, and muscle preparations were dissected and pinned to the bottom of silicone elastomer (Sylgard)-coated plastic
dishes containing a
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)-buffered control physiological solution, which contained (in mM) 159 NaCl, 2.5 KCl, 1.0 CaCl2, 4.2 MgCl2, and 5.0 HEPES, pH 7.3 (Coniglio et al.
1993; Connor et al. 1984
, 1997
). Transmitter release was induced by exposure to a potassium depolarizing solution in
which 35 mM NaCl was replaced by 35 mM potassium propionate (KP). The
35 mM KP solution with "control" Ca2+ contained (in
mM) 126 NaCl, 35 K propionate, 1.0 CaCl2, 4.2 MgCl2, 5.0 CsCl, and 5.0 HEPES, pH 7.3 (Connor et
al. 1997
). The cesium chloride was included to facilitate
voltage clamping depolarized muscle fibers to hyperpolarized potentials
(Coniglio et al. 1993
; Connor et al. 1984
,
1997
). For most experiments in the present study, the 35 mM KP
solution contained an "elevated" Ca2+ concentration
(3.6 mM) and no added magnesium. In a few experiments, 1.6 mM magnesium
was present with 3.6 mM Ca2+ in the 35 mM KP solution.
Because the results were not affected by the presence of magnesium,
data obtained using solutions with or without magnesium were presented
together. In one series of experiments, sodium (Na+) was
replaced by lithium (Li+) in the 35 mM KP solution
containing 3.6 mM Ca2+ (Li-KP) to minimize Ca2+
extrusion by Na+-Ca2+ exchange from the motor
neuron terminals (Blaustein 1988
; Missiaen et al.
1993
).
For other experiments, muscle preparations were treated with oligomycin (10 µM, a mixture of oligomycin A, B, and C; Sigma, St. Louis, MO) to inhibit the mitochondrial ATP synthase. Oligomycin was dissolved in dimethyl sulfoxide (DMSO) as 10 mM stock aliquots, frozen, and diluted each day to 10 µM in either the control physiological solution or the 35 mM KP solution. Generally, the muscle preparations were pretreated with oligomycin in the control physiological solution for ~20 min before exposure to the 35 mM KP solutions, which also contained oligomycin. In other experiments, 15 mM glucose was added to the 35 mM KP solution.
Electrophysiology
Twitch muscle fibers were identified using criteria described in
previous reports (Coniglio et al. 1993; Connor et
al. 1984
, 1997
; Dionne and Parsons 1981
). There
are two types of twitch fibers in snake muscle: slower twitch and
faster twitch fibers (Lichtman and Wilkinson 1987
;
Wilkinson and Lichtman 1985
). In these experiments, we
did not distinguish between twitch fiber types. MEPCs were recorded
from individual twitch fiber endplates bathed in the control
physiological solution or kept for various lengths of time in 35 mM KP
(Coniglio et al. 1993
; Connor et al. 1984
,
1997
). The fibers were voltage clamped to
150 mV to increase the driving force for MEPCs and thus increase the signal-to-noise ratio. Current records were stored on a PCM recorder (A. R. Vetter, Rebersburg, PA) for subsequent digitization and analysis.
Current records were digitized using the SCAN program (generously
provided by Dr. John Dempster, University of Strathclyde, Glasgow,
Scotland), and the frequency of MEPCs for a given recording was
determined by counting events displayed on computer traces
(Connor et al. 1997
). Averaged data are expressed as
means ± SE.
Optical identification of nerve terminals and assay of synaptic vesicle recycling
Twitch neuromuscular junctions were visualized by staining with
rhodamine-conjugated peanut agglutinin (PNA, Sigma), which marks
synaptic and terminal Schwann cell basal laminae (Connor et al.
1997; Ko 1987
). Muscle preparations were exposed
to PNA (33 µg/ml; dissolved in the control physiological solution)
for ~15 min and then rinsed. Optical estimates of vesicle release and
recycling were made with the use of K+-stimulated
transmitter release coupled with accumulation of the fluorophore
FM1-43 (2 µM; Molecular Probes, Eugene, OR) into recycling synaptic
vesicles (Betz and Bewick 1992
, 1993
; Betz et al.
1992
). Muscle preparations were exposed to FM1-43 during the
last 5-6 min of an ~300-min exposure to the 35 mM KP solutions, then
washed for
15 min in the control physiological solution before
viewing.
Nerve terminals were 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. Background levels of fluorescence were established by
examining endplates that had been exposed to FM1-43 for 5-6 min in
physiological solution, a condition in which snake nerve terminal
boutons are not stained by FM1-43 (Connor et al. 1997). FM1-43 staining was used at twitch nerve terminals maintained for
>300 min in a 35 mM KP solution, which contained control
Ca2+, to establish positive FM1-43 staining. The FM1-43
was present for the final 5-6 min of exposure to the KP solution.
Previously Connor et al. (1997)
demonstrated that
transmitter release continued at high rates for many hours in a 35 mM
KP solution containing control Ca2+ (1.0 mM
Ca2+) and that all twitch nerve terminals were uniformly
stained by FM1-43. In each experiment, results from endplates stained
with FM1-43 after exposure to the experimental 35 mM KP solutions were compared with endplates maintained only in the control physiological solution or endplates exposed to the 35 mM KP solution with control Ca2+. Endplates were examined visually and scored, as in
our previous study, as uniformly stained, partially stained, or
unstained (Connor et al. 1997
). A nerve terminal was
considered uniformly stained if all boutons within a nerve terminal
were stained and considered partially stained if at least one, but not
all boutons, were stained.
Electron microscopy
Nerve-muscle preparations were fixed and prepared for
ultrastructural examination. The muscles used for electron microscopy were taken from the same snakes that had been used to provide muscles
for MEPC recordings. The methods used to examine the ultrastructure of
the motor neuron terminals at twitch fiber endplates followed those
described previously (Coniglio et al. 1993). Snake
muscles were pinned to the bottom of Sylgard-coated plastic dishes and exposed for different durations of time either to the control physiological solution or to 35 mM KP solutions. Endplates from at
least two different muscle preparations from two different snakes were
examined for each condition. Preparations were fixed for 15 min in 2%
glutaraldehyde, washed in fresh Millonig's phosphate buffer, postfixed
for 30 min in 1% osmium tetroxide, and washed again in buffer. The
preparations were dehydrated in a graded series of ethanols to 100%
and en bloc stained with 2% uranyl acetate for 5 min. After en bloc
staining, muscles were returned to 100% ethanol, then into propylene
oxide, and finally embedded in a resin mixture of Embed 812-Araldite
502 (hard) between microscope slides that had been precoated with
liquid releasing agent. After polymerization, whole mounts were removed
from between the slides and examined on a compound microscope to
identify individual endplates. Areas of the whole mount that contained
motor endplates were cut away and re-embedded into precast
Embed-Araldite blocks. These blocks then were polymerized and thick
sectioned (5 µm sections). The thick sections were placed on
Teflon-coated microscope slides and examined on a compound light
microscope to identify thick sections that contained endplates.
Appropriate thick sections were remounted onto the ends of precast
Embed-Araldite blocks. The blocks were polymerized, trimmed, and
ultrathin sectioned (78-80 nm). Thin sections were examined and
photographed on either a JEOL 100CX or 100S electron microscope.
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RESULTS |
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MEPC frequency and nerve terminal morphology during prolonged exposure to 35 mM KP with elevated Ca2+
MEPC frequency remained elevated for many hours in muscles bathed in 35 mM KP with control Ca2+ (Fig. 1A). In contrast, for muscles kept in 35 mM KP with elevated Ca2+ (3.6 mM), the MEPC frequency initially rose to high values but then declined. The time course of this decline in MEPC frequency is presented in Fig. 1A. These results were obtained by recording MEPCs from different twitch fiber endplates at different times in the KP solution.
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The morphology of the nerve terminals from muscle preparations maintained for ~480 min in 35 mM KP with normal Ca2+ (Fig. 1F) was similar to that of twitch nerve terminals in muscles maintained in the physiological solution (Fig. 1B). Generally, synaptic vesicles still were present in abundant numbers, and the mitochondria, although often larger, were not extensively swollen or distorted. In addition, the majority of the mitochondria were located some distance from the side of the boutons facing the synaptic cleft. In contrast, there was a marked time-dependent change in the morphology of the mitochondria in twitch motor neuron terminals exposed to 35 mM KP with elevated Ca2+. The mitochondria were consistently swollen but were readily discernible after ~150 min in 35 mM KP with high Ca2+. The mitochondria became more swollen (Fig. 1D) with longer exposures and often were distorted to such an extent that their internal organization was no longer apparent (Fig. 1E). In these same preparations, there was no obvious change in the morphological appearance of mitochondria in the muscle fibers (Fig. 1C). Thus the progressive swelling of the mitochondria was limited to mitochondria located in the nerve terminal. In addition, in many boutons, numerous synaptic vesicles were present both after an ~150- and ~480-min exposure to 35 mM KP with elevated Ca2+. However, the number of synaptic vesicles varied between terminals and in some boutons the density of synaptic vesicles was reduced.
The consistent change in mitochondrial morphology provided support for the hypothesis that a gradual loss of mitochondrial function might be a factor contributing to the progressive decline in MEPC frequency that occurred when preparations were kept in 35 mM KP with elevated Ca2+. Consequently, additional electrophysiological and morphological experiments were done to test this hypothesis further. In all subsequent experiments, the exposure to the 35 mM K+ solution with 3.6 mM Ca2+ ranged between 300 and 360 min because both the decline in MEPC frequency and alteration in mitochondrial morphology were evident by this time.
Exocytosis and mitochondrial morphology with lithium (Li+) substituted for sodium (Na+) in 35 mM KP with elevated Ca2+
A number of organelles and cellular processes contribute to the
regulation of the intracellular Ca2+ concentration
(Gunter et al. 1994; Missiaen et al.
1993
). With sustained intraterminal Ca2+ loading,
an important pathway for Ca2+ extrusion should be the
low-affinity, high-capacity surface membrane Na+-Ca2+ exchange mechanism (Blaustein
1988
). Substitution of Li+ for extracellular
Na+ is known to effectively inhibit
Na+-Ca2+ exchange (Park et al.
1996
). We proposed that elimination of this high-capacity
extrusion pathway in the motor neuron terminal might facilitate
intraterminal Ca2+ loading and lead to a more rapid
accumulation of Ca2+ into mitochondria and dissipation of
the mitochondrial membrane potential. Thus mitochondria might become
compromised more quickly, leading to a more rapid decline in MEPC
frequency. Consequently we initiated experiments to determine the time
course of decline in MEPC frequency when Li+ was replaced
for Na+ in the 35 mM KP solution containing elevated
Ca2+ (Li-KP).
We recorded MEPCs from different twitch fibers during continuous
exposure to Li-KP. After ~20 min in Li-KP, the MEPC frequency appeared to be <100 s1 (Fig.
2A). However, even with
exposure times <20 min, many MEPCs were very small. With longer
durations in Li-KP, the amplitude of most MEPCs continued to decline
(Fig. 2, B-D). Eventually, it became virtually impossible
to accurately estimate the MEPC frequency, although inward current
fluctuations in the noise, like those shown in Fig. 2D, were
evident in recordings from twitch endplates exposed to Li-KP for
periods
300 min. Comparable current fluctuations were not observed
when recordings were made from nonjunctional areas of the muscle fiber
(Fig. 2E).
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We compared FM1-43 incorporation into recycling synaptic vesicles at
twitch nerve terminals after an ~300-min exposure to either 35 mM KP
with elevated Ca2+ or Li-KP, also with elevated
Ca2+. The FM1-43 was included in the respective KP
solutions for the final 5-6 min of exposure, and the muscle
preparations then were bathed in physiological solution containing PNA
for ~15 min to locate the endplates on individual muscle fibers
(Connor et al. 1997). All PNA-identified nerve terminals
innervating twitch endplates in preparations maintained for >300 min
in Li-KP were stained by FM1-43. Intensity of the staining varied
between nerve terminals with 94% of the terminals uniformly stained
and 6% partially stained (Table 1). In
contrast, as previously shown by Connor et al. (1997)
,
only 58% of the nerve terminals innervating twitch endplates in
preparations exposed to 35 mM KP with elevated Ca2+ were
stained by FM1-43. Of the positively stained terminals, 44% appeared
to be uniformly stained, whereas 14% were stained partially. Forty-two
percent of the terminals did not appear to be stained by FM1-43.
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Ultrastructural studies were completed to compare the morphology of motor neuron terminals innervating twitch endplates in preparations that had been kept for ~300 min in 35 mM KP with control Ca2+ (Fig. 3A), 35 mM KP with elevated Ca2+ (Fig. 3B), or Li-KP (Fig. 3C). Synaptic vesicles were present in all three examples. Omega figures commonly were noted in nerve terminals maintained in 35 mM KP with control Ca2+ and Li-KP (Fig. 3, A and C). This latter observation indicated exocytosis still was occurring, consistent with the high percentage of terminals stained by FM1-43 under these two conditions (Table 1). The morphology of mitochondria varied among the different nerve terminals. The mitochondria were not markedly enlarged in terminals maintained in 35 mM KP with control Ca2+ for ~300 min (Fig. 3A). In contrast, the majority of mitochondria in terminals maintained in 35 mM KP with elevated Ca2+ or Li-KP for ~300 min were swollen. Furthermore in some boutons, some mitochondria were distorted to the extent that the internal organization could no longer be discerned. The distortion appeared to be greater for terminals maintained in 35 mM KP with elevated Ca2+ than in terminals kept in Li-KP. In addition, for preparations kept in Li-KP, the morphology of individual mitochondria within the same bouton varied noticeably with some only slightly swollen, whereas others appeared to be completely distorted.
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Stimulation of glycolysis can sustain transmitter release during prolonged exposure to 35 mM KP with elevated Ca2+
The dependence of transmitter release on ATP production within the nerve terminals was investigated by testing whether inhibition of the mitochondrial ATP synthase or stimulation of glycolysis affected the decline in MEPC frequency. Preparations were treated with 10 µM oligomycin alone to inhibit the mitochondrial ATP synthase, oligomycin and 15 mM glucose to inhibit the ATP synthase and to stimulate glycolysis, or glucose alone. The muscle preparations were pretreated with 10 µM oligomycin in the control physiological solution for ~15 min before exposure to the KP solution, which also contained oligomycin. Glucose (15 mM) was present only during exposure to the 35 mM KP solution.
During the initial exposure to 35 mM KP with elevated Ca2+,
MEPC frequency at twitch endplates in untreated muscle preparations reached values >350 s1. The frequency then declined
progressively during a 360-min recording period (Fig. 1A),
remaining elevated above ~200 s
1 at many endplates
until ~120 min and then falling progressively to much lower values.
For example, the MEPC frequency recorded from 10 twitch endplates in
untreated muscles during the first 120 min of exposure to 35 mM KP with
high Ca2+ averaged 209 ± 31.7 s
1.
However, after ~200 min in the 35 mM KP solution with elevated Ca2+, MEPC frequency was consistently <50 s
1
(Table 2).
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The MEPC frequency recorded at twitch endplates from muscles
treated with oligomycin alone or oligomycin plus glucose also was >200
s1 during the first 120 min in 35 mM KP containing
Ca2+. In those preparations treated with only oligomycin,
the MEPC frequency after 200 min declined to values even lower than
those recorded just in 35 mM KP with elevated Ca2+ (Table
2). In contrast, the combination of oligomycin treatment and addition
of glucose significantly reduced the decline in MEPC frequency at many
endplates (Table 2). At 15 of 24 oligomycin-treated endplates, the MEPC
frequency during a 240- to 360-min exposure to 35 mM KP with oligomycin
and glucose remained >150 s
1; whereas MEPC frequency was
<50 s
1 at only eight endplates. After a 240-to 360-min
exposure to 35 mM KP with glucose alone, the MEPC frequency remained
>150 s
1 at 10 of 28 endplates, whereas the frequency was
<50 s
1 at 15 endplates (Table 2).
FM1-43 incorporation was determined at twitch nerve terminals after an ~300-min exposure to the 35 mM KP with elevated Ca2+ in muscle preparations that had been treated with 10 µM oligomycin alone, oligomycin in combination with 15 mM glucose, or just 15 mM glucose. Approximately 60% of the nerve terminals innervating twitch endplates in the three preparations treated with oligomycin alone and maintained for >300 min in KP with high Ca2+ were stained uniformly by FM1-43 (Table 1). Another 3% were partially stained, and the remaining 37% did not accumulate FM1-43. In contrast, all twitch nerve terminals in the three preparations treated with oligomycin and glucose accumulated FM1-43. The staining was generally very strong with 95% of the nerve terminals uniformly stained and 5% partially stained (Table 1). For those preparations bathed in 35 mM KP containing just glucose, ~77% of the twitch nerve terminals were stained by FM1-43: 74% uniformly and 3% partially. The remaining PNA-identified twitch nerve terminals did not appear to be stained by FM1-43.
Ultrastructural studies also were done to compare the morphology of motor neuron terminals innervating twitch endplates for the aforementioned three treatments. All muscle preparations were fixed after an ~300 min exposure to 35 mM KP with elevated Ca2+. There was no qualitative difference in the morphology of the boutons within twitch motor neuron terminals in these three groups of muscles (Fig. 3, D-F). Synaptic vesicles were present within most nerve terminals. In all cases, the mitochondria were swollen (Fig. 4), suggesting that none of the conditions reversed the progressive alteration of mitochondrial morphology that occurred with prolonged exposure to 35 mM KP with elevated Ca2+.
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DISCUSSION |
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The objective of the present study was to elucidate mechanisms underlying the Ca2+-induced inhibition of transmitter release at 35 mM KP-depolarized motor neuron terminals. Two key observations were that coincident with the decline in MEPC frequency was a progressive swelling and distortion of nerve terminal mitochondria and stimulation of glycolysis allowed transmitter release to continue at high rates in many K+-depolarized terminals even though mitochondria were distorted. On the basis of these observations, we propose that a major factor in the Ca2+-induced progressive decline in MEPC frequency was a gradual decrease in mitochondrial ATP production and consequent decrease in the cytosolic ATP/ADP ratio, which progressively impaired ATP-dependent processes in the nerve terminal.
Mitochondria can accumulate significant amounts of Ca2+ as
cytosolic levels exceed 500 nM (Friel and Tsien 1994;
Gunter and Gunter 1994
; Gunter et al.
1994
; Herrington et al. 1996
; Kiedrowski and Costa 1995
; Park et al. 1996
; Wang
and Thayer 1994
; White and Reynolds 1995
).
However, if mitochondrial Ca2+ exceeds mM levels,
morphological alterations, similar to those observed in this study,
were observed in cerebellar granule cells (Isaev et al.
1996
). Thus when bath Ca2+ was elevated in
the present study, Ca2+ accumulation within the nerve
terminal must have been high enough to exceed the various
Ca2+-buffering mechanisms within the nerve terminal. The
result of the excess Ca2+ in the terminal cytosol was a
progressive Ca2+ overload of the mitochondria. In contrast,
even though Ca2+ influx continued with prolonged
depolarization in 35 mM KP with control Ca2+, the
Ca2+ buffering mechanisms that regulate intraterminal
Ca2+ must have been sufficient to avoid extensive
Ca2+ accumulation.
Accumulation of FM1-43 into the nerve provided evidence that
endocytotic retrieval of synaptic vesicle membrane was occurring (Betz and Bewick 1992, 1993
). Previously, we
demonstrated that if transmitter release is not occurring at rates well
above the resting level, then constitutive endocytosis of vesicle
membrane is not sufficient to stain snake motor neuron terminals
(Connor et al. 1997
). Thus we have interpreted positive
FM1-43 staining to indicate that both exocytosis and endocytosis were
occurring at a highly accelerated rate. Under most experimental
conditions used in the present study, the percentage of twitch motor
neuron terminals positively stained by FM1-43 was consistent with the MEPC frequency values obtained after >300 min in the 35 mM KP with 3.6 mM Ca2+; i.e., the higher the mean MEPC frequency, the
greater the percentage of terminals positively stained (compare results
in Tables 1 and 2). The exceptions to this general correlation were
that in the oligomycin-treated preparations, ~60% of the terminals were FM1-43 positive and in the Li-KP preparations 100% of the terminals were stained. In both cases, FM1-43 staining occurred when
MEPC frequency was below the rate normally required to stain terminals.
It could be, although we consider it unlikely, that in the
oligomycin-treated nerve terminals, endocytosis was stimulated independently of exocytosis. However, the number of synaptic vesicles present in the nerve terminals in the oligomycin-treated preparations was not greater than that seen in nerve terminals in other
preparations, which also were exposed to 35 mM KP with elevated
Ca2+ but not treated with oligomycin (Fig. 3). A greater
number of vesicles would be expected if endocytosis was occurring at a
high rate but exocytosis was not equally enhanced. We favor an
alternative explanation. We suggest that exocytosis was occurring at
numerous terminals, but the synaptic vesicles had not been refilled,
and the only MEPCs recorded were those from vesicles still remaining in
preformed quantal stores. ATP is required for refilling of recycling
synaptic vesicles (Parsons et al. 1993
). Given that the
preparations were pretreated with oligomycin for
15 min in control
solution, mitochondrial ATP production should have been inhibited
before exposure to 35 mM KP. We propose that with ATP production
inhibited, intraterminal ATP levels very likely would have fallen
substantially after ~240 min in the elevated K+ solution,
and as a consequence, recycling vesicles would not have been refilled
with acetylcholine (ACh) in these terminals.
MEPC amplitudes decreased progressively during exposure to the Li-KP
solution, which also contained elevated Ca2+. We attributed
this to a gradual depletion of choline stores in the nerve terminal and
a reduced filling of synaptic vesicles. Choline uptake into nerve
terminals is a Na+-dependent process and is inhibited when
Li+ is substituted for Na+ (van der
Kloot and Molgo 1994). Therefore we suggest that the apparent
decrease in MEPC frequency resulted mostly from the progressive release
of increasingly smaller quanta of ACh until most events were lost in
the recording noise and not from an inhibition of exocytosis. This very
progressive decrease in MEPC amplitude is similar to that produced when
choline uptake is inhibited pharmacologically, such as with
hemicholinium (Elmqvist and Quastel 1965
). Evidence for
continued exocytosis of partially filled or empty vesicles was obtained
both from the positive FM1-43 staining (Table 1) and presence of
synaptic vesicles and omega figures (Fig. 3). Also we suggest that the
current fluctuations recorded after prolonged exposure to Li-KP most
likely represented current responses produced by ACh released from
partially filled synaptic vesicles.
At present, we only can speculate why exocytosis continued in motor
neuron terminals exposed to Li-KP. From inspection of mitochondria in
nerve terminals exposed to the different conditions, it appeared that
the numbers and extent of severely distorted mitochondria might be less
in the terminals exposed to the Li+-substituted solution.
Consequently, it is possible that exocytosis continued simply because
there was slightly less extensive physical disruption of the
mitochondria and ATP production continued for longer periods. Even
though this possibility could not be adequately quantitated with the
present data, a potential mechanism by which mitochondria might be
spared somewhat in Li-KP can be considered. External Na+ is
required to sustain cell membrane Na+-H+
exchange, which would transport H+ out of the nerve
terminal to maintain cytoplasmic pH within a physiological range. With
prolonged exposure to the Li+-substituted solution, the
intraterminal H+ concentration should increase
progressively, causing the intraterminal pH to decrease. A decrease in
pH protects against activation of the mitochondrial transition pore,
thus decreasing the likelihood of mitochondrial swelling and loss of
membrane potential (Bernardi et al. 1994; Nicolli
et al. 1993
). Therefore in Li-KP, the integrity of some
mitochondria might be prolonged and ATP production might have continued
longer even though the mitochondria had accumulated amounts of
Ca2+ that normally would cause activation of the transition
pore. If this explanation is valid, then the cytoplasmic ATP/ADP ratio might have been maintained longer, which could support ATP-dependent mechanisms.
The most extensive decline in MEPC frequency was observed when the preparations were pretreated with oligomycin to inhibit the ATP synthase (Table 2). The mitochondria in motor neuron terminals in these preparations were very distorted. We postulate that when the ATP synthase was inhibited, the mitochondria had no mechanism available to maintain the membrane potential. Thus the combination of the extreme Ca2+ accumulation and decline in membrane potential facilitated activation of the transition pore and led to the marked alteration in mitochondrial morphology and loss of function.
MEPC frequency remained high at many terminals when glucose was added
to the 35 mM KP solution with elevated Ca2+, even though
marked morphological changes in the mitochondria were still evident. We
suggest that the presence of glucose did not protect mitochondrial
function but rather stimulated glycolysis, which helped to maintain
cytoplasmic ATP levels. The combination of glucose and pretreatment
with oligomycin further enhanced the ability of terminals to maintain
transmitter release at high rates. Therefore when the ATP synthase was
inhibited and thus could not operate in a reverse mode, cytosolic ATP
was not consumed by the mitochondria in an attempt to maintain the
membrane potential (Budd and Nichols 1996) and the
glucose-stimulated glycolysis must have sustained cytosolic ATP levels
more effectively.
In recent years, evidence has accumulated that strongly suggests that
an alteration in mitochondrial function may be a critical factor in
programmed cell death and in the etiology of many neurodegenerative diseases (Beal 1992; Kristal and Dubinski
1997
; Orrenius and Nicotera 1994
; Petit
et al. 1996
; Zamzami et al. 1997
). Impairment of
energy metabolism and loss of Ca2+ buffering by
mitochondria have been suggested to be key factors in excitotoxic
neuronal death (Kiedrowski and Costa 1995
;
Schinder et al. 1996
; White and Reynolds
1996
). Mitochondria are a critical Ca2+ buffering
mechanism during glutamate-induced Ca2+ loading
(Kiedrowski and Costa 1995
; Wang and Thayer
1996
; White and Reynolds 1995
). However, if the
buffering capacity of mitochondria is overwhelmed, then the
mitochondrial membrane potential can collapse and marked
ultrastructural changes in the mitochondria occur (Isaev et al.
1996
; Schinder et al. 1996
). Thus
accumulation of excess Ca2+ by mitochondria and subsequent
activation of the permeability transition pore may be a critical early
event in the development of glutamate-induced excitotoxicity of neurons
within or derived from the CNS (Kristal and Dubinski
1997
; Zamzami et al. 1997
). The alteration in
mitochondrial morphology and disruption of ATP metabolism produced in
cultured neurons during glutamate-induced excitotoxicity are similar to
those observed in the present study for motor neuron terminals exposed
to the 35 mM KP solution with elevated Ca2+ (Isaev
et al. 1996
; Schinder et al. 1996
).
It is also well documented that after disruption of blood flow to areas
within the brain, ischemia develops and extracellular K+
levels rise dramatically in the affected area (Kristian and
Siesjo 1996; Sweeney et al. 1995
). Therefore in
addition to becoming deprived of oxygen and glucose, the elevation in
extracellular K+ very likely causes a prolonged
depolarization of neurons and axons passing through this region. The
effects of prolonged depolarization on the nerve terminals in an
ischemic region within the brain would be extremely difficult to study
because of their small size. Thus we suggest that the isolated motor
neuron nerve terminal is a convenient model system to analyze changes
in presynaptic function by conditions that produce mitochondrial
dysfunction.
In conclusion, the results of the present study show that a Ca2+-dependent disruption of mitochondrial morphology and function could be produced in isolated, chronically depolarized motor neuron terminals. The alteration in presynaptic function was probably not due to a direct effect of the elevated intraterminal Ca2+ concentration per se but more likely was a secondary consequence of the Ca2+-induced disruption of mitochondrial metabolic activity.
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ACKNOWLEDGMENTS |
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We thank Dr. John Dempster for providing the program SCAN. We also acknowledge helpful discussions during the course of this study and critical comments on the manuscript by L. Merriam and Drs. Ian Marshall and Chris Prior and thank L. Merriam for expert technical assistance in some experiments and with data analysis.
This work was supported in part by National Institute of Neurological Disorders and Stroke Grant NS-23978 to R. L. Parsons and a North American Treaty Organization Grant to R. L. Parsons and Dr. I. Marshall.
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FOOTNOTES |
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Address for reprint requests: R. L. Parsons, Dept. of Anatomy and Neurobiology, College of Medicine, University of Vermont, Burlington, VT 05401.
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 indicated this fact.
Received 28 July 1998; accepted in final form 29 October 1998.
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REFERENCES |
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