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The Journal of Neuroscience, August 1, 2002, 22(15):6491-6498
The Zebrafish Motility Mutant twitch once Reveals
New Roles for Rapsyn in Synaptic Function
Fumihito
Ono1,
Anatoly
Shcherbatko1,
Shin-ichi
Higashijima2,
Gail
Mandel2, and
Paul
Brehm1
1 Department of Neurobiology and Behavior and
2 Howard Hughes Medical Institute, State University of New
York at Stony Brook, Stony Brook, New York 11794
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ABSTRACT |
Upon touch, twitch once zebrafish respond with one
or two swimming strokes instead of typical full-blown escapes. This
use-dependent fatigue is shown to be a consequence of a mutation in the
tetratricopeptide domain of muscle rapsyn, inhibiting formation
of subsynaptic acetylcholine receptor clusters. Physiological analysis
indicates that reduced synaptic strength, attributable to loss
of receptors, is augmented by a potent postsynaptic depression not seen
at normal neuromuscular junctions. The synergism between these two
physiological processes is causal to the use-dependent muscle fatigue.
These findings offer insights into the physiological basis of human
myasthenic syndrome and reveal the first demonstration of a role for
rapsyn in regulating synaptic function.
Key words:
tetratricopeptide repeats; synaptic depression; myasthenia gravis; synapse development; muscle fatigue; rapsyn
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INTRODUCTION |
The emergence of zebrafish as a
vertebrate model for the study of nervous system development is
attributable primarily to the readily available supply of
genetic mutants (Granato and Nusslein-Volhard, 1996 ). From a
physiological standpoint, a critical advantage offered by this species
is the speed at which the nervous system develops. Thus, the functional
consequences of many mutations in neuronal and synaptic proteins that
are lethal in mammals can be examined in fish before death occurs. Such
has been the case for functional knock-outs of voltage-dependent sodium
channels (Ribera and Nusslein-Volhard, 1998), nicotinic acetylcholine
receptors (AChRs) (Westerfield et al., 1990; Ono et al., 2001a), and
acetylcholinesterase (Behra et al., 2002). Despite these proofs of
principal, relatively few studies have used physiological approaches to
determine the underpinnings of mutant behavior. This seems paradoxical
in light of the fact that so many of the mutants were originally
identified on the basis of defects in swimming behavior (Granato et
al., 1996). Any incertitude about whether the culprit gene could
eventually be identified purely on the basis of physiological
investigations has been quelled by the success of studies on paralytic
mutant fish. Physiological analyses of the paralytic mutant lines
nic-1 (Westerfield et al., 1990), sofa potato,
and relaxed (Ono et al., 2001a) have identified defects in
nicotinic receptors and muscle dihydropyrine receptors. In fact, the
successful identification of mutations underlying defects in motility
has stemmed, in part, from the large body of knowledge about key
proteins involved in nerve-muscle communication. In particular, more
is known about the neuromuscular junction than any other synapse, and
many of the genes encoding key players have been cloned (Burden, 1998; Sanes and Lichtman, 1999, 2001). However, even more important than
identification of the mutation, physiological analyses of the mutant
phenotypes have revealed new functional roles played by well
characterized postsynaptic proteins (Ono et al., 2001a).
Here we used physiological analysis of twitch once fish
neuromuscular synaptic transmission to identify the gene responsible for fatigue in swimming. Our findings identify a mutation in the muscle
protein rapsyn that is responsible for subsynaptic clustering of the
AChR. Disruption of the rapsyn-receptor interaction leads to a diffuse
organization of receptors within the muscle membrane, consistent with
the well established role of rapsyn in receptor clustering (Froehner et
al., 1990; Phillips et al., 1991). However, disruption of
rapsyn-receptor interaction in vivo revealed an additional,
unexpected role for rapsyn in synaptic function. In addition to
lowering synaptic strength through a reduction in subsynaptic receptor
density, interference with rapsyn-receptor interaction led to
pronounced frequency-dependent synaptic depression. This important
additional role played by rapsyn likely went undetected because rapsyn
knock-out mice die before functional synapse formation (Gautam et al.,
1995; Nguyen et al., 2000). Thus, the use of mutant zebrafish has
disclosed a new role for rapsyn.
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MATERIALS AND METHODS |
The distribution of muscle rapsyn and AChRs was determined using
fluorescence microscopy. Rapsyn distribution was measured using either
stable or transient expression. For stable expression of rapsyn
fluorescence, a line of fish was used that expressed a murine
rapsyn-green fluorescent protein (GFP) fusion protein under control of
a muscle  -actin promoter (Ono et al., 2001a). For transient
expression, cDNA coding for fish rapsyn-GFP fusion protein was injected
into 1-16 cell stage fertilized eggs. Observation of GFP and
rhodamine-based fluorescence was done as described previously (Ono et
al., 2001a). For imaging of fish swimming, digital images were taken at
the rate of 1000 frames/sec.
To clone the gene encoding zebrafish rapsyn, PCR primers corresponding
to conserved amino acids were synthesized. The amplified PCR product
from 5-d-old zebrafish cDNA was subcloned and sequenced. 5' rapid
amplification of cDNA ends (RACE) and 3' RACE (Clontech, Palo
Alto, CA) were performed to obtain the whole coding region. The GenBank
accession number for zebrafish rapsyn is AB070208. This gene was used
to genotype individual fish after confocal imaging or video imaging. To
do this, decapitated fish were placed in solution containing 10 mM Tris, pH 8.5, 0.2% Triton X-100, and 0.2 mg/ml
Proteinase K. After 1 hr incubation at 50°C, the reaction tube was
boiled for 1 min and centrifuged at 10,000 × g for 1 min. The extracted genomic DNA was amplified by PCR with rapsyn primers
flanking the region of G130E mutation. Amplified products were run on
agarose gel, isolated, purified, and sequenced.
To construct plasmids for transient rescue experiments, the zebrafish
-actin promoter (Higashijima et al., 1997), Kozak sequence, zebrafish rapsyn gene, enhanced GFP (EGFP), and simian virus 40 early polyadenylation signal were ligated such that rapsyn and EGFP are
in frame. The fish rapsyn sequence with G130E mutation was amplified
from twitch once
(two /) fish cDNA using Pfu DNA
polymerase (Stratagene, La Jolla, CA). Wild-type (WT) rapsyn
sequence was made by mutating the 130 E back to G. The regions coding
for rapsyn were sequenced for both wild-type and G130E constructs.
Plasmids were linearized upstream of -actin promoter sequence. DNA
(25 ng/µl) was pressure injected into 1-16 cell stage embryos
following methods described previously (Higashijima et al., 1997).
Evoked and spontaneous synaptic currents were recorded from muscle as
described previously, with slight modifications (Ono et al., 2001a).
Decapitated 7- to 10-d-old fish were fixed on sylgard with tungsten
needles in 100% HBSS. Skin was peeled from one side of the fish
with tungsten needles. The fish was treated with 2 M
formamide for 5 min to stop movement of muscle cells. After treatment,
the fish was washed vigorously in HBSS and placed in a recording
solution containing the following (in mM): 110 NaCl, 4 CaCl2, 2 KCl, 1 MgCl2, 4 glucose, and 5 HEPES, pH 7.4. The pipettes were filled with an internal
solution containing the following (in mM): 120 KCl, 5 HEPES, and 5 BAPTA, pH 7.1. Muscle cells were voltage clamped at  90
mV for miniature endplate current (mEPC) recordings and at 50
mV for EPC recordings. Holding muscle at 50 mV minimizes any possible
contamination by muscle sodium current in response to electrical stimulation.
For stimulation, two insulated 0.005-inch-diameter platinum-iridium
electrodes were positioned above and below the fish so that the current
flow was directed through the spinal cord. Care was taken to ensure
that the stimulating and recording electrodes were located in the same
muscle segment. A Grass stimulator (model SD5) was used to deliver the
current. The pulse duration was set to 0.3 msec, and the amplitude was
gradually increased until no additional incremental increase in EPC
amplitude was seen. This ensured that all of the motor units
innervating that muscle cell were recruited. The stimulus intensity
required for complete recruitment of the motor neurons generally
corresponded to 30-50 V. All experiments were performed in compliance
with institutional guidelines.
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RESULTS |
Homozygous twitch once
(two/) mutants failed to exhibit
the spontaneous swimming behavior normally acquired at 3 d of age
(Granato et al., 1996). These mutant fish were also unable to mount a
full-blown escape response to tail touch, which is acquired normally
within the first 3 d of age (Fig.
1). High-speed cinematic analyses of swimming patterns showed that
two/ fish executed only one or
two bilateral flips of the tail before the escape response was abruptly
terminated (Fig. 1). An inactive period followed, during which the fish
were unresponsive to additional mechanical prodding. The lack of
control over voluntary responses results in death when the fish reach
~14 d of age. The truncated escape behavior of the mutant fish
differs from wild-type behavior wherein a series of repetitive tail
flips in response to tail touch rapidly propels the fish out of the
field of view (Fig. 1). Such obvious and consistent differences in
behavior between the mutant and wild-type fish provided an easy initial
screen for identification of
two/ fish for additional
experimentation.
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Figure 1.
Comparisons of wild-type and
two/ fish escape responses. Five-day-old
wild-type fish (top) and
two/ mutant fish
(bottom) were each mechanically prodded to elicit escape
responses. The swimming response by each fish was recorded at the
indicated times (in milliseconds) after the stimulus. Superimposed
images of selected frames are indicated to the
right.
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Synaptic currents are reduced in
two/ fish
Recordings of synaptic currents in myotomal muscle revealed
striking differences between wild-type and
two/ fish. Using whole-cell
recordings from individual muscle cells, both the spontaneous (mEPC)
and evoked (EPC) synaptic currents in mutants were compared with those
of wild-type fish (Fig. 2). The evoked
endplate currents were generated in response to suprathreshold extracellular stimulation of the spinal cord, thus ensuring that all of
the primary and secondary motor neuron inputs to the individual muscle
cell were activated (Ono et al., 2001a). The mean EPC amplitudes corresponded to 3.7 nA (n = 12) for wild-type and 0.48 nA (n = 13) for
two/ fish (Fig. 2). This average
difference of 7.7-fold was highly statistically significant.
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Figure 2.
Spontaneous and evoked synaptic currents in
wild-type and two/ muscle. The
properties of EPCs and mEPCs are compared for wild-type
(left) and two/
(right) fish. Top, Representative EPCs
and mEPCs. The arrow indicates the position of the EPC
after the stimulus artifact. Calibration: EPC, 2 msec, 5 nA; mEPC, 100 msec, 100 pA. Middle, Frequency histograms of mEPC
amplitude for wild-type and two/
fish. Bottom, Scatter plot showing the largest EPC
amplitude from each recording in 12 wild-type and 13 two/ different fish. The overall
average ± SD for wild-type and
two/ fish are indicated by the
diamonds and bars.
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Both the amplitude and frequency of spontaneous mEPCs differed between
wild-type and two/ fish.
Specifically, the mEPC frequency was much lower in
two/ fish (Fig. 2). Furthermore,
as seen for the EPCs, the average mEPC event amplitude was greatly
reduced (Fig. 2). In wild-type fish, the mEPC amplitudes ranged from 23 to 752 pA, with the smaller-amplitude mEPCs contributing the majority
of the events. In two/ fish, it
was difficult to obtain large numbers of mEPCs in a single recording
attributable to a low frequency of detectable events. For those mEPCs
detected, the amplitudes ranged from 23 to 143 pA, representing an
~2.7-fold reduction in mean amplitude compared with wild-type muscle.
It is likely that the apparent reduction in mEPC frequency was a
consequence of this decreased amplitude, such that only the largest
events were able to be distinguished from the noise.
Postsynaptic ACh receptors are diffusely distributed in
two/ zebrafish
The postsynaptic distribution of ACh receptors was compared
between two/ and wild-type fish
to identify any differences that might account for the reduction in
synaptic response amplitude. For this purpose, the AChRs in myotomal
muscle were labeled with rhodamine-conjugated -bungarotoxin
(Rh--Btx) (Ono et al., 2001a). The muscle was exposed to toxin by
selective removal of skin on one side of the fish such that the muscle
on the other, intact side of the fish could be viewed using confocal
scanning microscopy. In this manner, the fluorescence distribution of
AChRs was determined without disturbing the muscle, nerve, or
endplates. For the purpose of orientation, a region of tail comprising
three to four myotomes and the individual muscle cells are shown in
Figure 3. The labeled receptors in
5-d-old wild-type fish showed well organized clusters on the muscle
cell surface. The predominant labeling is observed at the ends of
muscle cells, as well as punctate locations along the surface. These
regions correspond to the locations of neuromuscular junctions (Ono et
al., 2001a). In contrast, the receptors in 5-d-old two/ myotomal muscle were
diffusely distributed along the plasma membrane edge of muscle (Fig. 3)
(see Fig. 7). The apparent clusters within the muscle cells likely
represent optical slices through the invaginating T-tubular membrane
rather than true receptor clusters. Unlike the wild-type fish examined,
there was no evidence for formation of subsynaptic receptor aggregates
in two/ fish between the ages of
4 and 8 d.
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Figure 3.
Distribution of ACh receptors in the myotomal
muscle of wild-type and two/
fish. Top, A differential interference contrast image of
a section of the tail musculature is shown for orientation of the
fluorescence images. This image spans approximately four chevron-shaped
segments or myotomes and is are similar to the image area of the accompanying fluorescence photos. Layers of individual
muscle cells can be resolved between the myocomata that separate tail
segments. Confocal fluorescence image showing the distribution of
rhodamine-conjugated -bungarotoxin labeling in the tail of wild-type
(middle) and two/
mutant (bottom) fish. Scale bar, 50 µm.
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The lack of synaptic receptor aggregates could be a consequence of
reduced innervation by motor neurons, although some innervation would
have to be maintained because of the presence of synaptic responses. To
examine the innervation pattern, we exploited the availability of a
stable line of zebrafish expressing GFP under control of the
neuronal-specific Islet-1 gene promoter (Higashijima et al.,
2000). In this line, the majority of secondary motor neurons are
brightly fluorescent (Ono et al., 2001a). This line was crossed with
the twitch once zebrafish line to produce mutant fish with GFP fluorescent motor neurons. No obvious differences were observed in
the motor neuron patterns of wild-type and
two/ fish expressing GFP (data
not shown). Moreover, no clusters of AChRs colocalized beneath the
nerve terminals in two/ fish,
further distinguishing this mutant from wild-type fish (Ono et al.,
2001a). Rapsyn is a subunit associated with AChRs that is required for
subsynaptic clustering of the receptor (Burden et al., 1983; Froehner
et al., 1990; Phillips et al., 1991; Gautam et al., 1995). The unusual
AChR distribution in two/ muscle
offered a unique opportunity to test, in normally innervated muscle,
whether extrasynaptic AChRs have the ability to associate with rapsyn.
To test this idea, we once again exploited a preexisting stable line of
zebrafish. This line expresses exogenous murine muscle rapsyn fused to
GFP. The rapsyn-GFP fusion protein expresses to very high levels
specifically in skeletal muscle because it is under control of the
muscle actin promoter (Ono et al., 2001a). In wild-type fish
background, the rapsyn-GFP faithfully colocalized with the high-density
subsynaptic AChRs, and expression of either nonsynaptic AChR or
rapsyn-GFP was minimal (Ono et al., 2001a). In progeny from
matings of
two+// rapsyn-GFP fish
with two+/ fish, 50% of the fish
with two/ swimming patterns were
expected to also express the rapsyn-GFP gene. This assumes that the
genes sort independently. In fact, of 314 offspring tested, 16% of the
embryos showed a swimming pattern typical of
two/ fish, but in
none of these fish was rapsyn-GFP fluorescence detected. All fish
showing rapsyn-GFP-positive fluorescence in myotomal muscle were able
to mount escape responses. More careful measurements of swimming
patterns indicated that ~25% of the GFP-positive fish exhibited escape responses that were more sluggish than typical wild-type responses (Fig.
4A). These collective
findings suggested that the rapsyn-GFP transgene rescued the
twitch once phenotype. Subsequent genotypic analysis of the
sluggish zebrafish (see below) confirmed that these rescued fish were
two/. Additionally, Rh--Btx
labeling in these sluggish fish indicated a wild-type-like distribution
of AChRs. Specifically, the receptors were effectively clustered at
synaptic locations in the muscle (Fig. 4B).
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Figure 4.
Stable expression of a murine rapsyn-GFP fusion
protein rescues two/.
A, The escape response of a
two/ fish expressing murine
rapsyn-GFP is recorded at day 5. Sequential images taken at 20 msec
intervals are shown after the start of movement. B,
Confocal fluorescence images showing the red
fluorescence associated with AChRs (left) and the
green fluorescence associated with rapsyn-GFP
(middle) in the fish shown in A. The
merged images (right) reveal the striking colocalization
of the receptor-rapsyn distribution in this
two//rapsyn-GFP fish.
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Mutant rapsyn self-aggregates but fails to aggregate AChRs
To further test the idea that a rapsyn defect was causal to the
twitch once phenotype, rapsyn cDNA from wild-type zebrafish was cloned on the basis of homology with other species. Translated amino acid sequence of cloned zebrafish rapsyn showed 74% similarity with mouse rapsyn. Comparison of the amino acid sequences between wild-type and twitch once rapsyn revealed a single amino
acid difference at position 130 wherein a glycine (G) in the wild-type sequence was replaced by a glutamate residue (E) in the mutant sequence
(Fig. 5). It was the presence of this
mutation that allowed us to genotypically distinguish
two/ from wild-type or
two+/ fish in the behavioral and
cell biological analyses described above (Figs. 4, 5B)
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Figure 5.
Identification of the mutation in the
twitch once rapsyn gene. A, The rapsyn
protein motifs are shown, as well as the location of twitch
once (twoth26e) mutation in
the TPR domain. The 34 residue sequence of the fourth TPR domain is
shown. In twitch once rapsyn, the eighth residue in the
fourth TPR domain is mutated from glycine to glutamate.
B, Raw chromatogram of sequences used to genotype
wild-type fish and two/
fish.
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The G130E mutation corresponds to the eighth amino acid in the fourth
tetratricopeptide repeat domain in rapsyn (TPR) (Pont-ing and
Phillips, 1996), a position known to be important for the folding of
two helices within each TPR domain (Fig. 5A) (Das et
al., 1998). Our results suggested the possibility that the G130E
mutation was critical for rapsyn function. To confirm that rapsyn was
responsible for the mutant phenotype, wild-type (WT rapsyn-GFP) and
mutant (G130E rapsyn-GFP) rapsyn cDNAs fused with GFP were transiently
expressed in myotomal muscle of
two/ zebrafish embryos. The
rapsyn-GFP transgene was expressed specifically in myotomal muscle
because it was under control of the -actin promoter (Higashijima et
al., 1997). Plasmids were injected into 1-16 cell stage embryos, and
the fluorescence distribution from the rapsyn-GFP protein was compared
with Rh--Btx-labeled AChRs in 5-d-old fish. In transient assays such
as these, the transgene is expressed in a stochastic manner such that
there is mosaicism within the muscle, with some cells expressing the
transgene and others not. The mosaicism was fortuitous, permitting
comparison between effects of rapsyn-GFP expression in different muscle
cells of the same fish.
When rapsyn-GFP cDNA was injected into fish, transgene expression was
first observed at 2 d after the injection (Fig.
6). The level of transgene expression was
highly variable among the muscle cells. Clusters of rapsyn were easier
to identify in those muscle cells that expressed somewhat lower levels
of rapsyn-GFP (Fig. 6). Tail muscles in these fish were subsequently
colabeled with Rh--Btx to examine the distribution of the AChRs in
muscle cells with and without exogenous rapsyn-GFP. In >100
twitch once muscle cells that expressed exogenous wild-type
rapsyn-GFP, we observed well defined large AChR clusters (Fig. 6).
These clusters were similar in number and position to those present in
labeled muscle cells from wild-type fish. In the same
two/ fish, we examined the AChR
distribution in muscle cells that lacked GFP fluorescence, indicating
the absence of expressed WT rapsyn-GFP (Fig. 6). In all cells examined,
we observed no evidence of bona fide AChR clusters. Instead, the
receptors were homogenously and diffusely distributed over the entire
plasma membrane surface of the muscle. This distribution was
indistinguishable from that observed in
two/ fish without a transgene
(Fig. 3).
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Figure 6.
Transient expression of wild-type rapsyn in
two/ fish. 1-16 cell stage fish
embryos were microinjected with DNA constructs encoding wild-type
rapsyn-GFP. Fish embryos were examined by confocal microscopy on day 5 for distribution of rapsyn and receptors. Confocal fluorescence images
of red rhodamine-conjugated -bungarotoxin
(A), green rapsyn-GFP
(B), and merged images (C).
Note the presence of punctate clusters of both receptor and rapsyn that
appear to colocalize in the merged image. Scale bar, 50 µm.
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Distinctly different results were obtained when G130E rapsyn-GFP was
injected into two/ fish (Fig.
7). In all cells examined, the
distribution of AChRs remained diffuse, even in muscle cells expressing
the exogenous G130E rapsyn-GFP (Fig. 7). These results show
definitively that the G130E mutation was causal to the twitch
once phenotype. The distribution of the expressed G130E rapsyn-GFP
in two/ muscle also yielded
insights into the probable distribution of the endogenous mutant
rapsyn. The exogenously expressed G130E rapsyn-GFP was present at high
levels throughout the plasma membrane of muscle, even entering into the
T-tubules (Fig. 7C). High-resolution images revealed that,
unlike the homogenous diffuse organization of the AChRs, mutant rapsyn
formed some small microaggregates in the muscle membrane. These rapsyn
microaggregates failed to cluster AChRs along the edges of the muscle
cells (Fig. 7B,C), indicating that
the principal action of the mutation was to block the rapsyn-receptor
association. In other words, there is no difference of AChR
distribution between areas of plasma membrane with and without rapsyn
microclusters. However, the smaller size of the mutant rapsyn clusters,
compared with wild-type clusters, suggested that the point mutation in
the TPR domain also interfered with the ability to form large homomeric
clusters.
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Figure 7.
Transient expression of G130E rapsyn in
two/ fish. 1-16 cell stage fish
embryos were microinjected with DNA constructs encoding G130E
rapsyn-GFP. Fish embryos were examined by confocal microscopy on day 5 for distribution of rapsyn and receptors. A,
Low-power confocal fluorescence images of red
rhodamine-conjugated -bungarotoxin (left),
green rapsyn-GFP (middle), and merged
images (right). Note the absence of localized rapsyn and
receptor in the low-power images. Intermediate
(B) and higher (C)
-magnification images show small clusters of green
rapsyn, with no associated clusters of red receptor,
along the edge of muscle. The presence of green clusters
on a diffuse red background leads to
yellow clusters on the merge. The coincident
green and red fluorescence within the
muscle represents an optical slice through invaginating T-tubular
membrane. Scale bar: A, 50 µm; B, 10 µm; C, 5 µm.
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Synaptic depression underlies the use-dependent fatigue in
mutant fish
Is there a physiological effect of mutated rapsyn? The synaptic
responses observed for twitch once muscle were greatly
reduced in amplitude compared with those in wild-type fish (Fig. 2).
However, a reduction in amplitude alone could not account for the
defective swimming behavior in these mutant fish. Twitch
once fish generate one or two power strokes before the onset of
fatigue, suggesting that the initial muscle responses are stronger than
subsequent responses. To test for the ability of muscle to follow
high-frequency volley from the nervous system, motor neurons were
stimulated with 50 Hz trains, close to the native firing rate for the
motor swimming muscles (Fig. 8). Each 50 Hz train was composed of 10 pulses, and the trains were separated by 10 sec intervals. Whole-muscle patch-clamp recordings measured the
associated EPCs.
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Figure 8.
Endplate synaptic currents in
two/ muscle exhibit marked
depression in response to 50 Hz stimulation. A, Ten
consecutive endplate currents (top traces) recorded in
response to 50 Hz pulse train applied to the spinal cord. The timing of
the stimuli relative to the whole-cell muscle recordings is shown in
the bottom traces. The spinal cord was stimulated
extracellularly using 300 µsec, 30 V pulses. The muscle cells were
held at 50 mV to minimize contamination by sodium current. Note the
depression of endplate current amplitude in twitch once
muscle compared with wild type. Calibration: 20 msec, 100 (wild type)
or 50 (twitch once) pA. B, The
average ± SE ratio of each successive EPC to the amplitude of the
first EPC recorded. The EPC ratios for wild-type fish are represented
by filled circles (n = 80 from 8 cells), and two/ fish are
represented by open circles (n = 70 from 7 cells). Each average is based on 10 consecutive traces in a
single muscle cell. *p < 0.01; Student's
t test. C, The scatter plot relating
maximum EPC amplitude recorded from each
two/ fish versus the amount of
depression observed. The depression is indicated by the ratio of the
averaged 10th EPC amplitude to the averaged first EPC recorded in the
train. Each average is based on 10 consecutive traces in a single
muscle cell.
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Recordings from eight wild-type fish indicated no significant changes
in the average peak amplitudes of 10 successive EPCs (Fig.
8A,B). There was some variability
observed within the train, but the average of several individual trains
demonstrated that the successive EPCs were statistically invariant in
amplitude. Recordings from seven
two/ fish indicated that a
majority of muscle cells were not able to follow 50 Hz stimulation
without signs of synaptic depression (Fig.
8A,B). In six of seven recordings,
decreases in average peak EPC amplitude occurred with successive pulses
until an apparent plateau was reached by the end of the train (Fig.
8B). The tendency to depress was observed in all of
the individual runs, but the average of the individual runs shows a
smooth trend. The greatest depression in synaptic current amplitude
occurred between pulses 1 and 2. However, significant depression
continued throughout the successive EPCs in the train (Fig.
8B). In contrast, one of the seven
two/ muscle recordings exhibited
no statistically significant depression during the train. Thus, the
ability to exhibit depression is not shared by all of the mutant synapses.
The question arose as to whether differences in the ability of
wild-type and two/ muscle to
follow trains of 50 Hz stimuli resided in the differences in EPC
amplitude. As a test of this idea, the amount of depression for each
muscle recording was compared with the average EPC amplitude (Fig.
8C). In both wild-type and
two/ muscle, the average peak
EPC amplitude was quite variable, likely attributable to the
variability in synaptic receptor site densities. In particular, some of
the mutant synaptic responses were comparable with the
smaller-amplitude wild-type recordings (Fig. 2). Comparisons of peak
EPC amplitudes to the amount of depression observed showed that there
was no relationship between amplitude and the tendency to depress (Fig.
8C), supporting the idea that depression was not a simple
consequence of lowered EPC amplitude.
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DISCUSSION |
Multiple lines of evidence indicated that defects in rapsyn were
directly causal to the twitch once zebrafish phenotype.
First, stable expression of a murine rapsyn-GFP in
two/ fish greatly improved the
ability to mount escape responses to tail taps, and the behaviorally
rescued fish also exhibited wild-type synaptic clusters of ACh
receptors. Second, comparison of wild-type and twitch once
rapsyn sequences revealed an amino acid difference in the allele
twoth26e. At this location, the
predicted 130th amino acid of rapsyn was changed from a highly
conserved glycine to a glutamate residue. Third, transient expression
of wild-type zebrafish rapsyn-GFP in
two/ fish resulted in mosaic
animals wherein muscle cells expressing GFP fluorescence exhibited AChR
clusters. In contrast, when G130E rapsyn-GFP was transiently expressed,
no receptor clusters formed in muscle, despite the fact that the rapsyn
was still able to self-cluster. Additional comparisons between
wild-type and G130E rapsyn-GFP clusters indicated that the maximal
cluster size was smaller for the latter. Most important, however, was
the observation that G130E-GFP clusters occurred in the absence of AChR
clusters, indicating that the major effect of the mutation was a block
of the rapsyn-receptor interaction. One consequence of this blocked interaction was a widespread distribution of G130E rapsyn-GFP clusters.
Thus, as demonstrated previously in AChR-null mutant fish, rapsyn, in
the absence of interaction with receptor, was unable to effectively
localize to postsynaptic membrane (Ono et al., 2001a).
Rapsyn is a modular protein that is constructed of three functionally
distinct domains: tetratricopeptide repeat, coiled-coil, and RING-H2
domain (Scotland et al., 1993; Ramarao and Cohen, 1998; Ramarao et al.,
2001). Mutagenesis and deletional analyses of rapsyn have provided
tentative identification of the domains mediating self-aggregation and
receptor association (Ramarao and Cohen, 1998; Ramarao et al., 2001).
Disruption of the coiled-coil domain interferes with the ability of
rapsyn to aggregate AChRs in heterologous cells but does not interfere
with the self-clustering of rapsyn. The identification of the regions
mediating self-clustering of rapsyn is less clear, but the bulk of
evidence points to the involvement of the TPR domains (Ramarao and
Cohen, 1998; Ramarao et al., 2001). Our observation that a mutation in
a single TPR disrupts the size of clusters but not the ability to form
clusters is consistent with the published role of the TPR domain in
self-clustering. However, our finding that mutation in the TPR domain
disrupted interaction with AChRs appears to be at odds with studies
pointing to the coiled-coil domain as the site of interaction (Ramarao et al., 2001). It is possible that the mutation in the fourth TPR of
rapsyn caused global changes in protein conformation, thereby altering
the function of the coiled-coil region. The eighth residue has been
shown, through structural analysis, to represent a key structural
feature of TPR domains (Das et al., 1998), and mutations at this site
effectively disrupt function in protein phosphatases containing TPR
regions (Sikorski et al., 1993). Additional evidence for the importance
of this key position is reflected in the fact that the hereditary human
disorder chronic granulomatous disease results from a mutation in the
TPR of the p67 protein (de Boer et al., 1994). Remarkably, in the p67
protein, just as is found for the twitch once mutation in
rapsyn, glutamate is substituted for glycine in the eighth position.
Based on preliminary findings from our laboratory (Ono et al., 2001b),
mutations in TPR regions of rapsyn were subsequently identified in
congenital types of myasthenic syndrome (Ohno et al., 2002). Patients
afflicted with this disorder show use-dependent muscle fatigue that is
very similar in pattern to two/
swimming. As with two/ fish,
myasthenic patients exhibit reduced subsynaptic receptor densities and
elevated nonsynaptic receptor number (Fambrough et al., 1973). The
myasthenic phenotype in human rapsyn patients was proposed to result
from either lowered muscle cell resistance or reduced
Na+ channel activation attributable to
deformation of postsynaptic folds (Ohno et al., 2002). Our
physiological analyses of synaptic function in
two/ fish offer an alternative
explanation. In mutant fish, the weakened muscle response is a
consequence of the combined effects of lowered receptor density and
short-term depression in synaptic response amplitude. Repetitive 50 Hz
stimulation of two/ motor
neurons led to significant cumulative depression of endplate currents.
Because muscle cells are weakly excitable at early stages in
development (Buss and Drapeau, 2000), the force of contraction is
proportional to the magnitude of the synaptic potential. Thus, the
steep decline in amplitude that occurred within the first three EPCs in
mutant fish may have resulted in a peak response that was too small to
result in movement. In contrast, wild-type synaptic responses showing
no depression led to normal nonfatiguable responses.
How might a mutation in rapsyn augment synaptic depression? Synaptic
depression at neuromuscular junctions is common at newly formed
synapses in culture in which both presynaptic and postsynaptic differentiation are incomplete (Dan et al., 1995). However, depression at adult neuromuscular junctions is only observed during intense and
prolonged stimulation (Betz, 1970). The bulk of studies on neuron-neuron synapses point to presynaptic mechanisms as causal to
synaptic depression (Thomson, 2000). However, recent studies have
indicated that postsynaptic receptor desensitization can contribute to
depression at certain of those synapses (Trussell et al., 1993; Jones
and Westbrook, 1996). The ability to assign depression at the
neuromuscular junction to either presynaptic or postsynaptic mechanisms
has proven very difficult (Sandrock et al., 1997). In the case of
twitch once fish, the most likely mechanism underlying the
synaptic depression appears to be postsynaptic receptor
desensitization. Studies using fast application of ACh on
embryonic-type amphibian nicotinic receptors predict that frequencies corresponding to those in swimming fish will result in pronounced desensitization (Paradiso and Brehm, 1998).
A possible link between receptor desensitization and rapsyn is offered
by the finding that at least one of the principal sites of receptor
phosphorylation requires association with rapsyn for the modification
(Wallace et al., 1991; Gillespie et al., 1996; Apel et al., 1997;
Mittaud et al., 2001). Furthermore, tyrosine kinase phosphorylation, as
well as phosphorylation by protein kinase A, affects the kinetics of
desensitization (Hopfield et al., 1988; Paradiso and Brehm, 1998).
Thus, receptors in twitch once may be prone to
desensitization attributable to the absence of functional interactions
with rapsyn, and this desensitization may underlie the synaptic
depression. Until the mechanisms underlying depression in twitch
once fish have been identified, we cannot rule out indirect
effects on presynaptic release or alterations in synaptic morphology.
We can rule out the possibility that the depression is a simple
consequence of lowered receptor density based on the lack of
correlation between tendency to depress and postsynaptic response
amplitude (Fig. 8C). Although the mechanisms through which
rapsyn affects synaptic function are not known, it is clear that rapsyn
directly or indirectly plays a role in synaptic function.
Our studies on twitch once zebrafish may also offer a new
perspective on nonhereditary forms of myasthenia gravis. Eighty percent
of patients afflicted with this disease test positive for anti-AChR
antibodies (Lindstrom et al., 1976). However, important new insights
have been gained recently from a study of patients that failed to test
positive for receptor antibodies. A majority of these patients tested
positive for antibodies against either muscle-specific kinase (MuSK) or
rapsyn (Agius et al., 1998; Hoch et al., 2001). MuSK is required for
the proper anchoring of the rapsyn-AChR complex to the subsynaptic
site, as well as for the rapsyn-dependent phosphorylation of AChR
(DeChiara et al., 1996; Apel et al., 1997). A direct involvement of
rapsyn antibodies in the disease process was suggested by the onset of
myasthenia gravis-like symptoms in mice immunized with rapsyn (Agius et
al., 1998). Thus, based on our results from twitch once
mutant fish, the MuSK-rapsyn-AChR pathway may be involved in receptor
desensitization and associated synaptic depression at the neuromuscular
junction. Interference with any one of these components of receptor
localization may result in the use-dependent fatigue in movement
associated with this disease.
| |
FOOTNOTES |
Received April 1, 2002; revised May 15, 2002; accepted May 17, 2002.
This study was supported by National Institutes of Health Grant NS-8205
(P.B.). G.M. is an investigator of the Howard Hughes Medical Institute.
We thank Dr. Frohnhoefer (Max-Planck-Institute, Tuebingen,
Germany) for providing twitch once mutant
zebrafish (allele twoth26e). Dr.
Joseph Fetcho provided technical advice and insights throughout the
project. Joan Speh offered expert technical assistance with confocal
microscopy and Shelagh Palma provided expert care of the fish. We thank
Dr. Howard Sirotkin for critical reading of this manuscript.
Correspondence should be addressed to Paul Brehm, Department of
Neurobiology and Behavior, State University of New York at Stony Brook,
Stony Brook, NY 11794. E-mail: pbrehm{at}notes.cc.sunysb.edu.
| |
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ABSTRACT
INTRODUCTION
