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The Journal of Neuroscience, January 15, 1998, 18(2):672-678
Intracellular Calcium Regulates Agrin-Induced Acetylcholine
Receptor Clustering
Laura J.
Megeath1, 2 and
Justin R.
Fallon2
1 Department of Cell Biology, Graduate School of
Biomedical Sciences, University of Massachusetts Medical Center,
Worcester, Massachusetts 01655, and 2 Department of
Neuroscience, Brown University, Providence, Rhode Island 02912
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ABSTRACT |
Agrin is an extracellular matrix protein that directs neuromuscular
junction formation. Early signal transduction events in agrin-mediated
postsynaptic differentiation include activation of a receptor tyrosine
kinase and phosphorylation of acetylcholine receptors (AChRs), but
later steps in this pathway are unknown. Here, we have investigated the
role of intracellular calcium in agrin-induced AChR clustering on
cultured myotubes. Clamping intracellular calcium levels by loading
with the fast chelator BAPTA inhibited agrin-induced AChR aggregation.
In addition, preexisting AChR aggregates dispersed under these
conditions, indicating that the maintenance of AChR clusters is
similarly dependent on intracellular calcium fluxes. The decrease in
AChR clusters in BAPTA-loaded cells was dose-dependent and reversible,
and no change in the number or mobility of AChRs was observed. Clamping
intracellular calcium did not block agrin-induced tyrosine
phosphorylation of the AChR  -subunit, indicating that intracellular
calcium fluxes are likely to act downstream from or parallel to AChR
phosphorylation. Finally, the targets of the intracellular calcium are
likely to be close to the calcium source, since agrin-induced AChR
clustering was unaffected in cells loaded with EGTA, a slower-binding
calcium chelator. These findings distinguish a novel step in the signal transduction mechanism of agrin and raise the possibility that the
pathways mediating agrin- and activity-driven changes in synaptic architecture could intersect at the level of intracellular calcium fluxes.
Key words:
agrin; intracellular calcium; AChR phosphorylation; neuromuscular junction; synaptogenesis; postsynaptic
differentiation
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INTRODUCTION |
Synapses throughout the nervous
system are characterized by high concentrations of neurotransmitter
receptors in the postsynaptic apparatus (Fertuck and Salpeter, 1976 ;
Triller et al., 1985; Jacob et al., 1986; Nusser et al., 1994). Such
dense accumulations of receptors are necessary for efficient synaptic
transmission. Regulation of receptor number in the postsynaptic
membrane is a hallmark of synaptic development, and is likely to be an
important element underlying synaptic plasticity during learning and
memory (Bailey and Kandel, 1993; Weiler et al., 1995).
Agrin plays a pivotal role in synaptic differentiation at the
neuromuscular junction (Hall and Sanes, 1993; Bowe and Fallon, 1995).
Here, agrin secreted by motor neurons activates a receptor tyrosine
kinase, MuSK, to trigger synapse formation. Mutant mice lacking either
agrin or MuSK display three major abnormalities: grossly defective
presynaptic and postsynaptic differentiation, and a failure in synapse
selective transcription (De Chiara et al., 1996; Gautam et al., 1996).
The signaling pathways between MuSK activation and these three
endpoints must diverge, but in ways that currently are not understood
(Gautam et al., 1995; Wells and Fallon, 1996; Apel et al., 1997).
The best-characterized branch of the agrin signaling pathway leads to
the differentiation of the postsynaptic apparatus. Agrin secreted from
the nerve terminal induces the aggregation of acetylcholine receptors
(AChRs) and a host of other postsynaptic molecules on the muscle cell
surface, including the dystrophin/utrophin-associated protein complex
(Campanelli et al., 1994). The binding of agrin to a MuSK-containing
complex is the first known step, with activation of the kinase
occurring within minutes of agrin addition (Glass et al., 1996).
Increased tyrosine phosphorylation of the AChR -subunit is detected
~30 min later. The bench mark biological activity of agrin, the
clustering of AChRs, manifests ~2 hr after agrin addition (Wallace,
1988; Nastuk et al., 1991). The maximal number of AChR clusters and
level of AChR phosphorylation are achieved ~4 hr later (Wallace et
al., 1991; Nastuk and Fallon, 1993).
In addition to its role in initiating postsynaptic apparatus formation,
agrin also seems likely to be important for synaptic maturation,
maintenance, and plasticity. Two lines of evidence point to a
longer-term action in the agrin signaling pathway. First, agrin-induced
AChR clusters continue to mature for at least 1 d after agrin
addition in vitro. The AChR clusters become larger and more
stable, and cytoskeletal and basal lamina elements, including agrin
synthesized by muscle, accumulate with them (Wallace, 1988; Nitkin and
Rothschild, 1990; Lieth and Fallon, 1993). These events require the
ongoing action of agrin as well as new protein synthesis. Second, this
continued action is also likely to require sustained MuSK activation
(Glass et al., 1996). This long-term activation distinguishes MuSK from
many other receptor tyrosine kinases, which are activated only
transiently (Ullrich and Schlessinger, 1990).
Despite these advances in the understanding of the mechanisms of
agrin's activity, many questions remain. For example, there is no
direct evidence that tyrosine phosphorylation of AChRs is sufficient,
or even necessary, for their clustering by agrin. Moreover, additional
intracellular signal transduction events are likely to play a role in
agrin-induced postsynaptic differentiation, but their nature is
unknown. Of particular interest are elements that could be influenced
by synaptic activity. Although it is well established that activity can
shape synaptic architecture (Balice-Gordon and Lichtman, 1993; Kasai,
1993; Kirkwood and Bear, 1995; Koch, 1997), the interface between
activity and the biochemical machinery that organizes synaptic
structure is poorly understood.
In the present study we asked whether intracellular calcium fluxes
participate in the agrin signaling pathway. We provide evidence that
rapid calcium fluxes are required for agrin-induced AChR aggregation.
Moreover, these fluxes act downstream from or parallel to AChR
phosphorylation, which we demonstrate is not sufficient for AChR
clustering. These findings reveal a novel step in the agrin signal
transduction pathway. Intracellular calcium fluxes thus emerge as a
potential locus for the integration of agrin- and activity-mediated
changes in synaptic architecture.
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MATERIALS AND METHODS |
Myotube culture. Embryonic chick myotube
cultures were prepared as previously described (Nastuk et al., 1991).
Briefly, myoblasts from embryonic day 11 chick embryos were dissociated
and plated in medium containing MEM (Alpha Medium, Life Technologies,
Gaithersburg, MD) supplemented with 2% chick embryo extract, 10%
horse serum, 100 U/ml penicillin G, and 2 mM
L-glutamine. For ligand-binding and AChR phosphorylation
assays, cells were grown on plastic coated with 100 µg/ml gelatin
(Sigma, St. Louis, MO). For AChR clustering assays, cells were grown on
glass coverslips coated with 20 µg/ml poly-D-lysine
(MW > 300,000; Sigma) and gelatin. Myotubes were used 3-7 d
after plating.
Drug treatment. Stock solutions of the aminomethoxy ester of
BAPTA (BAPTA-AM; Molecular Probes, Eugene OR) were prepared in DMSO
(vehicle). The 10 mM BAPTA-AM stocks were stored at
 20°C. Aliquots were thawed immediately before experiments and were
not refrozen. BAPTA-AM or vehicle was diluted in serum-free medium (SFM) consisting of MEM (Alpha Medium, Life Technologies), 2 mM L-glutamine (Life Technologies), 0.5%
bovine serum albumin (BSA), 100 U/ml penicillin G, and 5 µg/l each
insulin, transferrin, and selenium (all from Sigma). To load cells with
BAPTA, we incubated cells in BAPTA-AM for 1 hr 37°C (final DMSO
concentration 0.5%) and then rinsed them with SFM. EGTA-AM (Molecular
Probes) was prepared and used in a similar manner.
AChR clustering assays. Recombinant rat agrin, containing
inserts of 12, 4, and 8 amino acids at the x, y,
and z splice sites, respectively, was produced in COS cells
as described previously (O'Toole et al., 1996). In some experiments,
agrin purified from Torpedo electric organ (Cibacron Pool;
Nitkin et al., 1987) was used with similar results. Native or
recombinant agrin was used at a concentration of 10 U/ml in SFM. One
unit is defined as the concentration of agrin at which half-maximal
AChR clustering activity is observed (Godfrey et al., 1984).
Cells grown on coverslips were incubated with agrin for 4 hr at 37°C.
Agrin was added immediately after BAPTA-AM treatment or 24 hr later
(see Washout, Fig. 3). To detect AChRs, we included 1 µg/ml rhodamine- -BTx in the final 45 min of the incubation. In
some experiments myotubes were incubated simultaneously with agrin and
KN-62 or K-252a (Calbiochem, La Jolla, CA) in DMSO. The 10 mM KN-62 and 1 mM K-252a stocks were stored at
4°C in the dark. The final DMSO concentration was  1%. Coverslips
were rinsed in HEPES-buffered MEM (MEM-H, Life Technologies), fixed in
methanol at 20°C for 5 min, mounted in Citifluor (Pella, Redding,
CA), and viewed on a Zeiss Axioplan (Oberkochen, Germany) or a Nikon Eclipse (Tokyo, Japan) microscope. For quantitation of AChR clustering, 20-30 myotube segments (200 µm in length) were chosen randomly from
two to three coverslips. AChR clusters (defined as AChR aggregates  4
µm in diameter) were scored under rhodamine optics (Nastuk et al.,
1991).
Antibody-induced AChR microclustering was performed as described by
Nastuk et al. (1991). Cells were loaded with BAPTA-AM or vehicle for 1 hr, rinsed, and then incubated with monoclonal antibody (mAb) 35 (Tzartos, 1983) for 30 min at 37°C, followed by goat anti-rat IgG
(Sigma) and rhodamine-coupled -bungarotoxin (-BTx; Molecular
Probes) for 30 min at 37°C. The distribution of AChRs was assessed
visually; three coverslips were surveyed for each condition.
Ligand-binding assays. Binding of -BTx and agrin to
myotubes was quantitated as previously described (Bowe et al., 1994). Myotubes grown on gelatin-coated removable 96-well strips (Immulon 4, Dynatech, Chantilly, VA) were blocked for 1 hr in MEM-H with 1% BSA
and 10% horse serum and incubated for 30 min with 10 nM 125I--BTx (10-20 µCi/µg, DuPont NEN, Boston, MA).
For agrin binding, wells were incubated with agrin for 2 hr, followed
by 1 µg/ml iodinated anti-agrin mAb 131 for 30 min. The mAb 131 (Hoch
et al., 1994) was iodinated by using IODO-GEN (Pierce, Rockford, IL)
per the manufacturer's instructions; the range of specific activities
was 5-8 µCi/µg. Wells were washed in MEM-H, immersed twice in HBSS
with 1% BSA and 1 mM calcium, dried, and counted. Nonspecific binding was determined by including 1 mM EGTA
(in agrin-binding experiments) or 100-fold excess competing unlabeled -BTx. In each experiment six individual wells were counted for each
condition, and then the results of multiple experiments were pooled.
Determination of AChR phosphorylation. AChRs from cultured
myotubes were purified according to the method of Wallace et al. (1991)
with minor modifications. Biotinylated -BTx (Molecular Probes) was
purified on an ImmunoPure Immobilized Monomeric Avidin column (Pierce).
Myotube cultures were loaded with BAPTA or vehicle, incubated for 4 hr
in agrin and 0.5 µg/ml biotinylated -BTx, washed twice in cold
PBS, harvested, and centrifuged; the cell pellet was resuspended in
extraction buffer containing (in mM) 5 EDTA, 5 EGTA, 20 Tris, pH 7.5, 20 glycine, 150 NaCl, 40 Na-pyrophosphate, 50 NaF, 10 Na-molybdate, 1 Na-orthovanadate, 5 benzamidine, 10 N-ethylmaleimide, and 1 phenylmethylsulfonyl fluoride, with
1% Triton X-100, 1 mg/ml bacitracin, and 50 µg/ml each chymostatin, pepstatin, aprotinin, leupeptin, and antipain. Samples were sonicated for 10 sec with a Branson 450 Sonifier at 70% power, incubated 15 min
at 4°C, and then spun for 20 min at 3000 × g.
Solubilized AChR-biotinylated--BTx complexes were incubated with
streptavidin-Sepharose beads (Sigma) for 2 hr with constant mixing at
4°C. Beads were washed four times in extraction buffer containing 1 M NaCl, twice in extraction buffer lacking NaCl and Triton
X-100, and eluted in SDS-PAGE sample buffer.
Isolated AChRs were electrophoresed on 5-15% gradient
SDS-polyacrylamide gels and transferred to nitrocellulose. Then the blots were blocked in PBS supplemented with 1% BSA. To detect AChR
- and - subunits, we probed blots with mAb 61 and mAb 111, respectively (Wallace et al., 1991) (generously provided by J. Lindstrom, University of Pennsylvania). In some experiments AChR  -subunit was detected with mAb 88b (Froehner et al., 1983; Qu and
Huganir, 1994) (generously provided by S. Froehner, University of North
Carolina). Tyrosine-phosphorylated polypeptides were detected with
anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY).
After incubation with primary antibody, blots were washed in PBS and
incubated for 1 hr with rabbit anti-mouse IgG (Sigma), followed by
100,000 cpm/ml 125I-protein A (2-10 µCi/µg, DuPont
NEN). Bound radioactivity was quantitated in each lane in regions of
equal area with a Molecular Dynamics PhosphorImager and software. The
amount of tyrosine phosphorylation that was detected was expressed
relative to the amount of AChR loaded, as determined by quantitation of
mAb 61 binding to the AChR -subunit. In some experiments blots
originally probed with 125I-protein A were stripped and
reprobed with anti-AChR antibodies. Then bound antibodies were detected
by biotinylated horse anti-mouse IgG (Vector Laboratories, Burlingame,
CA) and an alkaline phosphatase-based ABC kit (Vectastain ABC; Vector
Laboratories).
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RESULTS |
A rapid intracellular calcium chelator inhibits spontaneous and
agrin-induced AChR clustering
Both the AChR clustering activity of agrin and its binding
to the cell surface require extracellular calcium (Wallace, 1988; Nastuk et al., 1991). We used BAPTA to manipulate intracellular calcium
without depleting calcium outside the cells. Myotubes were loaded with
BAPTA, using its membrane-permeable non-calcium-binding AM ester
(BAPTA-AM) (Tsien, 1981). Upon traversing the plasma membrane, BAPTA-AM
is converted to BAPTA, which is membrane-impermeable, by intracellular
esterases. BAPTA binds calcium rapidly, selectively, and with high
affinity (KD ~100-180 nM), thus
serving to "clamp" intracellular calcium fluxes (Stern, 1992;
Roberts, 1993; Deisseroth et al., 1996).
Clamping intracellular calcium inhibited AChR clustering. The
number of agrin-induced AChR clusters was reduced >60% in cells loaded with 50 µM BAPTA-AM, the highest concentration
tested (Fig. 1). Significant inhibition
of agrin-induced AChR clustering was observed in all experiments
(n = 7) and was dependent on the BAPTA-AM concentration
used for loading (Fig. 2). The 50 µM BAPTA-AM concentration was chosen for all subsequent
experiments. The number of spontaneous AChR clusters (also known as
"hot spots"; Frank and Fischbach, 1979) was reduced by 40% in
BAPTA-loaded cells (Fig. 1). The inhibition of spontaneous clusters was
more variable than that seen for agrin-induced clusters. Although the
number of spontaneous clusters decreased in BAPTA-loaded cells in all
experiments (n = 7), the inhibition was significant in
only five of them. Treatment with vehicle alone had no effect on either
spontaneous or agrin-induced clusters. AChR clusters looked similar in
control and BAPTA-loaded myotubes, indicating that BAPTA prevented the
formation of clusters rather than causing them to form more diffusely.
These results indicate that intracellular calcium fluxes are necessary
for both the maintenance and the formation of AChR clusters.
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Figure 1.
The number of spontaneous and agrin-induced AChR
clusters is decreased in BAPTA-loaded cells. A, Myotubes
were incubated with 50 µM BAPTA-AM (bottom
panels) or vehicle only (top panels), washed, and incubated with or without agrin for 4 hr, as indicated. Cultures were then incubated in rhodamine--BTx and examined by fluorescence microscopy to reveal the distribution of AChRs. B,
Quantitation of AChR clusters revealed that significantly fewer
spontaneous and agrin-induced clusters are observed in BAPTA-loaded
cells. AChR clusters were quantitated as described in Materials and
Methods. Values are mean ± SEM averaged from seven separate
experiments. *p < 0.05, paired Student's
t test. Scale bar, 20 µm.
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Figure 2.
Quantitation of agrin-induced and spontaneous AChR
clusters in myotubes loaded with varying concentrations of BAPTA-AM.
Myotubes were incubated with the indicated concentrations of BAPTA-AM
for 1 hr and then incubated in media with or without agrin for 4 hr. Data shown are from one representative experiment and are expressed as
mean ± SEM. Similar results were seen in three additional
experiments.
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We were unable to detect any deleterious effects of BAPTA loading on
these cells. Myotubes loaded with BAPTA were morphologically indistinguishable from controls, as judged by phase-contrast
microscopy. Moreover, the effects of drug treatment were reversible.
After wash-out, the numbers of agrin-induced and spontaneous AChR
clusters returned to control levels (Fig.
3).
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Figure 3.
Inhibition of AChR clustering in BAPTA-loaded
cells is reversible. Cells were loaded with 50 µM
BAPTA-AM or vehicle for 1 hr, washed, and then incubated with agrin
either immediately or 24 hr later (Washout). After the
wash-out period, the numbers of spontaneous and agrin-induced AChR
clusters returned to levels similar to vehicle-treated cells. Values
are expressed as mean ± SEM from one experiment. Similar results
were observed in four other experiments.
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The results presented above suggest that clamping intracellular
calcium may interfere directly with the signal transduction pathway of
agrin. However, it is also possible that the inhibition is attributable
to indirect effects, such as altering the level of agrin-binding sites
or AChRs on the cell surface. Therefore, to test these possibilities,
we measured the levels of AChRs and of agrin binding. There was no
statistical difference in the number of surface AChRs in BAPTA-loaded
cells (102% ± 10 of control, n = 5; p = 0.62, paired Student's t test). Similarly, no differences in agrin binding were observed (90% ± 11 of control,
n = 4; p = 0.25, paired Student's
t test).
The formation of antibody-induced AChR microclusters is unaffected
by clamping intracellular calcium
It is possible that clamping intracellular calcium could
inhibit AChR clustering by immobilizing AChRs in the myotube membrane. Such immobilization of AChRs, with concomitant inhibition of
agrin-induced receptor clustering, has been reported in myotubes
treated with tyrosine phosphatase inhibitors (Meier et al., 1995). To
assess AChR mobility, we tested the ability of anti-AChR antibodies to drive the formation of AChR microclusters (Nastuk et al., 1991). This
manipulation is distinct from agrin-induced clustering because it
relies on the direct antibody-mediated cross-linking of AChRs. Antibody-driven AChR microclustering was equally robust in both untreated and BAPTA-loaded myotubes (Fig.
4). Together with the binding data
presented above, these observations support the hypothesis that
intracellular calcium fluxes play a direct role in the agrin signaling
pathway.
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Figure 4.
Antibody-driven AChR microclustering is unaffected
in BAPTA-loaded cells. Myotubes were incubated with vehicle only
(A, B) or 50 µM BAPTA-AM
(C) and then directly incubated in buffer alone (A) or anti-AChR antibody mAb 35 and anti-Rat IgG
for 1 hr at 37°C (B, C). The
distribution of AChRs was then determined by labeling with
rhodamine--BTx. In the absence of anti-AChR antibody incubation,
AChRs were distributed diffusely on the myotube surface (A). Incubation with anti-AChR antibodies caused
extensive AChR microclustering in both vehicle
(B) and BAPTA-loaded (C)
cells. Scale bar, 20 µm.
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A slower-binding calcium buffer does not inhibit agrin-induced
AChR clustering
To better characterize the mode of action of calcium in the
agrin signaling pathway, we tested another calcium chelator, EGTA. This
chelator binds calcium with a similar affinity to BAPTA, but with a
400-fold slower on rate. As a result, calcium issuing into the cytosol
can be buffered to within ~0.1 µm of the membrane in a BAPTA-loaded
cell but only to ~1 µm in the presence of EGTA (Stern, 1992;
Roberts, 1993; Deisseroth et al., 1996). In contrast to the results
observed in BAPTA-loaded cells, the numbers of spontaneous and
agrin-induced AChR clusters were unaffected in myotubes loaded with
EGTA (Fig. 5). Different AM ester
compounds may load into cells at different rates (Deisseroth et al.,
1996). Therefore, we tested a wider range of EGTA-AM concentrations. We
observed no inhibition of either agrin-induced or spontaneous AChR
clusters when we used an EGTA-AM concentration ranging from 25 to 100 µM (the highest dose tested; data not shown). These results indicate that global buffering of calcium is unlikely to
account for the BAPTA-mediated inhibition of AChR cluster formation. Moreover, these results demonstrate that the inhibition of AChR clustering is not the result of nonspecific side effects resulting from
the use of AM ester compounds. Finally, these findings indicate that
AChR clustering is likely to rely on calcium-sensitive effectors localized <1 µm from the calcium source.
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Figure 5.
Spontaneous and agrin-induced AChR clusters in
EGTA-loaded cells. Myotubes were loaded with EGTA by incubating them
with 50 µM EGTA-AM for 1 hr at 37°C and then incubating
them with or without agrin, as in Figure 1. The numbers of neither
spontaneous nor agrin-induced AChR clusters were significantly
different in EGTA-loaded, as compared with vehicle-loaded cells.
Mean ± SEM from one representative experiment;
p = 0.392 and 0.360 for spontaneous and
agrin-induced clusters, respectively, Student's t test.
Similar results were obtained by using cells loaded with 25 or 100 µM EGTA-AM.
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One candidate effector is calcium/calmodulin-kinase II (CaM-KII). To
test whether this enzyme plays a role in AChR cluster formation, we
incubated myotubes with agrin in the presence of CaM-KII inhibitors
(either 10-100 µM KN-62 or 0.01-10 µM
K-252a). No effects on AChR clusters were observed (data not shown),
suggesting that CaM-KII does not participate in the agrin signaling
pathway.
Clamping intracellular calcium does not perturb agrin-induced
AChR phosphorylation
We next wished to position intracellular calcium fluxes relative
to known events in the agrin signaling pathway. As discussed above,
early steps in this pathway include the agrin-induced activation of the
receptor tyrosine kinase MuSK, followed by the tyrosine phosphorylation
of AChR -subunits (Wallace et al., 1991; Glass et al., 1996). We
therefore assessed AChR phosphorylation in BAPTA-loaded cells.
Phosphorylation levels of AChRs were assayed 4 hr after agrin addition,
the time at which AChR clustering was assessed in the above experiments
and at which maximal agrin-induced tyrosine phosphorylation is achieved
(Wallace et al., 1991). As shown in Figure
6A, agrin induced the
tyrosine phosphorylation of AChRs in both BAPTA- and vehicle-loaded
cells. Quantitation of phosphotyrosine levels indicated that BAPTA
loading did not significantly change agrin-induced AChR -subunit
phosphorylation (Fig. 6B). Further, BAPTA treatment
did not alter the basal level of AChR phosphorylation in these
myotubes. These results demonstrate that tyrosine phosphorylation of
AChR -subunits is not sufficient to induce their clustering and
indicate that intracellular calcium fluxes act downstream or parallel
to AChR phosphorylation.
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Figure 6.
Agrin-induced tyrosine phosphorylation of
AChR -subunit in BAPTA-loaded cells. Myotubes were loaded with BAPTA
or vehicle and then incubated with or without agrin for 4 hr, as
indicated. Surface AChRs were affinity-purified with biotinylated
-BTx and separated by SDS-PAGE. A, Immunoblots were
probed with mAb 4G10 to visualize tyrosine phosphorylated proteins
(p-tyr) or mAb 61 to visualize the AChR
-subunit (anti-). In parallel blots, mAb 124 was
used to identify the AChR -subunit (data not shown). Agrin induced
tyrosine phosphorylation of AChR -subunit in both the presence and
absence of BAPTA. A second polypeptide of slightly slower mobility was
phosphorylated also. This polypeptide was identified tentatively as
AChR -subunit, on the basis of immunoreactivity with mAb 88b (data
not shown) and previous reports showing that agrin also induces the
phosphorylation of this subunit (Qu and Huganir, 1994).
B, Phosphotyrosine levels of the AChR -subunit were
measured and expressed relative to total AChR levels, as described in
Materials and Methods. Results were derived from three separate
experiments, each normalized to untreated controls. Agrin-induced AChR
-subunit tyrosine phosphorylation was equivalent in vehicle, as
compared with BAPTA-loaded cells. The basal level of AChR -subunit
tyrosine phosphorylation was also not significantly different in
BAPTA-loaded cells (p > 0.05; Newman-Keuls
multiple comparison test, after repeated measures ANOVA of three
separate experiments; data not normalized).
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DISCUSSION |
The goal of this study was to test the role of intracellular
calcium in the agrin signaling pathway. Our findings indicate that
locally acting intracellular calcium fluxes are necessary for
agrin-induced AChR clustering. The calcium-sensitive step or steps are
downstream of, or parallel to, agrin-induced tyrosine phosphorylation
of AChRs. We also show that agrin-induced AChR phosphorylation is not
sufficient for receptor aggregation.
We used BAPTA to buffer intracellular calcium. This compound was
designed by Tsien (1981) to bind calcium with high selectivity, affinity, and speed. The efficacy and specificity of this drug have
been documented in numerous studies (Stern, 1992; Roberts, 1993). For
example, BAPTA has been used to probe rapid calcium-signaling events
mediating exocytosis (Tsien, 1981; Penner and Neher, 1988; Adler et
al., 1991). Loaded via its AM- ester form, this compound has been used
to manipulate calcium-activated potassium channels (Robitaille et al.,
1993), to distinguish among classes of evoked EPSPs (Cummings et al.,
1996), and to probe calcium transients regulating different aspects of
neuronal differentiation (Gu and Spitzer, 1995).
Several lines of evidence indicate that the clamping of
intracellular calcium by BAPTA perturbs a step in the signaling pathway of agrin, rather than working via indirect mechanisms. We found no
evidence that BAPTA-AM or BAPTA caused toxicity in these studies. The
treated myotubes were indistinguishable from controls, as judged by
phase-contrast microscopy, and the effects of BAPTA were reversible
(see Fig. 3). The levels of surface agrin-binding sites and AChRs were
unchanged in the treated cells. It should be noted that although we
used the highly active, MuSK-activating isoform of agrin in these
assays (agrin 4, 8; Glass et al., 1996), it is likely that a
substantial portion of the observed binding was attributable to
interaction with dystroglycan on the cell surface (Bowe et al., 1994;
O'Toole et al., 1996). The normal levels of both basal and
agrin-induced AChR tyrosine phosphorylation observed in BAPTA-treated
myotubes also indicate that basic cell functions were uncompromised and
further suggest that the level of MuSK on the cell surface is not
altered substantially under these conditions. AChRs in the membrane
remained mobile, as judged by the robust AChR microclustering driven by
anti-AChR antibodies (see Fig. 4). However, the possibility remains
that BAPTA causes relatively small changes in receptor mobility that
could be beyond the sensitivity of this assay. Finally, nonspecific
side effects stemming from the use of AM esters are unlikely, because
loading cells with EGTA-AM had no effect on spontaneous or
agrin-induced AChR clusters (see Fig. 5).
The results presented here provide new insights about the roles of MuSK
activation and AChR tyrosine phosphorylation in agrin-induced AChR
clustering, and their places in the signaling pathway. Activation of
the MuSK receptor complex by agrin is essential for synaptic differentiation (De Chiara et al., 1996). Subsequent to this step, and
dependent on MuSK activation (Apel et al., 1997), is the tyrosine phosphorylation of AChR -subunits. Because AChR tyrosine
phosphorylation proceeds normally in BAPTA-loaded cells (see Fig. 6),
neither MuSK activation nor the phosphorylation of AChR -subunit is
sufficient for agrin-induced AChR aggregation. Therefore, additional
components of the agrin signaling pathway, at least some of which rely
on intracellular calcium fluxes, must come into play to achieve and to
maintain postsynaptic differentiation.
Our studies show clearly that agrin-induced tyrosine phosphorylation of
the AChR -subunit is not sufficient for clustering AChRs. However,
it is not known if this phosphorylation is necessary for agrin-induced
AChR clustering. Accordingly, we present two possible models for the
agrin signaling pathway (Fig. 7). In one model, the calcium-dependent step occurs downstream of agrin-induced AChR phosphorylation. In the second, agrin-induced AChR phosphorylation is a step in a parallel pathway that does not play a direct role in
clustering.
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Figure 7.
Two models of the signaling pathway of agrin. The
first step in the signaling pathway is the activation of the MuSK
receptor tyrosine kinase by agrin. The subsequent tyrosine
phosphorylation of the AChR is dependent on MuSK activation. The
results presented here are consistent with two models. (1) The
intracellular calcium-regulated step occurs downstream of AChR
phosphorylation. (2) The AChR phosphorylation is on a pathway parallel
to (but not necessarily required for) agrin-induced AChR clustering.
See Discussion for details.
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Calcium is likely to play a role at both extracellular and
intracellular loci during agrin-induced postsynaptic differentiation. Extracellular calcium is necessary for nerve- and agrin-induced AChR
clustering (Henderson et al., 1984; Wallace, 1988). Removal of
extracellular calcium destabilizes AChR clusters (Connolly, 1984;
Wallace, 1988; Caroni et al., 1993; Dmytrenko and Bloch, 1993), and
raising extracellular calcium can promote AChR clustering (Mook-Jung
and Gordon, 1995). In addition, a large fraction of agrin binding to
the cell surface and to the major agrin-binding protein on the cell,
dystroglycan, is dependent on extracellular calcium (Nastuk et al.,
1991; Bowe et al., 1994).
The source of the intracellular calcium transients that are necessary
for AChR aggregation is not known. The requirements for extracellular
calcium noted above are consistent with the possibility that the
extracellular pool could be one source. Both the AChR itself and
voltage-dependent calcium channels are potential conduits for such
extracellular calcium influx. However, they are unlikely to be the sole
sources, because neither -BTx nor tetrodotoxin inhibits AChR
clustering induced by neurons or agrin (Anderson et al., 1977; Godfrey
et al., 1984). However, calcium entering from these sources could
modulate postsynaptic differentiation (see below). Alternatively, the
calcium fluxes may arise from intracellular stores, such as
IP3-mediated release from the endoplasmic reticulum (Verma et al.,
1990).
The experiments comparing the effects of EGTA and BAPTA provide
information about the location of the calcium targets relative to the
sources. As a consequence of its slower-binding kinetics, EGTA, even
when present at saturating concentrations, can buffer a calcium
transient only to within 1-2 µm of its source. On the other hand,
BAPTA is estimated to buffer calcium ions within 0.1 µm from a source
(Stern, 1992; Roberts, 1993; Schweizer et al., 1995; Deisseroth et al.,
1996). For example, neurotransmitter release mediated by voltage-gated
calcium channels is blocked by BAPTA, but not by EGTA (Adler et al.,
1991; Augustine et al., 1992). Further, BAPTA, but not EGTA, blocks
activity-mediated cAMP response element binding protein (CREB)
phosphorylation in hippocampal neurons (Deisseroth et al., 1996).
Deisseroth and colleagues also showed that equivalent levels of
intracellular EGTA and BAPTA were achieved when the loading
concentration of EGTA-AM was threefold greater than that of BAPTA-AM.
In the present study we observed significant inhibition of
agrin-induced AChR clustering in cell loaded with 25 µM
BAPTA-AM, but we observed no effects when the cells were loaded with
100 µM EGTA-AM (see Figs. 2, 5). The inability of EGTA to
inhibit agrin-induced or spontaneous AChR clustering thus indicates
that the sources of the calcium fluxes are likely to be close to their
targets.
A possible connection between the intracellular calcium
requirements observed here and the pathogenesis of muscular dystrophies deserves comment. Many varieties of muscular dystrophy, including the
most prevalent forms, Duchenne and Becker, are the result of
deficiencies in the dystrophin-associated protein complex. Along with
clustering AChRs, agrin also induces the aggregation of many members of
this complex. Moreover, altered intracellular calcium levels and
calcium channel properties in dystrophic muscle have been reported (for
review, see Gillis, 1996). It is also of interest that limb-girdle
Muscular Dystrophy type 2A is due to a defect in calpain, a
calcium-activated protease (Richard et al., 1995; van Ommen, 1995).
We speculate that the organization of the
dystrophin-associated protein complex involves a
calcium-dependent step. Together, these observations raise the
possibility that there may be links between agrin's mechanism of
action and the molecular pathophysiology of muscular dystrophies.
Finally, the requirement for intracellular calcium fluxes in the
AChR clustering activity of agrin presents an attractive locus for
activity-mediated regulation of synaptic structure. Activity has
far-reaching effects on synaptic structure and function (Balice-Gordon
and Lichtman, 1993; Kasai, 1993; Kirkwood and Bear, 1995; Koch, 1997).
Many of these events have been linked to changes in intracellular
calcium. For example, neurotransmitter receptor synthesis in muscle is
inhibited by electrical activity in a pathway initiated by calcium
influx through voltage-dependent calcium channels (Huang et al., 1994).
Calcium also plays a central role in the mechanisms underlying LTP and
LTD at neuronal synapses (Bear and Malenka, 1994). At the neuromuscular
junction such calcium fluxes could feed into the cellular and molecular
machinery of the agrin signaling pathway to shape the synapse in an
activity-dependent manner. It is tempting to speculate that similar
mechanisms may underlie the structural plasticity of synapses in the
CNS.
| |
FOOTNOTES |
Received June 11, 1997; revised Oct. 28, 1997; accepted Nov. 3, 1997.
This work was supported by grants from the Muscular Dystrophy
Association and the National Institutes of Health (HD23924). We thank
J. Lindstrom and S. Froehner for the generous gifts of antibodies,
Richard Tsien for insightful discussions, and Beth McKechnie for superb
technical assistance. We also thank Katherine Deyst for valuable
comments on this manuscript.
Correspondence should be addressed to Dr. Justin R. Fallon, Department
of Neuroscience, Brown University, Box 1953, 190 Thayer Street,
Providence, RI 02912.
| |
REFERENCES |
-
Adler EM,
Augustine GJ,
Duffy SN,
Charlton MP
(1991)
Alien intracellular calcium chelators attenuate neurotransmitter release at the squid giant synapse.
J Neurosci
11:1496-1507[Abstract].
-
Anderson MJ,
Cohen MW,
Zorychta E
(1977)
Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells.
J Physiol (Lond)
268:731-756[Abstract/Free Full Text].
-
Apel ED,
Glass DJ,
Moscoso LM,
Yancopolous GD,
Sanes JR
(1997)
Rapsyn is required for MuSK signaling and recruits synaptic components to a MuSK-containing scaffold.
Neuron
18:623-635[Web of Science][Medline].
-
Augustine GJ,
Adler EM,
Charlton MP,
Hans M,
Swandulla D,
Zipser K
(1992)
Presynaptic calcium signals during neurotransmitter release: detection with fluorescent indicators and other calcium chelators.
J Physiol (Paris)
86:129-134[Web of Science][Medline].
-
Bailey CH,
Kandel ER
(1993)
Structural changes accompanying memory storage.
Annu Rev Physiol
55:397-426[Web of Science][Medline].
-
Balice-Gordon RJ,
Lichtman JW
(1993)
In vivo observations of pre- and postsynaptic changes during the transition from multiple to single innervation at developing neuromuscular junctions.
J Neurosci
13:834-855[Abstract].
-
Bear MF,
Malenka RC
(1994)
Synaptic plasticity: LTP and LTD.
Curr Opin Neurobiol
4:389-399[Medline].
-
Bowe MA,
Fallon JR
(1995)
The role of agrin in synapse formation.
Annu Rev Neurosci
18:443-462[Web of Science][Medline].
-
Bowe MA,
Deyst KA,
Leszyk JD,
Fallon JR
(1994)
Identification and purification of an agrin receptor from Torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans.
Neuron
12:1173-1180[Web of Science][Medline].
-
Campanelli JT,
Roberds SL,
Campbell KP,
Scheller RH
(1994)
A role for dystrophin-associated glycoproteins and utrophin in agrin-induced AChR clustering.
Cell
77:663-674[Web of Science][Medline].
-
Caroni P,
Rotzler S,
Britt JC,
Brenner HR
(1993)
Calcium influx and protein phosphorylation mediate the metabolic stabilization of synaptic acetylcholine receptors in muscle.
J Neurosci
13:1315-1325[Abstract].
-
Connolly JA
(1984)
Role of the cytoskeleton in the formation, stabilization, and removal of acetylcholine receptor clusters in cultured muscle cells.
J Cell Biol
99:148-154[Abstract/Free Full Text].
-
Cummings DD,
Wilcox KS,
Dichter MA
(1996)
Calcium-dependent paired-pulse facilitation of miniature EPSC frequency accompanies depression of EPSCs at hippocampal synapses in culture.
J Neurosci
16:5312-5323[Abstract/Free Full Text].
-
De Chiara TM,
Bowen DC,
Valenzuela DM,
Simmons MV,
Poueymirou WT,
Thomas S,
Kinetz E,
Compton DL,
Rojas E,
Park JS,
Smith C,
Distefano PS,
Glass DJ,
Burden SJ,
Yancopoulos GD
(1996)
The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo.
Cell
85:501-512[Web of Science][Medline].
-
Deisseroth K,
Bito H,
Tsien RW
(1996)
Signaling from synapse to nucleus: postsynaptic CREB phosphorylation during multiple forms of hippocampal synaptic plasticity.
Neuron
16:89-101[Web of Science][Medline].
-
Dmytrenko GM,
Bloch RJ
(1993)
Evidence for transmembrane anchoring of extracellular matrix at acetylcholine receptor clusters.
Exp Cell Res
206:323-334[Web of Science][Medline].
-
Fertuck HC,
Salpeter MM
(1976)
Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscopy autoradiography after 125I-alpha bungarotoxin binding at mouse neuromuscular junctions.
J Cell Biol
69:144-158[Abstract/Free Full Text].
-
Frank E,
Fischbach GD
(1979)
Early events in neuromuscular junction formation in vitro: induction of acetylcholine receptor clusters in the postsynaptic membrane and morphology of newly formed synapses.
J Cell Biol
83:143-158[Abstract/Free Full Text].
-
Froehner SC,
Douville K,
Klink S,
Culp WJ
(1983)
Monoclonal antibodies to cytoplasmic domains of the acetylcholine receptor.
J Biol Chem
258:7112-7120[Abstract/Free Full Text].
-
Gautam M,
Noakes PG,
Mudd J,
Nichol M,
Chu GC,
Sanes JR,
Merlie JP
(1995)
Failure of postsynaptic specialization to develop at neuromuscular junctions of rapsyn-deficient mice.
Nature
377:232-236[Medline].
-
Gautam M,
Noakes PG,
Moscoso L,
Rupp F,
Scheller RH,
Merlie JP,
Sanes JR
(1996)
Defective neuromuscular synaptogenesis in agrin-deficient mutant mice.
Cell
85:525-535[Web of Science][Medline].
-
Gillis JM
(1996)
Membrane abnormalities and Ca homeostasis in muscles of the mdx mouse, an animal model of the Duchenne muscular dystrophy: a review.
Acta Physiol Scand
156:397-406[Web of Science][Medline].
-
Glass DJ,
Bowen DC,
Stitt TN,
Radziejewski C,
Bruno J,
Ryan TE,
Gies DR,
Shah S,
Mattsson K,
Burden SJ,
Distefano PS,
Valenzuela DM,
Dechiara TM,
Yancopoulos GD
(1996)
Agrin acts via a MuSK receptor complex.
Cell
85:513-523[Web of Science][Medline].
-
Godfrey EW,
Nitkin RM,
Wallace BG,
Rubin LL,
McMahan UJ
(1984)
Components of Torpedo electric organ and muscle that cause aggregation of acetylcholine receptors on cultured muscle cells.
J Cell Biol
99:615-627[Abstract/Free Full Text].
-
Gu X,
Spitzer NC
(1995)
Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients.
Nature
375:784-787[Medline].
-
Hall ZW, Sanes JR (1993) Synaptic structure and development:
the neuromuscular junction. Neuron 10:[Suppl]99-121.
-
Henderson LP,
Smith MA,
Spitzer NC
(1984)
The absence of calcium blocks impulse-evoked release of acetylcholine but not de novo formation of functional neuromuscular synaptic contacts in culture.
J Neurosci
4:3140-3150[Abstract].
-
Hoch W,
Campanelli JT,
Harrison S,
Scheller RH
(1994)
Structural domains of agrin required for clustering of nicotinic acetylcholine receptors.
EMBO J
13:2814-2821[Web of Science][Medline].
-
Huang CF,
Flucher BE,
Schmidt MM,
Stroud SK,
Schmidt J
(1994)
Depolarization-transcription signals in skeletal muscle use calcium flux through L channels, but bypass the sarcoplasmic reticulum.
Neuron
13:167-177[Web of Science][Medline].
-
Jacob MH,
Lindstrom JM,
Berg DK
(1986)
Surface and intracellular distribution of a putative neuronal nicotinic acetylcholine receptor.
J Cell Biol
103:205-214[Abstract/Free Full Text].
-
Kasai H
(1993)
Cytosolic Ca2+ gradients, Ca2+ binding proteins, and synaptic plasticity.
Neurosci Res
16:1-7[Web of Science][Medline].
-
Kirkwood A,
Bear MF
(1995)
Elementary forms of synaptic plasticity in the visual cortex.
Biol Res
28:73-80[Medline].
-
Koch C
(1997)
Computation and the single neuron [news].
Nature
385:207-210[Medline].
-
Lieth E,
Fallon JR
(1993)
Muscle agrin: neural regulation and localization at nerve-induced acetylcholine receptor clusters.
J Neurosci
13:2509-2514[Abstract].
-
Meier T,
Perez GM,
Wallace BG
(1995)
Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation.
J Cell Biol
131:441-451[Abstract/Free Full Text].
-
Mook-jung I,
Gordon H
(1995)
Acetylcholine receptor clustering in C2 muscle cells requires chondroitin sulfate.
J Neurobiol
28:482-492[Web of Science][Medline].
-
Nastuk MA,
Fallon JR
(1993)
Agrin and the molecular choreography of synapse formation.
Trends Neurosci
16:72-76[Web of Science][Medline].
-
Nastuk MA,
Lieth E,
Ma J,
Cardasis CA,
Moynihan EB,
McKechnie BA,
Fallon JR
(1991)
The putative agrin receptor binds ligand in a calcium-dependent manner and aggregates during agrin-induced acetylcholine receptor clustering.
Neuron
7:807-818[Web of Science][Medline].
-
Nitkin RM,
Rothschild TC
(1990)
Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR-aggregation.
J Cell Biol
111:1161-1170[Abstract/Free Full Text].
-
Nitkin RM,
Smith MA,
Magill C,
Fallon JR,
Yao YM,
Wallace BG,
McMahan UJ
(1987)
Identification of agrin, a synaptic organizing protein from Torpedo electric organ.
J Cell Biol
105:2471-2478[Abstract/Free Full Text].
-
Nusser Z,
Mulvihill E,
Streit P,
Somogyi P
(1994)
Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization.
Neuroscience
61:421-427[Web of Science][Medline].
-
O'Toole JJ,
Deyst KA,
Bowe MA,
Nastuk MA,
McKechnie BA,
Fallon JR
(1996)
Alternative splicing of agrin regulates its binding to heparin alpha-dystroglycan, and the cell surface.
Proc Natl Acad Sci USA
93:7369-7374[Abstract/Free Full Text].
-
Penner R,
Neher E
(1988)
Secretory responses of rat peritoneal mast cells to high intracellular calcium.
FEBS Lett
226:307-313[Web of Science][Medline].
-
Qu Z,
Huganir RL
(1994)
Comparison of innervation and agrin-induced tyrosine phosphorylation of the nicotinic acetylcholine receptor.
J Neurosci
14:6834-6841[Abstract].
-
Richard I,
Broux O,
Allamand V,
Fougerousse F,
Chiannilkulchai N,
Bourg N,
Brenguier L,
Devaud C,
Pasturaud P,
Roudaut C,
Hillaire D,
Passos-Bueno MR,
Zatz M,
Tischfield JA,
Fardeau M,
Jackson CE,
Cohen D,
Beckmann JS
(1995)
Mutations in the proteolytic enzyme calpain 3 cause limb-girdle muscular dystrophy type 2A.
Cell
81:27-40[Web of Science][Medline].
-
Roberts WM
(1993)
Spatial calcium buffering in saccular hair cells.
Nature
363:74-76[Medline].
-
Robitaille R,
Garcia ML,
Kaczorowski GJ,
Charlton MP
(1993)
Functional colocalization of calcium and calcium-gated potassium channels in control of transmitter release.
Neuron
11:645-655[Web of Science][Medline].
-
Schweizer FE,
Betz H,
Augustine GJ
(1995)
From vesicle docking to endocytosis: intermediate reactions of exocytosis.
Neuron
14:689-696[Web of Science][Medline].
-
Stern MD
(1992)
Buffering of calcium in the vicinity of a channel pore.
Cell Calcium
13:183-192[Web of Science][Medline].
-
Triller A,
Cluzeaud F,
Pfeiffer F,
Betz H,
Korn H
(1985)
Distribution of glycine receptors at central synapses: an immunoelectron microscopy study.
J Cell Biol
101:683-688[Abstract/Free Full Text].
-
Tsien RY
(1981)
A non-disruptive technique for loading calcium buffers and indicators into cells.
Nature
290:527-528[Medline].
-
Tzartos S,
Langeberg L,
Hochschwender S,
Lindstrom J
(1983)
Demonstration of a main immunogenic region on acetylcholine receptors from human muscle using monoclonal antibodies to human receptor.
FEBS Lett
158:116-118[Web of Science][Medline].
-
Ullrich A,
Schlessinger J
(1990)
Signal transduction by receptors with tyrosine kinase activity.
Cell
61:203-212[Web of Science][Medline].
-
van Ommen GJ
(1995)
A foundation for limb-girdle muscular dystrophy.
Nat Med
1:412-414[Web of Science][Medline].
-
Verma A,
Hirsch DJ,
Hanley MR,
Thastrup O,
Christensen SB,
Snyder SH
(1990)
Inositol triphosphate and thapsigargin discriminate endoplasmic reticulum stores of calcium in rat brain.
Biochem Biophys Res Commun
172:811-816[Web of Science][Medline].
-
Wallace BG
(1988)
Regulation of agrin-induced acetylcholine receptor aggregation by Ca2+ and phorbol ester.
J Cell Biol
107:267-278[Abstract/Free Full Text].
-
Wallace BG,
Qu Z,
Huganir RL
(1991)
Agrin induces phosphorylation of the nicotinic acetylcholine receptor.
Neuron
6:869-878[Web of Science][Medline].
-
Weiler IJ,
Hawrylak N,
Greenough WT
(1995)
Morphogenesis in memory formation: synaptic and cellular mechanisms.
Behav Brain Res
66:1-6[Web of Science][Medline].
-
Wells DG,
Fallon JR
(1996)
Neuromuscular junction: the state of the union.
Curr Biol
6:1073-1075[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/182672-07$05.00/0
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|
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|
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|
|
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|
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Top
Abstract
Introduction
