Neuron, Vol. 46, 569–579, May 19, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.neuron.2005.04.002
Neurotransmitter Acetylcholine Negatively
Regulates Neuromuscular Synapse Formation
by a Cdk5-Dependent Mechanism
Weichun Lin,1Bertha Dominguez, Jiefei Yang,
Prafulla Aryal, Eugene P. Brandon,
Fred H. Gage, and Kuo-Fen Lee*
The Salk Institute
10010 North Torrey Pines Road
La Jolla, California 92037
postsynaptic apparatus. However, recent genetic studies
suggest that postsynaptic differentiation of the neuro-
muscular synapse is initiated by a nerve-independent
mechanism and is prepatterned within the muscle (Lin
et al., 2001; Yang et al., 2001; Yang et al., 2000). During
the initial stage of synaptogenesis, the nascent, post-
synaptic acetylcholine receptor (AChR) clusters are
formed along a narrow central region of muscle and are
not closely apposed by nerve terminals (Lin et al., 2001;
Lupa and Hall, 1989; Yang et al., 2001). We have pos-
tulated that, at subsequent stages of synaptogenesis,
the navigating nerve simultaneously provides both pos-
itive and negative signals to regulate proper differentia-
tion and connection of the postsynaptic apparatus with
a nerve terminal (Lin et al., 2001). The positive signals
promote apposition and stability of the postsynaptic
apparatus with a nerve terminal, whereas the negative
signals disperse the postsynaptic apparatus that fails
to make the connection. As a result of the interplay of
these positive and negative signals, individual postsyn-
aptic apparatuses on each muscle fiber are innervated
What is the molecular nature of these positive and
negative signals that maintain or disperse the postsyn-
aptic apparatus during embryonic development? Act-
ing in part through the muscle-specific kinase (MuSK)
receptor complex, agrin plays a positive role in post-
synaptic differentiation by inducing and maintaining
AChR clustering in vitro and in vivo (Ferns et al., 1992;
Gautam et al., 1996; Lin et al., 2001; McMahan, 1990;
Nitkin et al., 1987; Ruegg et al., 1992; Yang et al., 2001).
Interestingly, analysis of agrin and MuSK mutant mice
revealed that MuSK, but not agrin, is required for initiat-
ing postsynaptic differentiation (Herbst et al., 2002; Lin
et al., 2001; Yang et al., 2001). However, agrin is pos-
tulated to play a role in stabilizing the nascent postsyn-
aptic apparatus that it encounters as well as in inducing
new postsynaptic sites (Lin et al., 2001; Yang et al.,
2001). Consistent with this idea, AChR clusters are ini-
tially formed in agrin-deficient mice but become dis-
persed at subsequent stages. We have hypothesized
that negative signals from the nerve and/or accompa-
nying Schwann cells induce the dispersion of the post-
synaptic apparatus in agrin mutant mice (Lin et al.,
2001). Both trophic factors and neural activity may play
a role in negatively regulating postsynaptic differentia-
tion by disassembling the postsynaptic apparatus in
cultures (Bloch, 1979; Bloch, 1986; Loeb et al., 2002;
Trinidad and Cohen, 2004; Wells et al., 1999). Agrin has
been shown to cause inhibition of motor axon growth
and to promote the assembly of presynaptic specializa-
tion in vitro (Campagna et al., 1997; Campagna et al.,
1995). Interestingly, both MuSK and agrin mutant mice
display excess axon growth along muscle fibers (DeChi-
ara et al., 1996; Gautam et al., 1996; Lin et al., 2001).
Thus, agrin functions as a positive signal to promote
both presynaptic and postsynaptic development. Fi-
nally, several lines of evidence from studies using phar-
macological inhibitors of cytoplasmic protein kinases
and phosphatases suggest that these molecules are in-
Synapse formation requires interactions between pre-
and postsynaptic cells to establish the connection of
a presynaptic nerve terminal with the neurotransmitter
receptor-rich postsynaptic apparatus. At developing
vertebrate neuromuscular junctions, acetylcholine
receptor (AChR) clusters of nascent postsynaptic
apparatus are not apposed by presynaptic nerve ter-
minals. Two opposing activities subsequently pro-
mote the formation of synapses: positive signals sta-
bilize the innervated AChR clusters, whereas negative
signals disperse those that are not innervated. Al-
though the nerve-derived protein agrin has been sug-
gested to be a positive signal, the negative signals
remain elusive. Here, we show that cyclin-dependent
kinase 5 (Cdk5) is activated by ACh agonists and is
required for the ACh agonist-induced dispersion of
the AChR clusters that have not been stabilized by
agrin. Genetic elimination of Cdk5 or blocking ACh
production prevents the dispersion of AChR clusters
in agrin mutants. Therefore, we propose that ACh
negatively regulates neuromuscular synapse forma-
tion through a Cdk5-dependent mechanism.
Synapses are specialized structural units for cellular
communication in the nervous system and therefore are
essential for executing complex nervous system func-
tions. A central question in neurobiology is how the pat-
terns of synaptic connection are established and main-
tained (Cohen-Cory, 2002; Goda and Davis, 2003; Shen
et al., 2004). One of the key features during patterning
and formation of synapses is the close apposition of
individual neurotransmitter receptor-rich postsynaptic
apparatus with a specialized nerve terminal. The verte-
brate neuromuscular junction (NMJ) is a well-studied
model for elucidating the mechanisms underlying pat-
terning and formation of synapses (Burden, 1998;
Sanes and Lichtman, 1999; Wyatt and Balice-Gordon,
Over the last several decades, a neurocentric model
of NMJ synaptic formation has been espoused, sug-
gesting that motor nerves are largely responsible for
both initiating and maintaining differentiation of the
1Present address: Center for Basic Neuroscience, UT Southwest-
ern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas
volved in the maintenance and dispersion of AChR
clusters (Cheng and Ip, 2003; Dai and Peng, 1998;
Ferns et al., 1996; Madhavan et al., 2005; Wallace,
In the present study, we demonstrate that cyclin-
dependent kinase 5 (Cdk5), a cytoplasmic serine/threo-
nine kinase, plays a role in the dispersion of AChR clus-
ters. We show that cholinergic agonists activate Cdk5
and disperse AChR clusters in myotube cultures that
are previously induced by agrin. Blockade of Cdk5 ac-
tivity by a selective inhibitor, roscovitine, ameliorates
ACh-induced dispersion of AChR clusters in cultures.
Moreover, blocking Cdk5 activity by in utero treatment
with roscovitine or by genetic deletion of the Cdk5 gene
in agrin mutant embryos leads to the maintenance of a
significant number of AChR clusters that would other-
wise have been dispersed. Similar results were ob-
served when production of the neurotransmitter ACh
was blocked in agrin mutant embryos. These combined
pharmacological and genetic results suggest a Cdk5-
dependent pathway by which ACh regulates the forma-
tion of synapses by disassembling the postsynaptic
apparatus that fails to make correct connections with
the presynaptic terminals.
Figure 1. Maintenance of AChR Clusters in Agrin Mutants following
Injection of Cdk5-Selective Inhibitor
In utero injection of the Cdk5-specific inhibitor roscovitine dis-
solved in solution containing DMSO. E16.5 diaphragm muscles
were collected from embryos of pregnant females injected daily
with DMSO (A and B) or roscovitine (C and D) starting at E14.5 and
stained for AChR clusters. In contrast to a few AChR clusters in
AGD mutants (B), numerous AChR clusters were present in agrin
(AGD) mutants treated with roscovitine (D). Scale bar, 50 ?m.
Blockade of Cdk5 Activity Retains
AChR Clusters in Agrin Mutants
It has been previously shown that AChR clusters are
present during the early stages of development and
dispersed subsequently in agrin mutant embryos (Lin
et al., 2001; Yang et al., 2001). The nerve then provides
negative signals to elicit pathways to disperse AChR
clusters in the absence of agrin (Lin et al., 2001). What
might be the molecular basis of such negative signal-
ing? Several lines of evidence suggest that protein ki-
nases and phosphatases may play a role in clustering
or dispersing AChRs. For example, protein kinase and
phosphatase inhibitors profoundly affect the formation
and stability of AChR clusters induced by agrin (Dai and
Peng, 1998; Ferns et al., 1996; Madhavan et al., 2005;
Wallace, 1995). Several cytoplasmic phosphatases and
kinases have been implicated in the formation and sta-
bility of AChR clusters, including phosphatase Shp-2
and members of the Src kinase family (Dai and Peng,
1998; Madhavan et al., 2005; Smith et al., 2001). Recent
results suggest that Cdk5 may be activated by nerve-
derived dispersion factors to antagonize agrin-medi-
ated AChR clustering. First, Cdk5 and its coactivator
p35 are expressed in muscle (Fu et al., 2001). Second,
Cdk5 can be activated by factors that have been shown
to induce the dispersion of AChR clusters in vitro, in-
cluding brain-derived neurotrophic
(Tokuoka et al., 2000; Wells et al., 1999) and neuregu-
lin-1 (NRG-1) (Fu et al., 2001; Fu et al., 2003; Trinidad
and Cohen, 2004). Finally, activation of Cdk5 reduces
the clustering of the scaffolding protein postsynaptic
density-95 (PSD-95) and ion channels in heterologous
cells, and Cdk5-deficient cortical neurons display in-
creased PSD-95 cluster size (Morabito et al., 2004),
suggesting a negative role for Cdk5 in regulating as-
sembly of the postsynaptic apparatus. Therefore, we
asked whether Cdk5 is required for dispersing AChR
clusters in agrin mutant embryos.
We began our analysis by pharmacologically block-
ing Cdk5 activity in agrin (AGD allele) mutants in utero.
Pregnant E14.5 female mice from an intercross of AGD
heterozygotes were injected daily with the Cdk5-spe-
cific inhibitor roscovitine to block the Cdk5 activity
(Meijer et al., 1997). Embryos were collected at E16.5,
two days after the start of roscovitine injection, and
were processed for AChR clustering analysis. Since
roscovitine was dissolved in DMSO solution prior to in-
jection, DMSO-containing solution was injected as a
control. As shown in Figure 1, few AChR clusters were
present in AGD mutant muscles treated with DMSO
alone (Figure 1B). In contrast, numerous AChR clusters
were present in AGD mutants treated with roscovitine
(Figure 1D). Control littermates treated either with
DMSO alone (Figure 1A) or roscovitine (Figure 1C) did
not exhibit a marked alteration in AChR clustering.
Quantitatively, the average areas of individual AChR
clusters were much greater in AGD mutants treated
with roscovitine than those observed in AGD mutants
treated with DMSO alone (Table 1).
Although roscovitine has been widely used to block
Cdk5 activity, it is possible that the inhibitor may have
other nonspecific effects in embryos. We therefore took
a genetic approach to further determine whether Cdk5
plays a role in the dispersion of AChR clusters in agrin
mutants. We analyzed NMJ development in Cdk5 mu-
tants (Xie et al., 2003) and agrin (AGD)/Cdk5 double
ACh Negatively Regulates Synaptogenesis via Cdk5
S1–S3 in the Supplemental Data available with this arti-
cle online). Consistent with the results from roscovitine-
treated AGD mutant embryos (Figure 1D), significantly
more AChR clusters were present in AGD/Cdk5 double
mutants (Figure 2D) compared to AGD single mutants
(Figure 2C) (Table 1). Furthermore, presynaptic ter-
minals in AGD/Cdk5 double mutants were densely la-
beled by antibodies against synaptophysin, a synaptic
vesicle protein, indicative of nerve terminal differentia-
tion (inset in Figure 2D), whereas the nerve terminals
failed to differentiate in AGD mutants (inset in Figure
2C). Thus, both pharmacological and genetic data sup-
port the hypothesis that Cdk5 plays a critical role in the
dispersion of AChR clusters in agrin mutants.
Table 1. Analysis of AChR Clusters in E16.5 Controls and AGD,
Cdk5, and AGD/Cdk5 Mutants
Genotype Area Number/Field
32.2 ± 2.6 (97)
10.1 ± 2.2 (7)
33.3 ± 3.1 (88)
34.4 ± 2.4 (71)
6.3 ± 0.3 (5)
24.2 ± 1.6a(94)
33.5 ± 3.5 (125)
26.7 ± 2.7c(105)
265 ± 24 (3)
17 ± 2 (3)
274 ± 5 (3)
268 ± 11 (3)
11 ± 2 (3)
131 ± 28a(3)
319 ± 20b(3)
113 ± 15c(3)
Average area (?m2) of individual AChR clusters and numbers of
AChR clusters/field were measured as described in the Experimental
Procedures. Values are mean ± SEM; numbers in parentheses
indicate numbers of AChR clusters or animals measured.
aCompared with AGD:DMSO, p < 0.01.
bCompared with control, p < 0.05.
cCompared with AGD, p < 0.01.
Cholinergic Agonist Activates Cdk5
What might be the nerve-derived signals that activate
Cdk5 to induce the dispersion of AChR clusters? Sev-
eral lines of evidence suggest that ACh may be a physi-
ological candidate. First, Cdk5 is activated by synaptic
activity and plays a role in synaptic transmission (Li et
al., 2001). For example, Cdk5 activation is induced by
glutamate agonists in neostriatal slices (Liu et al., 2001).
Second, cholinergic agonists cause the dispersion of
spontaneous AChR clusters that are present in myo-
tube explant cultures (Bloch, 1979; Bloch, 1986). How-
ever, it is not known whether cholinergic agonists can
induce dispersion of AChR clusters that have been pre-
viously induced by agrin or whether agrin can antago-
nize the cholinergic agonist-induced AChR dispersion
if both are present in the culture. Finally, electrical stim-
ulation affects the stability of AChR aggregates in adult
muscles injected with agrin (Bezakova and Lomo,
2001), raising the possibility that agrin stabilizes embry-
onic AChR clusters that would otherwise be dispersed
by ACh released from motor axons (Hume et al., 1983;
Young and Poo, 1983). Thus, we sought to determine
whether cholinergic agonists activate Cdk5 to elicit a
signaling cascade that disassembles AChR clusters.
To determine whether cholinergic agonists activate
Cdk5, mouse C2C12 myotubes were treated with car-
bachol, agrin, or both together. Cell lysates were then
immunoprecipitated with anti-Cdk5 antibodies and
subjected to a kinase assay using histone H1 as the
substrate. As shown in Figure 3A, the levels of phos-
pho-H1, indicative of Cdk5 activity, were increased fol-
lowing the AChR agonist carbachol treatment in C2C12
myotubes (3.8 ± 0.86 fold; n = 3; Figure 3B). Agrin treat-
ment alone did not significantly affect Cdk5 activity
(1.36 ± 0.38 fold; n = 3; Figure 3B). Addition of agrin
slightly reduced carbachol-induced Cdk5 activation
(3.37 ± 0.21 fold; n = 3; Figure 3B). Cdk5-selective inhib-
itor roscovitine (Meijer et al., 1997) inhibited carbachol-
induced Cdk5 activation (Figure 3A). The levels of Cdk5
(Figure 3A) and its coactivator p35 (data not shown)
were not altered following carbachol or agrin treatment.
These results demonstrate that Cdk5 can be activated
by cholinergic agonists.
mutants. We found that the number of AChR clusters
in Cdk5 single mutant mice (Figure 2B) is significantly
greater compared to controls (Figure 2A) (Table 1) (for
the analysis of other aspects of NMJ development in
Cdk5 mutants, see Supplemental Results and Figures
Figure 2. Genetic Inactivation of the Cdk5 Gene Maintains AChR
Clustering in AGD Mutants
E16.5 whole-mount diaphragm muscles from controls (A) and Cdk5
(B), AGD (C), and AGD/Cdk5 (D) mutants were double stained with
Texas red-conjugated α-BTX and anti-synaptophysin antibodies. In
contrast to AGD mutants, numerous AChR clusters were detected
in AGD/Cdk5 double mutants. Compared to control and Cdk5 mu-
tants, the number of AChR clusters in AGD/Cdk5 double mutants
was reduced, and some of them were smaller in size. (Inset) Synap-
tophysin-rich nerve terminals (green) were apposed by AChR clus-
ters (red) (yellow color indicates colocalization) in controls (A) and
Cdk5 (B) and AGD/Cdk5 (D) mutants, indicating the differentiation
of presynaptic terminals. In contrast, presynaptic differentiation
was not seen in AGD mutants (C), as evidenced by the uniform
distribution of synaptophysin immunoreactivity along the entire
nerves. Scale bar, 100 ?m.
Agrin Prevents the Cholinergic Agonist-Induced
Dispersion of AChR Clusters
Next, we determined whether cholinergic agonists dis-
perse AChR clusters in the absence of agrin. Mouse
Figure 3. Cholinergic Agonist Activates Cdk5
(A) Kinase assay: C2C12 myotubes were treated for 15 min with
carbachol (10−4M), agrin (10 ng/ml), or carbachol together with
agrin. Cell lysates were immunoprecipitated with anti-Cdk5 anti-
bodies and subjected to kinase activity assay using histone H1 as
substrates. The levels of phospho-histone H1 (p-histone H1) were
indicative of Cdk5 activities. Western blotting with total lysates
showed that equal amounts of Cdk5 proteins were used for kinase
assay. Carbachol treatment increases Cdk5 kinase activity. Addi-
tion of roscovitine inhibited carbachol-induced Cdk5 activity.
(B) Quantification of kinase assay. The results were expressed as
mean ± SEM. **p < 0.01, control versus carbachol;##p < 0.01, car-
bachol versus carbachol + roscovitine.
Figure 4. Agrin Antagonizes Cholinergic Agonist-Dependent Dis-
persion of AChR Clusters in Mouse Myotube Cultures
(A–D) C2C12 myotubes stained with Texas red-conjugated αBTX to
detect AChR clusters. Myotubes were treated overnight without (A)
or with (B, C, and D) agrin, washed with medium to remove agrin,
and then treated with (C) or without (B) carbachol or with agrin
and carbachol (D) for an additional 16–18 hr. AChR clusters were
dispersed in the cultures treated with carbachol (C) but maintained
in the absence of carbachol (B) or when agrin was added together
with carbachol (D). (E) The number of AChR clusters per micro-
scopic field (400×) for each condition. In addition to carbachol,
some myotube cultures were treated with acetylcholine, muscarine,
or dTc. Only carbachol and ACh induced dispersion of AChR clus-
ters. (F) Half-life of AChR clusters following the removal of agrin or
addition of carbachol or of carbachol together with agrin to myo-
tubes previously treated with agrin. The results were expressed as
mean ± SEM (E and F). **p < 0.01, compared with the cultures
in which agrin is removed;##p < 0.01, compared with carbachol
C2C12 myotube cultures were treated with agrin over-
night to induce AChR clustering. Agrin was then re-
moved by washing the cultures with medium followed
by the treatment with (Figure 4C) or without (Figure 4B)
carbachol overnight. As shown in Figure 4, following
the removal of agrin, in agreement with previous results
(Grady et al., 2000; Smith et al., 2001), AChR clusters
remain stable in cultures (Figure 4B). By contrast, agrin-
induced AChR clusters were markedly dispersed fol-
lowing carbachol treatment (Figure 4C). Similar results
were obtained when ACh was used (Figure 4E; control,
6.31 ± 0.82 clusters/field, n = 14; agrin removed, 19.62 ±
1.02 clusters/field, n = 24; carbachol, 9.48 ± 0.72 clusters/
field, n = 27; ACh, 8 ± 1.63 clusters/field, n = 11). In
contrast, neither AChR antagonist curare (dTc) nor
muscarinic AChR agonists caused dispersion of AChR
clusters (Figure 4E; muscarine, 17.36 ± 2.01 clusters/
field, n = 13; dTc, 20.5 ± 2.60 clusters/field, n = 14).
We then asked whether agrin antagonizes ACh-
induced dispersion of AChR clusters by adding agrin
back together with carbachol in the cultures. As shown
in Figure 4D, significant numbers of AChR clusters were
maintained when both agrin and carbachol were added
into cultures as compared to the cultures treated with
carbachol alone (Figure 4E; 16.5 ± 1.2 clusters/field;
n = 14). These results demonstrate that ACh, acting
through nicotinic AChRs, induces the dispersion of
AChR clusters previously induced by agrin. Importantly,
agrin antagonizes the dispersion activity elicited by
ACh on AChR clusters. We further determined the half-
life of AChR clusters without agrin (agrin removed) or
with carbachol or carbachol plus agrin. As shown in
Figure 4F, the half-life of AChR clusters after the re-
moval of agrin and without carbachol treatment was
greater than 12 hr as compared to 1.2 hr for carbachol-
treated clusters. When agrin was added together with
carbachol, the half-life was increased to approximately
5–6 hr. The number of AChR clusters was not further
reduced, possibly in part due to formation of new clus-
ters induced by agrin that was added back to the cul-
tures. These results are consistent with the idea that
ACh and agrin play opposing roles in the maintenance
of AChR clusters.
Cdk5 Is Required for the Cholinergic Agonist-
Induced Dispersion of AChR Clusters
To determine whether Cdk5 activation is required for
the carbachol-induced dispersion of AChR clusters,
ACh Negatively Regulates Synaptogenesis via Cdk5
Figure 5. Cdk5 Is Required for Carbachol-Induced Dispersion of AChR Clusters in Myotube Cultures
(A) The Cdk5-specific inhibitor roscovitine blocked carbachol-induced dispersion of AChR clusters in C2C12 myotubes. **p < 0.01, carbachol
versus carbachol + agrin.
(B–D) Primary myotubes from controls and Cdk5 mutants were treated with or without agrin overnight, washed with medium to remove agrin,
and then treated with or without carbachol for another 16–18 hr. AChR clusters were dispersed in the cultures treated with carbachol in
control myotubes (C) but maintained in the Cdk5 mutant myotubes. (C) Because AChR clusters were present in a more diffuse appearance
in primary cultures, the longest axis of AChR clusters per 100 ?m myotube was measured. Carbachol treatment markedly reduced the size
of clusters in control myotubes. In contrast, carbachol treatment increased the size of AChR clusters in Cdk5 mutant myotubes. Similar
results were observed when the average size of clusters was measured (D). The results were expressed as mean ± SEM (A, C, and D). **p <
0.01, compared to agrin removed for controls.##p < 0.05, compared to agrin removed for Cdk5 mutants.
C2C12 myotubes were treated with agrin overnight. Af-
ter removing agrin, the cultures were then treated with
medium, carbachol, or carbachol together with roscovi-
tine. As illustrated in Figure 5A, roscovitine treatment
blocked carbachol-induced dispersion of AChR clus-
ters (agrin, 26.7 ± 4.4 clusters/field, n = 15; carbachol,
12 ± 2.91 clusters/field, n = 21; carbachol + roscovitine,
25.13 ± 2.82 clusters/field, n = 25). Roscovitine alone
did not induce AChR clustering in myotube cultures.
To further test the role of Cdk5 in the cholinergic ago-
nist-induced dispersion of AChR clusters, primary myo-
tubes were generated from control and Cdk5 mutants
and treated with agrin. Following the removal of agrin,
the cultures were then treated with carbachol. In con-
trast to the individual AChR clusters in C2C12 myo-
tubes, which had an elongated shape and sharp
boundary (Figure 4), the AChR clusters in both control
and Cdk5 mutant primary myotubes were more diffused
(Figure 5B). Therefore, the longest axis of AChR clus-
ters per 100 ?m myotube was measured. As shown in
Figures 5B and 5C, the sizes of AChR clusters in Cdk5
mutant myotubes were not significantly reduced by
carbachol. In contrast, carbachol markedly reduced the
sizes of AChR clusters in control myotubes. Similar re-
sults were found when the average sizes of individual
clusters were compared between control and Cdk5 mu-
tant myotubes (Figure 5D). These results suggest that
Cdk5 is required for the cholinergic agonist-induced
dispersion of AChR clusters.
Blocking of ACh Production in Agrin Mutants
Leads to the Maintenance of AChR Clusters
The above in vitro data suggest that the inhibition of
ACh production in agrin mutant mice may lead to the
maintenance of AChR clusters that would otherwise be
dispersed. To test this hypothesis, we took a genetic
approach and analyzed double mutant mice deficient
in agrin (Lin et al., 2001) and choline acetyltransferase
(ChAT) (Brandon et al., 2003), the key enzyme for bio-
synthesis of ACh. As shown in Figure 6, a few small,
dim AChR clusters were observed in E17.5 AGD mutant
muscles (Figure 6B) (Table 2). In contrast, numerous
AChR clusters were present in the E17.5 AGD/ChAT
double mutants (Figure 6D). Furthermore, the presyn-
aptic nerve terminals in AGD/ChAT double mutants dif-
ferentiated, since they were intensely labeled by anti-
bodies against synaptophysin (inset in Figure 6D). In
contrast, the presynaptic nerve terminals failed to dif-
Table 2. Analysis of AChR Clusters in E17.5 Controls and AGD,
ChAT, AGD/ChAT, and HB9 Mutant Embryos
41.4 ± 3.8 (103)
12.7 ± 1.9 (6)
156.9 ± 14 (267)
105.7 ± 11.3a,b(55)
58.3 ± 4.7c(173)
275 ± 32 (3)
5 ± 1 (3)
304 ± 6 (3)
81 ± 14a,b(3)
359 ± 26c(4)
Average area (?m2) of individual AChR clusters and numbers of
AChR clusters/field were measured as described in the Experimental
Procedures. Values are mean ± SEM; numbers in parentheses
indicate numbers of AChR clusters or animals measured.
aCompared with AGD, p < 0.01.
bCompared with ChAT, p < 0.05.
cCompared with AGD/ChAT, p < 0.01.
ferentiate in AGD mutants (inset in Figure 6B). Although
AChR clusters were present in E17.5 AGD/ChAT double
mutants and distributed in a broader pattern when
compared to that in controls (Figure 6A), the number of
AChR clusters was smaller than that observed in ChAT
mutants (Figure 6C) (Table 2). Furthermore, the average
size and area of individual AChR clusters in double mu-
tants were larger than those in controls and AGD mu-
tants, but smaller than those in ChAT mutants (Table 2).
These results suggest that, in addition to ACh, other
factors may exist to disperse AChR clusters (Davey and
Cohen, 1986; Womble, 1986) (see Discussion). In the
absence of agrin, these additional dispersion factors
may cause the dispersion of AChR clusters in agrin mu-
tants and decrease the number of AChR clusters in
AGD/ChAT double mutants. Consistent with this idea,
we found that the number of AChR clusters in aneural
muscle from HB9 mutants (Lin et al., 2001) is greater
than that in AGD/ChAT mutants (Table 2). However,
these results cannot rule out that agrin is essential for
increasing and maintaining the sizes of AChR clusters.
Although AChR clusters were initially maintained in
AGD/ChAT double mutant muscle, the stability of these
clusters may be decreased in the absence of agrin. In
ChAT mutants, agrin is present to maintain and/or in-
crease the size of individual AChR clusters. We have
noticed that increased numbers of smaller AChR clus-
ters are present in ChAT/AGD and HB9 mutants as
compared to ChAT mutants. Nevertheless, these results
support the hypothesis that ACh plays a critical role in
the dispersion of AChR clusters in AGD mutant mice.
The interplay of opposing ACh- and agrin-mediated
signaling modulates the average area of individual
AChR clusters (Table 2).
On the basis of these results, we examined whether
other aspects of postsynaptic differentiation occur in
AGD/ChAT double mutants. As shown in Figure 6, ace-
tylcholine esterase (AChE) aggregates were present in
AGD/ChAT double mutant muscle (Figure 6H). In con-
trast, few, small AChE clusters were detected in AGD
single mutant muscle (Figure 6F). However, synapse-
specific AChRα gene expression is not restored in
double mutant muscle (Figure S4). These results are
consistent with the idea that ACh negatively regulates
multiple aspects of postsynaptic differentiation in agrin
Figure 6. Genetic Inactivation of the ChAT Gene in Agrin Mutants
Leads to Increased AChR and AChE Clustering
AChR clustering: E17.5 whole-mount diaphragm muscles from con-
trols (A), AGD (B), ChAT (C), and AGD/ChAT (D) mutants were
double stained with Texas red-conjugated α-BTX and anti-synapto-
physin antibodies. Numerous AChR clusters were detected in AGD/
ChAT double mutants, but not in AGD single mutants. Furthermore,
the presynaptic nerve terminals in AGD/ChAT double mutants were
densely labeled by synaptophysin antibody and were closely ap-
posed by the postsynaptic AChR clusters (inset in [D]), similar to
those observed in controls (A) and ChAT (C) mutants, indicating the
differentiation of presynaptic terminals. In contrast, no presynaptic
differentiation was seen in AGD (B) mutants. Scale bar, 100 ?m.
AChE distribution: E17.5 whole-mount diaphragm muscle from
controls (E), AGD (F), ChAT (G), and AGD/ChAT (H) mutants was
subjected to AChE histochemistry. Similar to AChR clustering, sig-
nificantly more AChE clusters were detected in AGD/ChAT double
mutants (H) compared to AGD single mutants (F). Note also that
AChE clusters were distributed across a broader region in AGD/
ChAT double mutants (H) and ChAT single mutants (G), compared
to the controls (E). Scale bar, 500 ?m.
ACh Negatively Regulates Synaptogenesis via Cdk5
Figure 7. A Model for Establishing Synaptic Connections of the Neuromuscular Synapses
Summary of genetic analysis of postsynaptic differentiation in controls, AGD, Cdk5, AGD/ChAT, and AGD/Cdk5 mutants. Formation of the
nascent AChR clusters is induced by a nerve-independent, muscle-intrinsic, and MuSK-dependent mechanism at early stage (E14.5). These
nascent AChR clusters are not apposed by nerve terminals. At subsequent stages (E16.5–E18.5), the nerve provides agrin and ACh as positive
and negative signals, respectively, to regulate the establishment of synaptic connections. AChR clusters that are apposed by nerve terminals
are stabilized locally by an agrin-dependent activity. In contrast, clusters that are not apposed by nerve terminals are dispersed by ACh
through a Cdk5-dependent mechanism. In agrin (AGD) mutants, AChR clusters are dispersed due to the lack of the positive signal agrin and
the presence of the negative signal ACh. However, genetic elimination of Cdk5, as in AGD/Cdk5 double mutants, or blocking ACh production
as in AGD/ChAT double mutants prevents the dispersion of AChR clusters. Note that the numbers of AChR clusters are smaller in both AGD/
Cdk5 and AGD/ChAT double mutants as compared to controls, suggesting the existence of other dispersion factors as well as Cdk5-
independent signaling molecules. Green, nerve; red, AChR clusters; white, muscle.
tor nerves simultaneously supply both ACh and agrin,
as a negative and a positive signal, respectively, to reg-
ulate the activities of protein kinases and phosphatases
and, thereafter, their downstream effectors to maintain
or destabilize the postsynaptic apparatus (Dai and
Peng, 1998; Ferns et al., 1996; Luo et al., 2002; Wallace,
1995; Weston et al., 2000). We have identified Cdk5 as
one of the key intracellular regulators. At developing
NMJs, both ACh and agrin released from the nerve may
be able to diffuse an appreciable distance from axons
(Figure 7). Because agrin may be partially bound to the
extracellular matrix near nerve terminals, it may form
a steeper gradient than ACh from nerve terminals. For
clusters that are apposed by a nerve terminal, a local,
higher concentration of agrin elicits a signal to antago-
nize ACh-mediated dispersion, thereby maintaining the
AChR clusters. In contrast, for clusters that are not ap-
posed by a nerve terminal, agrin does not diffuse in
sufficient concentrations to overcome the dispersion
effect of ACh. In agrin mutant mice, the nerve exhibits
excessive growth along muscle fibers (Gautam et al.,
1996; Lin et al., 2001) (Figure 7), raising the possibility
that excess ACh is released from the nerve, leading to
increased dispersion of AChR clusters.
Based on this model, our results suggest the exis-
tence of other ACh-independent dispersing factors that
may activate both Cdk5-dependent and -independent
pathways to regulate postsynaptic differentiation. Con-
sistent with this idea, the number of AChR clusters in
AGD/ChAT mutants is decreased relative to that in
ChAT mutants (Table 2). Interestingly, BDNF and NRG-1
have been recently shown to antagonize agrin-induced
AChR clustering in myotube cultures (Trinidad and Co-
hen, 2004; Wells et al., 1999) and in chick embryos
(Loeb et al., 2002). Toward this end, we have tested
whether BDNF, acting through trkB receptor, might
cause dispersion of AChR clusters in agrin mutants by
analyzing AChR clustering in agrin (AGD)/trkB double
mutants. As was noted in AGD single mutants, AChR
clusters were not maintained in AGD/trkB double mu-
tants (data not shown). Thus, BDNF-mediated signal is
In the present study, we elucidate the mechanisms by
which nerve-derived positive and negative signals reg-
ulate synaptic development. During the initial stages of
neuromuscular synaptogenesis, the nascent AChR-rich
postsynaptic apparatus is not apposed by the nerve
terminal and is distributed along a narrow central re-
gion of muscle fiber (Lin et al., 2001). At subsequent
stages of development, the nerve provides positive sig-
nals to stabilize the postsynaptic apparatus that it en-
counters and/or to induce new synaptic sites. Simulta-
neously, negative signals are released to disassemble
the postsynaptic apparatus that is not closely apposed
by a nerve terminal and hence is not stabilized by the
nerve-derived positive signals (Lin et al., 2001). Here,
we provide several lines of evidence demonstrating
that ACh elicits a negative pathway to disassemble the
postsynaptic apparatus via a Cdk5-dependent mecha-
nism. First, acting through muscle nicotinic AChRs, but
not muscarinic AChRs, ACh disperses AChR clusters
previously induced by agrin in cultures. Second, ACh-
induced dispersion of AChR clusters is antagonized by
agrin, and inhibition of ACh production by genetic dele-
tion of the ChAT gene in agrin mutant mice leads to the
maintenance of numerous AChR clusters. Third, Cdk5
is activated by the ACh agonist, and pharmacological
blockade of Cdk5 activity in vitro inhibits the ACh ago-
nist-induced dispersion of AChR clusters. Fourth, ACh
agonist does not induce the dispersion of AChR clus-
ters in Cdk5 mutant myotubes. Finally, significant num-
bers of AChR clusters are maintained following in utero
pharmacological blockade of Cdk5 activity or genetic
ablation of the Cdk5 gene in agrin mutant mice.
On the basis of these and previous results (Brandon
et al., 2003; Lin et al., 2001; Yang et al., 2001), we pro-
pose the following model for neuromuscular synapse
formation. First, during the initiation stage of synapto-
genesis, the nascent AChR clusters are induced by a
muscle-intrinsic mechanism that requires MuSK (Lin et
al., 2001; Yang et al., 2001) (Figure 7). Second, the mo-
not essential for inducing the dispersion of AChR clus-
ters in agrin mutants. Because NRG-1 induces the dis-
persion of AChR clusters in myotube cultures (Trinidad
and Cohen, 2004), our results showing that Cdk5 is re-
quired for the dispersion of AChR clusters raise the
possibility that Cdk5 activated by NRG-1 plays a role in
dispersing AChR clusters in agrin mutant mice (Fu et
al., 2001). Furthermore, because the number of AChR
clusters in AGD/Cdk5 mutants is smaller than that in
controls or Cdk5 mutants (Table 1), other kinases may
also play a similar role in AChR clustering. Interestingly,
Pftaire-1, a Cdk5-related protein kinase, is also ex-
pressed in tissues where Cdk5 is detected, including
muscle (Besset et al., 1998; Lazzaro et al., 1997). In ad-
dition, cytoplasmic phosphatase Shp-2 has been impli-
cated in inducing dispersion of AChR clusters. As
Shp-2 is activated by NRG-1 (Tanowitz et al., 1999), it
is possible that Shp-2 is another signaling molecule
that is involved in dispersing AChR clusters (Madhavan
et al., 2005). Finally, as agrin is required for maintaining
AChR clusters or inducing new clusters, the absence
of agrin may contribute to a decrease in AChR cluster
sizes and numbers in AGD/ChAT and AGD/Cdk5 double
mutant muscle (Tables 1 and 2).
How do ACh and agrin antagonize each other to
modulate AChR clustering? Because agrin does not ap-
pear to attenuate the activation of Cdk5 induced by
ACh agonist, it is possible that agrin antagonizes the
dispersion effect of Cdk5 through further downstream
targets. One possible mechanism is that Cdk5 activa-
tion leads to instability or impaired assembly or recycl-
ing of AChR clusters by causing serine or threonine
phosphorylation of AChR subunits. However, none of
the muscle AChR subunits contains the phosphoryla-
tion consensus sequence of Cdk5, (R/K)(S/T)PX(K/H/R)
(Beaudette et al., 1993; Shetty et al., 1993). Alterna-
tively, the effects of Cdk5 activation by ACh may reside
in other downstream effectors of Cdk5. Cdk5 activation
has been shown to phosphorylate the intermediate fila-
ment protein nestin, which is colocalized with AChR
clusters (Sahlgren et al., 2003). Phosphorylation of nes-
tin by activated Cdk5 increases its solubility and affects
its association with other intermediate filaments, in-
cluding vimentin. These intermediate filaments may be
important scaffolding proteins linked to AChR clusters.
Thus, phosphorylation of nestin and other scaffolding
proteins may regulate the stability of the postsynaptic
apparatus. Interestingly, it has been shown that agrin
regulates the organization of cytoskeletal proteins as-
sociated with AChR clusters (Bezakova and Lomo,
2001). Detailed cellular and biochemical mechanisms
await future studies to determine whether these scaf-
folding proteins are the targets regulated by ACh and
agrin. Finally, although our data from muscle cultures
(Figures 3, 4, and 5) suggest that the effect of Cdk5 to
disperse aneural AChR clusters occurs at the postsyn-
aptic site, we cannot rule out that the effect of Cdk5 in
vivo is mediated, in part, through modulating presynap-
tic activity (Dhavan and Tsai, 2001; Tan et al., 2003;
Tomizawa et al., 2003).
Finally, our current findings may be relevant to syn-
apse elimination or editing at the NMJ that occurs dur-
ing the first 2 weeks of postnatal life in mice (Sanes and
Lichtman, 1999; Wyatt and Balice-Gordon, 2003). There
are some similarities and dissimilarities between em-
bryonic and neonatal phases of synaptic development.
The present study focuses on elucidating the mecha-
nisms underlying embryonic elimination of the unin-
nervated postsynaptic apparatus. As a result, individual
synaptic sites on each muscle fiber are polyneuronal at
birth because they are innervated by axons from two or
more motor neurons. These polyneuronally innervated
synapses then undergo the neonatal synapse elimina-
tion through synaptic competition to ensure that each
muscle fiber is singly innervated. Like embryonic re-
modeling, both positive and negative factors play
essential roles in neonatal synapse elimination (Sanes
and Lichtman, 1999; Wyatt and Balice-Gordon, 2003).
Several lines of evidence demonstrated that neural ac-
tivity plays an essential role in synapse elimination dur-
ing early postnatal stages (Akaaboune et al., 1999; Avila
et al., 1989; Buffelli et al., 2003; Rotzler and Brenner,
1990). Active and strong synapses (the winners) are
maintained, while less active or inactive synapses (the
losers) are eliminated during this period. Thus, it seems
paradoxical that our results suggest that ACh-mediated
neural activity causes disassembly of the postsynaptic
apparatus during embryonic development. This para-
dox may reflect different requirements for establishing
proper location and numbers of future synapses during
the dynamic phases of synaptogenesis in embryos ver-
sus maintaining the synapses in postnatal animals once
the synapses are formed along the middle of muscle
fibers. ACh may thus play a dual role in fulfilling these
different requirements. The discrepancy may be due to
several distinct structural, biophysical, and biochemi-
cal properties between embryonic and postnatal neuro-
muscular synapses. First, embryonic and postnatal
AChR complexes display different compositions and
distinct biophysical properties. The composition of the
AChR pentamer complex undergoes a postnatal switch
from α2βδγ to α2βδ? (Gu and Hall, 1988; Mishina et al.,
1986). Embryonic channels have longer open times with
a smaller mean channel conductance, whereas adult
channels have shorter open times with a higher mean
channel conductance (Fischbach and Schuetze, 1980;
Siegelbaum et al., 1984). It has been suggested that
activity-induced stabilization or disassembly of syn-
apses depends on the firing frequency and synaptic
efficacy (Barber and Lichtman, 1999). Thus, these dif-
ferent biophysical characteristics between embryonic
and postnatal synapses may lead to different patterns
of ionic influx that in turn regulate the differential sta-
bility of the respective AChR clusters. Second, it is pos-
sible that the transmembrane and cytoplasmic scaf-
folding and signaling molecules associated with the
embryonic AChR complex are different from their post-
natal counterparts, thereby contributing to different ef-
fects of ACh action on the stability of the postsynaptic
apparatus. Third, there may be additional nerve-derived
stabilizing factors that are expressed at postnatal syn-
aptic sites. Finally, active postnatal synapses may
cause the release of significantly higher concentrations
of stabilizing factors (e.g., agrin) and/or lower concen-
trations of dispersing factors than their embryonic
In conclusion, we have identified molecules that play
positive and negative roles in synaptic connection dur-
ACh Negatively Regulates Synaptogenesis via Cdk5
ively with water, cleared in PBS containing 50% glycerol, and
mounted for photography.
ing embryonic development. Agrin elicits signals to sta-
bilize the innervated postsynaptic apparatus, whereas
ACh signaling eliminates those that are uninnervated.
These results also suggest the presence of additional
positive and negative neuronal factors acting through
multiple kinases and phosphatases to regulate synaptic
connection. Elucidation of these positive and negative
pathways may shed light on the mechanisms underly-
ing the postnatal phase of synaptic remodeling or elimi-
In Situ Hybridization
Diaphragm muscles were fixed in 4% PFA in 0.1 M phosphate
buffer at 4°C overnight, cryoprotected in 30% sucrose, and sec-
tioned at 20 ?m thickness. Sections were hybridized with a33P-
labeled AChRα and δ subunit riboprobes. Slides were dipped in
NTB2 liquid nuclear emulsion, exposed for 5 days, photograph-
ically processed, and counterstained with eosin and hematoxylin.
Mouse Myotube Cultures
C2C12 myocytes were cultured in DMEM containing 20% fetal calf
serum and induced to form myotubes by replacing the medium
containing 2% horse serum. Myotubes were treated with agrin (10
ng/ml; R&D) overnight. To remove agrin, the cultures were washed
with medium three times. Carbachol, acetylcholine, muscarine, or
curare (dTc) was added at a concentration of 10−4–10−7M. A Cdk5-
specific inhibitor (Meijer et al., 1997), roscovitine (Calbiochem), was
added at 2–10 ?M. To detect AChR clusters, the cultures were fixed
with 2% PFA, washed, and incubated with Texas red-conjugated α
BTX. The number of AChR clusters per microscopic field was
counted at 400× magnification. Primary muscle cultures were pre-
pared from E18.5 embryos (O'Malley et al., 1997). The total length
and the size of AChR clusters were measured in NIH Image. The
length of the myotube was defined as the longest axis of the myo-
tube in the particular field. The size of individual AChR cluster was
measured as the longest axis of the cluster, which was usually near
parallel to the axis of myotubes. Only the AChR clusters of 1 ?m
or longer were measured.
The use of animals is in compliance with the guidelines of the Insti-
tute Animal Care and Use Committee of the Salk Institute. ChAT
(Brandon et al., 2003), agrin (AGD), HB9 (Lin et al., 2001), and Cdk5
(Xie et al., 2003) mutant mice have been described previously. Stan-
dard breeding methods were used to generate double mutants.
Diaphragm muscles were fixed in 2% paraformaldehyde (PFA) in
0.1 M phosphate buffer (pH 7.3) overnight at 4°C, rinsed briefly with
phosphate-buffered saline (PBS; pH 7.3), incubated in 0.1 M gly-
cine in PBS for 1 hr, rinsed briefly with PBS, and then washed with
0.5% Triton X-100 in PBS. The muscles were blocked in dilution
buffer (500 mM NaCl, 0.01 M phosphate buffer, 3% BSA, 5% goat
serum, and 0.01% thimerosal) overnight at 4°C, and then incubated
with primary rabbit antibodies against neurofilament-150 (1:1000;
Chemicon), synaptophysin (1:1000; DAKO), or S100-β (1:000;
Swant) in dilution buffer overnight at 4°C. After being washed three
times for 1 hr each in 0.5% Triton X-100 in PBS, the muscles were
incubated with fluorescein-conjugated goat anti-rabbit IgG (1:600;
Cappel) and Texas red-conjugated α BTX (Molecular Probes) over-
night at 4°C. The muscles were then washed three times for 1 hr
each with 0.5% Triton X-100 in PBS and once with PBS and flat
mounted in Vectorshield solution (Vector). The area of individual
AChR clusters was measured from digitized images with the NIH
Cdk5 Kinase Assay
C2C12 myotubes were treated with carbachol and/or agrin for 5–
15 min. Cell lysates were immunoprecipitated with rabbit anti-Cdk5
antibodies (Santa Cruz) for a Cdk5 assay as described previously
(Nikolic et al., 1998). Histone H1 was used as the substrate for the
Cdk5 kinase assay.
In Utero Roscovitine Injection
Timed pregnant females from AGD heterozygote intercrosses were
injected intraperitoneally with roscovitine in saline at 5–15 mg/kg
body weight once at E14.5 and E15.5. Roscovitine was initially dis-
solved in DMSO (15 mg/ml DMSO). Therefore, DMSO in saline was
used as a control. Diaphragm muscles were collected from E16.5
embryos and processed for AChR staining as described above.
Quantitative Analysis of AChR Clusters
Z-serial images were collected from whole-mount diaphragm sam-
ples with a 20× or 60× oil objective using an Olympus confocal
laser scanning microscope. The dorsal right side of the diaphragm
was used for quantitative analysis. A projected image was created
by overlaying each set of z series and was saved as a JPEG file.
The numbers of clusters were counted by two different methods
with similar results. The 20× images were adjusted and printed for
manual counting. Alternatively, the NIH Image Analysis program
was used to analyze both the numbers and sizes of clusters. Briefly,
images were opened with the NIH Image Program and changed to
grayscale. We used the threshold tool to isolate individual AChR
clusters. The areas of individual clusters were measured and ex-
pressed as pixels. The clusters with areas smaller than 15 pixels
were removed from quantification. A macro program was written
for repeated measurement of images. Pixel values of individual
clusters and the number of total clusters from each image were
transferred to an Excel spreadsheet. The numbers of clusters were
expressed as number of clusters/field. For the sizes of clusters,
60× images were used. The numbers of pixels were converted to
the area (?m2). Student’s t test was used to determine statistical
The Supplemental Data include Supplemental Results and four
supplemental figures and can be found with this article online at
We thank Chris Kintner, Sam Pfaff, John Thomas, Mary Lynn Gage,
and members of the Lee laboratory for helpful comments on the
manuscript. We thank Li-Huei Tsai for providing Cdk5 mutant mice;
Qiang Wang and Lin Mei for advice and protocols on the Cdk5
kinase assay; and Steve Burden for the in situ probes. This work
was supported by grants from the NIH (HD34534, NS41994,
NS44420, NS47345, and AG104435) and the Muscular Dystrophy
Received: February 27, 2004
Revised: December 23, 2004
Accepted: April 3, 2005
Published: May 18, 2005
AChE was visualized based upon the method described previously
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