the formation of neuromuscular junctions. Here we show that MuSK is expressed in the brain, particularly in neurons, as well as in
non-neuronal tissues. We also provide evidence that MuSK expression in the hippocampus is required for memory consolidation,
protein ? (C/EBP?) expression, suggesting that the role of MuSK during memory consolidation critically involves the CREB–C/EBP
pathway. Furthermore, we found that MuSK also plays an important role in mediating hippocampal oscillatory activity in the theta
frequency as well as in the induction and maintenance of long-term potentiation, two synaptic responses that correlate with memory
Muscle-specific tyrosine kinase receptor (MuSK) is a receptor
zuela et al., 1995), mouse (Ganju et al., 1995), Xenopus (Fu et al.,
on Northern blot analyses and in situ hybridizations have re-
tissues of mouse, chicken, and Xenopus (Ganju et al., 1995; Fu et
al., 1999; Ip et al., 2000), it has been generally accepted that this
receptor is mainly expressed and functional only in muscle, in
formation (DeChiara et al., 1996; Sanes and Lichtman, 1999).
Recently, however, MuSK has been reported to also be expressed
in sperm (Kumar et al., 2006).
At the NMJ, MuSK is critically involved in redistributing the
acetylcholine receptors (AChRs) in the postsynaptic apparatus.
form the agrin receptor complex (DeChiara et al., 1996; Sanes
and Lichtman, 1999; Hoch, 2003). In muscle, the activation of
MuSK via the ErbB–neuregulin pathway results in an increase of
AChR expression and triggers the activation of signaling path-
ways that lead to postsynaptic differentiation (Sandrock et al.,
formation and specifically for AChR organization at the NMJs,
because knock-out mice of agrin or MuSK do not form neuro-
muscular synapses, lack AChRs clustering, and die soon after
birth (DeChiara et al., 1996; Gautam et al., 1996). Agrin is ex-
pressed in several tissues, including the CNS, in which it partici-
1995; Ferreira, 1999; Smith and Hilgenberg, 2002). Despite agrin
isoforms with high AChR aggregating activity being widespread
throughout the brain, their receptor complex has not yet been
identified (Smith and Hilgenberg, 2002).
In the brain, synaptogenesis and remodeling of existing syn-
apses are abundant throughout development but also occur in
adulthood after injury and during long-term synaptic plasticity-
associated functions, including memory formation (Scheff et al.,
2005; Waites et al., 2005).
New memories become stable through a process known as
consolidation, which requires the activation of a cascade of gene
expression that critically involves the cAMP response element
binding protein (CREB) and CCAAT enhancer binding protein
(C/EBP) transcription factors (Alberini, 1999) and is accompa-
nied by modifications of synaptic structure and/or number
TheJournalofNeuroscience,July26,2006 • 26(30):7919–7932 • 7919
mitter, hormonal, or neurohumoral systems (Power et al., 2000;
Izquierdo and McGaugh, 2000), which include, among others,
the nicotinic cholinergic system (Gold, 2003; Hogg et al., 2003).
developing and adult tissues other than muscle (Ganju et al.,
CNS (Smith and Hilgenberg, 2002), we investigated the expres-
sion and the functional role of MuSK in the brain.
21- to 28-d-old male Long–Evans rats were used, as specified in Results.
Animals were individually housed and maintained on a 12 h light/dark
All rats were allowed ad libitum access to food and water. All protocols
complied with the National Institutes of Health Guide for the Care and
Use of Laboratory Animals and were approved by the Mt. Sinai School of
Medicine Animal Care Committees.
DNA amplifications. Total RNA was isolated with TRIzol (Invitrogen,
Carlsbad, CA), and reverse transcription was performed using oligo-dT.
Five sets of primers were designed based on the sequence of rat muscle
MuSK (GenBank accession number U34985) and used for PCR amplifi-
cations. Primers were as follows: starting from the 5? end of MuSK se-
quence, forward (F) 1, 5?-TTACAGATGCTCACCCTGGT-3?; reverse
(R) 1, 5?-CTTAATCCAGGACACGGATGG-3?; F2, 5?-CAAGCCATC-
CGTGTCCTGGAT-3?; R2, 5?-ACAGTAGCCTTTGCTTTCTT-3?; F3,
5?-AGTATAGCAGAATGGAGCAA-3?; R3, 5?-GGAAGGCAATGTG-
GTGAGGGT-3?; F4, 5?-CTGCCGAAGGAGGAGAGAGTG-3?; R4, 5?-
GTTTCCATCAGCTTTGTAGTA-3?; F5, 5?-AGGAACATCTACTCCG-
CAGAC-3?; R5, 5?-TGAAAAGATCCTCCTGGGTG-3?. All reactions
were run on an iCycler (Bio-Rad, Hercules, CA) with the following cycle
parameters: 1 cycle of 95°C for 2 min, 40 cycles of 95°C for 30 s followed
used the following: F, 5?-ATGAGAGAGCTCGTCAACAT-3?; R, 5?-
TGAAAAGATCCTCCTGGGTG-3?. Amplification reactions were per-
formed in the iCycler using the following parameters: 1 cycle of 95°C for
sity (New Haven, CT).
Hippocampal neuronal cell culture. Hippocampal neuronal cell cul-
tures (HNCs) were prepared from hippocampi of embryonic day 18
(E18) Long–Evans rats as described previously by Goslin and Banker
(1991) with some modifications. Cells were dissociated by treatment
with 0.25% trypsin for 15 min at 37°C followed by trituration through a
Pasteur pipette. Cells were plated at a density of 1.8 ? 104cells/cm2on
poly-L-lysine-coated coverslips in minimum essential medium (Invitro-
gen; Carlsbad, CA) containing 10% horse serum. After 3 h, when cells
had attached, coverslips were transferred to dishes containing Neuro-
basal medium supplemented with B-27 (Invitrogen), where they were
maintained for the entire time of culture. Cells were used at 12–14 d in
Riboprobe synthesis and in situ hybridization. Three cDNA fragments
rat MuSK (GenBank accession number U34985), subcloned into the
pPCR-Script Amp SK (?) plasmid (Stratagene, La Jolla, CA) were used.
and sense digoxigenin (DIG)-labeled riboprobe synthesis (Roche Diag-
nostics, Indianapolis, IN). Transcription was performed using T3 or T7
polymerases (Roche Diagnostics), and the synthesized riboprobes were
purified with ProbeQuant G-50 Micro Columns (GE Healthcare Bio-
Sciences, Piscataway, NJ). DIG was visualized with anti-DIG antibody
coupled to alkaline phosphatase (AP) and detected with AP Conjugate
paraformaldehyde (PFA) for 30 min at room temperature (RT). Cells
were washed with PBS-diethyl pyrocarbonate (DEPC), permeabilized
ing 0.1% active DEPC. The cells were then equilibrated twice for 10 min
in 2? SSC. Prehybridization was performed at 56°C for 1 h with the
hybridization buffer containing 50% formamide, 5? SSC, 2% blocking
reagent (Roche Diagnostics), and 40 ?g/ml salmon sperm DNA.
Twenty-micrometer rat brain tissue sections were obtained with a cryo-
and postfixed with 4% PFA-DEPC for 15 min at RT. After rinsing with
PBS-DEPC, sections were treated with 0.2 M HCl for 10 min and perme-
abilized with 0.5% Triton X-100 for 10 min at RT. Sections were finally
equilibrated in 0.1 M triethanolamine (TEA) for 5 min and acetylated in
freshly prepared 0.25% acetic anhydrite in 0.1 M TEA for 10 min. Sense
and antisense riboprobes (200 ng/ml for cell cultures and 400 ng/ml for
tissue sections) were diluted in hybridization buffer, denatured at 75°C
for 10 min, and incubated with either HNCs or brain sections overnight
at 56°C. The next day, the samples were rinsed with 2? SSC twice for 10
min at RT, treated with RNase A (20 ?g/ml) for 30 min at 37°C, and
washed sequentially with 2? SSC for 30 min at RT, 2? SSC for 1 h at
65°C, and 0.1? SSC for 1 h at 65°C. After rinsing with Tris-NaCl buffer
(100 mM Tris, 150 mM NaCl), the samples were incubated with blocking
Tris-NaCl buffer] for 30 min at 37°C. The hybridization was detected
using AP-conjugated anti-DIG antibody (Roche Diagnostics) diluted in
at RT. The reaction was revealed by nitroblue tetrazolium/5-bromo-4-
chloro-indolyl-phosphate solution (Roche Diagnostics) diluted in 0.1 M
Tris pH 9.5/0.1 M NaCl containing 1 mM levamisole incubated at RT in
the dark for 3–8 d. Images were acquired with Zeiss (Oberkochen, Ger-
many) Axioscope at the Mount Sinai School of Medicine Microscopy
Shared Resource Facility.
(generously provided by Steven Burden, Skirball Institute of Biomolec-
ular Medicine, New York University, New York, NY) were plated in
six-well plates at 3 ? 105cells per well. Synthetic double-stranded small
interfering RNA (siRNA) targeting MuSK (siRNA-MuSK: sense, 5?
CAGUACUtt) and the nonsilencing siRNA used as negative control
(sense, 5? AGUACUGCUUACGAUACGGtt; antisense, 5? CCGUAU-
CGUAAGCAGUACUtt) were generously provided by Ambion (Austin,
TX). siRNAs were resuspended in RNase-free water at 100 ?M.
HEK293M cells were transfected with Lipofectamine 2000 (Invitrogen)
and siRNAs (200 nM) according to the protocol provided by Invitrogen.
Forty-eight hours after transfection, cells were harvested, lysed, and
analyzed for their MuSK expression levels using quantitative Western
Western blot analysis. Fifty micrograms of total protein extract/lane
were resolved using 7.5% SDS-PAGE and analyzed by Western blot as
incubated in TBS overnight at 4°C. Primary antibodies: goat anti-MuSK
N-terminal (1:300; R&D Systems, Minneapolis, MN), mouse anti-agrin
AGR-520 (1:500; Stressgen, Victoria, CA), rabbit anti-GluR1 (1:2000;
Chemicon Temecula, CA), rabbit anti-C/EBP? (1:10,000; Santa Cruz
Biotechnology, Santa Cruz, CA), rabbit anti-pCREB (1:1000; Upstate
Biotechnology, Lake Placid, NY), rabbit anti-CREB (1:1000; Cell Signal-
ing Technology, Beverly, MA), mouse anti-nuclear pore protein 62
glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:4000; Chemi-
con), and mouse anti-actin (1:15 ? 104; Chemicon). Horseradish
peroxidase-coupled specific secondary antibodies (1:4000) were incu-
ECL detection reagents (GE Healthcare Bio-Sciences). Recombinant ex-
tracellular domain of MuSK (R&D Systems) and recombinant insulin-
like growth factor II (IGF-II) (Chemicon) were incubated at 250 ng/ml.
Actin or GAPDH were used for sample normalization and data are ex-
pressed as mean percentage ? SEM of the 0 h? (100%) control mean
values. Statistical analyses were performed as specified in Results. Data
7920 • J.Neurosci.,July26,2006 • 26(30):7919–7932Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain
compared, a t test was used.
Two-dimensional gel electrophoresis. To compare MuSK proteins
two-dimensional (2D) electrophoresis followed by Western blotting
with anti-MuSK antiserum (R&D Systems). Isoelectric focusing (IEF)
was performed on an Ettan IPGphor II (GE Healthcare, Buckingham-
shire, UK) using 7 cm broad-range nonlinear immobilized pH gradient
brain or muscle were diluted in 7 M urea, 2 M thiourea, 4% CHAPS
1% DTT (1,4-dithiothreitol). IPG buffer (pH 3–10; GE Healthcare) and
DeStreak reagent (GE Healthcare) were added to the protein mixtures
second dimension by SDS-PAGE, IPG strips were equilibrated in 1%
DTT, followed by 4% iodoacetamide. Second dimension was performed
MuSK antiserum as described above in Western blot analysis (R&D
Inhibitory avoidance training. Inhibitory avoidance (IA) training was
performed as described previously (Taubenfeld et al., 2001a). During
training, each rat received a foot shock (0.6 mA for 2 s) in the dark
compartment and was then returned to the home cage. Memory was
tested by measuring the latency to enter the shock chamber. Foot shock
540 s. Training and testing procedures were performed blind to treat-
ments. Different control groups were used in different experiments, as
detailed in the Results section. Statistical analysis was performed using
specified in Results. After testing, rats were anesthetized, and their hip-
blot analyses. Both hippocampi from each animal were pooled and ho-
mogenized; therefore, each hippocampal sample, unless otherwise spec-
conditions used for the IA training are specified in Results and consisted
of temporally matched unpaired or nonshocked conditions. Unpaired
controls received the exposure to the training apparatus in the same way
as the trained rats but were not shocked in the dark chamber. They
returned to their home cage and, 1 h later, were placed directly onto the
returned to their home cage. These unpaired experiences of context and
shock do not create an association between the two stimuli and, in fact,
the animals that undergo this protocol never show IA memory when
tested. The nonshocked controls consisted of rats that were exposed to
the training apparatus in the same way as the trained rats but did not
receive the shock in the dark chamber and were then returned to their
those of trained rats.
Surgeries and oligodeoxynucleotide injections. Bilateral hippocampal
surgeries (4.0 mm posterior to bregma; 2.6 mm lateral from midline; 2.0
mm ventral) were performed as described by Taubenfeld et al. (2001a),
MuSK antisense oligodeoxynucleotide (M-ODN) (5?-GAATGTTGA-
CGAGCTCTCTCATG-3?), scrambled ODN (S-ODN) (5?-TACTA-
TGGATCGTCTGCGCATAG-3?) or vehicle solution (PBS) were in-
jected bilaterally into the hippocampi. M-ODN was specific for the se-
quence that includes MuSK translational starting site. S-ODN, which
served as a control, contained the M-ODN base composition but in a
randomized order and showed no homology to any sequence in the
GenBank database, as confirmed by a BLAST (basic local alignment
search tool) search. Both ODNs were phosphorothioated on the three
were reverse phase cartridge-purified and purchased from Gene Link
(Hawthorne, NY). ODN injections were performed as described previ-
ously (Taubenfeld et al., 2001a). Briefly, at the indicated time points
before or after IA training, animals received bilateral injections (2 nmol/
?l) of M-ODN, S-ODN, or vehicle (PBS, pH 7.4). As detailed in Results,
either single or double injections were performed. Biotinylated M-ODN
were perfused with 4% PFA in PBS. Brains were postfixed overnight in
the same fixative with 30% sucrose. Forty-micrometer coronal sections
were stained using an Immunopure ABC kit according to the manufac-
turer’s protocol (Pierce, Rockville, IL).
TaqMan real-time quantitative reverse transcription-PCR. Hippocam-
pal total RNA was extracted with TRIzol (Invitrogen) and reverse tran-
scribed into cDNAs using Omniscript reverse transcriptase (Qiagen,
Hilden, Germany). TaqMan 5? nuclease fluorogenic quantitative PCR
assays were performed using the TaqMan Universal PCR Master Mix kit
(PE Applied Biosystems, Foster City, CA) according to manufacturer’s
instructions. Probes and primers were designed using PrimerExpress3
M17701) was used as a reference gene for comparative analysis. The
and data normalization, respectively: 6-carboxy-fluorescein (FAM) (re-
porter for MuSK), hexachlorofluoresceine (HEX) (reporter for
Amplifications were generated by using the following primers and hy-
bridization probes: MuSK-F, 5?-TGCGCCTATGTTGGAGCAA-3?;
MuSK-R, 5?-CCTCTGCTCTCTCGCACATG-3?; probe, 5?/FAM/TGC-
CATCCA-3?; GAPDH-R, 5?-CCAGTGAGCTTCCCGTTCA-3?; probe,
5?-/HEX/CTTGCCCACAGCCTTGGCAGCA). The cycle threshold
expression in trained and control rats, as described in PE Applied Bio-
systems User Bulletin Number 2 (P/N 4303859). Before comparative
mine whether the amplification efficiencies of the target and reference
gene are approximately equal. The amplification reactions were per-
formed using 1 ?l of cDNA, TaqMan universal PCR Master Mix (PE
Applied Biosystems), and 100 nM of fluorogenic internal probe. Three
TaqMan assays with triplicates were performed for each cDNA sample.
Relative quantification was expressed as fold change values in the 20 h?
group. Statistical analysis was performed using Student’s t test.
Northern blot analysis. Northern blot analyses were performed as de-
scribed previously (Taubenfeld et al., 2001b). The rat agrin probe con-
The same membrane was first hybridized with the agrin probe and then
stripped and rehybridized with a full-length rat cyclophilin cDNA that
was used as a control for normalization. Probes were labeled with ran-
dom oligonucleotides primers (Prime-It II kit; Stratagene) and
[?-32P]dCTP (GE Healthcare). Quantitative densitometry analysis was
performed using NIH Image software. Data were expressed as mean
percentage ? SEM of the 0 h? (100%) control mean values. Statistical
analyses were performed using one-way ANOVA followed by Newman–
Keuls post hoc test.
Electrophysiology. In all electrophysiology experiments, rats were in-
iments, we performed double injections in each hippocampus, with the
after the first set of ODN injections. The experimenter was blind to the
assignment of injection sites. Acute hippocampal slices were prepared as
described previously (Tsokas et al., 2005). Briefly, the rats were deeply
anesthetized with halothane and decapitated. The brain was rapidly re-
moved and placed in ice-cold artificial CSF (ACSF) containing the fol-
lowing (in mM; all from Sigma-Aldrich, St. Louis, MO): 118 NaCl, 3.5
bubbled with 95% O2/5% CO2. The hippocampus was then rapidly dis-
sected out and transverse 500-?m-thick slices were cut from its middle
Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain J.Neurosci.,July26,2006 • 26(30):7919–7932 • 7921
and humidified 95% O2/5% CO2atmosphere)
at room temperature (22–25°C) for at least 90
min before use. For recording, the slices were
in which they were perfused on a nylon mesh
with preheated, oxygenated ACSF. Slice health
was determined by delivering monophasic,
stainless steel electrode (FHC, Bowdoinham,
ME) placed in stratum radiatum of the CA3 re-
gion and measuring the evoked field EPSP
(fEPSP) in the stratum radiatum of the CA1 re-
gion with electrodes filled with ACSF (Re2
M?). fEPSPs were acquired by delivering stim-
uli at 0.033 Hz, and the signals were low-pass
acquisition, as well as amplitude and maxi-
mum initial slope measurement were per-
formed using either an Axobasic routine or
pClamp 9 (Molecular Devices, Foster City,
CA). After recording was completed, the slice
was removed from the recording chamber
and immediately frozen on microscope slides
on dry ice, for use in Western blotting
For the carbachol experiments, slices were
prepared from male Long–Evans rats aged
21–28 d and recorded (33–34°C) using a modi-
fication of the method of Williams and Kauer
(1997). After the maximal fEPSP was deter-
mined, the delivery of stimuli in area CA3 was
terminated, and slices were superfused contin-
uously with ACSF containing 2.5 mM KCl and
50 ?M carbachol (Sigma-Aldrich). Extracellular
recordings were obtained from the CA1 region
onset of carbachol treatment and stored using
pClamp 9 (Molecular Devices) for subsequent
off-line analysis. In our quantification of
carbachol-induced oscillations, we considered
only slices that were competent of producing
field events in response to carbachol treatment.
In our statistical analysis, we scored as “suc-
cesses” only those slices that displayed regular
periods of oscillatory activity, defined as at least
(Williams and Kauer, 1997).
For the LTP experiments, slices were pre-
of 100 Hz high-frequency stimulation (HFS), separated by 20 s, using a
stimulus intensity that evoked an fEPSP corresponding to 75% of the
maximal fEPSP. In all experiments, HFS was delivered when the basal
EPSP had been stable for at least 30 min, and recordings were concluded
baseline period and are presented as group means ? SEs.
To determine whether MuSK is expressed in rat brain, we used
reverse transcription-PCR amplifications of cDNAs obtained
from adult hippocampus, cortex, cerebellum or HNCs with sets
Bank accession number U34985) (Fig. 1A). Gel electrophoresis
analyses revealed that each set of primers generated fragments of
similar size across all cDNA preparations. For each cDNA, five
and 409 bp that covered the entire coding sequence of MuSK
were obtained and sequenced. The analysis of these sequences
revealed that the hippocampus, cortex, cerebellum and HNCs
express two MuSK isoforms (Fig. 1A). One isoform was 2644 bp
long, and its sequence was identical to an alternatively spliced
MuSK transcript (A454), described previously in muscle of hu-
(Hesser et al., 1999). Compared with the longest isoform cloned
in muscle (U34985), A454displays an alanine residue at position
454 that replaces an 8 aa deletion in the ectodomain. To our
knowledge, no functional significance has yet been associated
with this isoform.
The second MuSK transcript, also found in all brain regions
and HNCs, was shorter (2359 bp) and, compared with the
B, Electrophoretic analysis of PCR amplifications generated with the second set of primers (shown in green in Fig. 1A). M,
brain coronal sections and HNCs hybridized with antisense (AS) or sense (S) probes. DG, Dentate gyrus. CA1 regions of the
MuSK is expressed in adult rat brain. A–C, Cloning of MuSK in the brain. A, Schematic representation of MuSK
7922 • J.Neurosci.,July26,2006 • 26(30):7919–7932Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain
in Figure 1B, all PCR amplifications of cDNAs from the hip-
pocampus, cortex, cerebellum, and HNCs, obtained using prim-
ers designed between positions 564 and 1071 (U34985), gener-
ated two bands of 507 and 222 bp, respectively, whose sequences
corresponded to the two alternatively spliced MuSK transcripts.
An isoform lacking the same IgIII-like domain (MuSK-?IgIII)
had been found previously in denervated rat muscle by Hesser et
al. (1999) and reported to mediate muscle AChRs clustering.
These results were further confirmed by gel electrophoretic
analysis and sequencing of full-length MuSK fragments gener-
hippocampus, cortex, cerebellum and HNCs generated the am-
plification of two bands of 2644 and 2359 bp, respectively. Se-
tively spliced transcripts described above.
Next, the distribution of MuSK mRNA transcripts in the
brain, and particularly in neurons, was investigated using in situ
hybridizations. Adult brain sections and sister HNCs were hy-
bridized with either MuSK antisense or sense (control) probes.
Most brain regions, including the cortex, hippocampus, and cer-
ebellum, as well as most neurons of HNCs, exhibited labeling
with the antisense but not with the sense probe. As shown in
the antisense probe included the following: in the hippocampus,
most dentate gyrus granule cells, CA1, CA2 and CA3 pyramidal
neurons and hilar cells; in the cortex, the majority of neuronal
populations of most layers; and in the cerebellum, the Purkinje
cells and some elements of the granule cell layer. These results
were confirmed in several experiments by using three indepen-
dent sequences as probes, which hybridized to different regions
the transmembrane domain, or the C terminus as detailed in
Material and Methods).
Finally, the expression of MuSK protein levels in rat adult
brain, HNCs, developing brain and muscle, as well as several
non-neuronal tissues were investigated using Western blot anal-
yses. For this study, we used a commercially available goat anti-
MuSK antiserum (R&D Systems) raised against the purified re-
combinant rat MuSK extracellular domain (aa 22–453) and
previously shown to recognize rat and mouse MuSK in Western
blot after immunoprecipitation with other anti-MuSK antibod-
ies (Finn et al., 2003). We further examined the reactivity of this
antiserum against MuSK in Western blot to confirm its specific-
ity. Thus, we performed several tests using three independent
technical approaches. First, the antiserum was used in Western
blot against the recombinant N-terminal portion of MuSK,
which had been used as immunogen. Results depicted in Figure
2A showed that the antiserum reacted with the recombinant
truncated MuSK. Second, the antiserum was used in Western
blot to immunostain extracts obtained from either the HEK293
tag in position 249 (293M) or its relative nontransfected 293
control. As shown in Figure 2B, both anti-MuSK or anti-Myc
antisera revealed a similar pattern in the 293M, which consisted
nontransfected 293. This indicated that both bands correspond
to the MuSK-Myc fusion protein and that the 75 kDa band likely
represents a proteolytic fragment of the 100 kDa protein.
To further confirm that both bands specifically recognized by
specific siRNA or a relative control sequence. As depicted in Fig-
ure 2B, quantitative Western blot analyses of extracts obtained
from these transfections showed that the anti-MuSK but not the
anti-Myc antisera, confirming that these bands are indeed
MuSK-Myc proteins. Thus, together, all of the results described
above led us to conclude that the R&D Systems anti-MuSK anti-
serum specifically reacts to rat MuSK in Western blot.
This antiserum was then used in Western blot analyses to test
for the presence of MuSK in protein extracts obtained from sev-
eral tissues including hippocampus, cortex, cerebellum, 14 DIV
HNCs, E18 brain (brain E18), E18 muscle (muscle E18), adult
and Saxel, 1977) and 293M cell lines were included as positive
controls. Results showed that MuSK is ubiquitously expressed,
although to different degrees. As shown in Figure 2C, MuSK was
detected in several of these tissues. Its relatively strongest expres-
sion was found in E18 muscle and E18 brain extracts. A weaker,
but still detectable, expression was evident in the various adult
expression appeared to be that of adult muscle. To further con-
firm the specificity of the anti-MuSK labeling, Western blot
membranes containing the same extracts were immunostained
with the anti-MuSK antiserum preincubated with an excess of
either extracellular domain of recombinant MuSK or, as a con-
trol, an excess of the unrelated recombinant polypeptide IGF-II.
As shown in Figure 2C, the 100 kDa band/s recognized by the
not by recombinant IGF-II. Notably, some nonspecific bands at
?130–150 kDa (indicated by arrows in Fig. 2C) were not com-
peted. In contrast, in heart, lung, and spleen, additional bands
recombinant IGF-II, suggesting that they may represent either
cross-reacting proteins, MuSK isoforms complexed with other
proteins, or posttranslationally modified MuSK isoforms that
display a slower migration. RT-PCR amplifications of cDNAs
blot study confirmed the ubiquitous expression of MuSK (data
Interestingly, in some extracts, including hippocampus, cor-
performed in muscle and myotube extracts (Herbst and Burden,
2000), at least two closely migrating immunoreactive bands with
a molecular weight of ?100 kDa were detected (see representa-
tive examples in Fig. 2D). It is possible that these bands corre-
spond to differentially expressed splicing forms and/or differen-
tially phosphorylated isoforms of MuSK.
posttranslationally modified (likely to be phosphorylation)
forms, a 2D PAGE analysis of E18 rat brain and E18 rat muscle
extracts revealed that MuSK resolves in multiple spots separated
along the isoelectric point gradient and that E18 brain MuSK
is differentially modified compared with E18 muscle MuSK
Together, all of the results described above demonstrate that
adult brain, particularly in neurons, and in a variety of non-
(Fu et al., 1999; Ip et al., 2000; Kumar et al., 2006), showing that
Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrainJ.Neurosci.,July26,2006 • 26(30):7919–7932 • 7923
MuSK is expressed in tissues other than
skeletal muscle, including liver, lung,
heart, spleen, and sperm.
brain plays a role during functions that
involve synapse formation/remodeling;
thus, we tested whether MuSK is required
for memory formation. MuSK expression
in the hippocampi of adult rats was
knocked down by bilateral injections of
MuSK antisense (M-ODN) through ster-
eotactically implanted cannulas; S-ODN
was used as a control. The effect of these
treatments was determined on memory
Similar to previous studies showing the
distribution and stability of other hip-
McGaugh, 1997; Taubenfeld et al., 2001a),
we found that M-ODN diffused through-
out the entire dorsal hippocampus and its
temporal stability was quite brief. In fact,
injection (Fig. 3A). Therefore, we investi-
gated the effect of either single or double
injections of M-ODN, S-ODN, or vehicle
ter IA training.
As depicted in Figure 3B, single injec-
tions were administered either 8 h after
training or 2 h before testing (22 h after
training). Double injections, which were
performed to increase the effect of the
M-ODN treatment, involved delivery im-
8 h). All rats were killed immediately after
testing (24 h after training), and their hip-
ated by quantitative Western blot analysis.
As depicted in Figure 3C, at 24 h after
training, hippocampal MuSK expression
levels were reduced to 70.8% by the 8 h
more profoundly, to 58.8% by the double
(92.7%; n ? 8), compared with S-ODN
(100%; n ? 4) and PBS (100%; n ? 4)
controls performed at the same time
points. Both S-ODN- and PBS-injected
control groups showed very similar levels
of MuSK expression. Indeed, if the PBS-
injected groups were assigned the value of
100%, the single S-ODN injections at 8 or
22 h resulted in values of 96.3 ? 8.1 and 105.7 ? 3.0%, respec-
tively. Hence, the PBS and S-ODN-injected groups were com-
bined for statistical analysis.
To verify that M-ODN treatment specifically affected MuSK
expression, the concentration of an unrelated receptor protein,
same Western blot membranes. No change in GluR1 was found
across samples. Both MuSK and GluR1 were normalized against
the relative concentration of actin, which was also immuno-
ples relative to the equal concentrations loaded. An ANOVA
among all treatment and time point groups showed a significant
tissues. Western blot analysis was performed on extracts obtained from the indicated tissues. C2C12 and 293M were used as
7924 • J.Neurosci.,July26,2006 • 26(30):7919–7932 Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain
group effect (F(5,42)? 4.5; p ? 0.01), and a Newman–Keuls post
hoc comparison test revealed that MuSK levels were significantly
tion ( p ? 0.01) compared with all other groups. A single injec-
tion resulted in a consistent, although not significant, decrease.
No significant changes were found in the expression of GluR1
(Fig. 3C). Thus, M-ODN selectively downregulates the expres-
sion of MuSK in a dose-dependent manner.
The effect of M-ODN treatment on memory retention is de-
picted in Figure 3D. A one-way ANOVA comparing latencies
across all groups indicated a significant group effect (F(6, 82)?
30.1; p ? 0.0001). Newman–Keuls post hoc test revealed that the
8 h single M-ODN injection significantly impaired memory re-
iment, because no significant difference was observed between
the S-ODN- (221.4 ? 33.3 s) and PBS-injected groups (210.5 ?
14.3 s), the two were combined (215.9 ? 16.9 s) for statistical
analysis. The double-injection treatment of M-ODN more pro-
foundly and significantly impaired memory retention (M-ODN:
(M-ODN: 182.7 ? 38.1 s, n ? 8 compared
with S-ODN/PBS: 181.9 ? 31.5 s, n ? 8).
Because no significant difference was ob-
served between the S-ODN- (171.4 ?
62.1 s; n ? 4) and PBS-injected groups
bined for statistical analysis.
Together, these data indicate that
ory retention. Moreover, because the 22 h
single injection did not affect memory
M-ODN does not have toxic or nonspe-
cific effects on the expression of the mem-
ory. This is also supported by the fact that
the effect of the M-ODN on both MuSK
expression and memory retention is dose
Thus, we concluded that MuSK plays
an essential role during IA memory
Which molecular mechanisms are affected
by MuSK disruption during memory con-
solidation? Recent studies have shown
that, in hippocampal neurons, treatment
with the neuron-specific isoform of agrin,
but not with its ubiquitously expressed
counterpart, causes the phosphorylation
of the transcription factor CREB (Ji et al.,
1998). We have confirmed these results
and found that, in addition to CREB acti-
vation, agrin treatment of HNCs also re-
sults in increased expression of C/EBP?
(K. Gagnidze and C. M. Alberini, unpub-
lished data). Notably, the CREB–C/EBP-dependent pathway is
known to be required for the consolidation of different kinds of
memories, including IA (Yin and Tully, 1996; Silva et al., 1998;
al., 2002). In addition, previous work from our laboratory has
shown that the activation of the CREB–C/EBP pathway in the
feld et al., 1999, 2001b), which is known to carry modulatory
inputs to the hippocampus, among which the most prominent
ing down MuSK expression in the hippocampus influences the
activation of CREB and the induction of C/EBP?.
In the first set of experiments, we determined whether the
knockdown of MuSK affects the learning-induced phosphoryla-
tion of CREB in Ser133 (pCREB), a posttranslational modifica-
tion critical for the activation of this transcription factor. Thus,
we tested the levels of pCREB in the hippocampi of trained and
unpaired control rats that underwent either M-ODN or S-ODN
injections in their hippocampi. As described in Materials and
Methods, unpaired controls consisted of rats exposed to the
training apparatus but shocked 1 h after exposure, so that an
Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrainJ.Neurosci.,July26,2006 • 26(30):7919–7932 • 7925
and killed at the same time points as trained rats. Because hip-
pocampal pCREB significantly increases immediately after IA
ensure that MuSK disruption was achieved at this time, we in-
jected M-ODN or S-ODN bilaterally into the rat hippocampi 4 h
Hippocampal protein extracts were assessed for the concentra-
tion of pCREB using quantitative Western blot analyses. As de-
picted in Figure 4A, a one-way ANOVA showed a significant
group effect (F(3,12)? 6.1; p ? 0.01). Dunnett’s multiple-
and underwent IA training (S-ODN-train) had a significant in-
crease in hippocampal pCREB (222.6 ? 47.5%; n ? 4; p ? 0.05)
compared with unpaired S-ODN-injected rats (S-ODN-unp)
(100.0 ? 17.6%; n ? 4), confirming our previous findings
(Taubenfeld et al., 1999, 2001b). This increase was completely
blocked in trained rats that received M-ODN treatment (M-
ODN-train) (128.5 ? 34.9; n ? 4), and, in fact, in these rats
hippocampal pCREB levels remained comparable with those of
unpaired control rats injected with either S-ODN (100.0 ?
17.6%; n ? 4) or M-ODN (M-ODN-unp) (85.0 ? 14.6%). To
determine whether the pCREB blockade was attributable to de-
creased phosphorylation of preexisting CREB or a change in
CREB expression, the same membranes were stripped and
restained with anti-CREB antibody. As depicted in Figure 4A,
CREB concentrations were similar across all samples, and no
significant differences were found with a one-way ANOVA (S-
confirm the selectivity of the M-ODN effect, the same mem-
branes were stripped and stained for an unrelated nuclear pro-
tein, NP62. As shown in Figure 4A, NP62 also remained un-
changed (S-ODN-unp, 100 ? 10.3%; S-ODN-train, 127.2 ?
12.8%; M-ODN-unp, 128.1 ? 9.9%; M-ODN-train, 118.7 ?
experiment, pCREB, CREB, and NP62 levels were normalized
against the relative concentration of actin, which was used as a
We next tested whether hippocampal MuSK knockdown also
affects C/EBP? expression and/or its learning-dependent induc-
tion (Fig. 4B). Groups of rats underwent either IA training or
unpaired behavioral protocol followed by a double injection at 0
and 8 h of either M-ODN or S-ODN. Twenty-four hours after
training or context exposure, the rats were killed and their hip-
0.05). Similar to the effect found on pCREB, rats that received
S-ODN injection and underwent IA training had a significant
compared with unpaired S-ODN-injected rats (100.0 ? 6.9%;
ing our previous findings that training results in a significant
induction of this transcription factor (Taubenfeld et al., 2001b).
M-ODN treatment (115.4 ? 10.1; n ? 4). In the hippocampi of
M-ODN-treated rats, C/EBP? levels were similar to those of un-
paired control rats injected with either S-ODN (100.0 ? 6.9%;
n ? 4) or M-ODN (113.9 ? 10.2%; n ? 4). To confirm the
were stripped and stained for NP62. As shown in Figure 4B, the
concentration of this protein remained unchanged across sam-
ples (S-ODN-unp, 100 ? 4.1%; S-ODN-train, 114.9 ? 13.0%;
M-ODN-unp, 89.9 ? 22.3%; M-ODN-train, 107.8 ? 8.7%). In
each experiment, C/EBP? and NP62 levels were normalized
against the relative concentration of actin, which was used as a
Moreover, in agreement with these results, we found that the
C/EBP? hippocampal levels of the rats that were behaviorally
and killing time points. Quantitative Western blot analysis of hippocampal extracts from un-
(Ser133) antibody, stripped, and restained with anti-CREB, anti-NP62 antibodies and finally
ments (experiment described in Fig. 3). Blots were stained with anti-C/EBP? antibody,
normalization. A representative blot per condition is shown. Graphs represent the statistical
densitometric analysis of all data. Data are expressed as a mean percentage ? SEM of the
Hippocampal disruption of MuSK prevents the activation of the CREB–C/EBP?
7926 • J.Neurosci.,July26,2006 • 26(30):7919–7932Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain
analyzed in the previous experiment (Fig. 3D) were also signifi-
cantly and selectively reduced by the double M-ODN injection
levels of NP62, used as unrelated control, remained unaffected
(M-ODN, 93.8 ? 7.6%; S-ODN, 100 ? 12.3%). Both C/EBP?
and NP62 levels were normalized against the relative concentra-
tion of actin, which was used as loading control (Fig. 4C).
Together the results described above indicate that MuSK ex-
pressed in the hippocampus plays a key
role in the learning-dependent activation
of the CREB/C/EBP? pathway that medi-
ates memory consolidation.
Given the role of hippocampal MuSK on
IA, we next addressed a possible role of
MuSK in the cholinergic modulation of
hippocampal synaptic activity. Studies
from several groups have shown that the
cholinergic input to the hippocampus un-
derlies physiological theta rhythm in vivo
(for review, see Bland and Colom, 1993),
which has been implicated in diverse be-
havioral and cognitive functions, includ-
ing learning and memory (Winson, 1978;
strated that the cholinergic agonist carba-
chol produces a patterned oscillatory be-
havior in acute hippocampal slices that
MacVicar and Tse, 1989; Williams and
Kauer, 1997) and that such theta-like ac-
tivity can enhance synaptic plasticity in a
1995, 1996). We therefore tested whether
MuSK is involved in the modulation of
patterned cholinergic activity in the
rats (n ? 4). We confirmed, as described
below, that MuSK was selectively down-
regulated by injection of M-ODN through
cannulas that had been stereotaxically im-
planted 1 week earlier. We examined the
effects of this treatment on the extracellu-
larly recorded oscillatory activity induced
by carbachol in area CA1 of acute hip-
pocampal slices obtained from such ani-
mals. To control for possible effects of the
injection itself, in each animal we injected
one hippocampus with M-ODN and the
other hippocampus with control S-ODN.
We adopt here the terminology of Wil-
liams and Kauer (1997) and refer to single
events in the extracellular record as “field
events” and to groups of these events as
from hippocampi injected with S-ODN, bath application of 50
?M carbachol generated periodic bursts that occurred at approx-
imately theta frequency. These were similar, in terms of intra-
burst frequency (5–11 Hz), mean interburst interval (17.1 ?
2.7 s; n ? 6), and mean burst duration (5.2 ? 0.7 s; n ? 6), to
from hippocampi injected with M-ODN, such field events were
not organized as bursts but rather as unitary events. In most of
hippocampi. Representative experiment of six. Expanded traces (bottom) show that each burst consisted of a series of field
S-ODN slices displayed periodic bursting activity (left), whereas only 8.3% of the M-ODN slices exhibited such activity. The
displayed pacemaking (PACE) activity, with the remainder showing little or no such activity. Carbachol elicited pacemaking
activity in only 11.1% of the S-ODN slices (right). INACT, Inactivity. C, D, Hippocampal M-ODN injection did not affect basal
slices. Data are expressed as mean percentage ? SEM. E, Hippocampal M-ODN injection impaired long-term potentiation
(total AUC). Treatment with M-ODN affected neither the total AUC nor the NMDAR-mediated component. Calibration:
Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain J.Neurosci.,July26,2006 • 26(30):7919–7932 • 7927
these slices (6 of 11), the activity was periodic (pacemaking) and
occurred at frequencies generally ranging from 0.5 to 1 Hz (Fig.
ence between S-ODN- and M-ODN-treated slices in terms of
their ability to produce periodic bursts in response to carbachol
determined by Fisher’s exact test ( p ? 0.05). Western immuno-
blots performed on pooled homogenates of the slices used in
these experiments showed that the levels of MuSK protein were
matched S-ODN-injected controls.
synaptic plasticity, we examined the LTP induced in the
CA33CA1 synapses of acute hippocampal slices obtained from
treatments as those involved in the carbachol experiments out-
the downregulation of MuSK specifically in those slices that had
Basal synaptic function was not compromised by M-ODN, be-
group, two-way ANOVA) and degree of paired-pulse facilitation
(Fig. 5D) ( p ? 0.5, n ? 5 per group, two-tailed t test) were
indistinguishable from slices that had been treated with S-ODN
(Fig. 5C, input/output curves). In addition, the spike threshold
was unaffected by M-ODN (2.2 ? 0.3 mV, n ? 6 for S-ODN vs
2.2 ? 0.3 mV, n ? 6 for M-ODN; p ? 0.5).
LTP that can be maintained for at least 2 h (Osten et al., 1996;
Tsokas et al., 2005). As shown in Figure 5E, M-ODN-injected
slices showed an impairment of LTP relative to slices injected
with S-ODN, which displayed robust LTP that lasted for at least
2 h (two-way ANOVA of EPSP slope at 110–120 min post-HFS,
p ? 0.0001; n ? 6 for both S-ODN and M-ODN). In fact, the
6 for both S-ODN and M-ODN), indicating that MuSK contrib-
utes to the induction of LTP. However, MuSK also seems to be
required for the late phase of LTP, because the potentiation con-
tinued to decay in the M-ODN-treated slices relative to S-ODN
controls for the duration of the recording period. Thus, between
15 and 120 min after HFS, the size of LTP in M-ODN-treated
of only 18% in the S-ODN controls (from 89 to 73%).
It is known that, subsequent to induction, two phases of LTP
can be resolved: an early phase that involves only posttransla-
tional mechanisms and that decays within 1 h under our condi-
tions (Tsokas et al., 2005) and a protein synthesis-dependent
can persist for hours (Frey et al., 1988; Osten et al., 1996; Tsokas
et al., 2005). Our experiments indicate that normal expression of
effect was apparent during the first 5–15 min after HFS. In addi-
relative to scrambled ODN controls for the duration of the re-
To determine whether the effect of M-ODN on LTP could
reflect a change in the activation of NMDA receptors
(Harris et al., 1984), we analyzed the field potentials evoked by
HFS. Slices obtained from three independent rats that received
either M-ODN (n ? 7) or S-ODN (n ? 6) injection into their
hippocampi, as described above, underwent two-train stimula-
tions in the presence of 50 ?M D-amino-phosphonovaleric acid
(APV) to block NMDARs. As expected, no LTP was observed
delivered a second set of HFS trains. The field potential evoked
after washout exhibited a slowly developing negativity beyond
was more apparent in the second train, as reported previously
of the HFS-evoked potential was measured by subtracting the
area under the curve (AUC) in the presence of APV (second
train) from the AUC after washout (second train). As shown in
Figure 5F (right), M-ODN treatment had no effect on the total
field potential evoked in the absence of APV nor on the APV-
sensitive component of the potential. Thus, the ability of MuSK
antisense to inhibit LTP cannot be explained by an effect on
To determine whether MuSK expression is regulated after IA
training, we performed real-time PCR analyses to quantify the
trol rats using TaqMan nuclease fluorigenic quantitative PCR
assay. cDNAs were transcribed from pooled hippocampal ex-
tracts obtained from groups of rats that either underwent IA
training and were killed 20 h later (20 h?; n ? 10) or from
control groups that were exposed to the context without shock
administration and killed either immediately thereafter (0 h?;
n ? 10) or 20 h later (20 h?; n ? 10). The 20 h time point was
chosen because previous work showed that the activation of
and requirement for C/EBP? during IA consolidation lasts for
?20 h after training (Taubenfeld et al., 1999, 2001a,b).
As shown in Figure 6A, statistical analysis of these amplifica-
tions based on Student’s t test that compared 20 h? versus 0 h?
and 20 h? versus 0 h? showed that MuSK mRNA significantly
increased in the hippocampi of trained rats compared with con-
trols by a factor of 1.55 ? 0.02-fold.
underwent parallel increase after training and, for this investiga-
tion, we tested MuSK concentrations in the hippocampi of indi-
vidual rats. Controls included two groups, 0 h? and unpaired.
significantly lower compared with that of trained rats (453.7 ?
comparison test) but similar to that of the 0 h? group (4.9 ?
1.2 s). As depicted in Figure 6B, quantitative Western blot anal-
yses showed that, in trained rats, there was a trend toward an
increase in MuSK protein level (n ? 12; 130.9 ? 16.1%), which,
(0 h?, n ? 12, 100.0 ? 8.4%; 20 h unpaired, n ? 8, 105.2 ?
12.3%). Notably, the trend was observed in all experiments.
In the next sets of experiments, we tested whether agrin expres-
sion changes in the hippocampus after IA training. First, we de-
termined agrin mRNA concentration in the hippocampi of
trained or control groups of rats. Controls consisted of 0 h? and
unpaired groups (Fig. 6C). Quantitative Northern blot and sta-
7928 • J.Neurosci.,July26,2006 • 26(30):7919–7932Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain
tistical analyses, performed with a one-way ANOVA (F(2,12)?
11.14; p ? 0.01) followed by Newman–Keuls post hoc test, re-
vealed that trained rats had a significant increase in agrin mRNA
p ? 0.01) and unpaired controls (108.6 ? 15.0%; n ? 4;
p ? 0.05).
In the second set of experiments, we used quantitative West-
ern blot analyses to determine whether agrin protein levels also
increased. As shown in Figure 6D, the hippocampi of trained
animals exhibited a marked induction of agrin protein (258.5 ?
(100 ? 7.2% and 100.5 ? 17.5%, n ? 4 per group, respectively).
One-way ANOVA showed a significant group effect (F(2,13)?
6.26; p ? 0.05) and Newman–Keuls post hoc comparison test
revealed that agrin levels were significantly higher in the hip-
0.05 for both).
the consolidation of IA memory, because its disruption after
underlying CREB–C/EBP-dependent pathway. Hippocampal
oscillatory activity as well as both induction and maintenance of
LTP. We propose that MuSK expressed in the hippocampus me-
diates memory consolidation via the synchronization of theta
frequencies and recruitment of the CREB–C/EBP-dependent
patients suffering from myasthenia gravis (MG) have been re-
ported to have memory deficits (Bohbot et al., 1997). Because
antibody responses against both nicotinic receptors and MuSK
have been found in MG patients (Hoch et al., 2001), it is possible
Reverse transcription-PCR amplifications, in situ hybridization,
Western blot analyses, and antisense treatments, all provide evi-
dence that MuSK is expressed in the brain and particularly in
al., 2000), MuSK also appears to be expressed in other tissues
including liver, lung, heart, and spleen.
hippocampus, cortex, and cerebellum. This suggests a potential
function of MuSK in the context of the cholinergic network,
which is known to play an important role in neuropsychic activ-
to that of cholinergic or other types of receptors in the brain will
help to understand similarities and differences of MuSK roles in
muscle and CNS.
Why did previous work indicate that MuSK was expressed
selectively in muscle? The term muscle-specific tyrosine kinase
receptor was initially proposed by Valenzuela et al. (1995), who,
muscle development, in muscle fibers after denervation and in
NMJ but not in a variety of other adult rat tissues, including
brain. Similar results using Northern blot analysis were obtained
in mouse by Ganju et al. (1995), who, in the same study, also
showed that in situ hybridization detected the expression of
MuSK (Nsk2) transcripts in a number of developing tissues, in-
cluding the nervous system. Like Valenzuela et al. (1995) and
Ganju et al. (1995), we also found that Northern blot analysis is
not sufficiently sensitive to detect physiologic concentrations of
MuSK in a variety of tissues (data not shown).
In agreement, a relatively low level of expression is also found
by Western blot analysis in adult brain and muscle, compared
with higher levels in both tissues during development. Notably,
low levels of expression do not preclude that MuSK is function-
critical role at the NMJ (Kong et al., 2004; Hesser et al., 2006),
neither the mRNA nor the protein has yet been detected by
Northern or Western blot analyses. Therefore, it is likewise pos-
sible that, despite low levels of expression, MuSK plays a signifi-
When compared with the longest isoform cloned from mus-
cle, both MuSK isoforms expressed in the brain exhibit a 9 aa
0.05). B, Quantitative Western blot analysis of hippocampal extracts taken from 0 h?, un-
paired (unp), and IA trained (20 h?) individual rats. A representative blot per condition is
against actin and are expressed as mean percent ? SEM of the 0 h? control mean values
taken from 0 h?, unpaired, and 20 h? rats. A representative blot per condition is shown.
MuSK and agrin expression is increased in rat hippocampus after IA training.A,
Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrainJ.Neurosci.,July26,2006 • 26(30):7919–7932 • 7929
deletion with alanine substitution in the ectodomain (A454);
moreover, the shortest isoform also presents a deletion of the
IgIII domain (?IgIII). The A454isoform is identical to the iso-
The A454protein lacks a potential N-linked glycosylation site;
however, no functional significance has yet been associated with
this deletion. Both A454and ?IgIII deletions seem to originate
from alternative splicing of the longer sequence and, in fact, the
?IgIII has also been found previously in muscle and shown to
mediate muscle AChRs clustering similarly to the long isoform
(Hesser et al., 1999). A transcript bearing both deletions, to our
knowledge, has not been described previously.
of two distinct bands. As these bands migrate very closely, it is
unclear whether they represent both the A454and A454?IgIII iso-
forms. The molecular weight difference between these two iso-
forms, calculated on the basis of the deleted sequence (95 aa), is
10.3 kDa. However, posttranslational modifications may change
the migration of the corresponding band; thus, the two proteins
may ultimately migrate in more proximity to one another. It is
also possible that one of the two isoforms is more abundantly
expressed compared with the other and that the two closely mi-
underwent different posttranslational modification. In agree-
ment, our 2D gel studies suggest that MuSK expressed in the
brain undergoes posttranslational modification different from
those of MuSK expressed in muscle. The nature of these modifi-
muscle remain to be determined.
Interestingly, a recent finding indicates that ?3Na?/K?-
ATPase (?3NKA) is a neuronal receptor for agrin in the brain
(Hilgenberg et al., 2006). Whether MuSK expressed in the brain,
plex remains to be determined. Moreover, even in muscle, de-
of muscle MuSK had previously suggested that an additional
must exist as coreceptor for agrin at the NMJ (Glass et al., 1996).
which directly binds to agrin, may represent MASC or its brain
The antisense-mediated hippocampal knockdown of MuSK dis-
rupts memory consolidation. The effect of M-ODN on memory
after training and was even more profound with two injections,
one administered immediately after training and the second 8 h
later. In contrast, one M-ODN injection administered 2 h before
toxic nor caused nonspecific effects on memory expression. The
the same animals, demonstrated a selective and dose-dependent
prevented the learning-induced activation of the CREB–C/EBP
pathway, indicating that the role of MuSK in the hippocampus
during memory consolidation involves this molecular cascade.
Interestingly, previous work from our laboratory has shown
consolidation of IA memory requires an intact fornix (Tauben-
feld et al., 1999, 2001b). Fornix inputs to the hippocampus in-
clude cholinergic projections. Hence, a working model that we
propose is that, after learning, the cholinergic input to the hip-
through agrin. MuSK activation would then produce the induc-
tion of the CREB–C/EBP?-dependent gene cascade and hence,
CREB–C/EBP?-dependent cascade, via MuSK, would in turn
regulate the expression of MuSK and agrin, which, together with
additional late gene expression changes, would be critical for
maintaining MuSK function and/or restoring the homeostasis of
the system. In line with this model, MuSK expression has been
shown to be autoregulated (Moore et al., 2001), and putative
C/EBP? and CREB binding sites are present in the promoter
region of the MuSK gene (A. Garcia-Osta, unpublished observa-
tion) (Kim et al., 2003).
observed a small level of MuSK increase at 20 h after training.
tal role of MuSK in long-term synaptic plasticity and memory
formation and could be attributable to several reasons: (1) the
change in MuSK protein expression may occur only in a small
number of cells and therefore be too diluted in the whole hip-
increase of MuSK protein may indeed be very small because, as
suggested above in our working model, it simply reflects a
learning-induced turnover regulation process that maintains the
homeostatic pool of MuSK protein.
In slices treated with MuSK antisense, basal synaptic function
appeared normal: the input/output relationships, spike thresh-
guishable from controls. However, MuSK downregulation pro-
duced a clear deficit on two electrophysiological measures that
are relevant to learning.
First, normal MuSK expression is required for carbachol-
hippocampus of behaving rats, ongoing motor activity and cer-
tain types of sensory stimulation are associated with theta activ-
ity, a large-amplitude synchronized oscillation at a frequency of
?8–12 Hz (Buzsaki, 2002). Theta activity has been linked to
synaptic plasticity and learning; for example, LTP in area CA1 is
with a particular phase of the theta oscillation (Hyman et al.,
2003). Furthermore, the rate of acquisition for some behaviors is
accelerated when training is given during periods of relatively
intense oscillations (Berry and Thompson, 1978), and interven-
tions that eliminate theta activity, such as lesions of the medial
septum and fornix, can impair performance on hippocampus-
based tasks (Givens and Olton, 1990; Taubenfeld et al., 1999).
The precise role of theta activity in memory processing remains
speculative, but it has been proposed that the oscillations serve a
timing function, which governs the associativity of stimuli by
regulating neuronal excitability at a characteristic frequency
2003). Moreover, theta rhythms coordinate hippocampal–pre-
frontal interactions in a spatial memory task (Jones and Wilson,
2005), suggesting that theta rhythm may participate to both en-
7930 • J.Neurosci.,July26,2006 • 26(30):7919–7932Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain
coding and representing information integrated at the network
ganize carbachol-induced oscillations, converting the periodic
bursts to single field events that occur at 0.5–1 Hz or, in the case
of MuSK antisense, sometimes producing an almost complete
suppression of field events. Although this similarity suggests that
MuSK might modulate hippocampal nicotinic transmission
through effects on receptor clustering, it is notable that MuSK
recruits phosphotyrosine-binding proteins to regulate transcrip-
Moreover, the cytoplasmic PDZ [postsynaptic density-95 (PSD-
95)/Discs large/zona occludens-1] domain of MuSK suggests the
possibility of a direct interaction with PSD-95, a scaffolding pro-
tein that is critical for the organization and function of the gluta-
matergic synapses whose activation generates theta activity (El-
Husseini et al., 2000; Schnell et al., 2002).
In addition, in vivo MuSK knockdown impaired both induc-
tion and maintenance of hippocampal LTP. These results are in
line with the behavioral experiments and strengthen the conclu-
sion that MuSK plays a role in long-term synaptic plasticity un-
derlying memory formation. The critical role of MuSK during
above, which proposes that MuSK plays an important role that
begins during learning, and is also supported by the observation
that MuSK is required for the activation of the CREB–C/EBP-
dependent cascade. Furthermore, in the context of our behav-
ioral experiments in which MuSK disruption was targeted after
acquisition, the effect of antisense treatment on the maintenance
of LTP is also of particular interest. Indeed, these results confirm
the behavioral data indicating that the integrity of the memory
induction as well as the consolidation process of memory.
In conclusion, based on the results shown in this study, we
propose that hippocampal MuSK mediates learning-induced
synchronization activity, which requires an intact fornix, and is
likely mediated by cholinergic input. This rhythmic activity
would stabilize synaptic plasticity through the activation of the
CREB–C/EBP? pathway. This pathway, which includes MuSK
expression homeostatic regulation, will then lead to memory
Alberini CM (1999) Genes to remember. Exp Biol 202:2887–2891.
AthosJ,ImpeyS,PinedaVV,ChenX,StormDR (2002) HippocampalCRE-
mediated gene expression is required for contextual memory formation.
Nat Neurosci 5:1119–1120.
Bailey CH, Bartsch D, Kandel ER (1996) Toward a molecular definition of
long-term memory storage. Proc Natl Acad Sci USA 93:13445–13452.
BernabeuR,CammarotaM,IzquierdoI,MedinaJH (1997) Involvementof
hippocampal AMPA glutamate receptor changes and the cAMP/protein
kinase A/CREB-P signalling pathway in memory consolidation of an
avoidance task in rats. Braz J Med Biol Res 30:961–965.
Berry SD, Thompson RF (1978) Prediction of learning rate from the hip-
pocampal electroencephalogram. Science 200:1298–1300.
Bland BH, Colom LV (1993) Extrinsic and intrinsic properties underlying
oscillation and synchrony in limbic cortex. Prog Neurobiol 41:157–208.
BlandBH,ColomLV,KonopackiJ,RothSH (1988) Intracellularrecordsof
carbachol-induced theta rhythm in hippocampal slices. Brain Res
Blitzer RD, Gil O, Landau EM (1990) Long-term potentiation in rat hip-
pocampus is inhibited by low concentrations of ethanol. Brain Res
Bohbot VD, Jech R, Bures J, Nadel L, Ruzicka E (1997) Spatial and nonspa-
tial memory involvement in myasthenia gravis. J Neurol 244:529–532.
Burden SJ (2002) Building the vertebrate neuromuscular synapse. J Neuro-
Buzsaki G (2002) Theta oscillations in the hippocampus. Neuron
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.
El-Husseini AE, Schnell E, Chetkovich DM, Nicoll RA, Bredt DS (2000)
PSD-95 involvement in maturation of excitatory synapses. Science
Ferreira A (1999) Abnormal synapse formation in agrin-depleted hip-
pocampal neurons. J Cell Sci 112:4729–4738.
Finn AJ, Feng G, Pendergast AM (2003) Postsynaptic requirement for Abl
kinases in assembly of the neuromuscular junction. Nat Neurosci
FreyU,KrugM,ReymannKG,MatthiesH (1988) Anisomycin,aninhibitor
of protein synthesis, blocks late phases of LTP phenomena in the hip-
pocampal CA1 region in vitro. Brain Res 452:57–65.
Fu AK, Smith FD, Zhou H, Chu AH, Tsim KW, Peng BH, Ip NY (1999)
Xenopus muscle-specific kinase: molecular cloning and prominent ex-
pression in neural tissues during early embryonic development. Eur
J Neurosci 11:373–382.
Ganju P, Walls EJ, Brennan J, Reith AD (1995) Cloning and developmental
myogenesis. Oncogene 11:281–290.
(1996) Defective neuromuscular synaptogenesis in agrin-deficient mu-
tant mice. Cell 85:525–535.
Geinisman Y, Berry RW, Disterhoft JF, Power JM, Van der Zee EA (2001)
Associative learning elicits the formation of multiple-synapse boutons.
J Neurosci 21:5568–5573.
GivensBS,OltonDS (1990) CholinergicandGABAergicmodulationofme-
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.
Gold PE (2003) Acetylcholine modulation of neural systems involved in
learning and memory. Neurobiol Learn Mem 80:194–210.
Goslin K, Banker G (1991) Rat hippocampal neurons in low density cul-
tures. In: Culturing nerve cells (Banker G, Goslin K, eds), pp 251–278
GuzowskiJF, McGaugh JL (1997) Anti-sense
mediated disruption of hippocampal CREB protein levels impairs mem-
ory of a spatial task. Proc Natl Acad Sci USA 94:2693–2698.
HarrisEW,GanongAH,CotmanCW (1984) Long-termpotentiationinthe
hippocampus involves activation of N-methyl-D-aspartate receptors.
Brain Res 323:132–137.
Hasselmo ME, Bodelon C, Wyble BP (2002) A proposed function for hip-
pocampal theta rhythm: separate phases of encoding and retrieval en-
hance reversal of prior learning. Neural Comput 14:793–817.
Herbst R, Burden SJ (2000) (2000) The juxtamembrane region of MuSK
has a critical role in agrin-mediated signaling. EMBO J [Erratum (2000)
HesserBA,SanderA,WitzemannV (1999) Identificationandcharacteriza-
tion of a novel splice variant of MuSK. FEBS Lett 442:133–137.
HesserBA,HenschelO,WitzemannV (2006) Synapsedisassemblyandfor-
mation of new synapses in postnatal muscle upon conditional inactiva-
tion of MuSK. Mol Cell Neurosci 31:470–480.
Hilgenberg LG, Su H, Gu H, O’Dowd DK, Smith MA (2006) Alpha3Na?/
K?-ATPase is a neuronal receptor for agrin. Cell 125:359–369.
Hoch W (2003) Molecular dissection of neuromuscular junction forma-
tion. Trends Neurosci 26:335–337.
Hoch W, Ferns M, Campanelli JT, Hall ZW, Scheller RH (1993) Develop-
mental regulation of highly active alternatively spliced forms of agrin.
Hoch W, McConville J, Helms S, Newsom-Davis J, Melms A, Vincent A
Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrainJ.Neurosci.,July26,2006 • 26(30):7919–7932 • 7931
with myasthenia gravis without acetylcholine receptor antibodies. Nat
Hogg RC, Raggenbass M, Bertrand D (2003) Nicotinic acetylcholine recep-
tors: from structure to brain function. Rev Physiol Biochem Pharmacol
Huerta PT, Lisman JE (1995) Bidirectional synaptic plasticity induced by a
single burst during cholinergic theta oscillation in CA1 in vitro. Neuron
Huerta PT, Lisman JE (1996) Low-frequency stimulation at the troughs of
theta-oscillation induces long-term depression of previously potentiated
CA1 synapses. J Neurophysiol 75:877–884.
Hyman JM, Wyble BP, Goyal V, Rossi CA, Hasselmo ME (2003) Stimula-
tiation when delivered to the peak of theta and long-term depression
when delivered to the trough. J Neurosci 23:11725–11731.
(2000) Cloning and characterization of muscle-specific kinase in
chicken. Mol Cell Neurosci 16:661–673.
Izquierdo I, McGaugh JL (2000) Behavioural pharmacology and its contri-
JenningsCG,DyerSM,BurdenSJ (1993) Muscle-specifictrk-relatedrecep-
tor with a kringle domain defines a distinct class of receptor tyrosine
kinase. Proc Natl Acad Sci USA 90:2895–2899.
Ji RR, Bose CM, Lesuisse C, Qiu D, Huang JC, Zhang Q, Rupp F (1998)
Specific agrin isoforms induce cAMP response element binding protein
phosphorylation in hippocampal neurons. J Neurosci 18:9695–9702.
Jones MW, Wilson MA (2005) Theta rhythms coordinate hippocampal-
prefrontal interactions in a spatial memory task. PLoS Biol 3:2187–2199.
Kandel ER (2001) The molecular biology of memory storage: a dialogue
between genes and synapses. Science 294:1030–1038.
Kim CH, Xiong WC, Mei L (2003) Regulation of MuSK expression by a
novel signaling pathway. J Biol Chem 278:38522–38527.
Kong XC, Barzaghi P, Ruegg MA (2004) Inhibition of synapse assembly in
mammalian muscle in vivo by RNA interference. EMBO Rep 5:183–188.
Kumar P, Ferns MJ, Meizel S (2006) Identification of agrinSN isoform and
muscle-specific receptor tyrosine kinase (MuSK) [corrected] in sperm.
Biochem Biophys Res Commun [Erratum (2006) 344:453] 342:522–528.
MacVicar BA, Tse FW (1989) Local neuronal circuitry underlying cholin-
ergic rhythmical slow activity in CA3 area of rat hippocampal slices.
J Physiol (Lond) 417:197–212.
Moore C, Leu M, Muller U, Brenner HR (2001) Induction of multiple sig-
naling loops by MuSK during neuromuscular synapse formation. Proc
Natl Acad Sci USA 98:14655–14660.
O’Connell C, Gallagher HC, O’Malley A, Bourke M, Regan CM (2000)
Osten P, Valsamis L, Harris A, Sacktor TC (1996) Protein synthesis-
dependent formation of protein kinase M? in long-term potentiation.
J Neurosci 16:2444–2451.
Pape HC, Stork O (2003) Genes and mechanisms in the amygdala involved
in the formation of fear memory. Ann NY Acad Sci 985:92–105.
Power AE, Roozendaal B, McGaugh JL (2000) Glucocorticoids enhance-
ment of memory consolidation in the rat is blocked by muscarinic recep-
Sandrock Jr AW, Dryer SE, Rosen KM, Gozani SN, Kramer R, Theill LE,
FischbachGD (1997) Maintenanceofacetylcholinereceptornumberby
SanesJR,LichtmanJW (1999) Developmentofthevertebrateneuromuscu-
lar junction. Annu Rev Neurosci 22:389–442.
Synaptogenesis in the hippocampal CA1 field following traumatic brain
injury. J Neurotrauma 22:719–732.
Direct interactions between PSD-95 and stargazin control synaptic
AMPA receptor number. Proc Natl Acad Sci USA 99:13902–13907.
Seidenbecher T, Laxmi TR, Stork O, Pape HC (2003) Amygdalar and hip-
pocampal theta rhythm synchronization during fear memory retrieval.
Silva AJ, Kogan JH, Frankland PW, Kida S (1998) CREB and memory. Rev
SmithMA,HilgenbergLG (2002) AgrinintheCNS:aproteininsearchofa
function? Neuroreport 13:1485–1495.
StoneDM,NikolicsK (1995) Tissue-andage-specificexpressionpatternsof
alternatively spliced agrin mRNA transcripts in embryonic rat suggest
novel developmental roles. J Neurosci 15:6767–6778.
Strochlic L, Cartaud A, Cartaud J (2005) The synaptic muscle-specific ki-
nase (MuSK) complex: new partners, new functions. Bioessays
Taubenfeld SM, Wiig KA, Bear MF, Alberini CM (1999) A molecular cor-
relate of memory and amnesia in the hippocampus. Nat Neurosci
Taubenfeld SM, Milekic M, Monti B, Alberini CM (2001a) The consolida-
tion of new but not reactivated memory requires hippocampal C/EBP?.
Nat Neurosci 4:813–818.
Taubenfeld SM, Wiig KA, Monti B, Dolan B, Pollonini G, Alberini CM
enhancer-binding protein ? and ? co-localizes with phosphorylated
cAMP response element binding protein and accompanies long-term
memory consolidation. J Neurosci 21:84–91.
RD (2005) Localproteinsynthesismediatesarapidincreaseindendritic
elongation factor 1A after induction of late long-term potentiation.
J Neurosci 25:5833–5843.
Valenzuela DM, Stitt TN, DiStefano PS, Rojas E, Mattsson K, Compton DL,
Nunez L, Park JS, Stark JL, Gies DR (1995) Receptor tyrosine kinase
the neuromuscular junction, and after injury. Neuron 15:573–584.
Waites CL, Craig AM, Garner CC (2005) Mechanisms of vertebrate synap-
togenesis. Annu Rev Neurosci 28:251–274.
Williams JH, Kauer JA (1997) Properties of carbachol-induced oscillatory
activity in rat hippocampus. J Neurophysiol 78:2631–2640.
Winson J (1978) Loss of hippocampal theta rhythm results in spatial mem-
ory deficit in the rat. Science 201:160–163.
Yaffe D, Saxel O (1977) Serial passaging and differentiation of myogenic
cells isolated from dystrophic mouse muscle. Nature 270:725–727.
Yin YC, Tully T (1996) CREB and the formation of long-term memory.
Curr Opin Neurobiol 6:264–268.
7932 • J.Neurosci.,July26,2006 • 26(30):7919–7932 Garcia-Ostaetal.•MuSKExpressionandFunctionintheBrain