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INTRODUCTION |
Muscarinic acetylcholine receptors
(mAChR)1 are the predominant
cholinergic receptors in the central nervous system where they are
involved in learning and memory (1), in epileptic seizures (2), and in
processing the amyloid precursor protein (3, 4). Of the five subtypes
of these seven transmembrane, G protein-coupled receptors,
M1 is found in greatest abundance in the cortex and hippocampus where it constitutes 40-50% of the total mAChR (5). The
M1, M3, and M5 subtypes
preferentially couple via the Gq/11 protein family to
activate phospholipase C (PLC), stimulating phosphoinositide (PI)
hydrolysis (6). The resulting products, diacylglycerol and inositol
trisphosphate, activate PKC and trigger a release of
intracellular calcium, respectively. These events initiate an
overlapping network of signals, including the activation of
mitogen-activated protein kinase (MAPK) (7). M2 and
M4 preferentially couple via Gi to inhibit
adenylyl cyclase activity but in some cell types can also activate PLC
(8, 9). These subtypes can also activate MAPK by direct G protein

subunit-mediated mechanisms (10) and potentially also via
activation of PKC.
The MAP kinases ERK1 and ERK2 (p44 and p42) are known to play a pivotal
role in signaling events in the brain where they are involved in
neuronal plasticity (11), neuronal survival (12), and processing the
amyloid precursor protein (13, 14). They play a crucial role in
learning and memory, including long term potentiation (15, 16) and long
term spatial memory (17). In addition to activation by G
protein-coupled receptors, the ERKs can be stimulated by growth factors
signaling through receptor tyrosine kinases. Thus these kinases are
poised at a critical position allowing cross-talk between a variety of
signal transduction pathways.
Although the functional properties of the individual mAChR subtypes
have been well characterized in recombinant systems, the analysis of
endogenous mAChR function in cells and tissues expressing multiple
subtypes of mAChR is complicated by the lack of subtype-specific agonists and overlapping selectivity of antagonists. In addition, the
apparent affinity of a putative subtype-selective antagonist can vary
dramatically depending on membrane composition and lipid environment
(18), so that the same receptor polypeptide can potentially exhibit
different ligand affinities in different cell types. We have previously
shown that in mice with a targeted deletion of the gene encoding the
M1 receptor, mAChR-mediated suppression of the M current
potassium channel is completely lost in sympathetic neurons (19),
whereas mAChR-mediated suppression of the M current in hippocampal
neurons is unaltered (20). Thus, a given subtype of mAChR may not
couple to the same functional pathway in different cell types. In this
paper, we use mice lacking the M1 receptor to demonstrate
that M1 is the main receptor subtype responsible for
activation of PLC and MAPK in cultured cerebral cortical neurons.
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EXPERIMENTAL PROCEDURES |
Materials--
Neurobasal medium, B-27 supplement, and
penicillin-streptomycin were obtained from Life Technologies,
Inc. Carbamylcholine chloride (carbachol),
cytosine-
-D-arabinoside, phorbol 12-myristate 13-acetate
(PMA), tetrodotoxin, atropine sulfate, and papain came from
Sigma. Research Biochemicals International was the source of
oxotremorine methiodide (oxo), (+)-2-amino-5-phosphonopentanoic acid
(APV), nimodipine, and
trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD). ICN
Biomedicals Inc. supplied 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX).
Myo-[2-3H]inositol and ECL reagents came from
Amersham Pharmacia Biotech, poly-D-lysine hydrobromide
(high molecular weight) came from Collaborative Biomedical Products,
phospho-p44/42 MAPK antibody came from New England Biolabs, monoclonal
anti-MAPK (ERK1 + ERK2) antibody came from Zymed
Laboratories Inc., Immobilon-P membrane came from Millipore, and
ion exchange resin AG 1-X8 100-200 mesh (formate form) came from
Bio-Rad.
Immunoblot Analysis for Detection of M1
mAChR--
Membranes were prepared from mouse heart, cerebellum, and
forebrain as described by Luetje et al. (21). Dissected
mouse tissues were placed in phosphate-buffered saline (PBS) containing protease inhibitors, homogenized on ice 25 times in a glass-glass homogenizer, and centrifuged at 36 × g for 15 min to
remove debris. The supernatant was centrifuged at 8000 × g for 15 min to pellet membranes. After determination of
protein concentration, 55 µg of membrane protein was solubilized in
an SDS sample buffer containing 8 M urea and loaded onto a
3.5% stacking SDS-polyacrylamide gel and a 7% separating
SDS-polyacrylamide gel, each containing 4 M urea. Proteins
were transferred to Immobilon-P membrane, and immunoblot analysis was
carried out as described by McKinnon and Nathanson (22) using
affinity-purified polyclonal anti-M1 antibody directed
against the third cytoplasmic loop of the mouse M1
(19).
Primary Cortical Cell Culture--
Primary cortical cultures
were prepared as described by Chan et al. (23). Briefly, the
cerebral cortices of newborn mouse brains were digested with papain for
20 min at 37 °C. The rinsed tissue was then triturated 15 times and
allowed to settle. Supernatants were saved, and the trituration was
repeated on remaining tissue. Supernatants were combined, and cortical
neurons were seeded on plates treated with polylysine for a minimum of
2 h. Neurons for PI assays and determination of MAPK activation
were plated in 24-well (0.15 × 106 cells/well) and
6-well (1 × 106 cells/well) plates, respectively.
Cells were grown in neurobasal medium containing B-27 supplement,
glutamine (1 mM), penicillin (0.05 units/ml), and
streptomycin (0.05 µg/ml) and maintained at 37 °C in 5%
CO2. Medium was changed one and three days after plating
and contained 3.6 µM
cytosine-
-D-arabinoside to retard glial growth.
Quantitative Immunoprecipitation Analysis of M3
Expression--
The expression of the M3 mAChR was
determined by immunoprecipitation analysis using a polyclonal antibody
specific for the M3 mAChR as described (19).
Phosphoinositide Hydrolysis Assays--
After six to eight days
in culture, PI turnover was measured as described (24, 25). In brief,
cells were incubated overnight in culture medium containing 1 µCi/ml
myo-[2-3H]inositol (18.3 Ci/mmol). Cells were
washed and incubated 30 min at 37 °C with physiological saline
solution (118 mM NaCl, 4.7 mM KCl, 3 mM CaCl2, 1.2 mM
KH2PO4, 10 mM glucose, 0.5 mM EDTA, 20 mM HEPES, pH 7.4) containing 10 mM LiCl. Carbachol or ACPD was added for 15 min before the
reaction was terminated with ice-cold methanol. Total inositol
phosphates were quantified using ion exchange chromatography. Each drug
condition was performed in quadruplicate for each of four
cultures/genotype.
Determination of MAPK Activation--
MAPK analysis was
performed on cultures seven to 10 days after plating. Two h before
stimulation, cultures were treated with tetrodotoxin (1 µM), CNQX (40 µM), APV (100 µM), and nimodipine (5 µM) to decrease
endogenous synaptic activity (26). Cells were stimulated for the
intervals indicated with oxo (100 µM) and PMA (0.1 µM) or pretreated with atropine (3 µM) for
5 min before a 5-min stimulation with oxo. Medium was aspirated, and cells were washed two times with ice-cold PBS and lysed with 100 µl
4× SDS sample buffer. Scraped cells were placed in 1.5-ml
polypropylene tubes, sonicated 10 s, boiled 5 min, cooled, and
centrifuged. Samples (25 µl) were run on 10% SDS-polyacrylamide gels
and transferred to Immobilon-P membrane. Membranes were incubated with
phospho-p44/42 MAPK antibody (1:2000) overnight, and bands were
detected by ECL. Blots were stripped using the method supplied with
Immobilon-P membrane and reprobed with monoclonal anti-MAPK antibody
(1:2000) for 2 h, and bands were visualized by ECL. Autoradiograms
were scanned (Scan Wizard PPC 3.0.7, Microtek) and processed (Adobe Photoshop 5.0), and the p42 phospho-ERK band intensities were determined (NIH Image 1.62). Results were internally normalized and
applied to the intensity data obtained from the respective anti-phospho-MAPK blots to correct for differences in protein loading.
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RESULTS |
Functional M1 mAChR Protein Fragments Are Not
Reconstituted in M1-deficient Mice--
Previously, we
showed that the M1-KO mice do not express detectable
M1 protein as determined by immunoprecipitation of
radioligand-labeled, digitonin-solubilized receptors (19). Recent work
has shown that fragments of the mAChR can interact to form a
reconstituted functional receptor. The interaction between receptor
fragments containing transmembrane domains I-V and fragments
containing transmembrane VI-VII depends on the presence of the long
third intracellular loop, and chimeric receptors containing these
separate domains exhibited agonist-induced functional activity (27, 28, 29). Because our targeting construct used to generate M1-KO mice lacked the nucleotides coding for the first 55 amino acids of the
M1 receptor, it would be possible for protein synthesis to
initiate at a downstream methionine, resulting in the expression of a
truncated polypeptide containing most of the transmembrane domains as
well as the third cytoplasmic loop. If this truncated protein were
indeed synthesized, it could interact with one of the remaining mAChR
polypeptides to produce a protein exhibiting the functional coupling of
the M1 receptor but would not necessarily be stable to
detergent solubilization and subsequent detection by
immunoprecipitation. To determine whether this possibility was
occurring, we performed immunoblot analysis using an antiserum prepared
against the third cytoplasmic loop of the M1 receptor to
ensure that this portion of M1 was absent in our
M1-KO mice. In addition to mouse tissues, protein from the
mouse Y1 adrenal cell line and Y1 cells stably transfected with mouse
M1 (Y1-M1) under an inducible promoter (24)
were analyzed simultaneously. A strong signal was detected in the WT
forebrain, which was significantly reduced in forebrain from
heterozygote mice (Fig. 1). As expected from the negligible expression of the M1 receptor in
cerebellum and heart, no signal was detected in these tissues.
Immunoreactivity to M1 was not detectable in any of the
M1-KO tissues. The Y1-M1 cells express two
immunoreactive proteins, the lower corresponding to the band in the
M1-HET and WT forebrain samples with an apparent molecular
mass of 60 kDa. These results, combined with our previous data (19),
indicate that a polypeptide containing the functionally vital third
cytoplasmic loop of the M1 receptor is completely absent in
the M1-KO mice and that there most likely is no ectopic translation of a truncated protein.

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Fig. 1.
Immunoblot analysis of M1-mAChR
expression in WT, HET, and M1-KO mouse
tissues. SDS-polyacrylamide gel electrophoresis and immunoblot
analysis using an antibody raised against the third cytoplasmic loop of
the mouse M1 receptor was performed on equal amounts of
membrane protein (55 µg) from M1-KO ( / ),
M1-HET (+/ ), and WT (+/+). H, heart;
F, forebrain; and Cb, cerebellum. Membrane
protein (55 µg each) from untransfected Y1 cells (U) and
Y1 cells expressing M1 (T) were run as
controls.
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Decreased mAChR-mediated Activation of PLC in
M1-deficient Cortical Cultures--
To measure the effects
of inactivating the M1 receptor on mAChR-mediated
activation of PLC, we measured the stimulation of PI hydrolysis in
primary cortical cultures derived from WT and M1-KO mice in
response to the muscarinic agonist carbachol. The mean maximum increase
in inositol phosphates obtained with WT cultures was 250% above basal
level (Fig. 2A). In
M1-KO cultures this maximal level was reduced by a factor
of 2.6 with a mean stimulation of 95% above basal.

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Fig. 2.
Muscarinic receptor-mediated phosphoinositide
hydrolysis in cortical cultures from WT and M1-KO
mice. Primary cortical cultures from WT and M1-KO mice
were incubated with myo-[2-3H]inositol
overnight and subjected to 15 min stimulations with the mAChR agonist,
carbachol (A), or the metabotropic glutamate receptor
agonist, ACPD (B) at the concentrations indicated. Results
are expressed as percent stimulation above basal and represent
the mean ± S.E. of four sets of cultures analyzed in
quadruplicate at each concentration.
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We previously showed by quantitative immunoprecipitation analyses that
the level of the remaining mAChR subtypes was unaltered in the
forebrain of M1-KO mice (19). In addition to
M1, M3 couples via the Gq/11-family
and is present in significant amounts in the forebrain. To ensure that
levels of M3 were not altered in cortical cultures, we
carried out quantitative immunoprecipitation analyses with an
anti-M3-specific antibody. The levels of M3
were similar in cortical cultures prepared from WT (88 ± 17 fm/mg
protein) and M1-KO (73 ± 18 fm/mg protein) mice
(mean ± S.E.; n = 4).
Metabotropic glutamate receptors also activate PLC via
Gq/11 proteins. ACPD, a selective agonist for the
metabotropic glutamate receptor, was used as a positive control to
ensure that no general defect in G protein or PLC function existed.
Similar levels of maximal activation by ACPD were obtained from WT and
M1-KO cortical cultures (Fig. 2B), indicating
that the PLC signaling pathway downstream of the M1 mAChR
is normal in M1-KO mice.
Decreased mAChR-mediated MAP Kinase Activation in
M1-deficient Cortical Cultures--
Dual phosphorylation
of the MAP kinases ERK1 and ERK2 on Thr-202 and Tyr-204 by MAPK kinase
results in their activation; the detection of this dual phosphorylation
using a dual-phospho-specific antibody is a widely used, sensitive
method for the quantitation of the activation of MAPK in cultured cells
and intact tissues (13, 15-17). We performed SDS-polyacrylamide gel
electrophoresis and immunoblot analysis using antibodies specific to
the activated (dually phosphorylated) forms of ERK to measure
stimulation of MAPK in primary cortical cultures from WT and
M1-KO mice. In WT cultures the time course of treatment
with the agonist oxo showed maximal levels of phospho-ERK within 5-10
min of agonist stimulation with a return to basal level by 30 min (Fig.
3A, a). This blot was stripped
and reprobed with an antibody to the non-phosphorylated forms of ERK to
ensure that differences in band intensity observed with phospho-ERK
antibodies were not due to differences in protein loading (Fig.
3A, b). Immunoblot analyses comparing stimulation of WT and
M1-KO cultures showed a robust activation of MAPK by oxo in
WT cortical neurons (Fig. 3B, a) and low to nonexistent activation in the M1-KO cultures (Fig. 3C, a).
Quantitative analysis of the immunoblots showed that WT cultures
treated with oxo exhibited a mean stimulation of >300% above basal
compared with 31% for the M1-KO cultures
(n = 5 for each genotype; Fig.
4). This activation is decreased by
>75% when cultures are pretreated with the muscarinic-specific antagonist atropine, indicating that the effects of oxo are due to
mAChR activation. PMA, a direct activator of PKC, induced a similar
level of MAPK activation in WT (1050%) and M1-KO cultures (890%), respectively, indicating that the signaling pathway coupling stimulation of PKC to activation of the MAPK pathway is unaltered in
M1-deficient neurons.

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Fig. 3.
Immunoblot analysis of mAChR-mediated MAPK
activation in cortical cultures from WT and M1-KO
mice. A, immunoblot analysis of the time-course of MAPK
activation in WT cortical cultures. B and C,
immunoblots of MAPK activation in WT and M1-KO cultures,
respectively. Cultures were "turned down" for 2 h and treated
with PBS (10 min), PMA (10 min), atropine (5 min) followed by oxo (5 min) (atr/oxo), or oxo alone for the times
indicated. Membranes were probed with anti-phospho-p44/p42-MAPK
antibody (a) and then stripped and probed with anti-MAPK
antibody (b) to ensure similar levels of protein
loading.
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Fig. 4.
Quantitative analysis of mAChR-mediated MAPK
activation in WT and M1-KO primary cortical cultures.
Data represent the mean ± S.E. of five sets of cultures for each
genotype, which were treated with PBS (10 min), oxo (5, 10 min), and
atropine (5 min) followed by oxo (5 min), (atr/oxo), or PMA (10 min). Intensities of p42 phospho-MAPK bands on immunoblots were
corrected for variations in protein loading using the normalized p42
band intensities from the anti-MAPK immunoblots and expressed as
percent stimulation above basal.
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DISCUSSION |
A growing number of studies show that functionally significant
intermolecular interactions between muscarinic receptors can occur and
that the third cytoplasmic loop is vital for these potential heteromolecular interactions. In the original construct designed to
ablate the M1 gene product by homologous recombination,
nucleotides coding for the N terminus and first transmembrane domain
were deleted (19). However, the nucleotide sequence for >85% of the coding sequence, including the third cytoplasmic loop, is still present
in these mice. To ensure that this portion of the receptor was not
expressed, we performed immunoblot analyses using an
M1-specific antibody raised against the third cytoplasmic
loop of M1 (amino acids 233-332). Our results confirm that
this key region of the receptor is significantly reduced in the
forebrain of the M1-HET mice and is not detected in the
M1-KO mice.
Studies using recombinant M1 mAChR show that it couples via
the Gq/11 protein family to the activation of PLC (24, 30). The M1 receptor constitutes approximately half of the total
mAChR in adult mouse forebrain (19). In primary cortical cultures from
newborn M1-KO mice, agonist-stimulated PI hydrolysis was reduced by >60% compared with WT cultures. Thus, the M1
receptor is the predominant mAChR subtype responsible for PLC
activation in mouse cortical neurons. Despite the loss of
M1, there was still significant carbachol-stimulated
hydrolysis of PI. This is not unexpected because M3 also
couples to PLC and is expressed to similar extents in WT and KO
cortical cultures. The similar levels of PI hydrolysis obtained with
the metabotropic glutamate receptor agonist ACPD in both WT and
M1-KO cultures demonstrate that the G protein/PLC
interactions are unaltered in the M1-KO mice.
Although activation of mAChR in the brain has recently been shown to
activate MAPK in rat cortical cultures (16), most work demonstrating
the relationship between mAChR and MAPK has been performed in
established cell lines. In human neuroblastoma SH-SY5Y cells, carbachol
was found to activate MAPK via M3 and a
PKC-dependent pathway (31). When M1 is
overexpressed in PC-12 cells, agonist-induced stimulation leads to MAPK
activation (14). Although the predominant mAChR subtype in native PC-12
cells is M4 (~95%), carbachol-induced MAPK activation
was completely abolished with a highly selective M1 toxin
(32). However, pretreatment with pertussis toxin, which uncouples
members of the Gi/o family from their receptors, inhibited 70% of this agonist-induced activation, suggesting that either ectopic
coupling of M1 to Gi/o proteins or signal
transduction downstream of M4 also contributes to the
muscarinic agonist activation in this neuronal cell line.
MAPK activation in our WT primary cortical cultures ranged from 180 to
540% over basal values, similar to the values reported in rat cortical
cultures (Fig. 3A, Ref. 16)). The maximal MAPK activation
occurred in our WT mouse cultures within 5-10 min of agonist
stimulation and rapidly returned to baseline, in contrast to the more
persistent (30-60 min) activation reported for rat cultures (16). This
may be due to differences in species or tissue culture conditions. In
M1-KO cultures the range of MAPK stimulation by agonist was
reduced to
70 to 120%. The average magnitude of stimulation
decreased from 320% over basal in cultures from WT mice to 31% in the
cultures from M1-KO mice. Thus M1 is the mAChR
responsible for coupling to ERK activation in our primary cortical
neuron culture system. The M3 receptor is present in similar amounts in our WT and KO cultures and most likely accounts for
the agonist-induced PLC activation remaining in the M1-KO cultures. The much greater impairment in MAPK activation compared with
PLC activation observed in the M1-KO cultures suggests that the M3 receptor, although still able to evoke significant
activation of PLC, is relatively ineffective in mediating activation of
MAPK in these neuronal cultures. PMA-induced activation of MAPK was similar in WT and M1-KO cultures indicating that signaling
downstream of PKC is unaltered in mice lacking M1
mAChR.
In summary, these results show that there are significant defects in
mAChR-mediated activation of PLC and MAPK in the forebrains of mice
lacking the M1 receptor. Both the M1 and
M3 receptors couple to members of the Gq-family
of G proteins, although it is not known if there are subtle differences
in the specificity of the two receptors for either
or 
subunits; the results here suggest that the signaling pathways
activated by the two receptors in cortical neurons appear to be
different. The M1-KO mice will be useful in determining the
PKC-dependent and PKC-independent pathways by which mAChR
mediate secretion of soluble amyloid precursor protein. In addition,
these mice should be useful in determining the relationship between
M1 mAChR, MAPK activation, long-term potentiation, and
learning and memory. Indeed, preliminary data indicate that mice
lacking the M1 mAChR exhibit defects in the consolidation
of hippocampus-dependent learning and show a deficit in
long term potentiation (33).