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The Journal of Neuroscience, March 15, 2003, 23(6):2371
Regulation of Extracellular Signal-Regulated Kinase by
Cannabinoids in Hippocampus
Pascal
Derkinderen1, *,
Emmanuel
Valjent1, 2, *,
Madeleine
Toutant1,
Jean-Christophe
Corvol1,
Hervé
Enslen1,
Catherine
Ledent3,
James
Trzaskos4,
Jocelyne
Caboche2, and
Jean-Antoine
Girault1
1 Institut National de la Santé et de la
Recherche Médicale/Université Pierre et Marie Curie U536,
Institut du Fer à Moulin, Paris, France 75005, 2 Laboratoire de Neurobiologie des Processus Adaptatifs,
Centre National de la Recherche Scientifique and Université
Pierre et Marie Curie, UPMC Unité Mixte de Recherche 7102, Paris, France 75005, 3 Institut de Recherche
Interdisciplinaire en Biologie Humaine et Nucléaire,
Université Libre de Bruxelles, B1070, Brussels, Belgium, and
4 Bristol-Myers Squibb Company, Wilmington, Delaware
19880-0400
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ABSTRACT |
Endocannabinoids form a novel class of intercellular messengers,
the functions of which include retrograde signaling in the brain
and mediation or modulation of several types of synaptic plasticity.
Yet, the signaling mechanisms and long-term effects of the stimulation
of CB1 cannabinoid receptors (CB1-R) are poorly understood. We show
that anandamide, 2-arachidonoyl-glycerol, and  9-tetrahydrocannabinol
(THC) activated extracellular signal-regulated kinase (ERK) in
hippocampal slices. In living mice, THC activated ERK in hippocampal
neurons and induced its accumulation in the nuclei of pyramidal cells
in CA1 and CA3. Both effects were attributable to stimulation of CB1-R
and activation of MAP kinase/ERK kinase (MEK). In hippocampal slices,
the stimulation of ERK was independent of
phosphatidyl-inositol-3-kinase but was regulated by cAMP. The endocannabinoid-induced stimulation of ERK was lost in Fyn knock-out mice, in slices and in vivo, although it was insensitive
to inhibitors of Src-family tyrosine kinases in vitro,
suggesting a noncatalytic role of Fyn. Finally, the effects of
cannabinoids on ERK activation were dependent on the activity of
glutamate NMDA receptors in vivo, but not in hippocampal
slices, indicating the existence of several pathways linking CB1-R to
the ERK cascade. In vivo THC induced the expression of
immediate-early genes products (c-Fos protein, Zif268, and BDNF mRNAs),
and this induction was prevented by an inhibitor of MEK. The
strong potential of cannabinoids for inducing long-term alterations in
hippocampal neurons through the activation of the ERK pathway may be
important for the physiological control of synaptic plasticity and for
the general effects of THC in the context of drug abuse.
Key words:
hippocampus; cannabinoids; 2-AG; anandamide; CB1-R; THC; LPA; ERK; phosphorylation; Fyn; immediate-early genes; c-Fos; Zif268; BDNF; slices; rat; mouse
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Introduction |
The endocannabinoid system, which
has recently emerged as a major player in the control of synaptic
plasticity, is the pharmacological target of cannabis, the most widely
used illicit drug of abuse. Cannabinoid receptors of the CB1 subtype
(CB1-Rs) are highly expressed in the brain (Matsuda et al.,
1990 ). They are the targets for endogenous ligands (endocannabinoids),
including anandamide and 2-arachidonoyl glycerol (2-AG) (for review,
see Di Marzo et al., 1998). In hippocampus, CB1-Rs are abundant and
enriched in nerve terminals, especially those of a subpopulation of
inhibitory interneurons (Tsou et al., 1998, 1999; Katona et al., 1999,
2000). The production of 2-AG is increased after stimulation of
Schaeffer collaterals (Stella et al., 1997). Massive activation of
CB1-Rs decreases long-term potentiation (LTP) and depression (LTD)
(Misner and Sullivan, 1999) and impairs short-term memory tasks
associated with the firing of hippocampal neurons (Hampson and
Deadwyler, 2000). Endocannabinoids play a major role in the modulation
of synaptic transmission: they are released after depolarization of
postsynaptic neurons and act backward on presynaptic CB1-Rs to suppress
inhibitory neurotransmitter release (Ohno-Shosaku et al., 2001; Wilson
and Nicoll, 2001), a phenomenon called depolarization-induced suppression of inhibition. Thus, in physiological conditions the endocannabinoid system facilitates the induction of LTP (Carlson et
al., 2002).
Despite the recent progress in understanding the actions of
endocannabinoids on synaptic transmission, the signal transduction pathways regulated by
Gi/o-coupled CB1-Rs in
hippocampus are poorly characterized. Most of the data were obtained in
non-neuronal cell lines, in which stimulation of CB1-Rs activates
the extracellular-regulated kinase (ERK) subtype of
mitogen-activated protein (MAP) kinases (Bouaboula et al., 1995a;
Wartmann et al., 1995), leading to the expression of the
immediate-early gene (IEG) Zif268 (also known as
egr-1, NGFI-A, or Krox-24) (Bouaboula
et al., 1995a). CB1-Rs are also coupled to the activation of protein
kinase B/Akt (PKB) through the phosphatidylinositol 3 (PI3)-kinase
pathway (Gomez del Pulgar et al., 2000, 2002). Little is known about
the signaling pathways regulated by cannabinoids in the adult
nervous system. Intraperitoneal injection of cannabinoid agonists
increases the expression of IEGs, products including Zif268, c-Fos, and
c-Jun, in rat forebrain (Mailleux et al., 1994; Glass and
Dragunow, 1995), but the mechanism of these responses is not known. We
have shown previously that cannabinoids augment tyrosine
phosphorylation of the neuronal splice isoform of focal adhesion kinase
(FAK) (Derkinderen et al., 1996, 2001b; Burgaya et al., 1997) and its association with the Src family tyrosine kinase Fyn (Derkinderen et
al., 2001b). Cannabinoids also activate p38-MAPK, but not c-Jun N-terminal kinase, in hippocampal slices (Derkinderen et al., 2001a).
The aim of the present study was to determine whether cannabinoids
could regulate ERK in hippocampus, to examine the signaling pathways
involved, and to determine the role of this pathway in IEG expression.
We report that stimulation of CB1-Rs activates the ERK cascade both in
hippocampal slices and in vivo where it controls the
expression of IEGs.
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Materials and Methods |
Reagents. Anandamide, cremophor El,
9-tetrahydrocannabinol (THC), diethyl pyrocarbonate,
lysophosphatidic acid (LPA), protein A-Sepharose, and tetrodotoxin
were purchased from Sigma (Saint Quentin Fallavier,
France). 2-Arachidonoyl glycerol and WIN55212-2 were from
Research Biochemicals (Saint Quentin Fallavier, France). CP55940 was from Pfizer.
4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) was from Calbiochem (Meudon, France). LY290004 was
from Biomol (Le Perray en Yvelines, France). PD98059 was
from New England Biolabs (Ozyme, Orsay France). SR141716A
was from Sanofi (Montpellier, France). U0126 and SL327
were kindly provided by Dr. James Trzaskos (DuPont
Merck). Artificial CSF (ACSF) contained (in
mM): 125 NaCl, 2.4 KCl, 0.83 MgCl2, 1.1 CaCl2, 0.5 KH2PO4,
Na2SO4, 27 NaHCO3, 10 glucose, 11 HEPES, pH 7.4. Ca2+-free ACSF had the same composition
except for MgCl2, which was 1.93 mM. The following commercially available
phosphospecific antibodies were used for Western blotting: monoclonal
anti-MAP kinase, activated (clone MAPK-YT,
Sigma; 1:10,000), anti-diphospho-ERK (Promega; 1:5000), or anti-diphospho-ERK (New
England Biolabs; diluted 1:1000), anti-phospho-Ser218-Ser222
MEK1/2 (New England Biolabs; diluted 1:1000). Mouse
monoclonal antibodies (Zymed; 1:1000) or rabbit
affinity-purified IgG (Upstate Biotechnology, Lake Placid,
NY; 1 µg/10 ml) was used to detect the total amounts of ERK 1 and 2 proteins in homogenates. Monoclonal antibodies to phosphotyrosine
(4G10) were purchased from Upstate Biotechnology (diluted
1:4000). For immunocytochemistry, polyclonal anti-phospho-Thr/Tyr-ERK antibodies (New England Biolabs; diluted 1:200) and
polyclonal anti-c-Fos antibodies (Santa Cruz
Biotechnology, Santa Cruz, CA; diluted 1:500) were used.
Rat and mice hippocampal slices. Rat hippocampal slices (300 µm thick) were prepared from young male Sprague Dawley
rats (150-200 gm) with a McIlwain tissue chopper, as described
previously (Siciliano et al., 1994). Briefly, slices were dissected in
ice-cold, Ca2+-free ACSF and placed for 10 min in polypropylene tubes (three slices per tube) containing 1 ml of
Ca2+-free ACSF at 35°C, equilibrated at
pH 7.4 in O2/CO2 (95:5,
v/v). They were then incubated at 35°C in 900 µl ACSF containing
1.1 mM Ca2+ and 1 µM TTX, for 45 min before pharmacological
treatment. Treatment of control slices was performed by the addition of
vehicle. TTX was added to prevent indirect effects attributable to
neuronal firing and had no effects on tyrosine phosphorylation by
itself (data not shown). At the end of the experiment, ACSF was
aspirated, and slices were immediately frozen on dry ice and kept at
 80°C. Hippocampal slices from wild-type, CB1-R knock-out (Ledent et al., 1999), and Fyn knock-out mice (Grant et al., 1992) were prepared as rat slices except for the use of a Vibratome instead of a McIllwain chopper.
Western blot analysis. Slices were lysed by sonication in
1% SDS (v/v) containing 1 mM sodium
orthovanadate at 100°C. Equal amounts of lysate slices (60 µg) were
separated by SDS-PAGE (8 or 10%) before electrophoretic transfer onto
nitrocellulose membrane (Hybond Pure, Amersham, Orsay,
France). Membranes were blocked for 1 hr at room temperature in
Tris-buffered saline (TBS) (100 mM NaCl, 10 mM Tris, pH 7.5) with 0.1% Tween 20 or 5%
nonfat dry milk for the detection of phosphorylated and
nonphosphorylated proteins, respectively. Membranes were incubated
overnight at 4°C with the primary antibodies. Bound antibodies were
detected with horseradish peroxidase-conjugated anti-rabbit or
anti-mouse antibodies (Amersham; diluted 1:4000) and
visualized by enhanced chemiluminescent detection (ECL,
Amersham). When necessary, membranes were stripped in
buffer [containing 100 mM glycine, pH 2.5, 200 mM NaCl, 0.1% Tween 20 (v/v) and 0.1% (v/v)
 -mercaptoethanol] for 45 min at room temperature, followed by
extensive washing in TBS before reblocking and reprobing. The relevant
immunoreactive bands were quantified with laser-scanning densitometry
using Scion Image software. To allow comparison between
different autoradiographic films, the density of the bands was
expressed as a percentage of the average of control (untreated) slices.
The value of active diphospho-ERK2 (P-ERK2) was normalized to the
amount of total ERK2 in the same sample and expressed as a percentage
of controls. Statistical analysis was done by ANOVA followed by
t test using Prism 3.02 software.
In vivo experiments. Male CD-1 mice (Charles
River) weighing 20-22 gm were used. They were housed in cages
in groups of five in a temperature-controlled room (21 ± 1°C) 1 week before the experiments were started. The mice were given access to
food and water ad libitum and were maintained on a 12 hr
light/dark cycle. Animal care was conducted in accordance with standard
ethical guidelines (Guide for the Care and Use of Laboratory
Animals, National Academy Press, 1996) and approved by the local
ethics committee. THC was purchased as a 10 mg/ml solution in ethanol, which was diluted 1:100 in distilled water containing 5% ethanol and
5% cremophor El. A volume of 0.1 ml per 10 gm of body weight of this
mixture containing 0.1 mg/ml THC was injected intraperitoneally. The
CB1-R antagonist SR141716A (3 mg/kg) was dissolved in a solution of
10% ethanol, 10% cremophor El, and 80% distilled water, and injected
intraperitoneally in a final volume of 0.2 ml per 10 gm of body weight
15 min before THC injection. SL327 (100 mg/kg in DMSO) or DMSO was
injected intraperitoneally 1 hr before THC injection. At specified
times after receiving THC (1 mg/kg, i.p.) brains were fixed by
intracardiac perfusion of 4% paraformaldehyde (PFA) in 0.1 M
Na2HPO4/NaH2PO4
buffer, pH 7.5, as described previously (Valjent et al., 2000). Brains
were removed and postfixed in the same fixative solution overnight,
sectioned (30 µm) on a Vibratome (Leitz), and then kept
in a solution containing 30% ethylene glycol, 30% glycerol, 0.1 M phosphate buffer, 0.1% diethyl pyrocarbonate at 20°C until processed for in situ hybridization or immunohistochemistry.
Immunohistochemistry. Detection of active ERKs or c-Fos
proteins was performed as described previously (Atkins et al., 1998; Valjent et al., 2000). Briefly, free-floating sections were rinsed in
TBS (0.25 M Tris, 0.5 M NaCl, pH 7.5), incubated for 5 min in TBS
containing 3% H2O2 and
10% methanol, and rinsed three times 10 min each in TBS (0.1 mM NaF was included in all buffers and incubation
solutions). After 15 min incubation in 0.2% Triton X-100
in TBS, sections were rinsed three times in TBS and incubated overnight
with the primary antibody (see below) at 4°C. After three rinses in
TBS, sections were incubated for 2 hr at room temperature with the
secondary biotinylated antibody (anti-IgG) using a dilution twice that
of the first antibody in TBS. After three rinses in TBS, sections were
incubated overnight in avidin-biotin-peroxidase complex (ABC)
solution (Vector Laboratories; final dilution 1:50). Sections were then washed two times in TBS and two times in TB (Tris
0.25 M, pH 7,5), 10 min each, placed in a
solution of TB containing 0.1% 3-3' diaminobenzidine (50 mg/100 ml),
and developed by adding
H2O2 (0.02%). After
processing, tissue sections were mounted onto gelatin-coated slides and
dehydrated through alcohol to xylene for light microscopic examination.
In situ hybridization. The antisense (complementary to
cellular mRNA) probes were
35S-radiolabeled riboprobes. For Zif268
and BDNF riboprobes, murine cDNA subclones were used. Zif268 insert
corresponding to 1.6 kbp was linearized after HindIII
digestion and transcribed with T7 RNA polymerase. The BDNF (327 bp)
riboprobe was transcribed with T7 RNA polymerase after linearization
with SmaI. Transcription reactions contained 1 µM [ 35S]-UTP
(1000 Ci/mmol; Isotopchim), 250 µM
ATP, CTP, GTP, and unlabeled UTP (10.5 µM) and
were incubated at 39°C for 2 hr. After DNase I digestion, the labeled
RNA was purified by phenol/chloroform/isoamyl alcohol (25:24:1)
extraction and ethanol precipitation. Gel electrophoresis showed the
transcripts to be predominantly full length. Free-floating sections
were mounted on SuperFrost/plus slides (Menzel-Gläser) in
RNase-free conditions. Once dried, mounted sections were rinsed in PBS
and treated for 10 min with 0.1 M glycine in 0.1 M Tris-HCl, pH 7.4. Sections were rinsed for 5 min at 37°C in 0.1 M Tris-HCl, pH 8, 50 mM EDTA, and treated for 15 min at 37°C with 1 mg/ml proteinase K in the same buffer. Before hybridization, sections were subjected to the following treatment: postfixation for 15 min in
4% PFA, 5 mM MgCl2 in PBS
at room temperature, acetylation for 20 min in acetic
anhydride/triethanolamine, pH 8, at room temperature, and stepwise
dehydration in alcohol. The following hybridization solution was
applied to sections, which were then covered with GelBond Film
(FMC Bioproducts). The hybridization mixture contained 200 ng/ml (4 ng per section) of 35S-RNA probe
in 20 mM Tris-HCl, pH 8, 300 mM NaCl, 5 mM EDTA, 10% dextran sulfate, 1× Denhardt's solution (0.02% Ficoll, 0.02%
polyvinyl pyrolidone, 10 mg/ml BSA), 0.5 mg/ml Escherichia
coli tRNA, 0.1 M dithiothreitol (DTT), and
50% formamide. Hybridization was performed at 60°C in humid chambers
for 16 hr. After the GelBond coverslips were removed in 4× SSC (1×
SSC is 0.15 M NaCl/0.015 M
Na citrate), 10 mM DTT, the slides were washed in
the same solution for 1 hr at room temperature and then in 50%
formamide, 10 mM Tris-HCl, pH 8, 75 mM NaCl, 2.5 mM EDTA.
Sections were treated with RNase A (20 µg/ml; Sigma) in
400 mM NaCl, 10 mM
Tris-HCl, pH 7.5, 50 mM EDTA for 1 hr at 37°C,
and then rinsed for 15 min at 60°C in 2× SSC followed by 0.1× SSC.
After dehydration, sections were air dried and exposed with Biomax MR
films (Kodak) for 3 d.
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Results |
Cannabinoids activate ERK in the hippocampus
ERKs are present in brain and are particularly abundant in the
hippocampus (Fiore et al., 1993; Flood et al., 1998). Anandamide (1 µM), 2-AG (1 µM), synthetic CB1-R agonists
(1 µM CP 55940 and 100 µM WIN 55212-2),
and THC increased the active form of ERK1 and ERK2 in hippocampal
slices (Fig.
1A,B).
These effects were completely prevented by preincubation of slices with
100 µM SR141716A, a CB1-R antagonist (Fig.
1A,B). As a control, we used
lysophosphatidic acid, a lipidic intercellular messenger unrelated to
cannabinoids and known to activate ERK in several cell lines (Kumagai
et al., 1993). In hippocampal slices LPA (0.2 µM) increased ERK phosphorylation, and these
effects were not blocked in the presence of SR141716A (Fig.
1A,B). It should be noted that
although ERK1 and ERK2 were found at similar levels in hippocampal
slices, the signal was always much stronger for P-ERK2 than for P-ERK1,
which was below the detection threshold in some experiments. However,
when detected, the effects on ERK1 were qualitatively comparable with
those on ERK2. Because 2-AG is the endocannabinoid produced in
hippocampal slices after electrical stimulation (Stella et al., 1997),
we studied the time course and time dependence of the effects of 2-AG
on ERK phosphorylation. The activation of ERK2 phosphorylation by 2-AG
was rapid (half-maximal effect at ~1 min) (Fig. 1C) and concentration dependent (half-maximal effect at ~20 nM)
(Fig. 1D).
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Figure 1.
Cannabinoid agonists stimulate ERK phosphorylation
in rat hippocampal slices. A, Rat hippocampal slices
were incubated at 35°C, as described in Materials and Methods, for 50 min before the addition of vehicle (Control), 1 µM anandamide, 1 µM 2-AG, 1 µM CP 55940, 100 µM WIN 55212-2, 0.2 µM LPA, or 0.1 µM 9-THC for 5 min, in
the absence or in the presence of 100 µM SR 141716A
applied 30 min before. Slices were homogenized in SDS; 60 µg of
protein per sample were subjected to immunoblot analysis using
antibodies specific for the dually phosphorylated (active) forms of
ERK1 and ERK2 (Blot P-ERK). After stripping, the
membranes were reprobed with anti-ERK (Blot ERK)
antibodies. B, For quantification the optical densities
of P-ERK2-immunoreactive bands were measured, normalized to the optical
densities of total ERK2 in the same samples, and expressed as
percentages of controls. Data correspond to means ± SEM.
Statistical analysis was done with ANOVA
(F(13,24) = 22.8; p < 0.0001) followed by t test (treated vs control:
***p < 0.001, **p < 0.01;
treated in the presence of SR141716A vs in its absence:
°° p < 0.01, ° p < 0.05). C, D, Quantification of the
effects of 2-AG on ERK2 active form: time course (drug concentration 1 µM) (C); concentration-response
curve (treatment for 5 min) (D). Immunoreactivity
was quantified by scanning densitometry using NIH image 1.62 software.
Values are means ± SEM of four to eight independent experiments
and are expressed as percentages of the maximal increase above
unstimulated control values.
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ERK is not activated by endocannabinoids in CB1 knock-out mice
Endocannabinoids have a potential for acting on other targets
besides CB1-R, including CB2-R and possibly other receptors (Di Marzo
et al., 2000; Al-Hayani et al., 2001). The effects of 2-AG and
anandamide on ERK phosphorylation were mimicked by synthetic CB1-R
agonists CP 55940 and WIN55212-2 and blocked by the specific CB1-R
antagonist SR141716A (Fig. 1A,B);
however, the required concentration of WIN55212-2 and
SR141716A for complete effects was very high (100 µM). This could indicate a poor penetration of
WIN55212-2 and SR141716A in slices, or the fact that the receptors involved in ERK activation were atypical. To assess the exact contribution of CB1-R to the effects of anandamide and 2-AG, we used
hippocampal slices prepared from genetically altered mice lacking CB1-R
(Ledent et al., 1999). In wild-type mice, anandamide and 2-AG increased
ERK phosphorylation as in rats (Fig.
2A,B). In contrast, endocannabinoids had no effect on ERK activation in CB1-R
knock-out mice, although the expression levels of ERK1 and ERK2 were
unchanged (Fig. 2A,B). The ERK
pathway was normally functional in the hippocampus of CB1-R mutant mice
because LPA was capable of activating ERKs to a similar level in
knock-out and wild-type mice (Fig.
2A,B). These results, combined with
the pharmacological experiments, demonstrate that the effects of
endocannabinoids on ERK in hippocampus are mediated through activation
of the CB1-R.
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Figure 2.
The regulation of ERKs by endocannabinoids is
absent in hippocampal slices from CB1-R knock-out mice.
A, Hippocampal slices from wild-type (CB1-R
+/+) and CB1-R knock-out (CB1-R /) mice
were incubated at 35°C, as described in Materials and Methods, for 50 min before the addition of vehicle (Control), 1 µM anandamide, 1 µM 2-AG, or 0.2 µM LPA for 5 min. ERK phosphorylation was assayed as
described in the legend to Figure 1. B, Quantification
of the results for P-ERK2 as described in the legend to Figure 1. Data
correspond to means ± SEM. Statistical analysis was done with
ANOVA (anandamide: F(3,20) = 40.2, p < 0.0001; 2-AG and LPA:
F(5,24) = 8.2, p < 0.0001) followed by t test (treated vs control:
***p < 0.001, **p < 0.01, *p < 0.05; treated in knock-out vs wild type:
° p < 0.05).
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Endocannabinoids activate MEK and ERK independently
of PI3-kinase
Activation of ERKs results from the phosphorylation of their
activation loop, on a threonine and a tyrosine, by dual-specificity MEKs, MEK1 and MEK2. On the other hand, it has been shown
that in some systems, activation of ERKs results from the
inhibition of their dephosphorylation (Haneda et al., 1999). To
determine the mechanism of action of endocannabinoids, we examined the
phosphorylation state of MEK1/2 using a phosphospecific antibody
reacting only with the active phosphorylated form of these
enzymes (Fig. 3A). Anandamide
and 2-AG increased the phosphorylation of MEK1/2. Moreover, the effects
of endocannabinoids on ERK phosphorylation were completely inhibited by
U0126 and partially by PD98059 (Fig.
3B,C), two MEK inhibitors (Alessi
et al., 1995; Favata et al., 1998). In contrast, both of these
compounds fully antagonized the activation of ERKs by LPA (Fig.
3B,C). The lower efficacy of
PD98059 as compared with U0126 to inhibit ERK activation by
endocannabinoids might indicate a preferential involvement of MEK2 in
the activation of ERKs by cannabinoids, because PD98059 is 10-fold more
active on MEK1 than on MEK2, whereas U0126 has the same potency on both kinases (Alessi et al., 1995; Favata et al., 1998).
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Figure 3.
Role of MEK in the effects of endocannabinoids in
rat hippocampal slices. A, Rat hippocampal slices were
incubated as described in Materials and Methods, for 50 min before the
addition of vehicle (Control), 1 µM
anandamide, or 1 µM 2-AG for 5 min. Homogenates (60 µg
of protein per sample) were analyzed by immunoblotting with antibodies
specific for the phosphorylated forms of MEK1/2. Quantification of
P-MEK immunoreactivity (mean ± SEM): controls 100 ± 8, anandamide 857 ± 29, 2-AG 514 ± 57 (F(2,5) = 226, p < 0.001; t test treated vs control: p < 0.0001). B, Slices were treated with the same
compounds as in A, or with 0.2 µM LPA, in
the absence or in the presence of 50 µM PD98059 or 30 µM U0126, two MEK inhibitors. Homogenates were analyzed
for active dually phosphorylated ERK by immunoblotting.
C, Quantification of the results for P-ERK2 as described
in the legend to Figure 1. Data correspond to mean ± SEM.
Statistical analysis was done with ANOVA
(F(11,45 = 7.4; p < 0.0001) followed by t test (treated vs control:
***p < 0.001; treated in the presence of MEK
inhibitor vs in its absence: ° p < 0.05).
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Multiple pathways lead from G-protein-coupled receptors to the
activation of MEKs and ERKs (for review, see Derkinderen et al., 1999).
In Chinese hamster ovary (CHO) cells transfected with CB1-R,
wortmannin, a specific inhibitor of PI3-kinase, prevented the
activation of ERK by cannabinoid agonists (Bouaboula et al., 1997).
This observation was consistent with other reports in which activation
of ERK by Gi-protein-coupled receptors required
the activation of PI3-kinase (Hawes et al., 1996). Moreover, it has been shown that CB1-R is capable of activating PKB through a
PI3-kinase-dependent pathway (Gomez del Pulgar et al., 2000, 2002). To
test the role of PI3-kinase in the activation of ERK by
endocannabinoids in hippocampal slices, we pretreated the slices with a
specific inhibitor of this kinase, LY294002 (Vlahos et al., 1994). The
effects on ERKs of 2-AG (Table 1) and
anandamide (data not shown) were unaffected by the presence of
LY294002. We verified that LY294002 was able to block
insulin-induced activation of ERK as reported in cell lines
(Shepherd et al., 1998) (data not shown). These results reveal that, in
contrast to CB1-R-transfected CHO cells, the activation of ERKs by
endocannabinoids in hippocampus does not require an active
PI3-kinase.
Role of cAMP in the control of ERK activation by cannabinoids in
hippocampal slices
Stimulation of CB1-R inhibits adenylyl cyclase, thus decreasing
cAMP levels (Howlett, 1995). Depending on the cell type and experimental conditions, cAMP can either stimulate or inhibit the ERK
pathway, through various signaling pathways (Stork and Schmitt, 2002).
In various non-neuronal cells, cAMP prevents the activation of ERK by
mitogens, whereas in neuronal cells such as PC12, an increase in the
intracellular concentration of cAMP activates the ERK pathway. Because
we had demonstrated previously that cAMP was involved in the effects of
endocannabinoids on protein tyrosine phosphorylation in hippocampal
slices (Derkinderen et al., 1996, 2001b), we examined its role in ERK
regulation. We treated hippocampal slices with 8-Bromo
(Br)-cAMP, a cell-permeant analog of cAMP, or forskolin, a
stimulator of adenylyl cyclase, for different periods of time.
Treatment of slices with these compounds for <30 min induced an
increase in ERK phosphorylation, in agreement with previous reports
(Impey et al., 1998, 1999). In contrast, when slices were treated for
periods ranging from 30 to 45 min, ERK phosphorylation dramatically
decreased as compared with controls (Fig.
4A and unpublished
results). A 45 min pretreatment with 8-Br-cAMP completely prevented the
activation of ERK by endocannabinoids (Fig.
4B,C), whereas LPA was still
capable of activating ERKs in these conditions (data not shown).
Because a major and prolonged effect of cAMP is to activate
cAMP-dependent protein kinase (PKA), this result prompted us to test
whether inhibition of PKA could participate in ERK activation. We used
two unrelated PKA inhibitors: Rp-cAMPS, which prevents cAMP
binding to the regulatory subunit of the kinase, and H-89, an inhibitor
of the catalytic subunit of PKA. Remarkably, both compounds stimulated
ERK activity in hippocampal slices (Fig.
4B,C). Taken together, these
results underline the complex effects of cAMP on ERK phosphorylation in hippocampus, revealing a time-dependent combination of stimulatory and
inhibitory effects. They also suggest that a decrease in cAMP levels
and, consequently, in PKA activity, may participate in the effects of
CB1-R on the ERK pathway in hippocampus.
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Figure 4.
Role of cAMP in the effects of endocannabinoids on
ERK phosphorylation. A, Rat hippocampal slices were
incubated with forskolin (50 µM) for the indicated period
of time. Homogenates were analyzed for active dually phosphorylated ERK
by immunoblotting. B, Rat hippocampal slices were
incubated as described in Materials and Methods, in the presence or in
the absence of 4 mM 8-Br-cAMP for 45 min before the
addition of vehicle (Control), 1 µM
anandamide, or 2-AG for 5 min. In other experiments, slices were
incubated in the presence of the PKA inhibitors H-89 (100 µM) or Rp-cAMPS (1 mM) for 20 min.
Homogenates were analyzed for active dually phosphorylated ERK by
immunoblotting as described in the legend to Figure 1.
C, Quantification of the results for P-ERK2 as described
in the legend to Figure 1. Data correspond to mean ± SEM.
Statistical analysis was done with ANOVA (8-Br-cAMP:
F(5,18) = 14.5; H89 and RpcAMPs:
F(2,7) = 16.1; p < 0.01) followed by t test (treated vs control:
***p < 0.001, **p < 0.01, *p < 0.05; treated in the presence of
8-Br-cAMP vs in its absence: °° p < 0.01, ° p < 0.05).
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Role of Fyn in the effects of endocannabinoids
We have shown previously that stimulation of CB1-Rs in hippocampus
activates a protein tyrosine phosphorylation pathway, involving neuronal isoforms of FAK and an associated protein, p130-Cas
(Derkinderen et al., 1996, 2001b). In cell lines, after
autophosphorylation on Tyr-397, FAK recruits Src-family tyrosine
kinases, including Src and Fyn, which in turn phosphorylate multiple
residues in the kinase domain and C-terminal region of FAK, as well as
in p130-Cas (Girault et al., 1999; Schaller, 2001). Our recent results showed that, in hippocampal slices, endocannabinoids increase the
association of Fyn, but not Src, with FAK (Derkinderen et al., 2001b).
In several cell types, recruitment of Src-family kinases by FAK has
been shown to result in the activation of ERK through various signaling
mechanisms (Girault et al., 1999; Schaller, 2001). Moreover, other
pathways involving Src-family kinases can activate ERKs downstream of
G-protein-coupled receptors, independently of FAK (Luttrell et al.,
1996). To determine the role of Src-family kinases in the effects of
endocannabinoids, we used PP2, a compound that inhibits potently the
catalytic activity of this group of kinases (Hanke et al., 1996). As
reported previously (Derkinderen et al., 1996), endocannabinoids
increased tyrosine phosphorylation of a number of proteins in
hippocampal slices (Fig. 5A).
Pretreatment of slices with PP2 dramatically decreased the basal
tyrosine phosphorylation of most proteins and abolished the effects of
endocannabinoids (Fig. 5A), demonstrating that they are
almost completely accounted for by Src-family kinases. However, despite
the massive inhibition of protein tyrosine phosphorylation by PP2, the
activation of ERK by endocannabinoids was still present (Fig.
5A).
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Figure 5.
Role of Src-family kinases in the effects of
endocannabinoids in rat hippocampal slices. A, Rat
hippocampal slices were incubated as described in Materials and
Methods, in the presence or absence of 5 µM PP2 for 45 min before the addition of vehicle (Control), 1 µM anandamide, or 2-AG for 5 min. Homogenates were
analyzed by immunoblotting with antibodies specific for
anti-phospho-tyrosine (Blot P-Tyr), active dually
phosphorylated ERK (Blot P-ERK), or total ERK
(Blot ERK). The optical densities of P-ERK2 were
as follows: in the absence of PP2: control 100 ± 43, anandamide
767 ± 231, and 2-AG 413 ± 92; in the presence of PP2:
control 89 ± 28, anandamide 1358 ± 280, and 2-AG 861 ± 191 (F(5,14) = 7.1, p < 0.001; followed by t test,
p < 0.05 for endocannabinoid-treated vs control in
both groups). B, Hippocampal slices from
wild-type (Fyn +/+) and Fyn knock-out (Fyn
/) mice were incubated for 50 min before the addition of
either vehicle (Control) or 1 µM
2-AG for 5 min. The optical densities of P-ERK2 were as follows: in Fyn
+/+ slices: control 100 ± 8, 2-AG 1198 ± 351; in Fyn /
slices: control 115 ± 23, 2-AG 108 ± 63 (F(3,8) = 9.3, p < 0.01; followed by t test, p < 0.05, 2-AG vs control in wild-type slices, and p < 0.05, 2-AG in knock-out vs control slices).
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Because the endocannabinoids specifically increase the association of
Fyn with FAK (Derkinderen et al., 2001b), we examined whether the
effects of endocannabinoids on ERK were altered in Fyn knock-out mice.
In these mice the effects of 2-AG on protein tyrosine phosphorylation
were reduced dramatically (Fig. 5B), confirming the critical
role of Fyn in tyrosine phosphorylation in hippocampus (Grant et al.,
1995) and its specific involvement in the action of cannabinoids
(Derkinderen et al., 2001b). Moreover, endocannabinoids were no longer
capable of stimulating ERK (Fig. 5B), showing that Fyn is
required for this effect. It is important to note that the levels of
ERK (Fig. 5B) and CB1-R (Derkinderen et al., 2001b) were not
altered in these mice. Altogether these results strongly suggest that
Fyn in hippocampus is necessary for the activation of ERK by CB1-Rs,
but that this requirement is independent from acute Fyn catalytic activity.
THC, a cannabinoid agonist, activates ERKs in the hippocampus
in vivo
Our results demonstrated that endocannabinoids activate the ERK
pathway in hippocampal slices. To determine whether this effect could
be also induced by cannabinoids in vivo, we used THC, the cannabinoid agonist widely abused by humans, which has potent effects
when administrated at the periphery. As demonstrated above, THC was a
powerful stimulator of ERKs in hippocampal slices (Fig. 1). We analyzed
ERK activation in hippocampus by immunocytochemistry with a
phosphorylation state-specific antibody after injection of 9-THC (1 mg/kg, i.p.). A strong P-ERK-like immunoreactivity was detected in the
pyramidal cell layers of CA1 (Fig.
6A) and CA3 (Fig.
6B) 10 min after THC injection (number of positive
cells per section, mean ± SEM: vehicle 30 ± 4 vs THC
125 ± 7; n = 4 mice; t test;
p < 0.01). This effect was transient because the number of P-ERK-positive cells decreased rapidly: 57 ± 13 at 20 min, 31 ± 8 at 30 min, and 18 ± 4 at 60 min
(n = 4 mice per time point; no significant difference
with controls). No significant effect was observed in dentate gyrus
(data not shown). Analysis at a higher magnification indicated that ERK
was activated in the soma and dendrites of many neurons, although only
some principal neurons were labeled in response to THC (Fig.
6A,B). In CA1,
P-ERK-immunoreactivity was detected in pyramidal cells, whereas in CA3,
both pyramidal and nonpyramidal cells were labeled with a dense
plexiform dendritic network extending into the stratum radiatum. The
activation of ERK in response to THC was prevented in CB1-R knock-out
mice (Fig. 6C) as well as by pretreatment with SR141716A (3 mg/kg, i.p.) (Fig. 6D), demonstrating that THC
activates ERK by acting on CB1-R. Because a dramatic alteration in the
response to stimulation of CB1-R was observed in hippocampal slices of
Fyn/ mice, we examined whether the response to THC was also
hampered in vivo in these animals. In these mutant mice, no
P-ERK immunoreactivity was detected after injection of THC (Fig.
6E), revealing that Fyn is necessary for the
activation of ERK.
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Figure 6.
THC activates ERK in hippocampus in
vivo by stimulating CB1 receptors. Mice were injected with
vehicle (Veh) or THC (1 mg/kg, i.p.) 10 min before they
were killed. Active ERK1 and ERK2 were detected by peroxidase
immunocytochemistry in the hippocampus, using antibodies against the
doubly phosphorylated protein. Results in CA1 (A)
and CA3 (B) regions are shown. The right
panel corresponds to a higher magnification of the THC-treated
sections. In THC-treated mice, immunoreactive cells are mostly present
in the pyramidal cell layer of CA1 and CA3. A strong labeling is
visible in the cytoplasm (including the dendrites) and the nucleus.
C, THC failed to activate ERK in CA1 of CB1 / mice,
whereas it was active in CB1 +/+ matched controls. D, In
the presence of the CB1-R antagonist, SR141716A (3 mg/kg) injected
alone (Veh + SR141716A), or 15 min before THC injection
(THC + SR141716A), no activation of ERK was observed in
CA1. E, THC failed to activate ERK in CA1 of Fyn /
mice, whereas its effects were present in Fyn +/+ matched controls.
F, Stimulation of ERK phosphorylation by THC was
abolished by the NMDA receptor antagonist MK801 (0.1 mg/kg), injected
15 min before THC. The results obtained in CB1 /,
SR141716A-treated, Fyn /, and MK801-treated mice in CA3 (data not
shown) were similar to those illustrated here in CA1.
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In hippocampus, most CB1-Rs are located on GABA nerve terminals (Tsou
et al., 1998, 1999; Katona et al., 1999, 2000), and their stimulation
is capable of enhancing the activation of pyramidal cells by
glutamatergic inputs through a decrease in GABA inhibitory tone
(Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001). Therefore, we
tested the contribution of glutamate neurotransmission in the effects
of THC on the ERK pathway. In mice pretreated with MK801 (0.1 mg/kg,
i.p.), a noncompetitive antagonist of NMDA receptors that crosses the
blood-brain barrier, THC did not increase P-ERK immunoreactivity in
hippocampal neurons (Fig. 6F). This result suggests
that the effects of CB1-R on ERK activation in vivo either are mediated by an increase in NMDA receptor stimulation or require the
concomitant activation of these receptors. To determine whether the
activation of the ERK pathway by cannabinoids in hippocampus was
exclusively a consequence of an increased sensitivity to excitatory inputs, we examined the contribution of glutamate in the effects of
endocannabinoids in hippocampal slices. In this preparation, MK801 had
a small stimulatory effect on ERK phosphorylation by itself but did not
prevent ERK activation by either 2-AG (Table 1) or THC (data not
shown). In contrast, the stimulatory effect of glutamate on ERK
phosphorylation was prevented in the presence of MK801 (data not
shown). These results indicate that ERK activation by cannabinoids may
be achieved by several mechanisms, one of which depends on NMDA
receptors and appears predominant in vivo.
Role of ERK in the regulation of gene expression by THC
in vivo
The strong accumulation of P-ERK in nuclei (Fig.
7A) suggested its possible
role in gene regulation via phosphorylation of transcription factors.
To examine the consequences of ERK stimulation by THC in
vivo we used SL327, a drug that crosses the blood-brain barrier
and prevents the activation of ERK by inhibiting MEK (Atkins et al.,
1998; Valjent et al., 2000). This drug, closely related to U0126, which
inhibited completely ERK phosphorylation in hippocampal slices (Fig.
3), had a dramatic effect on P-ERK immunoreactivity in hippocampal
neurons and prevented its enhancement after THC treatment (Fig.
7B). ERK phosphorylates and activates transcription factors
of the ternary complex factor family, including Elk-1, that bind to the
serum response element (SRE) (Whitmarsh and Davis, 1996; Valjent et
al., 2001a). Moreover, ERK regulates genes that contain the
cAMP/Ca2+ response element (CRE) in their
promoter, possibly by phosphorylating and activating MAPK-activated
protein kinase 2, which phosphorylates cAMP response element-binding
protein (CREB) (Impey et al., 1999). Therefore, we examined the role of
ERK in the induction of IEGs by THC, focusing our attention on c-Fos
and Zif268, which contain both SRE and CRE in their promoter, and the
expression of which is known to increase in response to cannabinoids in
neurons (Mailleux et al., 1994; Glass and Dragunow, 1995). We analyzed
c-Fos protein by immunohistochemistry, with a specific antibody that
did not recognize Fos-related antigens (Fig.
7C), and we analyzed Zif268 mRNA by in situ hybridization (Fig. 7D).
THC increased the number and immunoreactivity of c-Fos-expressing
neurons in CA1 (Fig. 7C) and in CA3 (data not shown). Zif268
mRNA levels were increased in CA1 and CA3 but not in the dentate gyrus
(Fig. 7D,F). Both effects
were prevented by injection of SL327 before THC (Fig. 7C,D,F).
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Figure 7.
ERK-dependent induction of immediate-early genes
by THC in hippocampus in vivo. A, Mice
were injected with 1 mg/kg THC 10 min before they were killed, as in
Figure 6A, High magnification of a
peroxidase-labeled section shows the nuclear staining for dually
phosphorylated ERK. B, The effects of THC (middle
panel) were prevented in the CA1 region of mice injected
with SL327 (100 mg/kg) 60 min before THC (right
panel). Results shown are representative of four to six
animals for each group. C, c-Fos immunoreactivity
(peroxidase reaction) was examined in CA1 of mice injected with either
vehicle (Veh) or 1 mg/kg THC 60 min before they were
killed. SL327 (100 mg/kg) was injected 60 min before THC.
D, Zif268 mRNA expression was analyzed by in
situ hybridization in mouse hippocampus 1 hr after injection of
vehicle, THC (1 mg/kg, i.p.), or THC and SL327 (100 mg/kg, 60 min
before THC). Note the increased hybridization signals in the CA1 and
CA3 regions. E, BDNF mRNA expression was analyzed by
in situ hybridization in mouse hippocampus 1 hr after
injection of vehicle, THC (1 mg/kg, i.p.), or THC and SL327 (100 mg/kg,
60 min before THC). F, Signals for mRNA hybridization
were quantified using an image analyzer for six animals for each
treatment. Statistical analyses used one-way ANOVA followed by a
post hoc comparison with Newman-Keuls test;
*p < 0.001 when comparing THC-treated mice with
control mice; ^ p < 0.001 when comparing SL + THC with THC alone (n = 6 mice per group).
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We also examined the effects of THC on the expression of BDNF, a growth
factor that behaves as an IEG, transcribed in response to bursts of
neuronal activity through its CREB-binding sites (Shieh et al., 1998;
Tao et al., 1998), and plays an important role in hippocampal synaptic
plasticity (Kang and Schuman, 1995; Hartmann et al., 2001; Patterson et
al., 2001; Ying et al., 2002). We found a significant upregulation of
BDNF mRNA levels in CA1 and CA3 1 hr after injection of THC (Fig.
7E,F). This increase in BDNF
mRNA was prevented by pretreatment with SL327 (Fig.
7E,F). Thus, acute injection
of THC can induce BDNF mRNA transcription through the ERK signaling pathway.
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Discussion |
Regulation of ERKs by cannabinoids in hippocampus
The present study shows that cannabinoids activate ERKs in
hippocampus and in slices incubated in vitro, as well as in
living mice. In slices, 2-AG, an endocannabinoid produced in
hippocampus in response to stimulation of Schaeffer collaterals (Stella
et al., 1997), increased ERK phosphorylation rapidly and at low
concentrations. This effect was likewise achieved with other
cannabinoids, including synthetic agonists and THC. Injection of THC in
mice was also able to induce ERK activation in the hippocampus. The
effects of THC on ERK activation were mediated by CB1-Rs because they were prevented by pretreatment with SR141716A, a specific CB1-R antagonist, and they were not observed in CB1-R knock-out mice. In the
hippocampus, the neurons that expressed the strongest P-ERK immunoreactivity after in vivo administration of THC were
pyramidal cells in CA1 and CA3. In contrast, CB1-R-immunoreactive
neurons are located in all subfields in hippocampus and are mostly
interneurons, with a large predominance of cholecystokinin-positive
cells (Pettit et al., 1998; Katona et al., 1999; Tsou et al., 1999). In
fact, CB1-Rs are highly concentrated in nerve terminals, and
endocannabinoids have recently been shown to mediate retrograde
signaling at hippocampal synapses (Ohno-Shosaku et al., 2001; Wilson
and Nicoll, 2001). Although we cannot formally rule out a contribution
of CB1-Rs in principal cells, which express low levels of CB1-R mRNA
(Marsicano and Lutz, 1999), THC is likely to act on pyramidal cells
indirectly by modulating inputs at the presynaptic level. Because
CB1-Rs are mostly located on GABA terminals, they could modulate
indirectly the sensitivity to excitatory inputs by decreasing the
inhibitory tone on pyramidal neurons (Ohno-Shosaku et al., 2001; Wilson
and Nicoll, 2001). Stimulation of NMDA glutamate receptors is capable of activating the ERK pathway in vitro and in
vivo (Valjent et al., 2001a). Interestingly, however, the
contribution of NMDA glutamate receptors in the effects of THC appeared
different in vitro and in vivo. In living mice,
MK801, an NMDA receptor antagonist, prevented the effects of THC on the
ERK pathway, suggesting that THC effects on ERK in vivo
either were mediated by an increased stimulation or sensitivity of NMDA
receptors or required the concomitant activity of these receptors.
Signaling pathways involved in ERK regulation by CB1-Rs
Although the effects of in vivo CB1-R stimulation were
sensitive to a NMDA receptor inhibitor, a strong stimulation of ERK phosphorylation was observed in hippocampal slices in the presence of
TTX, which prevents neuronal firing. This effect was also resistant to
MK801, arguing in favor of a direct stimulation of the ERK pathway by
CB1-Rs in this preparation. It should be pointed out that the precise
cellular localization of the activation of ERK by CB1-Rs in hippocampal
slices is not known. Nevertheless, the mechanism by which stimulation
of CB1-Rs led to ERK activation in hippocampal slices appears to be
different from what has been reported in transfected cells. Studies in
CHO cells supported a role for  rather than for
i subunits in CB1-R-ERK coupling (Bouaboula
et al., 1995a,b). Activation of ERKs in cell lines by
Gi-coupled receptors can be mediated through
recruitment of PI3-kinase by subunits, independently of the
inhibition of adenylyl cyclase (Hawes et al., 1996; Lopez-Ilasaca
et al., 1997). However, in hippocampal slices, the cannabinoid-induced
activation of ERKs was insensitive to the PI3-kinase inhibitor
LY294002, strongly arguing against a role of the
subunits/PI3-kinase pathway in this effect. Other pathways linking
G-protein-coupled receptors to MAP kinases involve tyrosine kinases of
the Src family (Luttrell et al., 1997). For example, PP2, a potent
inhibitor of these kinases, blocked the effects of various
extracellular messengers on the activation of ERK in CHO cells (Igishi
and Gutkind, 1998). We have shown previously that stimulation of CB1-Rs
increases the tyrosine phosphorylation of the neuronal isoform of FAK
in hippocampus and its association with Fyn (Derkinderen et al., 1996,
2001b). Thus, FAK provides a possible link for the activation of ERK
through recruitment of Src-family kinases or PI3-kinase (Girault et
al., 1999; Schaller, 2001). However, the activation of ERK by CB1-Rs in
hippocampal slices cannot be accounted for by these known pathways
linking FAK to ERK, because it was resistant to inhibitors of
PI3-kinase and Src-family kinases. It was particularly striking that
PP2 did not prevent ERK activation in hippocampal slices, despite its
dramatic effect on protein tyrosine phosphorylation. Yet, the
Src-family kinase Fyn appeared to be critical for the activation of ERK
by CB1-Rs because this activation was absent in Fyn mutant mice, both
in vitro and in vivo. Although we verified that
CB1-R and ERK levels were unaltered in Fyn/ mice, we cannot rule
out the possibility that the absence of Fyn throughout development impaired indirectly the signaling pathways at other levels. An alternative explanation of our findings is that stimulation of CB1-Rs
leads to the recruitment of Fyn and to the activation of ERK by a
mechanism independent of Fyn catalytic activity. In support of this
hypothesis, the closely related tyrosine kinase Src is known to exert
some of its biological effects independently of its tyrosine kinase
activity (Kaplan et al., 1995; Schwartzberg et al., 1997).
The role of cAMP in ERK regulation in hippocampus
Several additional pathways have been described that link
G-protein-coupled receptors to ERK activation and could account for the
effects of endocannabinoids (Marinissen and Gutkind, 2001). It is well
established that CB1-Rs inhibit adenylyl cyclase, including in
hippocampus (Bidaut-Russell et al., 1990), and our observations reveal
that ERK activation by cannabinoids was closely associated with cAMP
regulation. The effects of endocannabinoids on ERK were prevented by
pretreatment of slices with cAMP analogs, suggesting that their
mechanism of action may involve an inhibition of cAMP production.
Similar effects of cAMP on the activation of ERK induced by anandamide
have been reported in a human breast cancer cell line (Melck et al.,
1999). The role of cAMP in the effects of CB1-Rs in hippocampus was
further supported by the fact that two different inhibitors of PKA,
acting on the regulatory or the catalytic subunit of PKA, also
activated ERK in hippocampal slices. These effects of cAMP are
reminiscent of those reported previously for FAK (Derkinderen et al.,
1996, 2001b). Interestingly, a negative effect of cAMP on both
FAK- and ERK-dependent responses has also been observed in a
completely different context, when adherent NIH3T3 cells were placed in
suspension (Howe and Juliano, 2000). These effects of cAMP might appear
paradoxical in light of the known stimulation of ERK by cAMP in
hippocampal slices (Impey et al., 1998, 1999). Indeed, we confirmed in
our experimental conditions that 8-Br-cAMP as well as forskolin were
also capable of activating ERK, in agreement with these previous
reports. These stimulatory effects lasted for a short period of time
and were followed by a decrease in ERK phosphorylation and its
insensitivity to endocannabinoids. The effects of cAMP on ERK in
hippocampus are complex, similar to what has been reported in other
cell types (Stork and Schmitt, 2002). cAMP appears to exert two
opposing effects on the ERK pathway in hippocampus, a stimulatory
effect that may be partly independent of PKA and, as indicated by our findings, an inhibitory effect mediated by PKA. Thus, in hippocampal slices, stimulation of CB1-Rs could be a critical modulator of cAMP
signaling, by opposing some of its effects and enhancing some others.
Further work will have to determine whether these apparently opposing
effects take place at different cellular or subcellular locations.
Functional implications of the regulation of ERK and
immediate-early genes by cannabinoids in hippocampus
Our findings have two types of implications concerning the
possible physiological function of the endocannabinoid system in hippocampus and the effects of THC in the context of drug abuse. Recent work from several laboratories has unveiled the important role
of the endocannabinoid system in hippocampus. Although massive stimulation of CB1-Rs inhibits several forms of synaptic plasticity in
hippocampus, in vivo and in vitro, including LTP
and LTD in CA1 neurons (Collins et al., 1995; Terranova et al., 1995;
Misner and Sullivan, 1999), it appears that focal stimulation of these receptors has more subtle effects. By decreasing locally the GABAergic inhibition of hippocampal neurons (Ohno-Shosaku et al., 2001; Wilson
and Nicoll, 2001), endocannabinoids facilitate the induction of LTP
(Carlson et al., 2002). In addition to these direct effects on synaptic
transmission, our results show that stimulation of CB1-Rs has potent
effects on signaling pathways that are known to be critical for
long-term synaptic plasticity. Stimulation of CB1-Rs activates ERK, a
protein kinase known to be critical in several forms of synaptic
plasticity and in learning and memory (English and Sweatt, 1997; Atkins
et al., 1998). The ERK pathway appears essential for some forms of LTP
in hippocampus (Winder et al., 1999) and for the regulation of gene
expression in the context of synaptic plasticity (Impey et al., 1999).
Remarkably, the activation of ERK by THC was absent in the hippocampus
of mice lacking the tyrosine kinase Fyn, which display altered synaptic plasticity (Grant et al., 1992).
CB1-R-mediated stimulation of ERK was responsible for the induction of
Zif268, a transcription factor essential for transition from short- to
long-term synaptic plasticity and for the expression of long-term
memories (Jones et al., 2001). Moreover, we demonstrate that THC also
induced the expression of BDNF, a neurotrophic factor that increases
synaptic efficiency (Kang and Schuman, 1995; Patterson et al., 2001;
Ying et al., 2002). Thus, stimulation of CB1-Rs is by itself capable of
activating signaling cascades known to be important for synaptic
plasticity. Localized increases in endocannabinoid production could
facilitate local synaptic efficiency not only by inhibiting presynaptic
release of GABA but also by activating signaling pathways important for
long-term modifications.
In contrast to this possible physiological role of the local activation
of CB1-Rs, their general pharmacological stimulation in the whole
tissue clearly has a negative effect on plasticity (Collins et al.,
1995; Terranova et al., 1995; Misner and Sullivan, 1999; Bohme et al.,
2000). The effects of THC on ERK phosphorylation in mice, reported
here, were observed at doses (1 mg/kg) that are low for experimental
studies in animals and may correspond to the amounts of THC absorbed by
human subjects during heavy intoxication (Barnett et al., 1985). This
dose of THC has minimal aversive effects in mice, and, given after a
priming injection in the home cage, is capable of inducing conditioned
place preference (Valjent and Maldonado, 2000). It should be pointed
out that general administration of THC also stimulates the ERK pathway
in other brain regions, including the dorsal striatum and nucleus
accumbens (Valjent et al., 2001b). In these brain regions, however, the activation of ERK was entirely dependent on activation of dopamine D1
receptors, whereas in hippocampus the effects of THC were still observed in the presence of SCH23390, a potent blocker of D1 receptors (our unpublished observations). Thus, the present study demonstrates that THC can activate ERK in brain by several pathways, and in regions
beyond those usually thought to be the major targets of drugs of abuse.
Therefore it will be important to determine whether effects of THC on
ERK in hippocampus and their consequences on gene expression may
participate in the cognitive and memory impairment reported in heavy
cannabis users (Pope et al., 2001; Solowij et al., 2002).
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FOOTNOTES |
Received Aug. 29, 2002; revised Dec. 18, 2002; accepted Dec. 24, 2002.
*
P.D. and E.V. contributed equally to this work.
P.D. was supported by a Poste d'accueil Institut National de la
Santé et de la Recherche Médicale. This work was supported in part by grants from Mission Interministerielle de Lutte contre les
Drogues et la Toxicomanie, Human Frontier Science Programme Organization, Fondation pour la Recherche Médicale, Fondation Schlumberger pour l'Enseignement et la Recherche, and Action
Concertée Incitative (Biologie du Développement et
Physiologie Intégrative). Prof. Seth Grant (University of
Edinburgh) is gratefully acknowledged for providing some of the Fyn
knock-out mice used in this study.
Correspondence should be addressed to Dr. Jean-Antoine Girault,
Institut National de la Santé et de la Recherche Médicale U536, Institut du Fer à Moulin, 17 rue du Fer à Moulin,
75005 Paris, France. E-mail:
girault{at}ifm.inserm.fr.
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References |
-
Alessi DR,
Cuenda A,
Cohen P,
Dudley DT,
Saltiel AR
(1995)
PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo.
J Biol Chem
270:27489-27494[Abstract/Free Full Text].
-
Al-Hayani A,
Wease KN,
Ross RA,
Pertwee RG,
Davies SN
(2001)
The endogenous cannabinoid anandamide activates vanilloid receptors in the rat hippocampal slice.
Neuropharmacology
41:1000-1005[ISI][Medline].
-
Atkins CM,
Selcher JC,
Petraitis JJ,
Trzaskos JM,
Sweatt JD
(1998)
The MAPK cascade is required for mammalian associative learning.
Nat Neurosci
1:602-609[ISI][Medline].
-
Barnett G,
Licko V,
Thompson T
(1985)
Behavioral pharmacokinetics of marijuana.
Psychopharmacology
85:51-56[Medline].
-
Bidaut-Russell M,
Devane WA,
Howlett AC
(1990)
Cannabinoid receptors and modulation of cyclic AMP accumulation in the rat brain.
J Neurochem
55:21-26[ISI][Medline].
-
Bohme GA,
Laville M,
Ledent C,
Parmentier M,
Imperato A
(2000)
Enhanced long-term potentiation in mice lacking cannabinoid CB1 receptors.
Neuroscience
95:5-7[ISI][Medline].
-
Bouaboula M,
Bourrié B,
Rinaldi-Carmona M,
Shire D,
Le Fur G,
Casellas P
(1995a)
Stimulation of cannabinoid receptor CB1 induces krox-24 expression in human astrocytoma cells.
J Biol Chem
270:13973-13980[Abstract/Free Full Text].
-
Bouaboula M,
Poinot-Chazel C,
Bourrié B,
Canat X,
Calandra B,
Rinaldi-Carmona M,
Le Fur G,
Casellas P
(1995b)
Activation of mitogen-activated protein kinases by stimulation of the central cannabinoid receptor CB1.
Biochem J
312:637-641.
-
Bouaboula M,
Perrachon S,
Milligan L,
Canat X,
Rinaldi-Carmona M,
Portier M,
Barth F,
Calandra B,
Pecceu F,
Lupker J,
Maffrand JP,
Le Fur G,
Casellas P
(1997)
A selective inverse agonist for central cannabinoid receptor inhibits mitogen-activated protein kinase activation stimulated by insulin or insulin-like growth factor 1: evidence for a new model of receptor/ligand interactions.
J Biol Chem
272:22330-22339[Abstract/Free Full Text].
-
Burgaya F,
Toutant M,
Studler JM,
Costa A,
Le Bert M,
Gelman M,
Girault JA
(1997)
Alternatively spliced focal adhesion kinase in rat brain with increased autophosphorylation activity.
J Biol Chem
272:28720-28725[Abstract/Free Full Text].
-
Carlson G,
Wang Y,
Alger BE
(2002)
Endocannabinoids facilitate the induction of LTP in the hippocampus.
Nat Neurosci
5:723-724[ISI][Medline].
-
Collins DR,
Pertwee RG,
Davies SN
(1995)
Prevention by the cannabinoid antagonist, SR141716A, of cannabinoid-mediated blockade of long-term potentiation in the rat hippocampal slice.
Br J Pharmacol
115:869-870[ISI][Medline].
-
Derkinderen P,
Toutant M,
Burgaya F,
Le Bert M,
Siciliano JC,
De Franciscis V,
Gelman M,
Girault JA
(1996)
Regulation of a neuronal form of focal adhesion kinase by anandamide.
Science
273:1719-1722[Abstract/Free Full Text].
-
Derkinderen P,
Enslen H,
Girault JA
(1999)
The ERK/MAP-kinase cascade in the nervous system.
NeuroReport
10:R24-34[ISI][Medline].
-
Derkinderen P,
Ledent C,
Parmentier M,
Girault JA
(2001a)
Cannabinoids activate p38 mitogen-activated protein kinases through CB1 receptors in hippocampus.
J Neurochem
77:957-960[ISI][Medline].
-
Derkinderen P,
Toutant M,
Kadaré G,
Ledent C,
Parmentier M,
Girault JA
(2001b)
Dual role of Fyn in the regulation of FAK+6,7 by cannabinoids in hippocampus.
J Biol Chem
276:38289-38296[Abstract/Free Full Text].
-
Di Marzo V,
Melck D,
Bisogno T,
De Petrocellis L
(1998)
Endocannabinoids: endogenous cannabinoid receptor ligands with neuromodulatory action.
Trends Neurosci
21:521-528[ISI][Medline].
-
Di Marzo V,
Breivogel CS,
Tao Q,
Bridgen DT,
Razdan RK,
Zimmer AM,
Zimmer A,
Martin BR
(2000)
Levels, metabolism, and pharmacological activity of anandamide in CB(1) cannabinoid receptor knockout mice: evidence for non-CB(1), non-CB(2) receptor-mediated actions of anandamide in mouse brain.
J Neurochem
75:2434-2444[ISI][Medline].
-
English JD,
Sweatt JD
(1997)
A requirement for the mitogen-activated protein kinase cascade in hippocampal long term potentiation.
J Biol Chem
272:19103-19106[Abstract/Free Full Text].
-
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ABSTRACT
Introduction
