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The Journal of Neuroscience, May 1, 2003, 23(9):3916
Increased Seizure Susceptibility and Proconvulsant Activity of
Anandamide in Mice Lacking Fatty Acid Amide Hydrolase
Angela B.
Clement1,
E.
Gregory
Hawkins1,
Aron H.
Lichtman2, and
Benjamin F.
Cravatt1
1 The Skaggs Institute for Chemical Biology and
Departments of Cell Biology and Chemistry, The Scripps Research
Institute, La Jolla, California 92037, and 2 Department of
Pharmacology and Toxicology, Medical College of Virginia Campus,
Virginia Commonwealth University, Richmond, Virginia 23298
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ABSTRACT |
A number of recent in vitro studies have described a
role for endogenous cannabinoids ("endocannabinoids") as
transsynaptic modulators of neuronal activity in the hippocampus and
other brain regions. However, the impact that endocannabinoid signals
may have on activity-dependent neural events in vivo
remains mostly unknown and technically challenging to address because
of the short half-life of these chemical messengers in the
brain. Mice lacking the enzyme fatty acid amide hydrolase [FAAH
( /) mice] are severely impaired in their ability to degrade the
endocannabinoid anandamide and therefore represent a unique animal
model in which to examine the function of this signaling lipid
in vivo. Here, we show that the administration of
anandamide dramatically augments the severity of chemically induced
seizures in FAAH (/) mice but not in wild-type mice.
Anandamide-enhanced seizures in FAAH (/) mice resulted in
significant neuronal damage in the CA1 and CA3 regions of the
hippocampus for the bicuculline and kainate models, respectively.
Notably, in the absence of anandamide treatment, FAAH (/) mice
exhibited enhanced seizure responses to high doses of kainate that
correlated with greatly elevated endogenous levels of anandamide in the
hippocampus of these animals. Collectively, these studies suggest that
both exogenously administered and endogenously produced anandamide
display FAAH-regulated proconvulsant activity and do not support a
general neuroprotective role for this endocannabinoid in response to
excitotoxic stimuli in vivo. More generally, these findings demonstrate that the disinhibitory actions of endocannabinoids observed in hippocampal slices in vitro may also occur
in vivo.
Key words:
anandamide; bicuculline; CB1 receptor; endocannabinoid; epilepsy; excitotoxicity; fatty acid amide hydrolase; kainate; seizure
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Introduction |
The primary psychoactive ingredient
of Cannabis sativa,
 9-tetrahydrocannabinol (THC), produces
many neuropharmacological effects in mammals primarily by activating a
single G-protein-coupled receptor CB1
(Ledent et al., 1999 ; Zimmer et al., 1999). The broad distribution of
the CB1 receptor in the CNS, coupled with
the identification of two natural CB1 ligands,
N-arachidonoyl ethanolamine (anandamide) (Devane et al.,
1992) and 2-arachidonoyl glycerol (2-AG) (Mechoulam et al., 1995;
Sugiura et al., 1995), has spurred considerable interest in elucidating
the function of the endogenous cannabinoid ("endocannabinoid")
system. Studies using CB1 agonists and
antagonists, in combination with CB1 (/)
mice, have indicated a role for this receptor in modulating several
behavioral processes, including pain (Zimmer et al., 1999), feeding (Di
Marzo et al., 2001), memory (Varvel and Lichtman, 2002), and locomotor
activity (Zimmer et al., 1999).
Many of the behavioral effects of cannabinoids could reflect an
inhibitory influence of these agents on neuronal firing in the CNS, as
has been observed in the cerebellum (Kreitzer and Regehr, 2001a) and
nucleus accumbens (Pistis et al., 2002). In the hippocampus, however,
slice electrophysiological studies have provided evidence that
endocannabinoids mediate a distinct form of transsynaptic communication
referred to as "depolarization-induced suppression of inhibition"
(Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001). In this form of
retrograde transmission, endocannabinoids disinhibit pyramidal neuron
activity by suppressing neurotransmitter release from GABAergic
interneurons (Katona et al., 1999), thus providing an example in which
the net effect of cannabinoids on neuronal firing is predicted to be
stimulatory in vivo (Wilson and Nicoll, 2002). However, the
limited number of in vivo studies conducted to date have not
described a disinhibitory function for cannabinoids in the hippocampus.
For example, THC was found to suppress the firing rates of hippocampal
neurons in response to sensory stimuli during a learning test (Hampson
and Deadwyler, 2000). These findings suggest that exocannabinoids and,
by extension, endocannabinoids may produce rather complex and even
contradictory effects on neuronal activity in the hippocampus, possibly
depending on the distribution of the CB1 receptor
in the circuits under examination. Accordingly, how cannabinoids impact
pathological events related to excess hippocampal excitation, such as
seizure and neurotoxicity, remains perplexingly difficult to predict. Indeed, although exocannabinoids are generally considered to be anticonvulsant (Adams and Martin, 1996), THC induces seizures in
certain strains of rabbits (Martin and Consroe, 1976).
A major technical challenge facing the study of endocannabinoids
in vivo is their rapid rate of catabolism. For example, the half-life of anandamide in the rodent brain is less than a few minutes
(Willoughby et al., 1997), primarily attributable to hydrolysis by the
membrane-associated enzyme fatty acid amide hydrolase (FAAH) (Cravatt
et al., 1996). FAAH is broadly expressed throughout the CNS (Thomas et
al., 1997), exhibiting a complementary cellular distribution to
CB1 receptors (Egertova et al., 1998; Tsou et al., 1998). Here, we took advantage of a mouse model in which FAAH has
been genetically deleted (Cravatt et al., 2001) to examine the role
that anandamide plays in regulating limbic seizures and hippocampal
excitotoxicity in vivo.
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Materials and Methods |
Animals. FAAH (+/+) and FAAH (/) mice were
generated by intercrossing 129vJ-C57BL/6 FAAH (+/) mice as described
previously (Cravatt et al., 2001). Studies were performed on littermate
FAAH (+/+) and FAAH (/) mice that had been backcrossed into the
C57BL/6 strain for at least five generations to limit potential
strain-dependent allelic variations that might contribute to behavioral
and physiological differences. CB(+/+) and (/) mice were bred from
CB(+/) mice backcrossed onto the C75BL/6 strain as described
previously (Zimmer et al., 1999). All experiments were performed with
mice from both genders, and no significant differences between female
and male mice were observed.
Pharmacological agents. Anandamide, N-oleoyl
ethanolamine (OEA), and N-palmitoyl ethanolamine (PEA),
synthesized as described previously (Cravatt et al., 1996),
(R)-(+)-WIN 55,212-2 (Sigma, St. Louis,
MO), (S)-()-WIN 55,212-3
(Sigma), and SR141716A (kindly provided by National
Institute on Drug Abuse) were dissolved in ethanol and diluted in a
final ratio of 1:1:18 in ethanol/Emulphor/saline. Kainate (Ocean
Produce International, Shelburne, Canada) was dissolved in saline.
Bicuculline (Sigma) was dissolved in a drop of 0.1 M warm HCl and further diluted in saline. Control
experiments were performed by injection of the corresponding vehicle
solutions. All drugs were administered by intraperitoneal injection.
Behavioral analysis. Animals were transferred into
individual cages the day before the experiments to allow them to
acclimate to the new environment. Animals were observed in these cages
for 3 hr after drug treatment. Seizures were scored according to a modified Racine scoring system from 0-6: 0, no motor seizures; 1, freezing, staring, mouth or facial movements; 2, head nodding or
isolated twitches, rigid posture; 3, tail extension,
unilateral-bilateral forelimb clonus; 4, rearing, in which the mice
sit in an immobile state on their rear haunches with one or both
forepaws extended; 5, clonic seizures with loss of posture, jumping,
and falling; 6, tonic seizure with hindlimb extension resulting in
death (Racine, 1972). Saline- or vehicle-injected animals did not show
any seizure behavior. All data are presented as means ± SEM.
Statistical significance for seizure scores was determined with the
Mann-Whitney U test unless otherwise indicated. For
comparison of mortality, Fisher's exact test was used.
Histochemistry. Brains were removed 3 d after drug
treatment and paraffin embedded. Serial 5-µm-thick sections were
stained with hematoxylin-eosin. Neuronal injury was evident as the
appearance of eosinophilic neurons with pyknotic nuclei (Liu et al.,
1999). Neuronal damage in the hippocampus was assessed in five randomly chosen sections from each animal.
FAAH enzyme activity assays. FAAH activity assays were
performed by measuring the conversion of 100 µM
14C-labeled OEA to oleic acid as described
previously (Cravatt et al., 2001). Data were expressed as picomoles of
oleic acid per minute per milligram of protein ± SEM.
Statistical significance was determined with the unpaired t test.
Determination of N-acyl ethanolamine levels.
N-acyl ethanolamine (NAE) levels in the hippocampus, cortex,
and cerebellum of FAAH (+/+) and FAAH (/) inbred mice were
quantified by isotope dilution liquid chromatography mass spectrometry
(LC-MS) as described previously (Di Marzo et al., 2000; Cravatt et al.,
2001). Briefly, mice were anesthetized by using
CO2-O2 and killed by
decapitation. Brain regions were removed within 1 min and immediately
homogenized in a 2:1:1 mixture of chloroform/methanol/50
mM Tris, pH 8.0, containing
d4-anandamide and d4-OEA as
standards (2.5 and 50 pmol per brain region, respectively). The organic
layer was removed and dried under N2 gas. The
remaining residue was solubilized in methanol and injected onto an
Agilent (Palo Alto, CA) 1100 series LC-MS. Levels of
endogenous anandamide and OEA were quantified by comparing the mass ion
peak heights with those of the corresponding d4
standards. PEA levels were quantified by comparing the peak height of
PEA with that of d4-OEA [a
d4-PEA internal standard could not be used
because of overlapping retention times and masses with endogenous OEA
(the mass of OEA equals the mass of the sodium adduct of
d4-PEA)]. NAE levels were expressed as picomoles
per gram of wet tissue ± SEM. Statistical significance was
determined with the unpaired t test.
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Results |
Anandamide exhibits proconvulsant activity in limbic seizure models
in FAAH (/) mice
The rapid rate at which anandamide is degraded in the brain
(Willoughby et al., 1997) has hindered experimental efforts to study
the function of this endocannabinoid in vivo. We recently described the generation and initial characterization of a mouse model
bearing a targeted disruption of the FAAH gene (FAAH (/) mice)
(Cravatt et al., 2001). FAAH (/) mice are severely impaired in
their ability to degrade anandamide and show exaggerated
CB1-dependent behavioral responses to this fatty
acid amide. Additionally, brain levels of anandamide are increased
>10-fold in FAAH (/) mice, a neurochemical phenotype that
correlates with enhanced CB1-dependent analgesia
in these animals. Interestingly, more recent studies have shown that
the hydrolytic rates of monoacylglycerols are unaltered in FAAH (/)
mice (Lichtman et al., 2002), indicating that these animals may provide
a means to discriminate the functions of amide (e.g., anandamide) and
ester (e.g., 2-AG) classes of endocannabinoids. Collectively, these
initial studies demonstrated that FAAH (/) mice represent a unique
animal model in which to examine the activity of both exogenously
applied and endogenously produced anandamide.
The effect of anandamide on chemically induced limbic seizures was
compared in FAAH (+/+) and (/) mice. As reported previously (Cravatt et al., 2001), FAAH (/) mice treated with anandamide at
doses ranging from 12.5 to 50 mg/kg (intraperitoneally) quickly entered
into an immobile state that was periodically interrupted during the
first 15 min after treatment by brief fits of spastic motion. After 15 min, FAAH (/) mice adopted a flattened, cataleptic state and
remained motionless for up to 3-4 hr unless startled by touch or
sound. In contrast, FAAH (+/+) mice showed no overt behavioral
responses to anandamide other than a modest degree of noncataleptic
hypomotility that dissipated within 20 min after injection. One hour
after treatment with anandamide, at a time when the effects of this
endocannabinoid were maximal in FAAH (/) mice (Cravatt et al.,
2001), both FAAH (+/+) and (/) mice were administered either
bicuculline (4 mg/kg, i.p.) or kainate (15 mg/kg, i.p.), and their
seizure responses were recorded using a standard scoring method (see
Materials and Methods). Compared with vehicle controls,
anandamide-treated FAAH (/) mice exhibited dramatically enhanced
seizures after treatment with either bicuculline (Fig.
1A) or kainate (Fig.
1C). In contrast, anandamide did not significantly impact
the seizure sensitivity of FAAH (+/+) animals. Anandamide was not found
to display anticonvulsant activity at lower doses (6.25 mg/kg, i.p.) in
either genotype, even when administered before higher doses of
bicuculline (6 mg/kg, i.p.) (Fig. 1B) or kainate (25 mg/kg, i.p.) (Fig. 1D), suggesting that this
endocannabinoid does not exhibit a dose-dependent paradoxical effect on
seizure threshold, as has been observed previously for opioid agonists such as morphine (Lauretti et al., 1994).
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Figure 1.
The effects of anandamide on bicuculline- and
kainate-induced seizures in FAAH (+/+) and (/) mice. Treatment with
anandamide (6.25-50 mg/kg, i.p.) 1 hr before administration of either
bicuculline (A; 4 mg/kg, i.p.) or kainate
(C; 15 mg/kg, i.p.) significantly enhanced the severity
of seizures in FAAH (/) mice (filled bars) but not in FAAH (+/+)
mice (open bars); n = 4 mice per group for 6.25 and
12.5 mg/kg anandamide, and n = 6-14 mice per group
for 25 and 50 mg/kg anandamide. Pretreatment with a low dose of
anandamide (6.25 mg/kg, i.p.) did not affect the seizure responses to
high doses of bicuculline (B; 6 mg/kg) or kainate
(D; 25 mg/kg) in FAAH (+/+) or (/) mice;
n = 6-14 mice per group. For A-D,
seizure scores are presented as mean ± SEM. All data were
compared with the Mann-Whitney U test.
 p < 0.05 for FAAH (+/+) versus FAAH (/)
mice receiving the same treatment. *p < 0.05 and
***p < 0.001 for FAAH (/) mice under different
treatment conditions.
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In the bicuculline model, the proconvulsant activity of anandamide (25 mg/kg, i.p.) in FAAH (/) mice was blocked by pretreatment with the
CB1 antagonist SR141716A (1 mg/kg, i.p.) (Fig.
2A), indicating that
CB1 receptors may mediate the effects of
anandamide in these animals. Consistent with a proconvulsant role for
CB1 receptors in the bicuculline model, the
CB1 agonist (R)-(+)-WIN
55,212-2, but not its inactive enantiomer
(S)-()-WIN 55,212-3, was found to enhance the
severity of bicuculline-induced seizures in both FAAH (+/+) and (/)
mice (Fig. 2B). Although the administration of
SR141716A alone did not affect bicuculline-induced seizures in either
FAAH (+/+) or (/) mice (Fig. 2A), pretreatment
with this CB1 antagonist did significantly
increase the severity of kainate-induced seizures in both FAAH (+/+)
and (/) mice (Fig. 2C). This proconvulsant activity of
SR141716A was not dependent on CB1 receptors,
because the agent also augmented kainate-induced seizures to an
equivalent magnitude in CB1 (/) mice (Fig.
2C). A two-way ANOVA supported this observation, as
reflected by a significant increase in seizure magnitude by SR141716A
compared with vehicle (F(1,76) = 30;
p < 0.001) and the effect of SR141716A being
independent of genotype. Collectively, these findings suggest that
SR141716A augments kainate-induced seizures through a
non-CB1 receptor mechanism of action. On this
note, recent studies of arterial relaxation in
CB1 (/) mice have also uncovered a
CB1-independent activity for SR141716A (Bukoski
et al., 2002), indicating that this compound may not be completely
selective for the CB1 receptor in
vivo. Regardless, the proconvulsant effects of SR141716A precluded its further use in the kainate-induced seizure model.
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Figure 2.
The effects of CB1 receptor agonists
and antagonists on chemically induced seizures in FAAH (+/+) and
(/) mice. A, FAAH (+/+) and (/) mice were
treated with the CB1 receptor antagonist SR141716A (1 mg/kg, i.p.) or vehicle 10 min before administration of anandamide (25 mg/kg, i.p.) or vehicle. Seizures were induced with (bicuculline, 4 mg/kg, i.p.) 60 min after anandamide injection. SR141716A blocked the
effects of anandamide on seizures in FAAH (/) mice but did not
affect seizure responses in vehicle-treated animals of either genotype
(SR group). n = 6-12 mice per group.
p < 0.05 for FAAH (+/+) versus FAAH (/)
mice receiving the same treatment. **p < 0.01 and
***p < 0.001 for FAAH (/) mice under different
treatment conditions (Mann-Whitney U test).
B, Treatment with the CB1 agonist
(R)-(+)-WIN 55,212-2, but not its inactive
enantiomer (S)-()-WIN 55,212-3 (15 mg/kg,
i.p.), 1 hr before administration of bicuculline (4 mg/kg)
significantly enhanced the severity of seizures in both FAAH (+/+) and
(/) mice. n = 6-12 mice per group.
*p < 0.05 and ***p < 0.001 for WIN 55,212-2-treated versus WIN 55,212-3-treated and
vehicle-treated mice of the same genotype, respectively (Mann-Whitney
U test). C, SR141716A augmented
kainate-induced seizures in wild-type (WT), FAAH (/), and
CB1 (/) mice. Subjects from each genotype were treated
with either vehicle or SR141716A (1 mg/kg, i.p.) 70 min before kainic
acid (15 mg/kg, i.p.). Data are depicted as means ± SEM
(n = 8-14 FAAH (/) and CB1 (/)
mice; 20-22 wild-type mice). ***p < 0.001 for
SR141716A-treated versus vehicle-treated animals (ANOVA).
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Unable to assess the CB1 dependence of the
effects of anandamide on kainate-induced seizures with
SR141716A, we sought indirect evidence for the involvement of
CB1 receptors by testing the activity of two
structurally related, non-CB1-binding NAEs: OEA
and PEA. Neither OEA nor PEA (50 mg/kg, i.p.) affected kainate-induced seizures in FAAH (+/+) or (/) mice (data not shown). The structural specificity of the proconvulsant activity of anandamide is consistent with this NAE acting at CB1 receptors as opposed
to other potential sites of action in the CNS such as gap junctions
(Guan et al., 1997), which exhibit less discrimination among endogenous
NAEs. Collectively, these results demonstrate that anandamide exhibits proconvulsant activity in two models of limbic seizures. These effects
are tightly regulated by FAAH and appear to be mediated by
CB1 receptors.
The proconvulsant effects of anandamide result in hippocampal
neuronal death in FAAH (/) mice
Anandamide and its precursor N-arachidonoyl
phosphatidylethanolamine are produced in a calcium-dependent manner in
response to brain injury and/or intense neuronal excitation (Hansen et al., 2001a,b), suggesting that these compounds may serve a
neuroprotective function in vivo (Hansen et al., 1998). In
support of this premise, CB1 agonists reduce
excitotoxic cell death in hippocampal neuronal cultures (Shen and
Thayer, 1998), apparently through the presynaptic inhibition of
glutamate release. Additionally, both anandamide and 2-AG reduce
ischemic damage in cortical neurons cultured under hypoxic conditions
(Sinor et al., 2000). Most recently, anandamide was found to reduce
neuronal damage in vivo in response to oubain-induced excitotoxicity (van der Stelt et al., 2001). Nonetheless, attempts to
block the neuroprotective effects of anandamide with
CB1 antagonists have produced mixed results
(Sinor et al., 2000; van der Stelt et al., 2001), suggesting that at
least a portion of the neuroprotection afforded by this endocannabinoid
may be attributable to the action of its metabolite, arachidonic acid,
which has also been shown to inhibit excitotoxicity (Lauritzen et al.,
2000).
If anandamide exhibited a neuroprotective effect in vivo in
response to excitotoxic stimuli, then one might anticipate that the
proconvulsant effects of this NAE in FAAH (/) mice would result in
minimal hippocampal neuronal damage. However, in FAAH (/) mice,
anandamide-dependent seizures induced by bicuculline and kainate
resulted in substantial hippocampal neuronal death in the CA1 and CA3
regions, respectively (Fig. 3). In the
bicuculline model, the neurotoxic effects of anandamide were blocked by
pretreatment with CB1 antagonist SR141716A (1 mg/kg, i.p.) (Fig. 3A). A more detailed examination did not
uncover a qualitative neuroprotective effect for anandamide, because
FAAH (/) mice that displayed similar seizure scores after treatment
with either a high dose of kainate (25 mg/kg, i.p.) or a low dose of
kainate (15 mg/kg) plus anandamide (50 mg/kg) showed similar degrees of
hippocampal damage (Fig. 3B). Collectively, these results do
not support a role for anandamide as an endogenous substance that
protects against excitotoxic neuronal death in the hippocampus.
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Figure 3.
The effects of anandamide on hippocampal
neurotoxicity associated with bicuculline- and kainate-induced seizures
in FAAH (/) mice. Brains from FAAH (/) mice
(n = 5-7 for each treatment group) were removed
3 d after drug treatment, and hematoxylin-eosin-stained slices
through the hippocampal region were examined for neuronal cell death.
Subthreshold doses of bicuculline (4 mg/kg) and kainate (15 mg/kg)
treatment that did not result in clonic-tonic seizures were not found
to induce neuronal cell death in the hippocampal region
(A, B, left panels). Administration of
anandamide (50 mg/kg, i.p.) 60 min before treatment with bicuculline or
kainate resulted in severe seizures (Fig. 1) that were
accompanied by prominent hippocampal neuronal death. In the
bicuculline-treated group, five of the seven surviving animals
exhibiting seizure scores of 4-5 displayed pyramidal cell death mainly
in the CA1 region of the hippocampus (A, middle panels).
Arrows point to examples of injured neurons, which appear as dark,
eosinophilic cells with shrunken nuclei. Administration of SR147161A
(SR) (3 mg/kg, i.p.) 10 min before treatment with anandamide blocked
the neurotoxic (A, right panels) effects of this
endocannabinoid. B, In the kainate model, three of the
five surviving FAAH (/) mice pretreated with anandamide that
exhibited seizure scores of 4-5 showed substantial neuronal damage
mainly in the CA3 region (B, middle panels). A similar
degree of neuronal damage was observed in a separate group of FAAH
(/) mice that exhibited strong seizure responses (scores of 4-5)
to a high dose of kainate (25 mg/kg, i.p.) (B, right
panels).
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Increased sensitivity to kainate-induced seizures in FAAH
(/) mice
FAAH (+/+) and (/) mice were compared for their seizure
responses to increasing doses of kainate and bicuculline. No genotype differences were observed in the severity of seizures induced by lower
doses of either agent (Fig. 4); however,
at the highest dose of kainate tested (30 mg/kg), all FAAH (/) mice
responded with intense clonic-tonic seizures that resulted in death
within 25 min (Fig. 4A,B). In
contrast, the majority of the FAAH (+/+) mice survived treatment with a
high dose of kainate, despite displaying relatively intense seizures
that lasted for up to 60 min (Fig. 4A,B). FAAH (+/+) and (/) mice
exhibited similar seizure responses to low or high doses of bicuculline
(Fig. 4C).
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Figure 4.
Seizure sensitivities of naive FAAH (+/+) and
(/) mice. In response to high (30 mg/kg, i.p.), but not low (15 and
25 mg/kg), doses of kainate, FAAH (/) mice exhibited more intense
seizures (A, filled bars) and reduced survival
(B, solid line) compared with FAAH (+/+) mice
(A, open bars; B, dashed line). No
genotype differences were observed in the severity of seizures induced
by low (4 mg/kg, i.p.) and high (6 mg/kg, i.p.) doses of bicuculline
(C). **p < 0.01 for
FAAH (+/+) versus (/) mice receiving the same treatment
(Mann-Whitney U test). The survival curve
(B) showed a significantly higher mortality for
FAAH (/) mice than for FAAH (+/+) mice
(p < 0.01; Fisher's exact test) after
treatment with 30 mg/kg kainate. n = 10 mice per
group.
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The heightened severity of kainate-induced seizures in FAAH (/)
mice prompted us to examine whether increased endogenous levels of
anandamide were present in the hippocampus of FAAH (/) mice that
might account for their altered behavior. Hippocampal levels of
anandamide, as well as other NAEs, were measured by isotope dilution
liquid chromatography mass spectrometry (Di Marzo et al., 2000) and
found to be >10-fold higher in FAAH (/) mice relative to FAAH
(+/+) mice (Table 1), consistent with the
virtual absence of fatty acid amide hydrolytic activity in the
hippocampus of FAAH (/) animals (Table
2). Additional brain regions examined, including the cortex and cerebellum, displayed similar increases in
endogenous NAE levels in FAAH (/) mice, although the absolute levels of anandamide in these regions were slightly lower than those
found in the hippocampus (Table 1). These data, in conjunction with the
proconvulsant activity observed for anandamide, but not other NAEs, are
consistent with an augmented anandamide-based endocannabinoid tone
contributing to the heightened seizure sensitivity of FAAH (/)
mice.
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Discussion |
Depending on the brain region and neural circuits under
examination, in vitro electrophysiological studies have
shown that the activity of cannabinoids can be either inhibitory
(Kreitzer and Regehr, 2001a; Pistis et al., 2002) or disinhibitory
(Kreitzer and Regehr, 2001b; Ohno-Shosaku et al., 2001; Wilson and
Nicoll, 2001). These apparently contradictory properties of
cannabinoids can be reconciled when one considers that both their
inhibitory and excitatory effects derive from an ability to suppress
neurotransmitter release from presynaptic terminals (Wilson and Nicoll,
2002). If the cannabinoid-sensitive synapse is glutamatergic, the net result is a suppression of excitation, or inhibition; if the
cannabinoid-sensitive synapse is GABAergic, the net result is a
suppression of inhibition, or excitation. Because the synthesis and
release of endocannabinoids appears to occur in an activity-dependent
manner from the postsynaptic cell, these lipid messengers may influence
neuronal cross-talk by a novel retrograde mode of action (Kreitzer and
Regehr, 2001b; Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001).
The context-dependent effects that cannabinoids exhibit on neural
circuits, in combination with the broad distribution of the
CB1 receptor in the CNS, make it difficult to
predict the net impact of CB1 activation on
complex pathological events such as seizure and neurotoxicity.
Additionally, evaluating the function of endocannabinoids is further
challenged by the rapid rate at which the compounds are catabolized
in vivo (Willoughby et al., 1997). Here, we attempted to
address these issues by evaluating seizure sensitivity and
excitotoxicity in mice lacking the protein FAAH (Cravatt et al., 2001),
the principal enzyme responsible for degrading the endocannabinoid
anandamide. FAAH (/) mice exhibit greatly reduced rates of
anandamide hydrolysis, and, as a consequence, anandamide produces an
array of intense, CB1-dependent behavioral
effects in these animals, including hypomotility, hypothermia, analgesia, and catalepsy (Cravatt et al., 2001). The robust cannabinoid activity of anandamide in FAAH (/) mice, which results in the appearance of a sedated state, might be anticipated to reflect a
widespread inhibition of neuronal activity in the CNS that could provide protection from seizure and excitotoxicity. In general support
of this notion, previous studies described an anticonvulsant activity
for exocannabinoids and endocannabinoids in mice in the maximal
electroshock model (Karler et al., 1974; Wallace et al., 2001, 2002),
suggesting that these agents might be useful clinically as
anti-epileptics. However, we found here that the endocannabinoid anandamide promotes rather than retards seizures induced by bicuculline and kainate in FAAH (/) mice. Additionally, untreated FAAH (/) mice exhibited enhanced seizure responses to high doses of kainate that
correlated with greatly elevated endogenous levels of anandamide in the
hippocampus and other brain regions. Collectively, these data uncovered
a previously unappreciated proconvulsant activity for anandamide in two
models of limbic seizures.
There are several possible explanations for the apparent discrepancy
between the results of the current study and those reported previously.
First, anandamide may augment seizure sensitivity in FAAH (/) mice
through non-CB1 mechanisms. Arguing against this
possibility, however, the proconvulsant effects of anandamide in the
bicuculline model were blocked by the CB1
receptor antagonist SR141716A. Additionally, the failure of
structurally related NAEs, such as PEA and OEA, to affect seizure
threshold suggests that the proconvulsant activity of anandamide
displays a degree of chemical specificity consistent with targeting the
CB1 receptor. Finally, the
CB1 agonist WIN 55,212-2, but not its inactive
enantiomer, also increased the severity of bicuculline-induced
seizures. A second potential explanation is that FAAH (/) mice, in
response to increased steady-state brain levels of anandamide, as well as other endogenous fatty acid amides and possibly their metabolites, exhibit compensatory changes in nervous system function that make them
responsive to anandamide in a manner unrepresentative of wild-type
animals. However, this hypothesis is not supported by previous studies,
which have shown that FAAH (+/+) and (/) mice exhibit functionally
equivalent CB1 receptor systems. For example, the
EC50 values for THC in the tetrad test are
equivalent in FAAH (+/+) and (/) mice (Cravatt et al., 2001), and
the brains from these animals display similar CB1
receptor densities (Lichtman et al., 2002). Instead, we speculate that
the apparently contradictory effects of cannabinoids on chemically
induced and maximal electroshock seizures may derive from the fact that
these models promote hyperexcitability in different brain regions. The
seizures caused by kainate and bicuculline are mostly limbic in origin
(Ben-Ari and Cossart, 2000), and therefore the distribution of the
CB1 receptor in brain regions such as the
hippocampus may be of primary importance for defining the effects of
cannabinoids in these models. With this consideration in mind, the
proconvulsant activity of anandamide observed herein is consistent with
in vitro electrophysiological studies describing a
disinhibitory function for endocannabinoids in the hippocampus
(Ohno-Shosaku et al., 2001; Wilson and Nicoll, 2001). In contrast,
seizures resulting from maximal electroshock reflect a more broad state
of hyperexcitability in the CNS (Follesa et al., 1994), and therefore
the anticonvulsant activity of cannabinoids in this model may derive
from an inhibitory activity of these agents in brain regions outside
the hippocampus. Thus, the current studies do not necessarily argue
against a role for cannabinoids as anti-epileptics but rather highlight
that certain forms of epilepsy, especially ones in which limbic
seizures are a major component (temporal lobe epilepsy) (Ben-Ari and
Cossart, 2000), may be exacerbated rather than ameliorated by the
action of endocannabinoids. Finally, it is important to emphasize that
our studies primarily pertain to the fatty acid amide subclass of
endocannabinoids (e.g., anandamide), and the effects of ester-based
endocannabinoids, such as 2-AG, which are catabolized by enzymes other
than FAAH in vivo (Dinh et al., 2002; Lichtman et al.,
2002), may be different.
The dramatic increase in endogenous levels of anandamide that
accompanies excitotoxicity and neurodegeneration (Hansen et al.,
2001b) has led to the suggestion that this endocannabinoid may display
a neuroprotective function in vivo. Consistent with this
notion, anandamide has been shown to reduce neuronal damage caused by
hypoxia in cerebral cortical neuron cultures (Sinor et al., 2000) and
oubain in rats in vivo (van der Stelt et al., 2001).
However, in these studies, the neuroprotective effects of anandamide
were not eliminated by SR141716A, suggesting either a
non-CB1 site of action for this fatty acid amide
or a possible neuroprotective function for its major metabolite
arachidonic acid (Lauritzen et al., 2000). If anandamide itself
possessed a neuroprotective function, a property that could conceivably be independent of its effects on seizure, we postulated that this activity should be augmented in FAAH (/) mice in which the effects of this endocannabinoid are greatly exaggerated. However, anandamide was not found to display a neuroprotective activity in FAAH (/) mice in response to either kainite- or bicuculline-induced seizures. Instead, in both of these seizures models, anandamide promoted hippocampal neuronal damage to a degree commensurate with its proconvulsant activity. In the bicuculline model, in which the effects
of SR141716A could be experimentally examined, both the proconvulsant
and neurotoxic activities of anandamide were blocked by pretreatment
with this CB1 receptor antagonist. Interestingly, a recent study examining the role of CB1
receptors in excitoxicity elicited by NMDA in the rat cortex also
uncovered a neuroprotective function for SR141716A (Hansen et al.,
2002). Collectively, these findings suggest that anandamide may promote
rather than inhibit neuronal damage caused by excitoxic stimuli
in vivo. However, for neuronal damage induced by other
stimuli, such as cerebral ischemia, endocannabinoids may display
neuroprotective effects, as is suggested by the increased severity of
stroke in CB1 (/) mice (Parmentier-Batteur et
al., 2002).
In summary, we found that, in two models of limbic
seizures, the endocannabinoid anandamide exhibits FAAH-regulated
proconvulsant and neurotoxic activity. These pharmacological findings,
coupled with the enhanced kainate sensitivity of FAAH (/) mice,
suggest that both exogenously applied and endogenously produced
anandamide can promote neuronal hyperexcitability in the hippocampus.
Accordingly, these experiments provide important evidence that the
disinhibitory activity of endocannabinoids observed in vitro
in electrophysiological studies of hippocampal slices also extends to
the manner in which these lipids signal in vivo.
Nonetheless, it is important to stress that anandamide and related
endocannabinoids are not likely to produce a general disinhibition of
neuronal activity in the hippocampus or, for that matter, other brain
regions. Indeed, a growing body of evidence indicates that, even within
the same brain region, endocannabinoids can exhibit either excitatory
or inhibitory activity, depending on the neural circuits under
examination (Kreitzer and Regehr, 2001b; Wilson and Nicoll, 2002).
Regardless, the studies described herein emphasize the central role
that FAAH plays in regulating the magnitude of the effects exerted by
anandamide in vivo and therefore further promote this enzyme
as a target for medicinal chemistry efforts aimed at manipulating
endogenous cannabinoid tone for basic research and/or therapeutic purposes.
| |
FOOTNOTES |
Received Oct. 22, 2002; revised Jan. 30, 2003; accepted Feb. 6, 2003.
This work was supported by National Institute of Drug Abuse of the
National Institutes of Health Grants DA13173 and DA15197, Allergan,
Inc., the Helen L. Dorris Institute for the Study of Neurological and
Psychiatric Disorders of Children and Adolescents, and the Skaggs
Institute for Chemical Biology. We thank Tamas Bartfai, Floyd Bloom,
and the entire Cravatt group for helpful discussions and critical
analysis of the work described in this manuscript.
Correspondence should be addressed to Dr. Benjamin F. Cravatt, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail: cravatt{at}scripps.edu.
| |
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TOP
ABSTRACT
Introduction
