Chronic monoacylglycerol lipase blockade causes functional antagonism of the endocannabinoid system.

Abstract

Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol. After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and functional antagonism of the brain cannabinoid system, respectively.

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The endogenous cannabinoid (endocannabinoid) system1 consists
of two G protein–coupled receptors, CB1 and CB2, and their nat-
ural lipid ligands, N-arachidonoylethanolamine (anandamide)2
and 2-arachidonoyglycerol (2-AG)3,4. The CB1 receptor is highly
expressed throughout the nervous system, where it mediates most
of the neurobehavioral effects of cannabinoid agonists, such as
Δ9-tetrahydrocannabinol (THC), the primary psychoactive com-
ponent of marijuana5. The CB2 receptor is only sparsely expressed
in the brain and is instead found mainly on immune cells5. Unlike
most other neurotransmitters, which are water soluble and stored in
membrane-delineated vesicles before release, the endocannabinoids
anandamide and 2-AG are hydrophobic neutral lipids that appear to
be biosynthesized and released at the moment of their intended action
(on-demand production6). These features indicate that the enzymes
involved in endocannabinoid production and degradation are impor-
tant regulators of signaling7–9. For instance, genetic10,11 or pharma-
cological12–15 disruption of fatty acid amide hydrolase (FAAH), the
principal degradative enzyme for anandamide16, elevates brain levels
of anandamide and produces CB1-dependent analgesia in multiple
pain assays. A similar outcome is observed following acute blockade of
the 2-AG–degrading enzyme MAGL, which raises 2-AG levels in the
nervous system and reduces pain behavior17,18. Inhibition of MAGL,
however, causes additional behavioral effects that are not observed fol-
lowing FAAH blockade, including hypomotility and hyperreflexia17,19,
which suggest that the enzyme has a broader effect on the brain
cannabinoid system. In further support of this premise, MAGL inhibi-
tors, but not FAAH inhibitors, augment depolarization-induced sup-
pression of inhibition (DSI)20 and excitation (DSE)20,21, forms of
synaptic plasticity that have been shown to require the CB1 receptor22
and the 2-AG biosynthetic enzyme diacylglycerol lipase-α23,24.
The overlapping, but distinct, behavioral effects of FAAH and MAGL
inhibitors raise provocative questions about the respective roles of ananda-
mide and 2-AG in the nervous system. Several of the behavioral pro-
cesses affected by FAAH inhibitors, including pain12–15 and anxiety12,25,
contain a substantial component of stress. In contrast, MAGL inhibi-
tors also appear to affect general neurological functions (for example,
locomotor activity)17,19. Could these pharmacological profiles point
to a broader role for 2-AG in the nervous system, with anandamide
functioning as a more restricted, stress-responsive endocannabinoid?
If so, what might be the effect of sustained elevations in anandamide
and 2-AG on the integrity of the endocannabinoid system?
We found that prolonged pharmacological or genetic inactivation
of MAGL caused profound alterations in the brain endocannabinoid
system in mice, as evidenced by a loss of analgesic responses to a
MAGL inhibitor, cross-tolerance to exogenous cannabinoid agonists,
and CB1 receptor downregulation and desensitization in specific brain
regions. In contrast, none of these effects were observed in mice with
chronically disrupted FAAH, which instead maintained an analge-
sic phenotype and intact CB1 receptor system. Our results suggest
that there are fundamental differences in the mode of signaling for
1Department of Pharmacology and Toxicology, Virginia Commonwealth University, Richmond, Virginia, USA. 2The Skaggs Institute for Chemical Biology and
Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA. 3Department of Pharmacology and Toxicology, Medical College of
Wisconsin, Milwaukee, Wisconsin, USA. 4Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. 5These authors contributed
equally to this work. Correspondence should be addressed to A.H.L. (alichtma@vcu.edu) or B.F.C. (cravatt@scripps.edu).
Received 6 July; accepted 19 July; published online 22 August 2010; doi:10.1038/nn.2616
Chronic monoacylglycerol lipase blockade causes
functional antagonism of the endocannabinoid system
Joel E Schlosburg1,5, Jacqueline L Blankman2,5, Jonathan Z Long2, Daniel K Nomura2, Bin Pan3,
Steven G Kinsey1, Peter T Nguyen1, Divya Ramesh1, Lamont Booker1, James J Burston1, Elizabeth A Thomas4,
Dana E Selley1, Laura J Sim-Selley1, Qing-song Liu3, Aron H Lichtman1 & Benjamin F Cravatt2
Prolonged exposure to drugs of abuse, such as cannabinoids and opioids, leads to pharmacological tolerance and receptor
desensitization in the nervous system. We found that a similar form of functional antagonism was produced by sustained
inactivation of monoacylglycerol lipase (MAGL), the principal degradative enzyme for the endocannabinoid 2-arachidonoylglycerol.
After repeated administration, the MAGL inhibitor JZL184 lost its analgesic activity and produced cross-tolerance to cannabinoid
receptor (CB1) agonists in mice, effects that were phenocopied by genetic disruption of Mgll (encoding MAGL). Chronic MAGL
blockade also caused physical dependence, impaired endocannabinoid-dependent synaptic plasticity and desensitized
brain CB1 receptors. These data contrast with blockade of fatty acid amide hydrolase, an enzyme that degrades the other
major endocannabinoid anandamide, which produced sustained analgesia without impairing CB1 receptors. Thus, individual
endocannabinoids generate distinct analgesic profiles that are either sustained or transitory and associated with agonism and
functional antagonism of the brain cannabinoid system, respectively.
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these two major endocannabinoid pathways in the nervous system
that result in either sustained agonism or functional antagonism. That
these effects occur through the same receptor (CB1) suggests that
ligand diversification is an important mechanism by which the endo-
cannabinoid system modulates mammalian physiology and behavior.
RESULTS
Mouse models for chronic inactivation of MAGL
We established complementary pharmacological and genetic models
to examine the consequences of sustained elevations in 2-AG in the
nervous system. We generated a chronic pharmacological model
by treating mice for six consecutive days with the MAGL inhibitor
JZL184 (40 mg per kg of body weight, intraperitoneal, one dose per
day), which has previously been shown to selectively inactivate MAGL
in the nervous system and increase the level of 2-AG in the brain
by up to tenfold that of control levels17. Mice treated with JZL184
acutely (single dose) or chronically showed highly elevated levels of
2-AG in the brain 2 h following final dosing (Fig. 1a). This increase in
brain 2-AG levels persisted for at least 26 h (Supplementary Fig. 1),
indicating that 2-AG remained elevated throughout the repeated
dosing regime. Chronic, but not acute, dosing also caused a modest
elevation in anandamide (~threefold) 2 h after final treatment
(Fig. 1a), likely reflecting a partial blockade of FAAH17 as a result
of cumulative exposure to JZL184 over the treatment regimen. This
change was, however, much lower than the 15-fold rise in brain
anandamide that we observed in mice treated for 1 or 6 d with the
selective FAAH inhibitor PF-3845 (10 mg per kg, intraperitoneal,
one dose per day; Fig. 1a)15 and was not of prolonged duration
(Supplementary Fig. 1). PF-3845 did not alter brain 2-AG levels after
acute or chronic treatment (Fig. 1a).
We also employed Mgll−/− mice as a complementary genetic model
for sustained elevations in 2-AG. We obtained Mgll−/− mice gener-
ated by gene trapping from the Texas A&M Institute for Genomic
Medicine (Fig. 1b,c) and confirmed by activity-based protein profil-
ing26,27 that these mice lack detectable MAGL activity without show-
ing alterations in other brain serine hydrolase activities, including
FAAH (Fig. 1d and Supplementary Fig. 2). We also confirmed the
absence of MAGL expression in Mgll−/− mice by in situ hybridization
and mass spectrometry–based proteomics (Supplementary Fig. 3).
Mgll−/− mice exhibited marked (~90%) reductions in brain 2-AG
hydrolytic activity (Fig. 1e and Supplementary Fig. 2) and about
tenfold elevations in brain 2-AG levels (Fig. 1f and Supplementary
Table 1). Brain arachidonic acid levels were also reduced in Mgll−/−
mice (Fig. 1f and Supplementary Table 1) or mice treated acutely
or chronically with JZL184 (Supplementary Fig. 2), consistent with
previous findings designating 2-AG as a physiological precursor for
arachidonic acid in the brain17,28. Anandamide levels were unaltered
in Mgll−/− mice (Fig. 1f and Supplementary Table 1). We observed
similar metabolic changes in a panel of peripheral tissues from
JZL184-treated29 or Mgll−/− mice, all of which showed reductions
in 2-AG hydrolysis and elevations in 2-AG, but not anandamide
(Supplementary Fig. 4). These data provide genetic confirmation that
MAGL is the principal 2-AG hydrolase in the mouse brain and many
peripheral tissues and establish Mgll−/− mice as a valid animal model
for examining the neurophysiological and behavioral consequences
of sustained elevations in 2-AG.
Chronic MAGL blockade causes tolerance in pain assays
Acute pharmacological blockade of MAGL or FAAH produced similar
efficacy in multiple pain assays (Fig. 2), including antinociception
in the acute thermal tail-withdrawal test (Fig. 2a) and reductions
in mechanical (Fig. 2b) and cold (Fig. 2c) allodynia in the chronic
constrictive injury of the sciatic nerve (CCI) model. In contrast, pro-
longed disruption of these enzymes resulted in a marked difference
in the expression of tolerance. Although mice treated repeatedly
with PF-3845 maintained hypoalgesic (Fig. 2a) and anti-allodynic
100
150
2-AG (nmol g–1)
0
JZL184
FAAH
75 kDa
50 kDa
37 kDa
25 kDa
ABHD12
MAGL
ABHD6
JZL184–––+
PF-3845
50
Vehicle
Acute
ChronicChronic
Acute
ab
Mgll genomic
GHSMG
1 2345678
Stop
ATG
Gene trap cassette
de
c
***
***
JZL184 PF-3845
***
***
***
60
80
Anandamide (pmol g–1)
0
430 bp
220 bp
Mgll+/+
Mgll+/–
Mgll–/–
Mgll+/+
Mgll+/–
Mgll–/–
Mgll–/–
Mgll+/+
Mgll+/–
Mgll–/–
Mgll–/–
Mgll+/+
Mgll+/–
Mgll–/–
Mgll+/+
Mgll+/–
Mgll–/–
Mgll+/+
Mgll+/–
Mgll–/–
20
40
Vehicle
Acute
ChronicChronic
Acute
**
60
2-AG hydrolysis
(nmol min–1 mg–1)
0
JZL184
–––+
20
10
30
40
50
f
***
*
200
2-AG (nmol g–1)
0
50
100
150
4
Anandamide (pmol g–1)
0
1
2
3
150
Arachidonic acid (nmol g–1)
0
50
100
***
ns
**
***
Figure 1 Characterization of endocannabinoid metabolism in mice with chronic
disruptions of MAGL or FAAH. (a) Brain levels of 2-AG and anandamide in mice
treated acutely or chronically with JZL184 (acute dosing regimen: 40 mg per kg,
intraperitoneal, 2 h; chronic dosing regimen: 6 d, one dose per day, evaluated 2 h
after final dose) or PF-3845 (acute dosing regimen: 10 mg per kg, intraperitoneal,
2 h; chronic dosing regime: 6 d, one dose per day, evaluated 2 h after final dose)
(n = 5–6 mice per group). (b) The genomic structure surrounding the integrated gene
trap vector in Mgll−/− mice. The gene trap cassette was inserted into the Mgll intron 3,
upstream of the catalytic exon E4 (GHSMG sequence containing the catalytic serine
nucleophile is shown). (c) PCR genotyping of Mgll+/+, Mgll+/−, and Mgll−/− mice from
genomic tail DNA. The 430-bp band corresponds to the wild-type allele and the
220-bp band corresponds to the gene-trapped allele. (d) Activity-based protein
profiling26,27 of brain membrane proteomes showing the selective loss of active MAGL
protein in Mgll−/− mice. (e) 2-AG hydrolytic activities of Mgll+/+, Mgll+/− and Mgll−/− brain membrane proteomes (n = 4 per genotype). Note that
treatment of the Mgll−/− brain proteome with JZL184 (5 μM) did not further decrease MAGL activity signals (d) or 2-AG hydrolysis (e), supporting the
complete loss of MAGL in this sample. (f) Brain levels of 2-AG, anandamide and arachidonic acid from Mgll+/+, Mgll+/− and Mgll−/− mice (n = 4–6 mice
per genotype). Data are presented as means ± s.e.m. *P < 0.05, **P < 0.01 and ***P < 0.001 versus vehicle-treated (a) or wild-type littermate control
mice (e,f) (Dunnett’s post hoc test).
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(Fig. 2b,c) responses, mice chronically treated with JZL184 showed
similar pain responses as control mice (Fig. 2a–c). Similarly, Mgll−/−
mice displayed equivalent tail-withdrawal latencies as Mgll+/+ and
Mgll+/− mice (Fig. 2d).
These findings indicate that the analgesic effects produced by acute
blockade of MAGL are lost following sustained inactivation of this
enzyme. We next investigated whether this form of tolerance was a
result of alterations in the endocannabinoid system.
Chronic MAGL blockade causes tolerance to CB1 agonists
We assessed the behavioral effects of cannabinoid receptor ago-
nists in mice with chronic disruptions in FAAH or MAGL. Faah−/−
mice10, as well as mice that were chronically treated with PF-3845
(Supplementary Fig. 5), exhibited wild-type responses to cannabinoids
in antinociception, hypothermia and catalepsy assays, indicating
that CB1 function was normal. In contrast, Mgll−/− mice or mice
treated chronically with JZL184 showed reduced responses to the
antinociceptive and hypothermic effects of THC (Supplementary
Fig. 6) and the full CB1 agonist WIN55,212-2 (Fig. 3). Chronic JZL184
treatment also elicited marked cross-tolerance to the anti-allodynic
effects of WIN55,212-2 and PF-3845 in the CCI model (Fig. 2e,f). CB1
agonist-induced catalepsy was less affected by sustained inactivation of
MAGL (Fig. 3c,f and Supplementary Fig. 6). These data indicate that
sustained inactivation of MAGL causes cross-tolerance to exogenous
CB1 agonists and to a FAAH inhibitor in a neuropathic pain model.
We next asked whether prolonged MAGL or FAAH blockade
produces physical dependence, a phenotype that has been observed
in rodents that have been exposed to repeated treatments with direct
CB1 agonists30. The CB1 receptor antagonist rimonabant precipitated
paw flutters in mice that were chronically treated with JZL184 to a
similar degree as mice subjected to a mild THC chronic dosing regimen
(10 mg per kg per day for 6 d; Supplementary Fig. 7). In contrast,
rimonabant did not precipitate paw tremors in mice that were chroni-
cally administered PF-3845.
Brain CB1 receptors are impaired by chronic MAGL blockade
The loss of analgesic responses and occurrence of cannabinoid cross-
tolerance in mice with sustained disruptions of MAGL suggested that
CB1 receptors might be downregulated and/or desensitized in these
mice. In support of this hypothesis, brain tissue from Mgll−/− mice
or mice chronically treated with JZL184 showed decreases in CB1
receptor number and function, as measured by specific binding of
1.6
1.4
1.2
1.0
0
Vehicle
JZL184 PF-3845
Acute
ChronicChronic
Acute
a
∆Latency to withdrawal (s)
bced
∆Latency to withdrawal (s)
***
**
###
**
5
4
3
2
1
0
PF-3845
Contralateral
paw
CCI paw
Chronic
Acute
Chronic
Acute
Mechanical theshold (g)
***
###
**
**
JZL184
15
10
5
0
JZL184 PF-3845
Contralateral
paw
CCI paw
Chronic
Acute
Chronic
Acute
Paw lifting (s)
***
###
***
**
1.6
1.4
1.2
1.0
0
VehicleJZL184
Mgll+/+
Mgll+/–
Mgll–/–
5
4
3
2
1
0
Contralateral
f
Vehicle
PF-3845
WIN55,21-2WIN55,21-2
PF-3845
Mechanical theshold (g)
***
###
###
**
VehicleJZL184
15
10
5
0
Contralateral
Vehicle
PF-3845
WIN55,21-2WIN55,21-2
PF-3845
Paw lifting (s)
***
###
###
***
Figure 2 Prolonged blockade of MAGL and FAAH causes differential analgesic tolerance. (a) Acute treatment with
JZL184 caused elevated withdrawal latencies in the tail-immersion test for thermal nociception, whereas this
hypoalgesic response was not observed following chronic treatment with JZL184 (P = 0.33). A similar magnitude
hypoalgesic effect was observed in mice treated acutely with PF-3845 and this effect was maintained following
chronic treatment with PF-3845. (b,c) Acute treatment with JZL184 or PF-3845 reduced mechanical (b) and
cold (c) allodynia in nerve-injured mice. The anti-allodynic effects of PF-3845, but not JZL184, were maintained
following chronic administration. (d) Mgll+/+, Mgll+/− and Mgll−/− mice had similar tail-withdrawal latencies.
(e,f) Chronic JZL184 treatment caused cross-tolerance to the anti-allodynic effects of WIN55,212-2 and PF-3845.
Data are presented as means ± s.e.m., with n = 6–8 per group in all studies. **P < 0.01 and ***P < 0.001 versus
vehicle-treated or wild-type littermate control mice (Dunnett’s post hoc test). ###P < 0.001 versus respective
acute drug treatment group (Bonferroni test).
100
37.5
0
0
Time immobile on bar (s)
Time immobile on bar (s)
15
15
30
30
45
45
60
60
37.5
32.5
32.5
Mgll+/+
Mgll+/–
Mgll–/–
∆Body temperature (°C)
∆Body temperature (°C)
30.0
30.0
35.0
35.0
27.5
27.5
80
Antinociceptive %MPE
60
40
20
0
0.10.1
JZL184
Vehicle
0.1
1.0 1.0
1.0
1.0 1.0
***
******
***
***
***
***
***
***
*
**
WIN55,212-2 (mg per kg)WIN55,212-2 (mg per kg)
WIN55,212-2 (mg per kg)
WIN55,212-2 (mg per kg)WIN55,212-2 (mg per kg)
100100
100
100 100
1010
10
1010
0.11.0
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100 10
ab
c
fe
100
80
Antinociceptive %MPE
60
40
20
0
d
Figure 3 Chronic disruption of MAGL produces behavioral cross-tolerance to
a subset of the pharmacological effects of the cannabinoid receptor agonist
WIN55,212-2. (a–c) Mgll−/− mice showed significant cross-tolerance to the
antinociceptive (a) and hypothermic (b), but not to the cataleptic (c), effects
of WIN55,212-2. (d–f) Similarly, chronic treatment with JZL184 caused
significant cross-tolerance to the antinociceptive (d) and hypothermic (e), but
not the cataleptic (f), effects of WIN55,212-2. Data are presented as means
± s.e.m., n = 7–8 per group. *P < 0.05, **P < 0.01 and ***P < 0.001 versus
vehicle-treated or wild-type littermate control mice (planned comparisons).
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3H-rimonbabant (Fig. 4a,b and Supplementary Fig. 8) and CB1
agonist (CP55,940)-stimulated [35S] GTPS binding (Fig. 4c,d and
Supplementary Fig. 8), respectively. In contrast, prolonged block-
ade of FAAH by PF-3845 did not affect CB1 receptor expression or
function (Fig. 4b,d and Supplementary Fig. 8). These findings are
consistent with previous work showing that CB1 receptor numbers
and function are not altered in Faah−/− mice31,32.
To provide further evidence that the behavioral tolerance and
CB1 receptor adaptations caused by chronic MAGL blockade were
the results of elevated 2-AG acting on CB1 receptors (as opposed
to other metabolic alterations, such as reductions in arachidonic
acid), we attempted to block these changes by concurrent chronic
treatment with rimonabant. For technical reasons, we focused on
antinociception for our behavioral measurements (Supplementary
Discussion). Over a 6-d period, we treated mice daily with vehicle,
JZL184 (40 mg per kg, intraperitoneal), rimonabant (3 mg per kg,
intraperitoneal), or both JZL184 (40 mg per kg, intraperitoneal)
and rimonabant (3 mg per kg, intraperitoneal), yielding four treat-
ment groups. As shown previously (Fig. 3), chronic JZL184–treated
mice produced marked tolerance to the anti-nociceptive effects of
WIN55,212-2 (Supplementary Fig. 9). In contrast, the rimonabant-
JZL184–treated mice exhibited greater antinociceptive responses
to WIN55,212-2 that were close in magnitude to those observed in
control (vehicle or rimonabant) mice (Supplementary Fig. 9). These
data indicate that daily treatment with rimonabant substantially pre-
vents the nociceptive adaptations caused by chronic MAGL blockade.
Rimonabant treatment (10 mg per kg, intraperitoneal) also amel-
iorated brain CB1 receptor adaptations in chronic JZL184–treated
mice, as determined by CP55,940-stimulated [35S]-GTPγS binding
(Supplementary Fig. 9).
A more extensive regional analysis of CP55,940-stimulated
[35S]GTPγS binding in mice treated chronically with either vehicle
or JZL184 revealed that sustained MAGL blockade produced a
heterogeneous reduction in CB1 function throughout the brain
(Fig. 5). Notable brain regions showing CB1 desensitization included
the cingulate cortex, hippocampus, somatosensory cortex and PAG
(Fig. 5b). In contrast, chronic JZL184 treatment did not elicit desen-
sitization in the caudate putamen or globus pallidus. These data,
taken together, indicate that prolonged inactivation of MAGL, but
not FAAH, causes marked changes in CB1 receptor expression and
function in specific brain regions, including those that participate
in pain perception (for example, the PAG) and cognitive/emotional
processing of pain33 (for example, cingulate cortex).
4
4
3
3
2
2
1
1
***
**
**
***
0
Mgll+/+
0
0
Mgll+/+
0
25
75
125
50
50
100
100
150
200
Mgll+/–
Mgll+/–
Mgll–/–
Vehicle
Vehicle
Vehicle
Vehicle
JZL184
JZL184
PF-3845
PF-3845
Mgll–/–
[3H]-SR141716A binding
Bmax (pmol mg–1)
[3H]-SR141716A binding
Bmax (pmol mg–1)
GTPγS Emax
(% CP55,940 stimulation)
GTPγS Emax
(% CP55,940 stimulation)
acdb
Figure 4 Chronic disruption of MAGL
produces CB1 receptor downregulation
and desensitization in the mouse brain.
(a,b) Comparison of the membrane-specific
CB1 receptor binding by the antagonist
[3H]-SR141716A, as evaluated by the best-
fit Bmax of binding curves from whole brain
homogenates. Mgll−/− mice and mice treated
chronically with JZL184, but not mice treated
chronically with PF-3845, had significantly
fewer CB1 receptors. (c,d) CP55,940-stimulated
[35S]-GTPγS binding. Mgll−/− mice and mice
treated chronically with JZL184, but not mice treated chronically with PF-3845, showed CB1 receptor desensitization. Data are presented as means ±
s.e.m. of nonlinear regression best-fit values of specific binding or sigmoidal dose-response curves (n = 4 tissue samples per group, run in separate
experiments with each individual sample run in triplicate for GTPγS and duplicate for receptor binding). **P < 0.01 and ***P < 0.001 versus vehicle-
treated or wild-type littermate control mice (determined by regression confidence intervals).
VEH
1
2
3
4
0
800
600
400
***
**
**
*
200
Net [35S]GTPγS binding (nCi g–1)
0
CG
CPU
GP
POA
HIPP
AMYG
HYPO
SS CTX
SN
PAG
CBLM
1693
Vehicle
JZL184
nCi g–1
JZL184
CG
CPU
SIM
HIPP
AMYG
PAG
CBLM
SN
a
b
Figure 5 Regional changes in cannabinoid agonist–stimulated [35S]GTPγS
binding following chronic disruption of MAGL. (a) Representative
autoradiograms showing CP55,940-stimulated [35S] GTPγS binding in
coronal brain sections following either chronic vehicle (left) or JZL184
(right) treatment. Pseudocolor images indicate levels of receptor-mediated
G protein activity and highlight decreases in CB1 receptor activation
in the cingulate cortex (CG, row 1), hippocampus (HIPP, row 2) and
periaqueductal gray (PAG, row 3). No differences were apparent
in the caudate putamen (CPU, row 1) or cerebellum (CBLM, row 4).
(b) Densitometric analysis of CP55,940-stimulated [35S]GTPγS binding
in selected regions, including cingulate cortex, caudate putamen, globus
pallidus (GP), preoptic area of the hypothalamus (POA), hippocampus,
amygdala (AMYG), hypothalamus (HYPO), somatosensory cortex (SS CTX),
substantia nigra (SN), periaqueductal gray and cerebellum. Data are
presented as means ± s.e.m., n = 8 brains per group, run in triplicate
slices for each targeted region. *P < 0.05, **P < 0.01 and ***P < 0.001
versus vehicle treatment for specific region (Student’s t test).
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Synaptic plasticity is impaired by chronic MAGL blockade
Endocannabinoids regulate several forms of synaptic plasticity34, includ-
ing DSI in the hippocampus20. Considering that CB1 receptors were
impaired in this brain region by sustained MAGL inactivation, we asked
whether DSI was also affected. In contrast with previous findings that
acute inhibition of MAGL by bath application of JZL184 potentiates DSI
in mouse hippocampal slices20, we observed decreases in the magnitude
and time constant (τ) of DSI in hippocampal slices from mice chroni-
cally treated with JZL184 when compared with slices from vehicle-
treated mice (Fig. 6a). We observed similar effects in layer V pyramidal
neurons of the cingulate cortex, where acute (Supplementary Fig. 10)
and chronic (Fig. 6b) treatment with JZL184 potentiated and disrupted
DSI, respectively. PF-3845 did not affect DSI in the hippocampus
(Fig. 6a) or cingulate cortex (Fig. 6b and Supplementary Fig. 10).
The attenuation of DSI by chronic JZL184 treatment is consistent
with desensitization of CB1 receptors in the affected neuronal cir-
cuits (Fig. 5). In support of this premise, CP55,940 (3 μM) induced
less depression of inhibitory postsynaptic currents (IPSCs) in the
hippocampal CA1 pyramidal neurons or layer V pyramidal neurons
of the cingulate cortex from chronically JZL184–treated mice than
in those from vehicle-treated mice (Fig. 6c,d). In contrast, repeated
in vivo administration of PF-3845 did not alter CP55,940-induced
depression of IPSCs in either hippocampus (Fig. 6c) or cingulate
cortex (Fig. 6d). The CB1 receptor antagonist AM251 (2 μM) com-
pletely blocked CP55,940-induced depression of IPSCs in both brain
regions (Supplementary Fig. 11). Notably, chronic JZL184 treatment
exerted only a modest effect that did not reach statistical significance
(P > 0.05) on CP55,940-induced depression of IPSCs in the caudate
putamen (Supplementary Fig. 12), a brain region that also showed
minimal CB1 receptor adaptations (Fig. 5).
Recent studies have suggested that CB1 receptors on glutamatergic
synapses mediate many of the behavioral effects of CB1 agonists35.
We therefore examined whether chronic JZL184 or PF-3845 treat-
ment altered CB1-mediated depression of glutamatergic excitatory
transmission in the hippocampus. Chronic JZL184, but not PF-
3845, treatment attenuated CP55,940-induced depression of field
excitatory postsynaptic potentials (fEPSPs) in the CA1 region of the
hippocampus (Supplementary Fig. 13).
These results, taken together, indicate that sustained inactivation of
MAGL, but not FAAH, impairs specific endocannabinoid-mediated
forms of synaptic plasticity. That we observed these effects for both
glutamatergic and GABAergic transmission is consistent with previ-
ous findings that acute MAGL, but not FAAH, blockade enhances
both DSE and DSI20 and that diacylglycerol lipase-α knockout mice
exhibit defects in both DSE and DSI23,24.
DISCUSSION
Prolonged treatment with THC and other cannabinoid receptor ago-
nists leads to the development of tolerance and physical dependence36
and these behavioral phenotypes have been shown to be mirrored by
substantial reductions in CB1 receptor expression and activity in the
brain37,38. We found that sustained elevations in brain 2-AG caused
by either genetic deletion or chronic pharmacological blockade of
MAGL also produced substantial functional antagonism of the brain
endocannabinoid system, as manifested by tolerance to the analgesic
effects of acute enzyme inhibition, cross-tolerance to CB1 receptor
agonists, a reduction in CB1 receptor expression and function, and
disruptions in endocannabinoid-dependent synaptic plasticity. This
profile markedly contrasted with that of sustained pharmacological
disruption of FAAH, which caused persistent analgesic effects with-
out evidence of tolerance or changes in CB1 receptor expression or
function. Thus, brain CB1 receptors undergo markedly different
adaptations in response to sustained elevations of the two principal
endocannabinoids, 2-AG and anandamide.
That the cannabinoid cross-tolerance and alterations in CB1 receptor
function caused by JZL184 were both attenuated by co-treatment
with rimonabant supports a model in which chronic MAGL block-
ade produces a sustained elevation in 2-AG that tonically activates
and eventually desensitizes CB1 receptors in the brain. We cannot,
however, rule out the possibility that other metabolic changes caused
by MAGL inhibition, such as reductions in arachidonic acid, also
contribute to alterations in brain endocannabinoid pathways. We
also note that chronic JZL184 treatment produced an evidently larger
degree of cross-tolerance to CB1 agonists (Fig. 3a,b) than genetic
disruption of Mgll (Fig. 3d,e). Although we do not yet understand
the basis for this difference, it could reflect differences in background
strain (C57BL/6J versus 129SvEv/C57BL/6J for the JZL184-treated
and Mgll−/− mice, respectively) or the existence of compensatory
mechanisms in the Mgll−/− mice that counteract the observed CB1
receptor adaptations.
Although there are other examples of functional antagonism of
receptor systems following deletion of a metabolic enzyme, including
reduced activity of nicotinic receptors39 in Ache−/− (acetylcholinesterase)
mice and impairments in 5-HT1A receptors in Maoa−/− (monoamine
oxidase A) mice40, we report the first instance, to the best of our
a
cd
b
Hippocampus
JZL184
2
Hippocampus
JZL184
Cingulate cortex
Vehicle
2
Cingulate cortex
Vehicle
JZL184
2
1
0 mV, 5 s
11
30 ms 0.3 nA
0.3 nA
22
JZL184PF-3845
PF-3845
2
Vehicle
2
Vehicle
2
1
1
1
CP 55,940 CP 55,940
1
2
2
01020
Time (min)Time (min)
3040010203040
2
1
1
1
2
100
50
100
50
100
50
100
50
020 40
Time (s)
80
Vehicle
JZL184
PF-3845
Vehicle
JZL184
PF-3845
Vehicle
JZL184
PF-3845
Vehicle
JZL184
PF-3845
60100 120 1400 20 40
Time (s)
8060 100 120 140
IPSC amplitude (%)
IPSC amplitude (%)
IPSC amplitude (%)
IPSC amplitude (%)
1
0 mV, 5 s
1
30 ms
30 ms 30 ms
2
2
1
1
11
2
2
0.2 nA
0.2 nA
PF-3845
PF-3845
Figure 6 Chronic disruption of MAGL impairs CB1-dependent forms
of synaptic plasticity. (a,b) Chronic treatment with JZL184, but not
PF-3845, attenuated DSI in hippocampal CA1 pyramidal neurons (a)
and layer V pyramidal neurons of the cingulate cortex (b) (P < 0.01
for JZL184- versus vehicle-treated groups for both magnitude and τ of
DSI, n = 11–15 mice per group). The lines superimposed are the single
exponential fitting curves of the decay of DSI. (c,d) Bath application of
the CB1 agonist CP55,940 (3 μM) induced significantly less depression
of IPSCs in the hippocampus (c) and cingulate cortex (d) from mice
chronically treated with JZL184 than in those from vehicle-treated control
mice (P < 0.05 in both brain regions). Brain regions from mice chronically
treated with PF-3845 did not differ significantly from vehicle controls
(P > 0.05; n = 6–7 mice per group). Data are presented as means ± s.e.m.
© 2010 Nature America, Inc. All rights reserved.

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1118 
VOLUME 13 | NUMBER 9 | SEPTEMBER 2010 nature neurOSCIenCe
artICleS
knowledge, in which disruption of an enzyme that degrades a lipid
transmitter leads to receptor downregulation and desensitization in
the nervous system. This finding indicates that, despite fundamen-
tal differences in the mechanisms of storage and release (vesicular
versus nonvesicular), classical neurotransmitters and lipid messengers
are both capable of causing tolerance and receptor desensitization
following chronic inactivation of their cognate degradative enzymes.
That behavioral and CB1 receptor adaptations occurred only in mice
with chronically inactivated MAGL, but not FAAH, indicates that
sustained elevations in 2-AG exert a greater effect than anandamide
on the integrity of the brain endocannabinoid system. How 2-AG is
more capable of causing substantial CB1 alterations in vivo remains
unclear, but our data would indicate that this effect is not necessarily
correlated with the induction of superior efficacy in behavioral assays.
Indeed, MAGL and FAAH inhibitors displayed similar relative analge-
sic activity in acute treatment procedures. One possibility is that 2-AG
and anandamide have differential effects on CB1 receptor desensitiza-
tion and/or recycling in the brain, as has been observed previously in
heterologous expression systems41. This differential desensitization
may be related to the higher efficacy of 2-AG as a full CB1 receptor
agonist (in contrast with anandamide, which acts as a partial CB1
receptor agonist)42, although previous research suggests that the mag-
nitude of CB1 receptor desensitization is not related to the intrinsic
activity of exogenous agonists43. Bulk brain levels of 2-AG are also
much higher than anandamide (see Fig. 1a; although the interstitial
levels of these endocannabinoid are similar44), and elevated 2-AG
may therefore achieve greater occupancy of CB1 receptors in vivo.
Finally, MAGL and FAAH are found in different neuronal populations
and subcellular compartments (pre- and postsynaptic, respectively)
throughout the brain and these anatomical distinctions might also
differentially affect endocannabinoid signaling pathways in the
nervous system. Regardless, our observation that chronic MAGL
blockade produced cross-tolerance to a FAAH inhibitor in the CCI
model (Fig. 2e,f) indicates that 2-AG and anandamide pathways can
crosstalk in the neural circuits that regulate pain behavior.
The endocannabinoid system regulates several forms of synaptic
plasticity, including DSI and DSE34. Acute MAGL, but not FAAH,
inhibition potentiates DSI in neurons of the hippocampus20 and cin-
gulate cortex (Supplementary Fig. 4). Notably, however, we found
that repeated administration of JZL184 led to profound DSI deficits in
these neuronal populations (Fig. 6). These impairments in short-term
synaptic plasticity are consistent with the observed alterations in CB1
receptor function in the hippocampus and cingulate cortex (Fig. 5),
as deletion or antagonism of this receptor has been shown to abol-
ish DSI22,45. That acute and chronic inhibition of MAGL produced
opposing effects on DSI in multiple brain regions supports a model
in which prolonged elevations of endogenous 2-AG cause functional
antagonism of CB1 receptors in the nervous system.
In contrast with direct CB1 agonists, which produce cross-tolerance
to the antinociceptive, hypothermic and cataleptic effects of THC
and WIN55,212-2 (ref. 46), chronic MAGL disruption only caused
strong cross-tolerance to the antinociceptive and hypothermic effects
of these drugs. The minimal cross-tolerance to cannabinoid-induced
catalepsy is consistent with the lack of CB1 receptor desensitization in
caudate putamen and globus pallidus (Fig. 5), which are associated
with cannabinoid-induced catalepsy47. Conversely, the tolerance
that we observed to the antinociceptive effects of JZL184 and the
occurrence of cross-tolerance to WIN55,212-2– and THC-induced
antinociception could be attributed to the desensitization of CB1
receptors in PAG (Fig. 5), a brain area that has been strongly impli-
cated in cannabinoid-induced antinociception48. We also note that
neither MAGL nor FAAH inhibitors alone cause catalepsy; however,
combined treatment with these inhibitors does promote cataleptic
behavioral responses19. It will be interesting to determine whether
sustained inactivation of both MAGL and FAAH causes cross-tolerance
to the cataleptic effects of other CB1 agonists and concomitant altera-
tions in CB1 receptor expression and activity in brain regions such as
the caudate putamen and globus pallidus.
In summary, our data support a model in which ligand diversifica-
tion is important for shaping the distinct functions and properties
of endocannabinoid signaling pathways in the nervous system. The
widespread behavioral and CB1 receptor adaptations caused by chronic
disruption of MAGL suggest a broad role for 2-AG throughout the
nervous system. In contrast, the preservation of analgesic phenotypes
and CB1 receptor function in mice with sustained inactivation of FAAH
may reflect a more limited, stress-dependent function for anandamide.
This idea is also consistent with the behavioral phenotypes observed
in FAAH-disrupted animals, which preferentially show reductions in
pain11 and anxiety25 procedures with strong stress components. These
discoveries may have important translational implications. Consider,
for instance, that acute inhibition of FAAH and MAGL produces similar
efficacy in multiple pain assays, but these effects are only sustained in
chronically disrupted FAAH systems. Might this imply that MAGL is
a less suitable target for treatment of pain disorders? Perhaps, but it
also may be possible to achieve prolonged analgesic responses through
partial MAGL blockade. In this event, one would still need to be con-
cerned about the potential tolerance and withdrawal effects of MAGL
inhibitors. That CB1 receptors, on the other hand, are surprisingly
nonadaptive to continuous elevations of brain anandamide suggests
that FAAH inhibitors are capable of producing sustained analgesic
activity without high risk for dependence.
METhODS
Methods and any associated references are available in the online
version of the paper at http://www.nature.com/natureneuroscience/.
Note: Supplementary information is available on the Nature Neuroscience website.
AcknowleDgmenTS
We thank S. Niessen and H. Hoover for assistance with proteomics studies,
I. Beletskaya and R. Abdullah for technical support and the Cravatt and Lichtman
laboratories for critical reading of the manuscript. This work was supported by
the US National Institutes of Health (grants DA017259, DA009789, DA025285,
DA005274, DA015683, DA03672, DA005274, DA07027, DA014277, DA023758
and DA024741), Ruth L. Kirschstein US National Institutes of Health Predoctoral
Fellowships (DA026261 to J.L.B., DA026279 to J.E.S., DA028333 to L.B. and
DA023758 to P.T.N.), the American Cancer Society (D.K.N.) and the Skaggs
Institute for Chemical Biology.
AUTHoR conTRIBUTIonS
J.E.S. performed behavioral and receptor adaptation experiments. J.L.B.
performed the metabolic biochemistry, proteomic and in situ hybridization
experiments and contributed to behavioral experiments. J.Z.L. contributed to
metabolic biochemistry and behavioral experiments. D.K.N., S.G.K., D.R. and
L.B. contributed to behavioral experiments. B.P. performed the electrophysiology
experiments. P.T.N. and J.J.B. contributed to receptor adaptation experiments.
E.A.T. contributed to the design and interpretation of in situ hybridization
experiments. D.E.S and L.J.S.-S. contributed to the design and interpretation
of receptor adaptation experiments. Q.-s.L. contributed to the design and
interpretation of electrophysiology experiments. A.H.L. and B.F.C. supervised the
design, execution and interpretation of the experiments and wrote the manuscript.
comPeTIng FInAncIAl InTeReSTS
The authors declare no competing financial interests.
Published online at http://www.nature.com/natureneuroscience/.
Reprints and permissions information is available online at http://www.nature.com/
reprintsandpermissions/.

© 2010 Nature America, Inc. All rights reserved.

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nature neurOSCIenCe
doi:10.1038/nn.2616
ONLINE METhODS
Animals. Subjects consisted of male C57BL/6J mice (Jackson Laboratories) as well
as male and female Mgll+/+, Mgll+/− and Mgll−/− mice on a mixed 129SvEv/C57BL/
6J background. MgllGt1(neo) mutant mice (TG0078; derived from OmniBank ES
cell line OST113734) containing a gene trap vector inserted into the third intron
of the Mgll gene were obtained from the Texas Institute of Genomic Medicine.
The gene trap vector insertion site was mapped to the sequence GCC TTG TGG
ACT GGA T(gene trap insertion)CT TGG GCC TTC TGT TC, which is upstream
of the Mgll catalytic exon 4. Mgll genotype was determined by PCR amplification
of genomic tail DNA using the following primers designed by Texas Institute of
Genomic Medicine: Mgll forward 5′-TTG CCT GCT TGC TCT TAA CTC TTG
C-3′, Mgll reverse 5′-GGG AGT CAA GAC ACT GGG GAA TCC T-3′, and gene
trap reverse 5′-ATA AAC CCT CTT GCA GTT GCA TC-3′, which amplified a
430-bp product for the wild-type allele and a 220-bp product for the gene-trapped
allele. Mice homozygous for the gene-trap (Mgll−/− mice) are viable, born at the
expected Mendelian frequency, and display normal cage behavior compared with
Mgll+/+ and Mgll−/− littermates. Animal experiments were conducted in accord-
ance with the guidelines of the Institutional Animal Care and Use Committees of
the Scripps Research Institute and Virginia Commonwealth University.
Drugs and chemicals. JZL184 and PF-3845 were synthesized as described pre-
viously15,17. WIN55,212 was purchased from Cayman Chemical. Rimonabant,
THC and CP55,940 were obtained from the Drug Supply Program of the National
Institute on Drug Abuse. AM251 was obtained from Tocris. GDP, GTPγS, adeno-
sine deaminase and bovine serum albumin (BSA) were purchased from Sigma-
Aldrich. [35S]GTPγS (1250 Ci mmol−1) was obtained from PerkinElmer Life
and Analytical Sciences. [3H]SR141716A (44.0 Ci mmol−1) was purchased from
Amersham Pharmacia. Scintillation fluid (ScinitSafe Econo 1) was purchased
from Thermo Fisher Scientific and Whatman GF/B glass fiber filters were
obtained through Fisher Scientific.
Drugs were dissolved via sonification in a vehicle consisting of ethanol,
Alkamuls-620 (Sanofi-aventis) and saline in a ratio of 1:1:18. All drugs were
administered via the intraperitoneal route of administration in a volume of
10 μl per g of body mass. For chronic drug administration, subjects received a
daily injection of JZL184 (40 mg per kg), PF-3845 (10 mg per kg), THC (10 mg
per kg), rimonabant (3 mg per kg for behavioral analysis, 10 mg per kg for CB1
receptor adaptation) or vehicle for 6 d.
Preparation of mouse brain homogenates. Membrane and soluble brain
homogenates from C57BL6/J and Mgll+/+, Mgll+/− and Mgll−/− mice (n = 4 per
genotype) were prepared as previously described29.
Activity-based protein profiling analysis. Analysis of brain proteomes pre-
treated with 5 μM JZL184 or DMSO vehicle (30 min at 25 °C) was performed as
described previously27.
2-Ag hydrolysis assays. 2-AG hydrolytic activity of Mgll+/+, Mgll+/− and Mgll−/−
brain homogenates (n = 4 per genotype) pretreated with either 1 μM JZL184 or
DMSO vehicle (30 min at 25 °C) was determined using a previously described
liquid chromatography–mass spectrometry assay27 on an Agilent 6520 QTOF MS.
Brain metabolite measurements. Brain lipid levels were determined as previ-
ously described29 except that free fatty acid levels in Mgll+/+, Mgll+/− and Mgll−/−
brains were measured on an Agilent 1100 series LC-MS and quantified compared
to a palmitic acid calibration curve.
In situ hybridization. Perfused brains from 12-week-old male Mgll+/+ and Mgll−/−
mice were postfixed, cryoprotected and frozen as previously described49. In situ
hybridization was performed on 25-μm-thick free-floating coronal sections as
described49 with [35S]UTP-labeled, single-stranded antisense and sense control
cRNA probes to Mgll cDNA (bases 285–600).
multidimensional lc-mS proteomic analysis. Mouse brain proteomes
(0.75 mg total protein) from Mgll+/+ and Mgll−/− mice (n = 3 mice per geno-
type) were precipitated with 1:4 chloroform:methanol and denatured with
25 mM ammonium bicarbonate in 6 M urea. Samples were reduced with 10 mM
dithiothreitol, alkylated with 40 mM iodoacetamide and diluted to 2 M urea
with 25 mM ammonium bicarbonate. Digestion with trypsin (0.5 μg μl−1) was
performed overnight at 37 °C in the presence of 1 mM CaCl2. The tryptic peptide
samples were acidified with 5% formic acid (wt/vol) and aliquots were frozen at
−80 °C until use. Multidimensional protein identification technology (MudPIT)
analysis was performed as previously described27 on an LTQ mass spectrometer
(ThermoFinnigan) coupled to an Agilent 1100 series HPLC (n = 2 per genotype,
30 μg protein, 5-step MudPIT) or an LTQ Orbitrap Velos mass spectrometer
(ThermoFinnigan) coupled to an Agilent 1200 series HPLC (n = 1 per geno-
type, 45 μg protein, 10-step MudPIT). The tandem mass spectrometry data were
searched against the mouse IPI database using the SEQUEST search algorithm
and results were filtered and grouped with DTASELECT. Peptides with cross-
correlation scores greater than 1.8 (+1), 2.5 (+2), 3.5 (+3) and delta CN scores
greater than 0.08 were included in the spectral counting analysis.
Behavioral assays. To control for stress of repeated injection, all acute treat-
ment groups received 5 d of daily vehicle injections, with acute drug treatment
occurring on day 6. Subjects were evaluated 2 h after acute drug administration
or the final chronic injection. Acute thermal antinociception was assessed in the
tail-immersion test at 56.0 °C using a 10-s cut-off19. Surgery for CCI model of the
sciatic nerve and allodynia assessment were performed as previously described18.
Subjects were assessed for mechanical allodynia using von Frey filaments (North
Coast Medical) and approximately 30 min later were evaluated for cold allodynia
in the acetone-induced paw lifting model, with a maximum cut-off time of 20 s.
Cross-tolerance studies in the CCI model were performed starting 26 h following
the final chronic drug injection.
Subjects were evaluated for cross-tolerance to WIN55,212-2 or THC 26 or
48 h after the final chronic injection. Cannabimimetic activity was assessed
by evaluating mice for catalepsy in the bar test19, antinociception in the tail
immersion test at 52.0 °C31 and hypothermia by inserting a thermocouple probe
2.0 cm into the rectum. To reduce the number of mice required for this study,
we evaluated dose-response relationships using a cumulative dosing regimen
in which baseline behavioral endpoints were assessed, injections were given
every 40 min and subjects were evaluated for each measure 30 min after each
injection, with the entire dose-response assessment being completed in less
than 4 h31.
For precipitated withdrawal, mice were challenged with rimonabant (10 mg
per kg) 2 h after the final chronic injection and the incidents of paw fluttering,
including any tremors or shaking of the front paws, were recorded for a 1-h
observation period50.
Binding assays. CP-55,940–stimulated [35S]GTPγS binding and [3H]-SR141716A
binding in whole brains were conducted as previously described31. For CP-
55,940–stimulated [35S]GTPγS autoradiographs, coronal sections (20 μm)
were cut on a cryostat at −20 °C, thaw-mounted onto gelatin-subbed slides and
stored desiccated at −80 °C until use. For assay, slides were brought to 20–25 °C,
incubated in assay buffer and 0.5% BSA (wt/vol) containing 0.04 nM [35S]GTPγS
in the presence or absence (basal) of maximally effective concentrations of
CP55,940 (3 μM) and/or vehicle for 2 h at 25 °C. After final incubation, slides
were rinsed twice in 50 mM Tris buffer (pH 7.4) at 4 °C and then rinsed in deion-
ized water. The rinsed slides were dried and exposed to Kodak BioMax MR film
with [14C] standards for 24–36 h. Films were digitized at 8-bits per pixel with a
Sony XC-77 video camera. Regions of interest were selected using anatomical
landmarks and measured using ImageJ (US National Institutes of Health).
electrophysiology slice preparation and testing. In the chronic experiments,
subjects were anaesthetized by isoflurane inhalation and decapitated 24–26 h
after the final injection. Hippocampal, cortical and caudate putamen slices
(300 μm thick) were cut using a vibrating slicer (Leica) and prepared as described
previously20. In the acute experiments, the slices were perfused with JZL184
(1 μM) or PF-3845 (10 μM) for 40–80 min.
Whole-cell voltage-clamp recordings were made using patch clamp amplifier
(Multiclamp 700B) under infrared differential interference contrast micro-
scopy. Data acquisition and analysis were performed using a digitizer (DigiData
1440A) and analysis software pClamp 10 (Molecular Devices). To record IPSCs,
we clamped the neurons at −60 mV and filled the pipettes with an internal solu-
tion containing 80 mM cesium methanesulfonate, 60 mM CsCl, 5 mM QX-314,
10 mM HEPES, 0.2 mM EGTA, 2 mM MgCl2, 4 mM MgATP, 0.3 mM Na2GTP
© 2010 Nature America, Inc. All rights reserved.

Page 9

nature neurOSCIenCe
doi:10.1038/nn.2616
and 10 mM sodium phosphocreatine (pH 7.2 with CsOH). Glutamate receptor
antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 20 μM) and d-2-
amino-5-phosphonovaleric acid (d-AP5, 25 μM) were present in the artificial
cerebrospinal fluid throughout the experiments. Series resistance (15–30 MΩ)
was monitored throughout the recordings, and data were discarded if the resist-
ance changed by more than 20%. To record fEPSPs, the pipettes were filled with
1 M NaCl, and picrotoxin (50 μM) was present in the artificial cerebrospinal fluid.
To evoke IPSCs or fEPSPs, a bipolar tungsten stimulation electrode was placed
in the in the stratum radiatum of the CA1 region of hippocampus, in layer V of
cingulate cortex or the caudate putamen. All recordings were performed at 32 ±
1 °C by using an automatic temperature controller.
Data analysis and statistics. All results are expressed as mean ± s.e.m. unless
otherwise noted. Results were considered to be significant at P < 0.05. All lipid
quantification and behavioral endpoints were initially evaluated by ANOVA
(treatment or genotype) or repeated-measures ANOVA (cumulative dose
responses). Following a significant ANOVA, Dunnett’s post hoc test was per-
formed for comparisons to treatment or genotypic control. Planned comparisons
and specific within-drug treatments are noted in figure captions when used,
using a Bonferroni test to correct for multiple comparisons. [35S]GTPγS binding
experiments were performed in triplicate and all data points are reported as mean
± s.e.m. of four experiments. Nonspecific binding was first subtracted from all
binding data. Stimulated binding was determined as agonist-stimulated binding
minus basal binding and values are reported as percentage stimulation above
basal. All receptor binding experiments were performed in duplicate and reported
as mean ± s.e.m. of four experiments. Nonspecific binding was first subtracted
from total binding, yielding specific binding data. Nonlinear regression analyses
of agonist concentration effect curves were performed with Prism 5.0 using a
sigmoidal dose-response model or specific binding of single site model (GraphPad
Software). Values from regressions are reported as mean ± s.e.m. for interpolated
results. Regional G protein stimulation autoradiography data are reported as
mean ± s.e.m. of triplicate sections from seven to eight brains per group. Net
[35S]GTPγS binding is defined as agonist-stimulated [35S]GTPγS binding − basal
[35S]GTPγS binding. Analysis was performed in GraphPad Prism Version 5 using
Student’s t test between the two treatments for each individual region analyzed.
For electrophysiological analysis, IPSC/fEPSP amplitude was normalized to
the baseline. The τ of DSI was measured using a single exponential function of
y = y0 + k × e−x/τ. The magnitude of DSI was calculated as DSI (%) = 100 ×
[1 − (mean amplitude of two IPSCs immediately after depolarization divided by
mean amplitude of five IPSCs before depolarization)]. Values of 2–3 DSI trials
were averaged for each neuron. The depression (%) of IPSCs/fEPSPs by CP55,940
was calculated as 100 × (mean amplitude of IPSCs/fEPSPs during the last 5-min
treatment divided by mean amplitude of baseline IPSCs/fEPSPs). Data sets were
compared with Student’s t test.
49. Thomas, E.A. et al. Clozapine increases apolipoprotein D expression in rodent brain:
towards a mechanism for neuroleptic pharmacotherapy. J. Neurochem. 76,
789–796 (2001).
50. Schlosburg, J.E. et al. Inhibitors of endocannabinoid-metabolizing enzymes reduce
precipitated withdrawal responses in THC-dependent mice. AAPS J. 11, 342–352
(2009).
© 2010 Nature America, Inc. All rights reserved.