Cannabinoid-Induced Tetrad in Mice UNIT 9.

59
Mathilde Metna-Laurent,1 Miguel Mondésir,2,3 Agnès Grel,2,3
Monique Vallée,2,3,4 and Pier-Vincenzo Piazza2,3,4
1
Aelis Farma, Neurocentre Magendie, Bordeaux, France
2
Physiopathology of Neuronal Plasticity, Neurocentre Magendie, INSERM U1215,
Bordeaux, France
3
University of Bordeaux, Bordeaux, France
4
M. V. and P.-V. P. equally supervised this work.

Cannabinoid-induced tetrad is a preclinical model commonly used to evaluate

if a pharmacological compound is an agonist of the central type-1 cannabinoid
(CB1) receptor in rodents. The tetrad is characterized by hypolocomotion,
hypothermia, catalepsy, and analgesia, four phenotypes that are induced by
acute administration of CB1 agonists exemplified by the prototypic cannabinoid
delta-9-tetrahydrocannabinol (THC). This unit describes a standard protocol in
mice to induce tetrad phenotypes with THC as reference cannabinoid. We
provide typical results obtained with this procedure showing a dose effect of
THC in different mouse strains. The effect of the CB1 antagonist rimonabant
is also shown. This tetrad protocol is well adapted to reveal new compounds
acting on CB1 receptors in vivo. 
C 2017 by John Wiley & Sons, Inc.

Keywords: cannabinoid r tetrad r CB1 receptors r mice

How to cite this article:

Metna-Laurent, M., Mondésir, M., Grel, A., Vallée, M., & Piazza,
P.-V. (2017). Cannabinoid-induced tetrad in mice. Current Protocols
in Neuroscience, 80, 9.59.1–9.59.10. doi: 10.1002/cpns.31

INTRODUCTION
Type-1 cannabinoid (CB1) receptors are potential targets for the treatment of several
central nervous system disorders, including psychiatric and neurodegenerative diseases.
Full agonists or antagonists of CB1 receptors show undesired effects that limit their
use in human (Pacher & Kunos, 2013; Pertwee, 2012). However, the discovery of new
CB1 ligands with more selective modes of action, including receptor allostery or ligand-
biased signaling, represents new opportunities to treat CB1-related pathologies with
unmet clinical needs (Picone & Kendall, 2015; Vallée et al., 2014).

The tetrad model has been successfully used to discover and classify agents with pharma-
cological activity on CB1 receptors. The tetrad model includes four specific phenotypic
effects of CB1 agonists that were first described following acute systemic administra-
tion of the plant-derived cannabinoid delta-9-tetrahydrocannabinol (THC) in rodents.
Within the same dose and temporal ranges, THC induces hypolocomotion (decrease of
spontaneous horizontal activity), hypothermia (decrease of body temperature), catalepsy
(impaired capacity to initiate movement), and analgesia (decrease of pain sensitivity)
(Howlett et al., 2002; Martin, 1986; Varvel, 2005).

Here we describe a procedure to induce this tetrad by THC administration in mice. This
protocol can be used to compare the effects of THC with other putative CB1 agonists
and can also characterize potential CB1 antagonist properties of new compounds for Preclinical Models
innovative clinical development. of Neurologic and
Psychiatric
Disorders

Current Protocols in Neuroscience 9.59.1–9.59.10, July 2017 9.59.1

Published online July 2017 in Wiley Online Library (wileyonlinelibrary.com).
doi: 10.1002/cpns.31
Copyright C 2017 John Wiley & Sons, Inc. Supplement 80
 BASIC THC-INDUCED TETRAD IN MICE
PROTOCOL
The THC-induced tetrad requires standard rodent manipulations and intraperitoneal (i.p.)
injections. Tetrad phenotypes are analyzed with specific equipment, including a rodent
thermometer and a hot plate.
Materials
2 to 3 month-old naı̈ve male C57Bl/6J mice
20% ethanol
Delta-9-tetrahydrocannabinol (THC) solution (see recipe)
Vehicle control solution for THC

Scale, to weight mice

Flexible rectal probe (e.g., Physitemp RET-3) and monitoring thermometer (e.g.,
Physitemp TH-5)
1-ml syringes
25-G needles, 16 × 0.5 mm
Behavioral testing room containing:
Open field area (made in house)
Video camera
Hot plate
Catalepsy cages (made in house)
Chronometer
NOTE: All protocols using live animals must first be reviewed and approved by an
Institutional Animal Care and Use Committee (IACUC) or must conform to governmental
regulations regarding the care and use of laboratory animals.

NOTE: The use of cannabinoid agents for preclinical research requires specific autho-
rization from local regulatory authorities. In general, the investigator needs authorization
before ordering and using cannabinoids that are listed as psychotropic drugs for animal
research. The application for authorization describes the purpose of the animal project
and the exact amount of drugs required for the development of the project. The use of
cannabinoid drugs is tracked and checked by the authorities.
Preparation and setup
1. Upon arrival, house the mice in single cages on a 12 hr light-dark cycle with ad
libitum access to food and water.
Mice that are 2 to 3 months old are used in this protocol. At this age mice should weight
25 g under standard laboratory diet.
Other mouse strains can be used (e.g., C57Bl/6N, CD-1 Swiss mice); however, the dose
range of THC inducing tetrad phenotypes may change. CD-1 Swiss mice are less sensitive
to the effect of THC on tetrad phenotypes (see Anticipated Results). Cannabinoid-induced
tetrad can be studied in female mice (Fride, Perchuk, Hall, Uhl, & Onaivi, 2006); however,
the present protocol has never been performed in female mice.
Cannabinoid-induced tetrad can also be studied in rats (McLaughlin et al., 2013). Equip-
ment and setup described in this unit are adapted for mice. For instance, open fields,
activity cages, and catalepsy cages are larger for rats.
Group-housed mice can be used, but particular attention should be paid to fighting and
social hierarchy, especially when mice arise from large colonies. Moreover, each mouse
should be carefully identified to avoid group assignment errors.

Cannabinoid- 2. Allow the mice to acclimate to the vivarium for at least 7 days before the start of
Induced the experiments. Weigh the mice on arrival and at least once during acclimation to
Tetrad
ensure normal body weight gain.
9.59.2
Supplement 80 Current Protocols in Neuroscience
 For experiments, do not include mice that do not gain weight during acclimation and/or
mice with apparent health problems.

3. Before beginning the experiment (2 days), take basal temperature to habituate the
mice to the rectal probe insertion. Immobilize the mouse and gently insert 1 cm of
the probe into the rectum until stabilization of temperature. Place the mouse back in
its home cage. Note the temperature of each mouse. Between each mouse clean the
probe with 20% ethanol, and dry with paper towel. Repeat this step 24 hr later.
An infrared probe can also be used to measure temperature in a less invasive way.
However, infrared thermometers are generally less accurate than rectal thermometer.

4. Randomly assign mice to the experimental groups (typically THC- and vehicle-
treated groups). Verify that body weight and body temperature means are equivalent
among groups. Otherwise, perform a novel randomization, using body weight after
acclimation.
In order to remain blind to the experimental conditions, a collaborator can mask the
group label by using a color or symbol to mark group assignments on the mouse file.

Tetrad induction and assessment

5. Before starting THC injection (1 hr), prepare THC and its vehicle solutions.
In order to remain blind to the experimental conditions, a collaborator can label each
solution vial with the corresponding color or symbol.

6. Take the basal temperature of the first four mice as described in step 3 and weigh
them.
7. Inject THC or vehicle (i.p.) in the first four mice (10 ml/kg), and 30 min later inject
the next four mice.
The syringe should be filled just before the injection. One needle per mouse should be
used. One syringe can be used per group.
It is important to maintain the same delay between THC administration and testing. The
longest test of the tetrad is locomotion assessment. The number of mice that are injected
at the same delay depends on the number of mice that are tested simultaneously in the
open field.

8. After THC administration (30 min), start locomotor activity assessment. Place each
of the four mice into the center of the respective open fields, and record video for
15 min. At the end of the session, place the mice back in their home cage, and clean
the open field with 20% ethanol.
The open field area is a square (100 × 100 cm) made of polyvinylchloride walls divided
into four open fields (50 × 50 cm each) allowing assessment of locomotion in four mice
simultaneously. Light intensity into the open field should be fixed at 10 lux. A video
tracking system is used to automatically score the distance travelled.
Alternatively, automatic activity cages (e.g., Imetronic) can be used. They are made of
a cabinet including eight identical cages (22 × 12 × 18 cm) allowing assessment of
locomotion in eight mice simultaneously. Each cage is equipped with infrared beam cells
spaced along the cage 3 cm from the floor. The light intensity in the activity cage is fixed at
20 lux. The activity cages are connected to a computer which records the number of break
cells for each mouse. This system is advantageous when a high number of experimental
mice is required, such as for pharmacological screening of compounds.

9. Turn on the hot plate, and set the temperature to 52°C to ensure that the plate is at
the correct temperature at the moment of pain sensitivity measurement. Preclinical Models
of Neurologic and
Psychiatric
Disorders

9.59.3
Current Protocols in Neuroscience Supplement 80
 Figure 9.59.1 Photograph of a catalepsy cage containing a mouse showing catalepsy following
the administration of THC (10 mg/kg).

10. After returning the mice to their home cage following open field testing (5 min),
take the rectal temperature of each mouse as described in step 3.
11. Start catalepsy assessment. Take the mouse by the tail, and place its forepaws gently
on the horizontal bar and its hind legs on the floor of the cage, ensuring that the
mouse is not lying down on the floor. Release the tail and start the chronometer.
Stop the chronometer when the mouse descends from the bar (i.e., when the two
forepaws touch the floor) or when 5 min have elapsed (i.e., cut-off time). Repeat
trials until reaching the cut-off time or a maximum of three trials. Place the mice
back into their home cage. When the four mice have completed the catalepsy test,
clean the catalepsy cages with 20% ethanol.
To make a catalepsy cage, in a standard single mouse plastic cage (33 × 15 × 13 cm),
fix a horizontal bar (1 cm diameter; made from a serological pipet cut to fit in the width
of the cage) across the width at 3.5 cm from the bottom. Figure 9.59.1 shows a picture of
a catalepsy cage containing a cataleptic mouse.
Two mice can be tested simultaneously by one experimenter.
Within each trial, if the mouse moves from the bar immediately after releasing the tail
(<2 sec), place the mouse again on the bar and restart the chronometer. Repeat this
procedure a maximum of three times (see Critical Parameters and Troubleshooting).

12. After testing catalepsy of the four mice, start pain sensitivity measurement. Place
the first mouse on the hot plate and start the chronometer. Stop the chronometer and
remove the mouse from the hot plate when the mouse shows the first signs of pain
(i.e., flinching, paw licking, or startle). Remove the mouse from the hot plate after
30 sec if the mouse does not show signs of pain to avoid any wounds to the mouse.
Between each mouse, clean the hot plate with a paper towel moistened with 20%
Cannabinoid- ethanol.
Induced
Tetrad Figure 9.59.2 illustrates the time schedule of steps 7 through 12.
9.59.4
Supplement 80 Current Protocols in Neuroscience
Figure 9.59.2 Time schedule of the tetrad procedure from THC administration.

BLOCKADE OF THC-INDUCED TETRAD BY THE CB1 RECEPTOR SUPPORT

ANTAGONIST RIMONABANT PROTOCOL
THC-induced tetrad is also a useful model to evaluate the activity of putative CB1
antagonists in vivo. Here we describe a procedure for inhibiting THC-induced tetrad
with the CB1 orthosteric antagonist rimonabant.

This protocol is the same as the Basic Protocol except that the CB1 antagonist rimonabant
is administered before THC.
Additional Materials (also see Basic Protocol)
Rimonabant (e.g., Bertin Pharma, cat. no 90000484)
0.9% saline
Dimethylsulfoxide (DMSO; e.g., Sigma Aldrich)
Tween 80 (e.g., Sigma Aldrich)
THC solution (see recipe)
Vehicle control solution for THC
Glass vials
Vortex
1. Perform steps 1 to 4 of the Basic Protocol.
2. Prepare 1 mg/ml rimonabant and its vehicle solution. Weigh rimonabant powder in
a glass vial. Dissolve in 0.9% saline containing 3% DMSO and 3% Tween 80 and
vortex. Prepare 0.9% saline containing 3% DMSO and 3% Tween 80 for the vehicle.
Vortex and homogenize under magnetic agitation at room temperature until use (at
least for 30 min).
3. Before starting THC injection (1 hr), prepare THC and its vehicle solution.
4. Take the basal temperature of the first group of mice as described in step 3 of the
Basic Protocol and weight them.
5. Inject the mice (10 ml/kg) with 1 mg/ml rimonabant (10 mg/kg, i.p.) or its vehicle.
6. After rimonabant administration (30 min), inject THC solution (10 mg/kg, i.p.) or its
vehicle (10 ml/kg, i.p.).
To limit injuries caused by repeated i.p. injections, inject THC or vehicle in the opposite
side of rimonabant injection. Preclinical Models
of Neurologic and
7. Perform steps 8 through 12 of the Basic Protocol (see Fig. 9.59.2). Psychiatric
Disorders

9.59.5
Current Protocols in Neuroscience Supplement 80
 REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. For common stock
solutions, see APPENDIX 2A.
Delta-9-tetrahydrocannabinol (THC) solution
1. Prepare a 50 mg/ml stock solution by dissolving THC (provided as dronabinol resin;
e.g., THC Pharm GMBH, ref. THC-1295S-250) in 100% ethanol.
Stock solutions of 50 mg/ml THC should be stored at –20°C.
2. To prepare 10 ml of solution, add 0.3 ml Tween 80 (3% final) to a 12-ml dark glass
vial.
3. Add 0.2 ml of 50 mg/ml THC and vortex.
4. Add 0.2 ml of 100% ethanol (2% final) and vortex.
5. Bring to 10 ml with 0.9% saline.
6. Vortex and stir under magnetic agitation at room temperature until use.
Exposure to light induces degradation of cannabinoids. THC solutions should be maintained
in the dark (e.g., cover vials with aluminum foil).
Diluted resin in ethanol can be provided by the same supplier at 10 mg/ml; however, the
percentage of ethanol in injectable solution is quite elevated. Total evaporation of ethanol is
then required before preparing injectable solutions.

COMMENTARY
Background Information
The tetrad model has been success-
fully used to characterize and classify
cannabimimetic compounds before the
cloning of type-1 cannabinoid (CB1) re-
ceptors and the use of in vitro binding
assays (Howlett et al., 2002; Matsuda,
Lolait, Brownstein, Young, & Bonner, 1990).
Cannabinoid-induced tetrad determines the
structure-activity relationship of cannabinoid
agents; the intensity of their effects in the
tetrad is highly correlated with their affinity
with CB1 receptors in in vitro binding
studies (Compton et al., 1993). Moreover,
cannabinoid tetrad is mediated by brain CB1
receptors (Compton, Aceto, Lowe, & Martin,
1996; Monory et al. 2007; Rinaldi-Carmona
et al., 1994; Varvel, 2005), albeit cannabinoid-
induced analgesia is also partially mediated by
peripheral CB1 and CB2 receptors (Monory
et al. 2007; Rahn et al., 2011). Overall, the
tetrad model is a suitable in vivo screen-
ing tool for putative central CB1 receptor
ligands.
Tetrad phenotypes can be induced by com-
pounds from other pharmacological classes.
Antipsychotics such as haloperidol and cloza-
pine are able to induce the four pheno-
typic effects of the tetrad but through CB1-
independent mechanisms (Compton et al.,
Cannabinoid- 1996; Wiley, 2003). Indeed, tetrad effects in-
Induced duced by putative CB1 agonists should be
Tetrad prevented by the administration of a CB1
9.59.6
Supplement 80
receptor antagonist to avoid false positive
errors.
The mechanisms underlying cannabinoid-
induced tetrad are complex and poorly de-
scribed. CB1 receptors are expressed on dis-
tinct brain cell populations (e.g., neurons, glia)
and organelles (e.g., mitochondria; Bénard
et al., 2012; Han et al., 2012; Monory et al.,
2007). Surprisingly, CB1 receptors expressed
on forebrain GABAergic neurons, which rep-
resent the highest receptor density, do not
mediate delta-9-tetrahydrocannabinol (THC)-
induced tetrad (Monory et al., 2007). Instead,
THC-induced tetrad phenotypes depend on
complex brain pathways involving glutamater-
gic, serotoninergic, and dopaminergic signal-
ing. Partial blockade of cannabinoid-induced
tetrad phenotypes can reveal specific prop-
erties of new compounds acting either on
specific CB1 receptors subpopulations (i.e.,
located on specific cell types or cell compart-
ments) or on specific downstream signaling
pathways of CB1 receptors (Ahn, Mahmoud,
Shim, & Kendall, 2013).
Critical Parameters
Expression of catalepsy results
The latency for moving from the catalepsy
bar is recorded with a cut-off time fixed at
300 sec (5 min). Each mouse is submitted to
a maximum of three consecutive trials. When
mice reach the cut-off time at trial 1 or 2, no
Current Protocols in Neuroscience
Figure 9.59.3 The effects of THC (10 and 15 mg/kg, i.p.) on tetrad phenotypes depend on
the mouse strain. Comparison of THC-induced tetrad phenotypes in C57Bl/6J and in CD-1 Swiss
mouse lines. (A) THC administration in C57Bl/6J mice induces a significant decrease in locomotion
compared to vehicle in the automatic activity cages. The effect of THC on locomotion is not
statistically significant in CD-1 Swiss mice. (B) THC administration induces a similar decrease
in body temperature compared to vehicle in both mouse lines. (C) THC administration induces a
weaker increase in latency for moving from the catalepsy bar in CD-1 Swiss mice than in C57Bl/6J.
(D) THC administration in CD-1 Swiss mice induces a weaker increase in latency for showing the
first sign of pain in the hot plate test than in C57Bl/6J mice. Data are mean ± s.e.m. N = 8 to 16.
*p < 0.05, **p < 0.01, ***p < 0.001 versus THC 0 mg/kg (vehicle) after Kruskall-Wallis test and
Dunn’s post hoc tests. # p < 0.05, ## p < 0.01, Mann-Whitney tests for strain comparisons.

additional trial is performed. According to this
protocol, the parameter selected as the mea-
sure of catalepsy for each mouse is the maxi-
mum immobility time among the three trials.
Because mice may not be submitted to the
same number of trials, the mean immobility
time is not a correct parameter.
Floor and ceiling effects in drug screening
In order to assess the effect of putative,
more potent CB1 agonists than THC, it is
preferable to increase the maximum delay
of immobility during the catalepsy evalua-
tion (i.e., 10 min) and of pain sensitivity (i.e.,
1 min) scoring. These protocol deviations ex-
tend the length of the experiment as well as the
delay between drug administration and testing.
It is thus important to take into consideration
the pharmacokinetics of the tested drugs. For
Current Protocols in Neuroscience
instance, THC concentrations in the brain de-
crease drastically 3 hr after i.p. administration
(Duverneuil-Mayer et al., 2011). We recom-
mend not to extend the delay between injection
and test beyond 2 hr.
Statistical analyses of tetrad results
Most of the tetrad measures are limited with
cut-off, and temperature has no true zero—
two features that prevent the use of paramet-
ric statistics. Data therefore should be ana-
lyzed with non-parametric tests. For instance,
pairwise comparisons can be performed us-
ing the Mann-Whitney test (see Figs. 9.59.3
[strain comparison] and 9.59.4). The Kruskall-
Wallis test can be used to analyze the effect of Preclinical Models
one variable (e.g., treatment) among at least of Neurologic and
three groups (e.g., different doses of THC; see Psychiatric
Disorders
Figs. 9.59.4 and 9.59.5).
9.59.7
Supplement 80
 Figure 9.59.4 The CB1 antagonist rimonabant (10 mg/kg, i.p.) fully prevents the effects of
THC (10 mg/kg, i.p.) on tetrad phenotypes in C57Bl/6J mice. (A) Rimonabant (Rimo) administra-
tion fully blocks the decrease in locomotion induced by THC as assessed in the activity cages.
(B) Rimonabant administration blocks the decrease in body temperature induced by THC. (C)
Rimonabant administration prevents the increase in catalepsy latency induced by THC. (D) Ri-
monabant administration blocks the increase in the latency for pain reaction induced by THC. Data
are mean ± s.e.m. compared with Mann-Whitney tests. N = 8. ***p < 0.001 for the group injected
with rimonabant and THC vehicles (VEHRimo -VEHTHC ; first columns) versus the group injected
with rimonabant vehicle and THC (VEHRimo -THC; middle columns). ### p < 0.001, for VEHRimo -
THC group versus with group injected with rimonabant and THC (Rimo-THC; last columns).

Troubleshooting mia, catalepsy, and analgesia induced by

Measure of catalepsy
Catalepsy assessment is sensitive to the
mouse’s arousal. After THC administration,
mice can show startle (usually called “pop-
corn” effect) to environmental stimuli such as
noise or contact with the experimenter. For
instance, mice can jump when the experi-
menter is placing it on the bar. This startle
reaction is not considered a voluntary move-
ment and can bias the evaluation of catalepsy.
Therefore, it is important to perform catalepsy
testing in a quiet and separate room to mini-
mize the occurrence of startle behaviors.
Cannabinoid- Anticipated Results
Induced The Basic Protocol is able to reveal a
Tetrad dose-dependent hypolocomotion, hypother-
9.59.8
Supplement 80
THC (3, 10, or 15 mg/kg) in C57Bl/6J mice
(Fig. 9.59.5). Under these experimental condi-
tions, THC administered at 10 mg/kg induces
strong effects on the tetrad phenotypes, which
is consistent with studies performed in other
laboratories (DeLong, Wolf, Poklis, & Licht-
man, 2010; Monory et al., 2007). Using this
procedure, it is possible to study the activ-
ity of molecules thought to bind CB1 recep-
tors by comparing their effects with those of
THC.
Under these conditions, CD-1 Swiss mice
are less sensitive to the effects of THC on
the tetrad (Fig. 9.59.3). It is thus important
to carefully select the dose range of the stud-
ied compounds according to the mouse strain
used.
Current Protocols in Neuroscience
Figure 9.59.5 Dose-dependent effects of THC administration (3, 10, or 15 mg/kg, i.p.) on the
tetrad phenotypes in C57Bl/6J mice. (A) THC administration induces a decrease in locomotion
as compared to vehicle in the open field test. (B) THC administration induces a decrease in body
temperature as compared to vehicle. For each mouse, the difference between basal temperature
(i.e., taken before THC or vehicle administration) and the temperature measured after THC or
vehicle administration is expressed. (C) THC administration induces an increase in the latency
for moving from the catalepsy bar. (D) THC administration induces an increase in the latency for
showing the first sign of pain in the hot plate test. In the four tetrad phenotypes, the effects of
THC are statistically significant at 10 and 15 mg/kg. At 15 mg/kg, THC abolishes locomotion and
induces maximal catalepsy time. Data are mean ± s.e.m. N = 9 to 13. **p < 0.01, ***p < 0.001
versus THC 0 mg/kg (vehicle) and #p < 0.05, ##p < 0.01 versus THC 3 mg/kg after Kruskall-Wallis
test and Dunn’s post hoc tests.

The Support Protocol has been included
in this unit to show the inhibition of THC-
induced tetrad phenotypes by an antagonist of
CB1 receptors (Fig. 9.59.4). This procedure
should be performed to demonstrate that tetrad
phenotypes induced by a compound are medi-
ated by CB1 receptors.
Time Considerations
THC-induced tetrad is a relatively fast pro-
cedure that is well suited for in vivo drug
screening. However, obtaining the authoriza-
tion for cannabinoid purchase and use often
requires several months. It is therefore impor-
tant to anticipate these administrative proce-
dures before the desired start of the study.
Following animal arrival, at least 9 days
of habituation to the animal facility and to
Current Protocols in Neuroscience
temperature measurements are required before
the tetrad study. This tetrad procedure is per-
formed in 90 min. Depending on the equip-
ment used for measuring locomotion (open
field or automatic activity cages), four or eight
mice can be tested simultaneously. Several
batches of mice can be tested successively at
a day. Typically, we can run three batches of
eight mice per day, allowing testing of up to
24 mice.
Acknowledgments
We thank all the personnel of the An-
imal Housing Facility of the Neurocentre
Magendie granted by INSERM and LabEX Preclinical Models
BRAIN ANR-10-LABX-43. This work was of Neurologic and
supported by INSERM, the University of Psychiatric
Disorders
Bordeaux, and Region Aquitaine funds and
9.59.9
Supplement 80
 by a LabEX BRAIN grant (CANNAPREG;
P.-V.P.).
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