Pain 139 (2008) 190–200

www.elsevier.com/locate/pain

Endocannabinoid and serotonergic systems are needed

for acetaminophen-induced analgesia
Christophe Mallet a,b, Laurence Daulhac a,c, Jérôme Bonnefont a,b, Catherine Ledent d,
Monique Etienne a,b, Eric Chapuy a,b, Frédéric Libert a,b,e, Alain Eschalier a,b,e,*
a
INSERM E9904, U766, Facultés de Médecine et de Pharmacie, 28, Place Henri Dunant, B.P. 38, F-63001 Clermont-Ferrand Cedex 01, France
b
Clermont Université, Université d’Auvergne, Faculté de Médecine, Laboratoire de Pharmacologie Médicale, F-63001 Clermont-Ferrand, France
c
Clermont Université, Université d’Auvergne, Faculté de Pharmacie, Laboratoire de Pharmacologie, F-63001 Clermont-Ferrand, France
d
IRIBHM, Université Libre de Bruxelles, B-1070 Bruxelles, Belgium
e
CHU Clermont-Ferrand, Service de Pharmacologie, Hôpital G. Montpied, F-63003 Clermont-Ferrand, France

Received 16 October 2007; received in revised form 18 January 2008; accepted 24 March 2008

Abstract

Acetaminophen is the most used analgesic/antipyretic drug. Its unclear mechanism of action could rely on cyclooxygenase inhi-
bition, NO synthesis blockade or reinforcement of the serotonergic system. Here we show that in thermal, mechanical and chemical
pain tests, AM-251, a specific CB1 receptor antagonist, abolished the analgesic action of acetaminophen, which was also lost in CB1
receptor knockout mice. Moreover, acetaminophen was shown unable to bind to CB1 receptors demonstrating an indirect involve-
ment of these receptors in the analgesic effect of this compound. Accordingly with these results, we also demonstrated that the inhi-
bition of FAAH, an enzyme involved in the cerebral metabolism of acetaminophen into AM404, known to reinforce the activity of
the endocannabinoid system, suppressed the antinociceptive effect of acetaminophen. In addition, similarly to the interaction of
acetaminophen with bulbospinal serotonergic pathways and spinal serotonin receptors, we observed that the antinociceptive activity
of ACEA, a CB1 receptor agonist, was inhibited by lesion of bulbospinal serotonergic pathways and antagonists of spinal 5-HT
receptors. We therefore propose that acetaminophen-induced analgesia could involve the following sequence: (1) FAAH-dependent
metabolism of acetaminophen into AM404; (2) indirect involvement of CB1 receptors by this metabolite; (3) endocannabinoid-
dependent reinforcement of the serotonergic bulbospinal pathways, and (4) involvement of spinal pain-suppressing serotonergic
receptors.
Ó 2008 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.

Keywords: Acetaminophen; Endocannabinoids; Fatty acid amide hydrolase; Nociception; Serotonin

1. Introduction ity of the descending serotonergic pathways on spinal

nociceptive processing, which has been supported by dif-
The mechanism of the antinociception elicited by ferent groups [5,43,48]. Indeed, the lesion of the bulbo-
acetaminophen is not yet elucidated. One main hypo- spinal descending serotonergic pathways abolishes the
thesis relies on the reinforcement of the inhibitory activ- antinociceptive action of acetaminophen [48]. More-
over, our group demonstrated that different spinal 5-
HT receptor subtypes are involved in the antinociceptive
*
Corresponding author. Address: INSERM E9904, U766, Facul- effect of acetaminophen in rats [6,39] and recently con-
tés de Médecine et de Pharmacie, 28, Place Henri Dunant, B.P. 38,
firmed this serotonergic mechanism in humans [42].
F-63001 Clermont-Ferrand Cedex 01, France. Tel.: +33 4 73 17 82 32;
fax: +33 4 73 27 71 62. However, those mechanisms or others do not explain
E-mail address: alain.eschalier@u-clermont1.fr (A. Eschalier). how the action of acetaminophen is initiated.

0304-3959/$34.00 Ó 2008 Published by Elsevier B.V. on behalf of International Association for the Study of Pain.
doi:10.1016/j.pain.2008.03.030
 C. Mallet et al. / Pain 139 (2008) 190–200 191

The discovery of an involvement of endocannabi- for Research and Ethical Issues of IASP [54] and to our Insti-
noids on pain modulation opens new mechanistic per- tutional Ethic Committee for animal experiments. Animals
spectives [12,44]. Anandamide and 2-arachidonoyl- were housed under controlled environmental conditions (21–
glycerol, two endogenous ligands of CB1 and CB2 recep- 22 °C; 55% humidity) and kept under a 12/12 h light/dark
cycle, with food and water ad libitum for a week prior to start-
tors, mainly metabolized by the fatty acid amide hydro-
ing the experiments in order to acclimatize.
lase (FAAH), and the monoacylglycerol lipase,
respectively, induce antinociceptive effects [24,51]. Simi-
2.2. Intrathecal injections
larly, activation of this system by exogenous ligands for
cannabinoid (particularly CB1) receptors induces antin- Intrathecal (i.t.) injections were performed under isoflurane
ociception in various acute pain tests in rodents anesthesia (4% induction, 2% maintenance) according to Mes-
[16,24,33] but also in several animal models of chronic tre et al. [34]. The anesthetized rat was held in one hand by the
pain [17]. Moreover, the combination of D9-THC and pelvic girdle and a 25-gauge  1-inch needle connected to a
cannabidiol is proposed in the treatment of pain for 25 ll Hamilton syringe was inserted into the subarachnoidal
patients with multiple sclerosis [46]. Several studies space between lumbar vertebrae L5 and L6, until a tail flick
reported that cerebral injection of cannabinoids in the was elicited. The syringe was held in position for few seconds
periaqueductal gray (PAG) or the rostroventral medulla after the injection of a volume of 10 ll/rat.
(RVM) elicits antinociception, therefore suggesting the
2.3. Lesion of the descending serotonergic pathways
modulation of descending pathways to inhibit pain pro-
cessing at the spinal level [30,32,33].
5,7-Dihydroxytryptamine (5,7-DHT, 100 lg/rat), dissolved
Interestingly, recent findings showed that acetamino- in saline containing 0.2 mg/ml ascorbic acid, was administered
phen could be metabolized in the brain into AM404, a intrathecally 7 days before the experiment. Desipramine
compound able to inhibit the reuptake of anandamide (10 mg/kg) was i.p. injected 30 min before 5,7-DHT to prevent
[14] thanks to FAAH [23] and that the antinociceptive the reuptake of 5,7-DHT by catecholaminergic neurons. On
activity of acetaminophen may rely on an interaction the day of the experiment, animals were treated with the active
with the endocannabinoid system [37], even if this last drug and submitted to the paw pressure test (before and 15, 30,
result has to be cautiously interpreted. We therefore 45, 60, 90 and 120 min after administration) before sacrificing
hypothesized that the interaction of acetaminophen with them and removing spinal cords in order to confirm 5,7-DHT
the endocannabinoid system could be on the basis of the efficacy by determining 5-HT lumbar levels by HPLC. Briefly,
lumbar dorsal horn samples were homogenized in a saturated
reinforcement of the serotonergic system.
KCl solution and centrifuged at 15,000g for 15 min. The
In this line, we investigated the possible interaction
resulting supernatants were mixed with 400 ll of an internal
between acetaminophen-induced antinoception and the standard (n-methyl-serotonin 0.6 mg/l, diluted in a pH 11 gly-
cannabinoid system by performing three experimental cin buffer) and extracted with 2.5 ml of dichloromethane/n-
series: (i) we first studied the involvement of CB1 butanol (75/25). The organic layer was back-extracted with
receptors on the acetaminophen-elicited antinocicep- 300 ll of 0.1 M phosphate buffer (pH 4.4). One-hundred
tion; (ii) we determined if this involvement was direct microliters of this buffer was injected in the HPLC system with
or not; and (iii) we assessed the involvement of the electrochemical detector.
serotonergic descending bulbospinal pathways and
spinal 5-HT receptors in the antinociceptive effect of 2.4. Behavioral pain tests
arachidonyl-20 -chloroethylamide (ACEA), a CB1 recep-
tor agonist to compare it with that demonstrated for 2.4.1. Formalin test
Rats and mice received 50 and 25 ll of 2.5% formalin
acetaminophen.
injected subcutaneously (s.c.) into the dorsal surface of the
Our results demonstrated that acetaminophen,
hind paw, respectively. Drugs were administered at different
devoid of any direct effect on CB1 receptors, needed a times before formalin (40 min in rats or 30 min in mice for
FAAH-dependent metabolism to exert its CB1-mediated acetaminophen; 10 min for ACEA in rats). Biting and licking
antinociceptive effect that could lead to the reinforce- of the injected paw was monitored by measuring the total
ment of the activity of the 5-HT bulbospinal pathways. duration of the response in seconds during the two typical
phases of nociceptive behavior (phase I: 0–5 min; phase II:
2. Methods 20–40 min for rats or 15–40 min for mice).

2.1. Animals 2.4.2. Paw pressure test

Rats were submitted to the paw pressure test using an Ugo
Adult male Sprague–Dawley rats weighing 175–200 g were Basile analgesimeter (Apelex, tip diameter of the probe: 1 mm,
purchased from Charles River. Male CB1 null mutant (Cb1/) weight: 30 g). Nociceptive thresholds, expressed in grams (g),
mice [28] and wild-type mice (CD1 background) were gener- were measured by applying an increasing pressure to the right
ously supplied by Dr Ledent. Animal care and experiments hind paw of the animals until a squeak (vocalization threshold)
were carried out according to the guidelines of the Committee was obtained (cut-off: 750 g). Treatments were done after the
192 C. Mallet et al. / Pain 139 (2008) 190–200

measurement of two consecutive stable vocalization threshold 2.6. Experimental procedure

values and testing was performed 15, 30, 45, 60, 90 and
120 min after drug administration. All experiments were performed blind by a single experi-
menter using a parallel group design. Treatments were ran-
2.4.3. Tail immersion test domized and administered according to the method of equal
Tail of the rats was immersed in a hot water bath main- blocks in order to assess the effect of the different treatments
tained at 46 °C. The time latency for tail withdrawal was then (blindly administered) at the same time interval to avoid
determined and a cut-off time of 30 s was applied to avoid unverifiable and time-variable environmental influences. One
injury. Treatments were done after the measurement of two block includes a number of animals corresponding to the num-
consecutive stable withdrawal latency values and testing was ber of the different treatments administered in each experi-
performed 15, 30, 45, 60, 90 and 120 min after drug ment; the number of blocks corresponds to the number of
administration. animals per treatment; treatments in the different blocks were
randomized, the order of treatments being different from one
2.5. Assessment of tetrad effect block to an other; all animals in a same block were tested in
the same short laps of time; different animals were used in each
The general behavioral tests performed to screen for typical experiments (n = 6–10 per treatment, according to the experi-
high dosage cannabinoid effects, namely, antinociception, ments) which were performed in a quiet room. When the
motor impairment, catalepsy and hypothermia [8] were dose–effect relationship was studied, the following groups were
conducted. injected with active drug (acetaminophen or ACEA, 3–4 doses
for each) or vehicle. When the influence of antagonists or
2.5.1. Antinociception inhibitors was tested, four groups of different animals were
Antinociception was assessed using the paw pressure test injected as follows: vehicle + vehicle; vehicle + active drug
described above. (acetaminophen or ACEA); antagonist or inhibitor + vehicle;
antagonist or inhibitor + active drug. Acetaminophen (100–
2.5.2. Spontaneous locomotor activity 600 mg/kg) was administered orally (p.o.) while ACEA (1–
The system used for recording spontaneous activity of the 10 mg/kg), URB597 (0.15 mg/kg) haloperidol (5 mg/kg), desi-
rats (VideoTrack; ViewPoint, Champagne au Mont d’Or, pramine (10 mg/kg) and AM-251 (3 mg/kg) were administered
France) comprised a digital video camera set above 4 dark intraperitoneally (i.p.) and PMSF (10 mg/kg) subcutaneously
25  25  40 cm enclosures. A computer was used to analyze (s.c.). WAY-100635 (40 lg/rat), tropisetron (0.5 lg/rat) and
digital pictures (25 frames/second). Animal activity was 5,7-DHT (100 lg/rat) were administered intrathecally.
assessed by the total number of pixels that changed color
between 2 successive pictures (from black to white and from 2.7. CB1 receptor binding assay
white to black, when an animal changed its position). Sponta-
neous locomotor activity was counted for 5 min at 15, 30, 60 Evaluation of the affinity of acetaminophen for the CB1
and 120 min post-administration. cannabinoid receptor in transfected CHO cells was performed
in a radioligand binding assay using [H3]CP 55940, according
2.5.3. Catalepsy to the protocol of Rinaldi-Carmona et al. [45].
Catalepsy was measured using the ‘‘ring test” described by
Pertwee [41] modified for the rat [9]. The apparatus consisted 2.8. Drugs
of a plastic ring (12 cm diameter) fixed horizontally at a height
allowing the hindpaws of rats to just touch the bench. The rat Acetaminophen was generously provided by Bristol-Myers-
was placed across the ring and the time (s) during which the rat Squibb (France). N-[2-[4-(2-methoxyphenyl)-1-piperazinyl]-
remained motionless on the ring was recorded. The test was ethyl]-N-2-pyridinylcyclohexane-carboxamide maleate salt
conducted for 4 min at 15, 30, 60 and 120 min post- (WAY-100,635), 5,7-dihydroxytryptamine creatinine sulfate
administration. (5,7-DHT), desipramine, haloperidol and Phenylmethylsul-
fonyl Fluoride (PMSF) were purchased from Sigma (France).
2.5.4. Body temperature N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-
Rats were placed individually into an environmental room methyl-1H-pyrazole-3-carboxamide (AM-251) and Arachido-
maintained at a constant temperature of 21 ± 0.3 °C. The ani- nyl-20 -chloroethylamide (ACEA) were obtained from Tocris
mals were allowed to acclimate for 60 min before the first tem- Cookson (UK), and tropisetron from Novartis (France). 30 -
perature reading. Prior to drug administration, baseline carbamoyl-biphenyl-3-yl-cyclohexylcarbamate (URB597) was
temperatures were taken every 30 min for 90 min using a from Cayman Chemical (France). PMSF, URB597, AM-251
thermistor probe (Ellab, Denmark), which was lubricated and ACEA were dissolved in DMSO, while the other drugs
and inserted approximately 7 cm into the colon. A digital ther- were dissolved in sterile physiological serum (0.9% NaCl).
mometer (Ellab, Denmark) was used to record body tempera-
ture. Rats were unrestrained throughout the experiment, with 2.9. Statistical analysis
only the tail being held gently between two fingers. Following
the baseline interval, treatments were administered. Body tem- Data are presented as the means ± SEM. Data from forma-
perature was recorded 15, 30, 60 and 120 min post- lin test, hypothermia and locomotor activity assessment (at 2 h
administration. and 60 min, respectively) were analyzed by a one-way ANOVA
 C. Mallet et al. / Pain 139 (2008) 190–200 193

followed by a Student–Newman–Keuls’ test, when the F-value tions as high as 1 mM (percentage of specific binding:
was significant. For paw pressure, tail immersion and tetrad 8.6 ± 1.2%; 6.9 ± 2.8% and 11.2 ± 3.0% for acetamino-
tests, a two-way ANOVA analysis was performed and, when phen concentrations of 107, 105 and 103 M, respec-
the F-value was significant, a Dunnett’s test was used to ana- tively) in transfected CHO cells expressing CB1
lyze the time-course of the effects. The level of statistical signif-
receptor. These results indicated that acetaminophen
icance was set at p < 0.05.
activates CB1 receptors via an indirect pathway.

3. Results 3.2.2. Acetaminophen-induced antinociception relies on

FAAH-dependent AM404 formation
3.1. Acetaminophen/endocannabinoid system relationship We assessed the influence of FAAH inhibitors on the
antinociceptive activity of acetaminophen. PMSF
3.1.1. Evidence of a CB1 receptor involvement in the (10 mg/kg, s.c), used at a dose shown to be able to abol-
antinociceptive effect of acetaminophen ish the FAAH-dependent metabolism of acetaminophen
Acetaminophen (300 mg/kg, p.o.) induced a signifi- [23], significantly inhibited the antinociceptive effect of
cant antinociceptive activity in the formalin, (30.2 ± acetaminophen in both the paw pressure and the forma-
7.8% and 44.3 ± 10.2% inhibition in phase I and II, lin tests (Fig. 4a and b). In addition, URB597 (0.15 mg/
respectively; Fig. 1a), paw pressure (maximal vocalization kg, i.p.), an irreversible brain-penetrating FAAH inhib-
threshold increase: 97.4 ± 14.1%; Fig. 1b) and tail immer- itor, also reversed the antinociception elicited by acet-
sion (maximal latency time increase: 37 ± 6%; Fig. 1c) aminophen (Fig. 4c and d).
tests. In each test, this effect was completely abolished
by AM-251 (3 mg/kg, i.p.), a selective CB1 receptor 3.3. CB1 receptor agonist, ACEA, elicits antinociception
antagonist. via a serotonergic bulbospinal mechanism
To confirm these observations, we examined the
antinociceptive activity of acetaminophen in CB1 recep- In order to demonstrate that the involvement of CB1
tor knockout mice. While acetaminophen reduced the receptors in the antinociceptive effect of acetaminophen
nociceptive behavior elicited by formalin in phase I was in line with its previously described serotonergic
(29.1 ± 6.1%) and phase II (37.1 ± 10.1%) in wild- mechanism, we studied the involvement of both bulbo-
type mice, it did not produce any significant effect when spinal serotonergic pathways and spinal 5-HT receptors
administered in Cb1/ mice (Fig. 1d). in the effect of a CB1 receptor agonist.
We first performed a dose–response effect of ACEA
3.1.2. Tetrad effects of acetaminophen (i.p.) in order to look at the antinociceptive activity of this
Those results suggested the involvement of CB1 CB1 agonist in the paw pressure test and compare its
receptors in the antinociceptive effect of acetaminophen. potency with acetaminophen. ACEA induced a dose-
We therefore looked at the ability of acetaminophen to dependent antinociceptive effect for a maximal duration
induce cannabinoid tetrad effects. Acetaminophen dose of 45 min for 10 mg/kg (Fig. 5a). ED50s were 125.9 and
dependently induced three of these effects: we observed 2.8 mg/kg for acetaminophen and ACEA, respectively,
a dose-dependent antinociceptive activity from 100 to at the peak effect time. The effect of ACEA was inhibited
300 mg/kg (Fig. 2a), as well as a reduction in locomotor by AM-251, a CB1 receptor antagonist (Fig. 5b).
activity and a hypothermia which were delayed in com- We then examined the action of ACEA (3 mg/kg)
parison with the antinociceptive effect and needed higher after lesioning the serotonergic bulbospinal pathways
doses (300–600 mg/kg; Fig. 2b, 2c). However, acetami- using 5,7-DHT. Such a treatment significantly reduced
nophen did not induce any catalepsy, even at 600 mg/ the 5-HT levels in the dorsal horn of the spinal cord
kg, while haloperidol, used as positive control, was by 95.5 ± 0.3% (2301 ± 114 vs 103 ± 7 ng/g of spinal
active in our conditions (Fig. 2d). cord tissue for control and 5,7-DHT-treated rats,
Locomotor depressive and hypothermic effects respectively) and led to the suppression of the effect of
induced by acetaminophen (300 mg/kg, p.o.) were not ACEA in the paw pressure test (Fig. 5c).
mediated by CB1 receptors as a pre-treatment with To further compare the serotonergic profile of acet-
AM-251 (3 mg/kg, i.p.) failed to abolish these two effects aminophen and a CB1 receptor agonist, we investigated
(Fig. 3a and b). whether ACEA-elicited antinociception was involving
the same spinal serotonergic receptors than those identi-
3.2. Interaction between acetaminophen and CB1 fied with acetaminophen. We previously showed that
receptors intrathecal injection of the 5-HT1A receptor antagonist
WAY-100,635 and tropisetron, a 5-HT3/4 receptor
3.2.1. Acetaminophen does not bind to CB1 receptors antagonist, reversed the antinociceptive effect of acet-
We observed no significant displacement of the bind- aminophen in the formalin test and the paw pressure
ing of [H3]CP 55940 by acetaminophen at concentra- test, respectively [1,5,6,40]. We observed that the
194 C. Mallet et al. / Pain 139 (2008) 190–200

Phase I Phase II
135 225

Biting & licking time (s)

Biting & licking time (s)

120 200
105 175
90 * 150
75 125 **
60 100
45 75
30 50
15 25
0 0
Aceta
a (p.o.) - + - + Aceta
a (p.o.) - + - +
AM251 (i.p.)
(i .) - - + + AM251 (i.p.)
(i .) - - + +

600 **
Veh
h - V eh
** Veh
h - Aceta
Vocalization Threshold (g)

500 AM -251 - Veh

AM -251 - Ace
Aceta
**
400
*
300

200
0
0 15 30 45 60 75 90 105 120
Time (min)

11 Veh
h - V eh
** **
** Veh
h - Aceta
Time withdrawal latency (s)

10 AM-251 - Veh
AM-251 - Ace
Aceta
9

6
0
0 15 30 45 60 75 90 105 120
Time (min)

Phase I Phase II
160 400
Biting & licking time (s)
Biting & licking time (s)

140 350
120 * 300
100 250 *
80 200
60 150
40 100
20 50
0 0
Acet a (p.o.) - + - + Aceta (p.o.) - + - +

WT C b1 - / - WT C b1 - / -

Fig. 1. The CB1 receptors are involved in the antinociceptive effect of acetaminophen. The effect of acetaminophen (Aceta; 300 mg/kg, p.o.) was
assessed (a) in the formalin, (b) the paw pressure, and (c) the tail immersion test in rats after i.p. administration of AM-251 (3 mg/kg) or vehicle. In all
tests, AM-251 was administered 10 min before acetaminophen. (d) Formalin test was performed in wild-type (WT) or in Cb1/ mice after treatment
by acetaminophen (Aceta; 300 mg/kg, p.o.) or vehicle. Error bars represent SEM. *p < 0.05, **p < 0.01 as compared with the vehicle-treated group;
n = 6–8 per group.

antinociceptive activity of ACEA was blocked by WAY- by tropisetron (0.5 lg/rat, i.t.) in the paw pressure test
100,635 (40 lg/rat, i.t.) in the formalin test (Fig. 6a) and (Fig. 6b).
 C. Mallet et al. / Pain 139 (2008) 190–200 195

Antinociception Motor impairment

600 * Veh
Aceta 100 mg/kg
* Veh
14

Vocalization Threshold (g)

Aceta 200 mg/kg

Locomotor activity (*100 cm)

500 Aceta 100 mg/kg 12 Aceta 300 mg/kg
* Aceta 200 mg/kg Aceta 600 mg/kg
* * Aceta 300 mg/kg 10
400 * 8

6
300 *
4 *

200 2 *

0 0
0 15 30 45 60 75 90 105 120 0 15 30 45 60 75 90 105 120
Time post dose (min) Time post dose (min)

Hypothermia Catalepsy
38.8

38.4 300
Temperature (°C)

38.0 * *
250
*
37.6
200

Latency( s)
* Veh
37.2 Aceta 100 mg/kg
150 * Aceta 200 mg/kg
36.8 Veh * Aceta 300 mg/kg
Aceta 100 mg/kg *
36.4 100 Aceta 600 mg/kg
Aceta 200 mg/kg Haloperidol5 mg/kg
36.0 Aceta 300 mg/kg 50
Aceta 600 mg/kg
*
0 0
0 15 30 45 60 75 90 105 120 0 15 30 45 60 75 90 105 120
Time post dose (min) Time post dose (min)

Fig. 2. Dose–effect relationship of acetaminophen in the behavioral tetrad effect. Animals were tested after administration (at time 0) of
acetaminophen (aceta; 100, 200, 300 or 600 mg/kg, p.o.) or vehicle (a) in the paw pressure test, (b) by video tracking, (c) in the ring test and (d) for
hypothermia. Haloperidol (5 mg/kg, i.p.) was used as a positive control for catalepsy (c). Error bars represent SEM. *p < 0.05, as compared with the
vehicle-treated group; n = 6–8 per group.

4. Discussion for instance, from the inhibition of cyclooxygenases

[36,38], to the inhibition of NO synthesis [4], to the
From its initiating molecular mechanism to the reinforcement of the serotonergic system [5,42,43,48].
mediators/receptors responsible for antinociception, They all failed to fully explain the action of the drug,
the singular mechanism of action of acetaminophen probably because some gaps remain to be filled
remains elusive and is still a matter of debate more between all those different systems. Here, we report
than one century after the discovery of this com- events that could serve as a new basis to resolve this
pound. Several hypotheses have been emitted going, mystery.

Before treatments
38.8 After treatments

38.4 8
Locomotor activity (*100 cm)

38.0 * 7
*
Temperature (ºC)

6 * *
37.6 **
5
37.2 ***
4
36.8
3
36.4
2
36.0 1

0 0
Veh Veh AM-251 AM-251 Veh Veh AM-251 AM-251
Treatments: Treatments:
Veh Aceta Veh Aceta Veh Aceta Veh Aceta

Fig. 3. The CB1 receptors are not involved in the motor impairment and hypothermia due to acetaminophen. (a) Rectal temperature was measured
in rats before and after treatments with an i.p. injection of AM-251 (3 mg/kg) or vehicle and an oral administration of acetaminophen (300 mg/kg) or
vehicle. Data shown were obtained 2 h after administration of acetaminophen. (b) Motor activity was assessed in rats treated with AM-251 (3 mg/kg,
i.p.) or vehicle and acetaminophen (300 mg/kg, p.o.) or vehicle. Data shown were obtained 1 h after administration of acetaminophen. In all tests,
AM-251 was administered 10 min before acetaminophen. Error bars represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with the same
treated group, before treatments (a) or with the vehicle-treated group (b); n = 6–8 per group.
196 C. Mallet et al. / Pain 139 (2008) 190–200

Phase I Phase II
135 250

Biting & licking time (s)

Biting & licking time (s)

120 225
105 200
90 * 175
75 150 *
125
60 100
45 75
30 50
15 25
0 0
Aceta (p.o.) - + - + Aceta (p.o.) - + - +
PMSF (s.c.) - - + + PMSF (s.c.) - - + +

700
** Veh - Veh
Vocalization Threshold (g) Veh - Aceta
600 **
PMSF - Veh
PMSF - Aceta
500

400

300

200
-15 0 15 30 45 60 75 90 105 120
Time (min)

Phase I Phase II
135 250
Biting & licking time (s)

Biting & licking time (s)

120 225
105 200
* 175
90
75 150 **
125
60 100
45 75
30 50
15 25
0 0
Aceta (p.o.) - + - + Aceta (p.o.) - + - +
URB597 (i.p.) - - + + URB597 (i.p.) - - + +

700
**
Veh - Veh
Vocalization Threshold (g)

600 ** Veh - Aceta

URB597 - Veh
500 URB597 - Aceta
**
400

300

200
-15 0 15 30 45 60 75 90 105 120
Time (min)

Fig. 4. The FAAH inhibitors, PMSF and URB597, block the antinociceptive effect of acetaminophen. The effect of acetaminophen (Aceta; 300 mg/
kg, p.o.) was assessed in the formalin (a and c) and the paw pressure tests (b and d) after administration of PMSF (10 mg/kg, s.c.) or vehicle (a and b)
and URB597 (0.15 mg/kg, i.p.) or vehicle (c and d). In all tests, PMSF and URB597 were administered 20 and 10 min before acetaminophen,
respectively. Error bars represent SEM. *p < 0.05, **p < 0.01 as compared with the vehicle-treated group; n = 6–8 per group.

Our results propose that the endocannabinoid system acetaminophen and AM-251, a specific antagonist of
would be a major component of the activity of acetami- CB1 receptors, which inhibited the effect of acetamino-
nophen and could be an essential link between some of phen. If our results are in accordance with a recent
the different hypotheses. Firstly, we observed that the report [37], it is noteworthy that this hypothesis needed
antinociceptive activity of acetaminophen relied on the to be verified using different noxious stimuli. Indeed, in
endocannabinoid system in different behavioral tests in this previous study, the effect of the drug was assessed
rats and mice. We demonstrated the involvement of only using high doses of acetaminophen (up to
CB1 receptors using both knockout mice insensitive to 1000 mg/kg, p.o.) in the thermal hot plate nociceptive
 C. Mallet et al. / Pain 139 (2008) 190–200 197

600 * *
ify this last point. Secondly, we looked at the effect of
acetaminophen on the classical ‘‘tetrad” which has
Veh
allowed to establish a cannabimimetic behavioral profile
Vocalization Threshold (g)

500 ACEA 1 mg/kg

* ACEA 3 mg/kg for the naturally occurring D9-THC and anandamide
* ACEA 10 mg/kg
400 *
[8,52] or several cannabinoid receptor agonists [17].
Using orally administered doses of acetaminophen from
300 100 to 600 mg/kg, we observed three of the four tetrad
effects (antinociception, motor impairment, hypother-
200 mia), but failed to observe catalepsy. According to Mar-
0 tin et al. [31] and Fride and Mechoulam [18] who
0 15 30 45 60 75 90 105 120 consider that only an activity of compounds in all four
Time (min)
tests would characterize them as cannabimimetics, we
600
could suspect that acetaminophen is not a cannabimi-
Veh - Veh
*** *** Veh - ACEA metic compound. This conclusion is reinforced by the
lack of inhibition of its locomotor depressive and hypo-
Vocalization Threshold (g)

AM - 251 - Veh
500
AM - 251 - ACEA thermic effects by a CB1 receptor antagonist and is in
400
line with the finding that non-cannabinoid compounds
(amphetamine, scopolamine, morphine, desipramine,
300
pimozide, pentobarbital, ethanol, and diazepam) can
induce some of the tetrad effects showing the lack of
200 specificity of this procedure [53].
0
Whatever, the results obtained here in pain tests dem-
0 15 30 45 60 75 90 105 120 onstrated that intact CB1 receptors are needed for the
Time (min) antinociceptive effect of acetaminophen. The question
would now be how CB1 receptors are activated. Indeed,
600 Veh - Veh
** Veh - ACEA
we did not observe any binding of acetaminophen to
CB1 receptors with a high affinity confirming the results
VocalizationT hreshold (g)

5,7 - DHT - Veh

500
**
5,7 - DHT - ACEA of Fowler et al. [15]. A recent study suggested that every-
**
thing could start from the liver where acetaminophen is
400
metabolized into p-aminophenol, which can be further
transformed into AM404 in the brain after a FAAH-
300
dependent conjugation with arachidonic acid [23].
200
AM404 is in turn able to reinforce the activity of the
0
endocannabinoid system through both the inhibition
0 15 30 45 60 75 90 105 120 of cellular reuptake [3,19] and the reduction of degrada-
Time (min) tion (by inhibiting FAAH) [20] of anandamide. Several
Fig. 5. Serotonergic bulbospinal pathways are needed for CB1 agonist- studies also reported that AM404 could induce a can-
induced antinociception. (a) Animals were tested after administration nabinoid-dependent antinociception [21,27]. In line with
of ACEA (1, 3, 10 mg/kg, i.p.) or vehicle in the paw pressure test. (b) those data, we studied the effect of FAAH inhibitors on
The effect of ACEA (3 mg/kg, i.p.) was then assessed in the paw the antinociceptive activity of acetaminophen to assess
pressure tests 10 min after i.p. injection of AM-251 (3 mg/kg) or
the involvement of this metabolic pathway. We want,
vehicle. (c) Vocalization thresholds were determined in rats after i.p.
injection of ACEA (3 mg/kg) or vehicle in healthy rats or in rats with first, to mention that the inhibition of FAAH can reduce
lesioned serotonergic bulbospinal pathways by 5,7-DHT. Error bars the degradation of anandamide [20], which could
represent SEM. *p < 0.05, **p < 0.01, ***p < 0.001 as compared with account for an antinociceptive effect [25,47]. In order
the vehicle-treated group; n = 6–8 per group. to avoid such an effect, we used low FAAH-inhibiting
doses of URB597 (0.15 mg/kg, i.p.) and PMSF
test. Those doses can induce toxicity [26], as well as (10 mg/kg, s.c.) that did not induce any intrinsic antino-
motor impairment and hypothermia, observed in the ciception in our conditions, as previously shown by
present work, which induce a false antinociception when Kathuria et al. [25] and Compton and Martin [7],
motor reflexes and thermal stimulus are used [29]. More- respectively. Using these inhibitors, in these conditions,
over, Haller et al. [22] failed to show any inhibitory we showed, for the first time, that this enzyme is needed
effect of a CB1 antagonist using phenylbenzoquinone- for the effect of acetaminophen. Thus FAAH-dependent
induced abdominal writhes which could suggest differ- formation of AM404 seems to be necessary for acetami-
ences in CB1 involvement due to the stimulus used or nophen-induced antinociceptive effect and could
to the site where the noxious stimulus was applied account for an indirect involvement of CB1 receptors
(somatic versus visceral). Further work is needed to ver- by the analgesic drug. However, further studies are
198 C. Mallet et al. / Pain 139 (2008) 190–200

Phase I Phase II
150 350

Biting & licking time (s)

Biting & licking time (s)

135 300
120
250 **
105 **
90 200
75
60 150
45 100
30
50
15
0 0
ACEA (i.p.) - + - + ACEA (i.p.) - + - +
WAY-100635 (i.t.) - - + + WAY-100635 (i.t.) - - + +

600 Veh - Veh

** **
Veh - ACEA

Vocalization Threshold (g)

Tropisetron - Veh
500
Tropisetron - ACEA

400 **

300

200

0
0 15 30 45 60 75 90 105 120
Time (min)

Fig. 6. Spinal 5-HT receptors are involved in CB1 agonist-induced antinociception in rats. The effect of ACEA (3 mg/kg, i.p.) was assessed (a) in the
formalin and (b) the paw pressure tests after i.t. injection of (a) WAY-100,635 (40 lg/rat) or vehicle and (b) tropisetron (0.5 lg/rat) or vehicle.
Intrathecal administrations were performed 5 min before acetaminophen or saline. Error bars represent SEM. **p < 0.01 as compared with the
vehicle-treated group; n = 6–8 per group.

needed to assess the antinociceptive effect and the mech- That would lead to suspect that acetaminophen would
anism of action of AM404. first reinforce the activity of the endocannabinoid sys-
Different studies reported the implication of the tem and then that of the serotonergic one. However,
endocannabinoid system in pain modulation either at the demonstration that AM404 can be synthesized in
spinal or supraspinal levels [35]. Notably, the stimula- the spinal cord after acetaminophen treatment and the
tion of CB1 receptors in PAG and RVM reduced GABA presence of spinal CB1 receptors [23] might also suggest
release from the presynaptic boutons of local interneu- an involvement of these receptors. Thus, further studies
rons which can reduce the GABAergic negative influ- will be needed to determine the site of the interaction of
ence on the inhibitory descending pathways [49,50]. acetaminophen (or its metabolite AM404) with the
Since the antinociceptive activity of acetaminophen endocannabinoid system, the actual modalities of this
has been shown to depend on spinal serotonergic recep- interaction and how this leads to the reinforcement of
tors [6,11,39,40] and that the source of spinal 5-HT the serotonergic system.
comes exclusively from supraspinal centers and mainly It also remains possible that AM404 mediates the
from RVM [35], we suspected that the participation of antinociceptive activity of acetaminophen through other
the endocannabinoid system in the effect of acetamino- mechanisms. For instance, AM404 could activate the
phen would occur through the reinforcement of the TRPV1 receptor, whose stimulation has been shown to
activity of the bulbospinal serotonergic pathways. elicit antinociception in the PAG [30] and to be active in
Accordingly, we demonstrated that the antinociceptive the tetrad tests [13]. In addition, AM404 has been shown
effect induced by a CB1 receptor agonist (ACEA) needed to inhibit COX activities in vitro, with a potency close to
intact descending bulbospinal serotonergic pathways. those of NSAIDs [23]. The involvement of a central COX
Furthermore, ACEA-dependent recruitment of the sero- inhibition during the acetaminophen-induced antinoci-
tonergic system involved similar spinal 5-HT receptors ceptive and/or antipyretic activities will have to be con-
to those involved in the antinociceptive action of acet- firmed in vivo. On the contrary, the fact that AM404 is
aminophen and 5-HT [1,2,5,6,40], i.e. the 5-HT1A recep- not found in blood after administration of acetamino-
tor in the formalin test and the 5-HT3/4 receptor in the phen [23] might explain why acetaminophen does not
paw pressure test. This similar 5-HT-dependent mecha- exert any significant peripheral anti-inflammatory effect.
nism and the inhibition of the effect of acetaminophen This could therefore further explain why acetaminophen
by inactivation of CB1 receptors suggest that the endo- does not exert any significant anti-inflammatory effect.
cannabinoid system is an important link between acet- Other studies stated that the antinociceptive activity of
aminophen and 5-HT to produce antinociception. acetaminophen may rely on a decrease in spinal NO
 C. Mallet et al. / Pain 139 (2008) 190–200 199

(grant 796/2004 to C. Mallet) and l’Institut Paul Hamel.

Acetaminophen The authors have no conflicting financial interests.

LIVER 1 References

[1] Alloui A, Chassaing C, Schmidt J, Ardid D, Dubray C, Cloarec

p-aminophenol
p-aminopheno
A, et al. Paracetamol exerts a spinal, tropisetron-reversible,
antinociceptive effect in an inflammatory pain model in rats. Eur
J Pharmacol 2002;443:71–7.
FAAH [2] Bardin L, Lavarenne J, Eschalier A. Serotonin receptor subtypes
BRAIN 2 involved in the spinal antinociceptive effect of 5-HT in rats. Pain
AM404 2000;86:11–8.
[3] Beltramo M, Stella N, Calignano A, Lin SY, Makriyannis A,
Piomelli D. Functional role of high-affinity anandamide transport,
as revealed by selective inhibition. Science 1997;277:1094–7.
CB1 receptor 3 [4] Bjorkman R, Hallman KM, Hedner J, Hedner T, Henning M.
PAG
Acetaminophen blocks spinal hyperalgesia induced by NMDA
RVM Serotonergic and substance P. Pain 1994;57:259–64.
descending 4 [5] Bonnefont J, Alloui A, Chapuy E, Clottes E, Eschalier A. Orally
pathways
administered paracetamol does not act locally in the rat formalin
test: evidence for a supraspinal, serotonin-dependent antinocicep-
tive mechanism. Anesthesiology 2003;99:976–81.
[6] Bonnefont J, Chapuy E, Clottes E, Alloui A, Eschalier A. Spinal
5-HT receptors 5
SPINAL CORD 5-HT1A receptors differentially influence nociceptive processing
according to the nature of the noxious stimulus in rats: effect of
WAY-100635 on the antinociceptive activities of paracetamol,
venlafaxine and 5-HT. Pain 2005;114:482–90.
Analgesia [7] Compton DR, Martin BR. The effect of the enzyme inhibitor
phenylmethylsulfonyl fluoride on the pharmacological effect of
anandamide in the mouse model of cannabimimetic activity. J
Fig. 7. Schematic representation of the proposed sequence of events Pharmacol Exp Ther 1997;283:1138–43.
going from an oral treatment with acetaminophen to its antinocicep- [8] Compton DR, Rice KC, De Costa BR, Razdan RK, Melvin LS,
tive activity. (1) Acetaminophen is deacetyled in p-aminophenol in the Johnson MR, et al. Cannabinoid structure-activity relationships:
liver; (2), then metabolized in the brain by FAAH into AM404. (3) correlation of receptor binding and in vivo activities. J Pharmacol
AM404 reinforces the activity of the supraspinal CB1 receptors, (4) Exp Ther 1993;265:218–26.
which in turn reinforce the activity of the serotonergic descending [9] Conti S, Costa B, Colleoni M, Parolaro D, Giagnoni G.
pathways. (5) Spinal release of serotonin (5-HT) stimulates either the Antiinflammatory action of endocannabinoid palmitoylethanola-
5-HT3/4 receptor in a mechanical pain test or the 5-HT1A receptor in a mide and the synthetic cannabinoid nabilone in a model of acute
chemical pain test to inhibit the transmission of noxious stimuli inflammation in the rat. Br J Pharmacol 2002;135:181–7.
towards higher centers and therefore produce antinociception. PAG: [10] Costa B, Siniscalco D, Trovato AE, Comelli F, Sotgiu ML,
periaqueductal gray; RVM: rostroventral medulla. Colleoni M, et al. AM404, an inhibitor of anandamide uptake,
prevents pain behaviour and modulates cytokine and apoptotic
[e.g. 4], which could also be a consequence of the rein- pathways in a rat model of neuropathic pain. Br J Pharmacol
2006;148:1022–32.
forcement of the endocannabinoid system [10]. [11] Courade JP, Chassaing C, Bardin L, Alloui A, Eschalier A. 5-HT
In conclusion, our present data together with results receptor subtypes involved in the spinal antinociceptive effect of
published by Högestätt et al. [23] suggest that the activity acetaminophen in rats. Eur J Pharmacol 2001;432:1–7.
of acetaminophen, orally administered, would require a [12] Di Marzo V, Bifulco M, De Petrocellis L. The endocannabinoid
multi-step process (Fig. 7). Firstly, the drug would need system and its therapeutic exploitation. Nat Rev Drug Discov
2004;3:771–84.
to be metabolized in two steps to form cerebral FAAH- [13] Di Marzo V, Bisogno T, De Petrocellis L, Brandi I, Jefferson RG,
dependent AM404. This metabolite could reinforce the Winckler RL, et al. Highly selective CB(1) cannabinoid receptor
activity of the endocannabinoid system through CB1 ligands and novel CB(1)/VR(1) vanilloid receptor ‘‘hybrid”
receptors, which would in turn reinforce the activity of ligands. Biochem Biophys Res Commun 2001;281:444–51.
the bulbospinal serotonergic inhibitory pathways. [14] Di Marzo V, Deutsch DG. Biochemistry of the endogenous
ligands of cannabinoid receptors. Neurobiol Dis 1998;5:386–404.
Finally, antinociception would occur at the spinal level [15] Fowler CJ, Stenstrom A, Tiger G. Ibuprofen inhibits the
where activation of stimulus-dependent 5-HT receptors metabolism of the endogenous cannabimimetic agent ananda-
would block the transmission of nociceptive influx. mide. Pharmacol Toxicol 1997;80:103–7.
[16] Fox A, Bevan S. Therapeutic potential of cannabinoid receptor
agonists as analgesic agents. Expert Opin Investig Drugs
2005;14:695–703.
Acknowledgements [17] Fox A, Kesingland A, Gentry C, McNair K, Patel S, Urban L,
et al. The role of central and peripheral Cannabinoid1 receptors in
This work was funded by ‘Association Nationale de the antihyperalgesic activity of cannabinoids in a model of
la Recherche Technique’ and Bristol-Myers-Squibb neuropathic pain. Pain 2001;92:91–100.
200 C. Mallet et al. / Pain 139 (2008) 190–200

[18] Fride E, Mechoulam R. Pharmacological activity of the cannab- [36] Muth-Selbach US, Tegeder I, Brune K, Geisslinger G. Acetami-
inoid receptor agonist, anandamide, a brain constituent. Eur J nophen inhibits spinal prostaglandin E2 release after peripheral
Pharmacol 1993;231:313–4. noxious stimulation. Anesthesiology 1999;91:231–9.
[19] Giuffrida A, Beltramo M, Piomelli D. Mechanisms of endocan- [37] Ottani A, Leone S, Sandrini M, Ferrari A, Bertolini A. The
nabinoid inactivation: biochemistry and pharmacology. J Phar- analgesic activity of paracetamol is prevented by the blockade of
macol Exp Ther 2001;298:7–14. cannabinoid CB1 receptors. Eur J Pharmacol 2006;531:280–1.
[20] Glaser ST, Abumrad NA, Fatade F, Kaczocha M, Studholme [38] Ouellet M, Percival MD. Mechanism of acetaminophen inhibition
KM, Deutsch DG. Evidence against the presence of an ananda- of cyclooxygenase isoforms. Arch Biochem Biophys
mide transporter. Proc Natl Acad Sci U S A 2003;100:4269–74. 2001;387:273–80.
[21] Guhring H, Hamza M, Sergejeva M, Ates M, Kotalla CE, Ledent [39] Pelissier T, Alloui A, Caussade F, Dubray C, Cloarec A,
C, et al. A role for endocannabinoids in indomethacin-induced Lavarenne J, et al. Paracetamol exerts a spinal antinociceptive
spinal antinociception. Eur J Pharmacol 2002;454:153–63. effect involving an indirect interaction with 5-hydroxytryptamine3
[22] Haller VL, Cichewicz DL, Welch SP. Non-cannabinoid CB1, non- receptors: in vivo and in vitro evidence. J Pharmacol Exp Ther
cannabinoid CB2 antinociceptive effects of several novel com- 1996;278:8–14.
pounds in the PPQ stretch test in mice. Eur J Pharmacol [40] Pelissier T, Alloui A, Paeile C, Eschalier A. Evidence of a central
2006;546:60–8. antinociceptive effect of paracetamol involving spinal 5HT3
[23] Högestätt ED, Jonsson BA, Ermund A, Andersson DA, Bjork H, receptors. Neuroreport 1995;6:1546–8.
Alexander JP, et al. Conversion of acetaminophen to the bioactive [41] Pertwee RG. The ring test: a quantitative method for assessing the
N-acylphenolamine AM404 via fatty acid amide hydrolase- ’cataleptic’ effect of cannabis in mice. Br J Pharmacol
dependent arachidonic acid conjugation in the nervous system. J 1972;46:753–63.
Biol Chem 2005;280:31405–12. [42] Pickering G, Loriot MA, Libert F, Eschalier A, Beaune P, Dubray
[24] Hohmann AG, Suplita Jr RL. Endocannabinoid mechanisms of C. Analgesic effect of acetaminophen in humans: first evidence of
pain modulation. Aaps J 2006;8:E693–708. a central serotonergic mechanism. Clin Pharmacol Ther
[25] Kathuria S, Gaetani S, Fegley D, Valino F, Duranti A, Tontini A, 2006;79:371–8.
et al. Modulation of anxiety through blockade of anandamide [43] Pini LA, Sandrini M, Vitale G. The antinociceptive action of
hydrolysis. Nat Med 2003;9:76–81. paracetamol is associated with changes in the serotonergic system
[26] Kikkawa R, Fujikawa M, Yamamoto T, Hamada Y, Yamada H, in the rat brain. Eur J Pharmacol 1996;308:31–40.
Horii I. In vivo hepatotoxicity study of rats in comparison with [44] Piomelli D, Giuffrida A, Calignano A, Rodriguez de Fonseca F.
in vitro hepatotoxicity screening system. J Toxicol Sci The endocannabinoid system as a target for therapeutic drugs.
2006;31:23–34. Trends Pharmacol Sci 2000;21:218–24.
[27] La Rana G, Russo R, Campolongo P, Bortolato M, Mangieri [45] Rinaldi-Carmona M, Calandra B, Shire D, Bouaboula M, Oustric
RA, Cuomo V, et al. Modulation of neuropathic and inflamma- D, Barth F, et al. Characterization of two cloned human CB1
tory pain by the endocannabinoid transport inhibitor AM404 [N- cannabinoid receptor isoforms. J Pharmacol Exp Ther
(4-hydroxyphenyl)-eicosa-5,8,11,14-tetraenamide]. J Pharmacol 1996;278:871–8.
Exp Ther 2006;317:1365–71. [46] Russo EB, Guy GW, Robson PJ. Cannabis, pain, and sleep:
[28] Ledent C, Valverde O, Cossu G, Petitet F, Aubert JF, Beslot F, et al. lessons from therapeutic clinical trials of Sativex, a cannabis-based
Unresponsiveness to cannabinoids and reduced addictive effects of medicine. Chem Biodivers 2007;4:1729–43.
opiates in CB1 receptor knockout mice. Science 1999;283:401–4. [47] Russo R, Loverme J, La Rana G, Compton TR, Parrott J,
[29] Lund A, Tjolsen A, Hole K. The apparent antinociceptive effect of Duranti A, et al. The fatty acid amide hydrolase inhibitor
desipramine and zimelidine in the tail flick test in rats is mainly URB597 (cyclohexylcarbamic acid 3’-carbamoylbiphenyl-3-yl
caused by changes in tail skin temperature. Pain 1989;38:65–9. ester) reduces neuropathic pain after oral administration in mice.
[30] Maione S, Bisogno T, de Novellis V, Palazzo E, Cristino L, Valenti J Pharmacol Exp Ther 2007;322:236–42.
M, et al. Elevation of endocannabinoid levels in the ventrolateral [48] Tjolsen A, Lund A, Hole K. Antinociceptive effect of paracetamol
periaqueductal grey through inhibition of fatty acid amide hydro- in rats is partly dependent on spinal serotonergic systems. Eur J
lase affects descending nociceptive pathways via both cannabinoid Pharmacol 1991;193:193–201.
receptor type 1 and transient receptor potential vanilloid type-1 [49] Vaughan CW, Connor M, Bagley EE, Christie MJ. Actions of
receptors. J Pharmacol Exp Ther 2006;316:969–82. cannabinoids on membrane properties and synaptic transmission
[31] Martin BR, Compton DR, Thomas BF, Prescott WR, Little PJ, in rat periaqueductal gray neurons in vitro. Mol Pharmacol
Razdan RK, et al. Behavioral, biochemical, and molecular 2000;57:288–95.
modeling evaluations of cannabinoid analogs. Pharmacol Bio- [50] Vaughan CW, McGregor IS, Christie MJ. Cannabinoid receptor
chem Behav 1991;40:471–8. activation inhibits GABAergic neurotransmission in rostral ven-
[32] Meng ID, Johansen JP. Antinociception and modulation of tromedial medulla neurons in vitro. Br J Pharmacol
rostral ventromedial medulla neuronal activity by local microin- 1999;127:935–40.
fusion of a cannabinoid receptor agonist. Neuroscience [51] Walker JM, Huang SM. Endocannabinoids in pain modulation.
2004;124:685–93. Prostaglandins Leukot Essent Fatty Acids 2002;66:235–42.
[33] Meng ID, Manning BH, Martin WJ, Fields HL. An analgesia [52] Wiley J, Balster R, Martin B. Discriminative stimulus effects of
circuit activated by cannabinoids. Nature 1998;395:381–3. anandamide in rats. Eur J Pharmacol 1995;276:49–54.
[34] Mestre C, Pelissier T, Fialip J, Wilcox G, Eschalier A. A method [53] Wiley JL, Martin BR. Cannabinoid pharmacological properties
to perform direct transcutaneous intrathecal injection in rats. J common to other centrally acting drugs. Eur J Pharmacol
Pharmacol Toxicol Methods 1994;32:197–200. 2003;471:185–93.
[35] Millan MJ. Descending control of pain. Prog Neurobiol [54] Zimmermann M. Ethical guidelines for investigations of experi-
2002;66:355–474. mental pain in conscious animals. Pain 1983;16:109–10.