Assessment of Anandamide's Pharmacological Effects in Mice Deficient of Both Fatty Acid Amide Hydrolase and Cannabinoid CB1 Receptors

European Journal of Pharmacology 557 (2007) 44–48 www.elsevier.com/locate/ejphar

Short communication Assessment of anandamide's pharmacological effects in mice deficient of both fatty acid amide hydrolase and cannabinoid CB1 receptors

Laura E. Wise a, Christopher C. Shelton a, Benjamin F. Cravatt b, ⁎ Billy R. Martin a, Aron H. Lichtman a, a Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0613, United States b The Skaggs Institute for Chemical Biology and Departments of Cell Biology and Chemistry, The Scripps Research Institute, 10550 N. Torrey Pines Rd. La Jolla, CA 92037, United States Received 12 September 2006; received in revised form 2 November 2006; accepted 6 November 2006 Available online 10 November 2006

Abstract

In the present study, we investigated whether anandamide produces its behavioral effects through a cannabinoid CB1 receptor mechanism of action. The behavioral effects of anandamide were evaluated in mice that lacked both fatty acid amide hydrolase (FAAH) and cannabinoid CB1 receptors (DKO) as compared to FAAH (−/−), cannabinoid CB1 (−/−), and wild type mice. Anandamide produced analgesia, catalepsy, and hypothermia in FAAH (−/−) mice, but failed to elicit any of these effects in the other three genotypes. In contrast, anandamide decreased locomotor behavior regardless of genotype, suggesting the involvement of multiple mechanisms of action, including its products of degradation. These findings indicate that the cannabinoid CB1 receptor is the predominant target mediating anandamide's behavioral effects. © 2006 Elsevier B.V. All rights reserved.

Keywords: Cannabinoid CB1 receptor; FAAH [Fatty acid amide hydrolase]; N-arachidonoyl ethanolamine (anandamide); Pain; Analgesia; Marijuana

1. Introduction 1998a,b; Smith et al., 1998, 1994). Thus, anandamide's weak and transient pharmacological effects in mice (Smith et al., 1994), While anandamide is most prominently known as the first rats (Lichtman et al., 1996), and dogs (Lichtman et al., 1998)after endogenous cannabinoid extracted from mammalian brain, it has exogenous administration is not altogether surprising. CB1 (−/−) activity at multiple targets including cannabinoid CB1 receptors, mice as well as mice pretreated with the cannabinoid CB1 re- cannabinoid CB2 receptors (Calignano et al., 1998; Sokal et al., ceptor antagonist rimonabant exhibit pharmacological effects 2003), TRPV1 receptors (Tognetto et al., 2001; Zygmunt et al., following intravenously injected anandamide, suggesting a non- 1999), potassium channels (Maingret et al., 2001), T-type calci- CB1 receptor mechanism of action (Adams et al., 1998; Di Marzo um channels (Chemin et al., 2001), and gap junctions (Venance et al., 2000; Smith et al., 1994). In contrast, anandamide given et al., 1995). The rapid catabolism and consequent short half-life through the intraperitoneal route of administration to either (Willoughby et al., 1997) of this lipid signaling molecule by fatty FAAH (−/−) mice or mice treated with FAAH inhibitors elicits acid amide hydrolase (FAAH) have undoubtedly contributed to profound pharmacological effects, which are blocked by discrepant findings from various investigations in whole ani- SR141716 (Cravatt et al., 2001, 2004; Kathuria et al., 2003; mals.(Adams et al., 1998; Calignano et al., 1998; Costa et al., Lichtman et al., 2004). Of course, a major difference between the 1999; Di Marzo et al., 2000; Farquhar-Smith and Rice, 2001; two strategies is that high doses of anandamide were required to Jaggar et al., 1998; Lichtman et al., 1996; Richardson et al., elicit a pharmacological effect in wild type mice and consid- erable quantities of metabolites are formed, whereas low doses of anandamide can produce pharmacological effects in FAAH − − ⁎ Corresponding author. Tel.: +1 804 828 8480. ( / ) mice. While products of metabolism could contribute to E-mail address: [email protected] (A.H. Lichtman). anandamide's effects in the wild type animals, it remains

0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.11.002 L.E. Wise et al. / European Journal of Pharmacology 557 (2007) 44–48 45 plausible that the effects of this endogenous cannabinoid are laboratory have previously shown that at 52 °C there are no mediated through a non-CB1 receptor mechanism of action. genotype differences in baseline hotplate latencies among The purpose of the present study was to investigate whether FAAH (+/+), (+/−), or (−/−) mice (Cravatt et al., 2001; Lichtman intact anandamide produces overt behavioral effects at sites et al., 2004). Rectal temperature was determined using a tele- other than cannabinoid CB1 receptors. In order to address this thermometer by inserting a thermocouple probe 2.0 cm into the question, we created mice that lacked both FAAH and canna- rectum. Following baseline assessments, each subject was given binoid CB1 receptor genes (DKO mice) by selectively breeding an i.p. injection of vehicle or 50 mg/kg anandamide. The mice FAAH (−/−) mice with CB1 (+/−) mice. We then investigated the were then placed into photocell activity cages (6.5×11 in.) effects of anandamide in DKO mice as compared to wild type, where locomotor activity was assessed from 10–20 min post- CB1 (−/−), and FAAH (−/−) mice in the tetrad test, a well injection by recording the total number of photocell beam in- established screen for cannabinoid activity (Compton et al., terruptions (Omnitech Electronics Inc., Columbus, OH). Imme- 1990, 1993; Martin et al., 1991). diately after locomotor activity assessment, hot plate latencies were evaluated. Catalepsy and rectal temperature were deter- 2. Methods mined 60 min post-injection (Cravatt et al., 2001; Lichtman et al., 2004). Catalepsy was determined using the bar test, in 2.1. Subjects which the front paws of each subject were placed on a rod (0.75 cm diameter) that was elevated 4.5 cm from the bench top. The subjects consisted of male and female mice on a C57BL/ The duration of time that the mice remained motionless (with the 6 background of the following four genotypes: 1) wild type: exception of respiratory movements) with their front paws on the FAAH (+/−)/CB1 (+/+); 2) CB1 (−/−) mice: FAAH (+/−)/CB1 bar for 10 s was scored. The same subjects were evaluated (−/−); 3) FAAH (−/−) mice: FAAH (−/−)/CB1 (+/−); and 4) following vehicle and anandamide administration in a counter- DKO: FAAH (−/−)/CB1 (−/−). Previous studies have demon- balanced fashion with at least 72 h between test days to control strated that FAAH (+/−) mice display virtually identical phe- for any order effects. notypes as wild type mice (Cravatt et al., 2001, 2004; Lichtman et al., 2004) and thus were used as a control in this study. All 2.4. Data analysis subjects were littermates derived from FAAH (−/−)/CB1 (+/−) and FAAH (+/−)/CB1 (+/−) breeding pairs. These breeding pairs Two-way analysis of variance (ANOVA) was used to eval- yielded a greater percentage of the desired genotypes for these uate whether baseline hot plate and body temperatures differed experiments than had the breeding pairs consisted of two mice among the four genotypes (CB1 vs. FAAH) and three-way with FAAH (+/−)/CB1 (+/−) genotypes. All mice were born ANOVA was used to analyze drug differences among the four in the Virginia Commonwealth University vivarium. Subjects genotypes (anandamide vs. CB1 vs. FAAH) in each assay. weighed between 18 and 30 g, and were housed four to five Scheffe test was used for post-hoc analyses. Differences were animals per cage in a temperature-controlled (20–22 °C) facility. considered significant at the Pb0.05 level. Mice were given unlimited access to food and water and were maintained on a 12/12-h light/dark cycle. The Institutional 3. Results Animal Care and Use Committee at Virginia Commonwealth University approved all experiments. No relevant differences were found among the different genotypes with respect to baseline hot plate latencies or body 2.2. Drugs temperatures (Table 1), though mice lacking cannabinoid CB1 receptors displayed a small, but a significant decrease in baseline Anandamide was synthesized as previously described rectal temperature, F(1, 39)=7.6, Pb0.01. Significant three- (Cravatt et al., 1996). The vehicle consisted of a mixture of way interactions were found among FAAH genotype, CB1 ge- ethanol:alkamuls-620 (Rhone-Poulenc, Princeton, NJ):saline in notype, and anandamide for analgesia (F(1,39)=37, Pb0.001), a ratio of 1:1:18, respectively. Mice were given an i.p. injection catalepsy (F(1,39)=10, Pb0.01), and hypothermia (F(1,39)= of vehicle or anandamide (50 mg/kg) in a volume of 10 μl/g body 26, Pb0.001). As shown in Fig. 1, anandamide elicited anal- weight. This dose of anandamide was previously found to pro- gesia, catalepsy, and hypothermia in FAAH (−/−)/CB1 (+/+) duce profound pharmacological effects in FAAH (−/−), but not in (+/−) or (+/+), mice (Cravatt et al., 2001, 2004; Lichtman et al., 2004) and, thus, was used in the present study to assess Table 1 anandamide's effects in each of the four genotypes. Baseline hot plate latencies and rectal temperatures

FAAH CB1 Hot plate latency Rectal temperature n 2.3. Behavioral measures genotype genotype (s) (°C) +/− +/+ 16.8±1.5 36.1±0.1 12 Subjects were assessed for basal responses in the hot plate test +/−−/− 17.9±1.5 35.9±0.1 12 and baseline rectal temperature. The hot plate that was main- −/− +/+ 17.2±1.8 36.1±0.1 12 tained at 52 °C, and the latency to jump or lick/shake a hind paw −/−−/− 16.5±1.9 35.6±0.2 7 within a 60 s observation period was recorded. Results from our All values reflect mean±S.E.M. 46 L.E. Wise et al. / European Journal of Pharmacology 557 (2007) 44–48

Fig. 1. The effect of anandamide (50 mg/kg, open bars) on locomotor activity (A), the hot plate test (B), the bar test (C), and body temperature (D) in FAAH (+/−)/CB1 (+/+) (n=12), FAAH (+/−)/CB1 (−/−)(n=12), FAAH (+/−)/CB1 (+/−)(n=12), and FAAH (−/−)/CB1 (−/−)(n=7) mice. Anandamide significantly decreased locomotor activity (A) in each genotype and regardless of drug treatment CB1 (−/−) mice displayed a small, but significant, decrease in locomotor activity (see text for statistics). Anandamide produced analgesia (B), catalepsy (C), and hypothermia (D) in FAAH (−/−)/CB1 (+/+) mice, but not in mice of the other genotypes tested, ⁎⁎⁎ indicating that these effects were regulated by FAAH and mediated by a cannabinoid CB1 receptor mechanism. Pb0.001 for anandamide versus each of the other groups (Scheffe post-hoc test). Results are shown as mean±S.E.M.

mice, but not in mice of any of the other three genotypes, motor activity (Crawley et al., 1993; Fride and Mechoulam, 1993; indicating that these effects were mediated by cannabinoid CB1 Romero et al., 1995a,b; Smith et al., 1994). Anandamide also receptors. Anandamide significantly decreased locomotor activ- produces a cannabinoid CB1 independent hypomotility in FAAH ity in each genotype, as reflected by a significant main effect of (+/−) mice and mice that possess FAAH restricted to the nervous drug, F(1,39)=94, Pb0.001. system and not in peripheral tissue (Cravatt et al., 2004). It should be noted that the FAAH (−/−) mice, which have at least a ten- 4. Discussion fold increase in endogenous anandamide do not exhibit any apparent changes in locomotor behavior. The high dose of As previously found (Cravatt et al., 2001), anandamide pro- anandamide used here was selected in order to optimize the duced analgesia, catalepsy, and hypothermia in genetically likelihood of detecting behaviors that are not mediated through modified mice that lack FAAH, but here we report that it had no cannabinoid CB1 receptors. Importantly, the effects elicited by significant effects in wild type (i.e., FAAH (+/−) and CB1 (+/+)), 50 mg/kg of anandamide do not resemble the effects of elevated CB1 (−/−) mice, and DKO mice. Thus, these effects of anan- endogenous anandamide observed in the FAAH (−/−) mice. In damide are regulated by FAAH and mediated through a canna- particular, arachidonic acid, which is produced by the rapid binoid CB1 receptor mechanism of action. Corroborating these FAAH-mediated catabolism of anandamide, has been shown to findings is previous work demonstrating that rimonabant blocks reduce locomotor activity in mice (Lichtman et al., 2002). each of these effects of anandamide in FAAH (−/−) mice Clearly locomotion is sensitive to many factors in addition to (Cravatt et al., 2001). products of anandamide metabolism and stimulation of can- In contrast, anandamide decreased locomotor activity in all nabinoid CB1 receptors. Alternative interpretations related to the mice, irrespective of genotype, indicating a non-CB1 receptor use of knockout models must also be considered, including mechanism of action. In contrast to the present findings, anan- hitchhiking genes that are derived from the original cell line, damide was previously reported to produce hypomotility in epistasis in which the effect of gene disruption is modified by the FAAH (−/−) mice that was blocked by rimonabant, but not by genetic background on which it is placed, and pleiotropic effects the cannabinoid CB2 antagonist SR144528, indicating that this in which other consequences of gene disruption indirectly affects effect was produced by a CB1-dependent mechanism of action the behavior of interest (Mogil and Grisel, 1998). Thus, the (Cravatt et al., 2001, 2004). Other studies have reported that locomotor depressant effects observed here may have been injections of anandamide or exogenous cannabinoids decrease mediated by anandamide's actions at the cannabinoid CB1 L.E. Wise et al. / European Journal of Pharmacology 557 (2007) 44–48 47

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