Social contact among male mice stimulates the mobilization of anandamide in the nucleus accumbens , a key brain region for reward signaling, in an oxytocin-dependent manner. Conversely, in isolated male mice, specifically stimulating oxytocin neurons in the paraventricular nucleus of the hypothalamus drives anandamide mobilization in the NAc. Consequent activation of CB1 receptors is necessary and sufficient to regulate social reward. Anandamide enhancement offsets the effects of oxytocin receptor blockade on social reward and NAc activity. In addition, human studies also support the notion that the endocannabinoid system affects social behavior. Acutely intoxicated marijuana users enjoy interacting more and feel more connected and empathetic. Chakrabarti and Baron-Cohen reported that a polymorphism in the CB1 cannabinoid receptor gene modulates social gaze. Based on those studies, we hypothesized that enhancement of anandamide-mediated signaling at CB1 receptors might offer a therapeutic strategy for ASD-related social impairment. As a first test of this idea, we examined the effects of a well-characterized FAAH inhibitor, the compound URB59719, in two established mouse models of ASD: BTBR mice, an inbred strain discovered through the Mouse Phenotype Project, which shows pronounced deficits in social approach, grow trays 4×4 reciprocal social interactions, and juvenile play, and fmr1-/- mutant mice – a model of Fragile X Syndrome, the most common monogenetic cause of ASD, which features persistent social deficits.
We show that pharmacological FAAH inhibition substantially improves social behavior in BTBR and fmr1-/- mice, and that this effect is independent of anxiety modulation.Animals. We used male mice bred at UC Irvine. The mice were weaned at P21 and group-reared in cages of 4-5 animals. Testing was done during the light cycle . Naïve mice were used for each behavioral experiment. All procedures met the National Institute of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at University of California, Irvine. Drug preparation and treatment. URB597 and AM251 were dissolved in a vehicle of saline/propylene glycol/Tween-80 . URB597, and AM251 were administered i.p. 3 h before starting the behavioral testing. Drugs were administered at 4 µL / g weight of the animal. FAAH assay. Procedures were described previously25. Briefly, reactions were conducted with tissue homogenate dissolved in 0.5 mL Tris buffer containing [3 H]-anandamide at 37°C for 30 min. Reactions were stopped with 1 mL of chloroform/methanol . Samples were centrifuged at 3000xg for 10 min at 4o C. Radioactivity in the aqueous layer was measured by liquid scintillation counting. Lipid analyses. Procedures were described previousl. Briefly, tissue samples were homogenized in methanol containing internal standards for H2 -anandamide, H2 -oleoylethanolamide and 2 H8-2-arachidonoyl-sn-glycerol .
Lipids were separated by a modified Folch-Pi method using chloroform/methanol/water and open-bed silica column chromatography. For LC/MS analyses, we used an 1100 liquid chromatography system coupled to a 1946D-mass spectrometer detector equipped with an electrospray ionization interface . The column was a ZORBAX Eclipse XDB-C18 . We used a gradient elution method as follows: solvent A consisted of water with 0.1% formic acid, and Solvent B consisted of acetonitrile with 0.1% formic acid. The separation method used a flow rate of 0.3 mL/min. The gradient was 65% B for 15 min, then increased to 100% B in 1 min and kept at 100% B for 14 min. The column temperature was 15°C. An isotope-dilution method was used for quantification. Three-chambered social approach task. Previously established methods were followed21. Test mice were habituated to an empty three-chambered acrylic box . Habituation included a 10-min session in the center chamber with doors closed, and then a 10-min session in all chambers with doors open. Test mice were then tested in a 10-min session. Subjects were offered a choice between a novel object and a novel mouse in opposing side-chambers. The novel object was an empty inverted pencil cup and the novel social stimulus mouse was a sex, age and weight-matched 129/SvImJ mouse. These mice were selected because they are relatively inert, and they were trained to prevent abnormal behaviors, such as biting the cup.
Weighted cups were placed on top of the pencil cups to prevent climbing. Low lighting was used – all chambers were measured to be 5 lux before testing. The apparatus was thoroughly cleaned with SCOE 10X odor eliminator between trials to preclude olfactory confounders. Object/mouse side placement was counterbalanced between trials. Chamber time scoring was automated using image analysis in ImageJ. Sniffing time was scored by trained assistants who were unaware of treatment conditions. Excluded were subjects with outlying inactivity. Elevated plus-maze. The procedure was based on previously published methods27. The maze was made of black Plexiglass and consisted of two open arms and closed arms . The arms extended from a center square . The maze was mounted on a Plexiglas base and raised 39 cm above the ground. Lighting consisted of two 40W incandescent bulbs, each hanging at a height of 1 m above the open arms of the test apparatus. The floor of the apparatus was cleaned with a SCOE 10X odor eliminator between trials. The animals were placed in the center square and allowed to freely explore for 5 min. The amount of time spent in each arm and the number of entries into each arm were quantified by Ethovision 3.1 video tracking system . Statistical analyses. Results are expressed as means ± SEM. Significance was determined by two tailed Student’s t-test, One-way or Two-way analysis of variance with Tukey’s posthoc test. Differences were considered significant if P<0.05. Analyses were conducted using GraphPad Prism .We used the CB1-receptor inverse agonist, AM251, or the FAAH inhibitor, URB59728, to probe the role of anandamide in social behavior of young adult mice. Social activity was evaluated in the widely used social approach test. The mice were placed in a dimly lit three-chambered apparatus, and given a choice between a novel, inert mouse restrained by an inverted pencil cup in one chamber or a novel object in the opposite chamber. We first evaluated maximally effective doses of AM251 29 or URB597 28 in socially normal C57Bl6J mice, and found that neither drug altered the two outcome measures of the test, namely the time spent in the social chamber and the time spent sniffing the target mouse . In contrast to C57Bl6J mice, BTBR mice show no social preference in the test . However, administration of URB597 significantly increased the time BTBR mice spent in the social chamber and sniffing, to levels that were comparable to those displayed by control, socially normal C57Bl6J mice . The effect of FAAH inhibition on social approach depended on CB1 receptors, and thus presumably on anandamide accumulation, because it was prevented by concomitant administration of AM251 . The results on time spent in the social chamber are summarized as an index . Using an enzymatic activity assay and liquid chromatography-mass spectrometry , we confirmed that URB597 inhibited FAAH and substantially increased the levels of anandamide in the forebrain of BTBR mice , without affecting levels of the other endocannabinoid 2-arachidonoyl-sn-glycerol . Together, the results suggest that elevated anandamide activity at CB1 receptors corrects social-approach behavior in BTBR mice, horticulture products whereas it does not alter social approach in control C57Bl6J miceThe endocannabinoids are important modulators of stress reactivity in humans and experimental animals. In rodent experiments, this modulation depends on the adverseness of environmental conditions, such as the intensity of ambient lighting. To test whether the prosocial actions of FAAH inhibition in BTBR mice might be due to a general reduction in anxiety, we asked whether URB597 exerted anxiolytic-like effects in the elevated plus-maze test under the same dim lighting conditions used for the social approach test . We found that URB597 administered to BTBR mice did not alter the time spent in the open arms, or the number of open-arm entries , suggesting that the prosocial effect of this compound cannot be attributed to reduced anxiety.Interpretation of data obtained with BTBR mice is limited by the possibly polygenetic contributions to the phenotype of this inbred strain. Therefore, we asked whether the prosocial effect of anandamide is translatable to a monogenetic model of ASD-related social impairment. Fmr1-/- mutant mice bred on an FVB/NJ background have been reported to exhibit a deficit in social approach. In our hands, however, this deficit was not statistically significant .
Nevertheless, we found that acute administration of URB597 , which did not alter the time spent in the social chamber or sniffing in wild-type fmr1+/+ mice, increased the time spent by fmr1-/- mice in the social chamber and the time spent sniffing,to levels to those displayed by control mice . These results suggest that the prosocial action of increased anandamide activity is generalizable across at least two distinct models of ASD-related social impairment, without affecting socially normal animals. We did not test fmr-/- mice in the elevated plus maze test because these mice display an innate preference for the open arms of the elevated plus-maze, which might confound data interpretation.For better or worse, desperate parents are treating their autistic children with various forms of marijuana. This societal experiment highlights the lack of scientific knowledge regarding the therapeutic utility and safety of cannabinoid agents in ASDs. The present study provides an initial test of the idea that enhancing anandamide-mediated endocannabinoid signaling might help to alleviate social impairment in ASD. We show that inhibition of the anandamide-deactivating enzyme FAAH corrects social impairment in two distinct ASD-related models – BTBR and fmr1-/- mice. We confirm that FAAH inhibition is an appropriate strategy to elevate levels of anandamide, and thus anandamide signaling, in BTBR mice. Furthermore, we show that the prosocial action of FAAH inhibition is independent of reducing anxiety in BTBR mice. Together, the results put forth FAAH as a novel therapeutic target for ASD-related social impairment. Separate lines of previous research suggest the general notion that abnormal endocannabinoid signaling might contribute to ASD. First, endocannabinoids play important roles in neurodevelopment, which is also affected by exogenous cannabinoids. Second, ASD-related alterations in synaptic signaling have been linked to the endocannabinoid system. For example,mutations in neuroligins, a family of ASD-linked synaptic adhesion proteins, impair tonic endocannabinoid signaling. In a related example, we found that deletion of FMRP, the mRNA-trafficking protein missing in Fragile X Syndrome, impairs the formation of a key signaling complex that links metabotropic glutamate receptor-5 and the 2-AGsynthesizing enzyme diacylglycerol lipase-α 27. Third, ASD-related insults disturb resting endocannabinoid levels or endocannabinoid system components. For instance, we found that chronic isolation increases 2-AG in the prefrontal cortex, without affecting anandamide, and increases both 2-AG and anandamide in the piriform cortex. Furthermore, developmental treatment with valproic acid reduces cerebellar mRNA levels of DGL-α and reduces hippocampal mRNA levels of the 2-AG-hydrolyzing enzyme monoacylglycerol lipase 36. Importantly, however, these lines of research have not addressed whether deficient endocannabinoid signaling contributes to the core component of ASD – social impairment. A limited literature has hinted at this possibility indirectly by suggesting a role for endocannabinoid signaling in normal social behaviors. Genetic removal of CB1 receptors alters social interactions in mice in a context-dependent manner, which may be related to social anxiety and/or cognition. CB1 agonists impair social play in rats. In contrast, genetic removal of FAAH in mice increases social interactions, and FAAH inhibition promotes social play in rats. Thus, the bidirectional modulation of social behavior likely depends on the dose and the identity of the affected circuits. We recently identified a signaling mechanism in male mice by which oxytocin drives anandamide-mediated endocannabinoid signaling to control social reward. In addition, human studies have found that marijuana may enhance sociability and a polymorphism in the CB1 cannabinoid receptor gene modulates social gaze.Given that endocannabinoid signaling has been linked to ASD and might play a role in normal social behavior, we focused the present investigation on the possible role of endocannabinoid signaling in the social impairment component of ASD. We found that that social impairment is corrected in two distinct mouse models by increasing anandamide activity through FAAH inhibition. This correction raises the immediate question of whether increasing anandamide activity in these mice is prosocial per se or simply anxiolytic. This question is particularly important in light of the known roles of endocannabinoids in stress modulation. In our case, we found that the prosocial action of anandamide is unlikely to be due to general anxiolysis, because FAAH inhibition did not alter BTBR performance in the elevated plus-maze when tested in the dim lighting conditions of the social approach test. This result is in line with previous reports showing that FAAH inhibition results in anxiolytic-like effects only under ‘high-light’ conditions.