Acetaminophen differentially enhances social behavior and cortical cannabinoid levels in inbred mice

Abstract

Supratherapeutic doses of the analgesic acetaminophen (paracetomol) are reported to promote social behavior in Swiss mice. However, we hypothesized that it might not promote sociability in other strains due to cannabinoid CB₁ receptor-mediated inhibition of serotonin (5-HT) transmission in the frontal cortex. We examined the effects of acetaminophen on social and repetitive behaviors in comparison to a cannabinoid agonist, WIN 55,212-2, in two strains of socially-deficient mice, BTBR and 129S1/SvImJ (129S). Acetaminophen (100 mg/kg) enhanced social interactions in BTBR, and social novelty preference and marble burying in 129S at serum levels of ≥ 70 ng/ml. Following acetaminophen injection or sociability testing, anandamide (AEA) increased in BTBR frontal cortex, while behavior testing increased 2-arachidonyl glycerol (2-AG) levels in 129S frontal cortex. In contrast, WIN 55,212-2 (0.1 mg/kg) did not enhance sociability. Further, we expected CB₁-deficient (+/−) mice to be less social than wild-type, but instead found similar sociability. Given strain differences in endocannabinoid response to acetaminophen, we compared cortical CB₁ and 5-HT_1A receptor density and function relative to sociable C57BL/6 mice. CB₁ receptor saturation binding (Bmax = 958 ± 117 fmol/mg protein), and affinity for [³H] CP55,940 (K_D = 3 ± 0.8 nM) was similar in frontal cortex among strains. CP55,940-stimulated [³⁵S] GTPγS binding in cingulate cortex was 136 ± 12, 156 ± 22, and 75 ± 9% above basal in BTBR, 129S and C57BL/6 mice. The acetaminophen metabolite para-aminophenol (1 μM) failed to stimulate [³⁵S] GTPγS binding. Hence, it appears that other indirect actions of acetaminophen, including 5-HT receptor agonism, may underlie its sociability promoting properties outweighing any CB₁ mediated suppression by locally-elevated endocannabinoids in these mice.

Introduction

Deficits in social interaction such as social anxiety, withdrawal, and inattentiveness are symptoms of many psychiatric conditions, including autism, schizophrenia, depression and post-traumatic stress disorder (Derntl et al., 2011, Feldman and Vengrober, 2011, Pinkham et al., 2008). Behaviors paralleling sociability impairments are inherent in several inbred strains of mice, including BTBR and 129S1/SvImJ (Defensor et al., 2011, Moy et al., 2007, Spencer et al., 2011). BTBR mouse social behavior is sensitive to changes in serotonin (5-HT) neurotransmission, since administration of the 5-HT reuptake inhibitor fluoxetine or 5-HT_1A agonist buspirone increased their sociability (Gould et al., 2011). However, local application of the endocannabinoid agonist anandamide (AEA) or high doses (50–200 mg/kg) of acetaminophen also promoted social interactions in Swiss mice (Umathe et al., 2009). Hence increases in central 5-HT transmission or endocannabinoid levels can promote social behaviors in mice.

Acetaminophen is normally metabolized through sulfation conjugation pathways, but at high doses its metabolic deacetylation products include para-aminophenol and/or N-arachidonoylphenolamine (AM404), an AEA uptake blocker (Beltramo et al., 1997). These metabolites may activate cannabinoid (CB) receptors directly, or indirectly by raising extracellular endogenous CB levels in the brain (Bertolini et al., 2006, Högestätt et al., 2005, Mallet et al., 2008, Ottani et al., 2006). CB agonists such as WIN 55,212-2 can inhibit presynaptic 5-HT release in brain by activating CB₁ receptors found on cell bodies and axons of 5-HT neurons (Lau and Schloss, 2008, Nakazi et al., 2000). Acetaminophen might also be expected to inhibit social behavior via this mechanism, except endocannabinoids have different ligand properties and may be released as mixtures in a region-specific manner, in contrast to systemically-administered agonists.

We aimed to determine if acetaminophen-induced enhancement of Swiss mouse sociability found by Umathe et al. (2009): 1) would occur in socially-deficient strains, and 2) if it is mediated indirectly by AM404, or directly by para-aminophenol action at CB₁ receptors, since its derivatives have some (Ki ~ 200 nM) affinity for them (Sinning et al., 2008). Hence, we examined acetaminophen's effects on social and repetitive behaviors in BTBR and 129S mice, and on endocannabinoid levels in frontal cortex.

Serotonergic tone in the frontal cortex is linked to anxiety and emotional states shaping social behavior (Bartolomucci et al., 2010, Boylan et al., 2007, File and Seth, 2003, Filipenko et al., 2002, Gerretsen et al., 2010). Cannabinoids modulate 5-HT signaling in this region, as exemplified by higher extracellular 5-HT levels in the frontal cortex of CB1 knock-out vs. wild-type mice (Aso et al., 2009). For this reason we measured 5-HT levels after acetaminophen or saline treatment, and compared the density and function of 5-HT_1A and CB₁ receptors in the frontal cortex of all strains.

Finally, several human CB₁ receptor gene polymorphisms alter their expression and/or function, and may be associated with social motivation and anxiety disorders (Chakrabarti et al., 2006, Lazary et al., 2009). In mice, CB₁ knock-outs exhibit similar social behavior to wild-types, but not under stressful conditions (Haller et al., 2004). Since CB₁ receptors are intricately involved in brain development, their absence may trigger confounding compensatory alterations in neural function or structure (Hoyle et al., 2011, Trezza et al., 2008). Hence, we compared the sociability of CB₁-deficient (heterozygous) mice to wild-type littermates to determine whether partial loss of CB₁ receptors alone would alter social or repetitive behaviors.