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 ≥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 (1μM) 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 CB₁mediated suppression by locally-elevated endocannabinoids in these mice.

2.1 Mouse Subjects

All procedures involving mice were performed in accordance with guidelines for care and use of laboratory animals (National Institutes of Health), and were approved by the institutional animal care and use committee. BTBR T+tf/J, 129S1/SvImJ and C57BL/6 mouse colony founders were obtained from Jackson Laboratory (Bar Harbor, ME). Mice were bred in the animal facilities of the University of Texas Health Science Center through 2 generations. After weaning (postnatal days 23–25), male littermates were housed in groups of 4–5 per cage until behavioral testing at 3–4 months of age. Mice had ad libitumaccess to food (Teklad, Harlan, Indianapolis, IN) and water in ventilated clear plastic cages lined with chipped wood bedding. The housing room had a 12h light/dark cycle (lights on/off at 7:00) and was maintained at 20–22°C.

CB₁ knock-out mice on a C57BL/6 background strain were the product of wild-type × heterozygous crosses. To determine their genotypes, a 0.5–0.75 cm section of tail tip was collected from each mouse. Tail tips were added to a solution of 200 μL DirectPCR (Viagen Biotech, Los Angeles, CA) and 20 μL of 20 μg/μL Proteinase K (Sigma Chemical Co., St. Louis, MO), then incubated at 55° C until fully digested (5–14 hours). To extract DNA, samples were centrifuged and the supernatants were mixed with 500 μL isopropanol. Samples were centrifuged again, the supernatant discarded, and 200 μL of genomic buffer was added to the pellet before storage at −20°C. PCR amplification was carried out using 1 μL of DNA sample, 6 μL water, 10 μL AccuStart PCR Supermix (Quanta Biosciences, Gaithersburg, MD), and 0.5 μM of each primer G50: 5′-GCTGTCTCTGGTCCTCTTAAA-3′, G51: 5′-GGTGTCACCTCTGAAAACAGA-3′, G54: 5′-CCTACCCGGTAGAATTAGCTT-3′ (Invitrogen, Carlsbad, CA). The PCR was carried out in a Techne TC 3000 thermo cycler (Barloworld Scientific, UK): 4 min at 94° C, 35 cycles of 45 sec at 94° C, 45 seconds at 51° C, 60 sec at 72°C, and concluded with 10 min at 72° C. Products were run on 1% agarose gel in 1X TBE buffer at 120V for two hours. The gel was transferred onto a UV Transilluminator (Spectroline, Westbury, NY) for analysis of band placement. A band at 342 bp signified homozygous knockout; 413 bp signified homozygous wild type; both bands signified a heterozygote.

2.2 Drug administration

Mice were administered acetaminophen (1 – 400 mg/kg, Sigma) or 0.9% saline solution by intraperitoneal (i.p.) injection. The cannabinoid agonist WIN55,212-2 (Ascent Scientific, Princeton, NJ) was initially dissolved in dimethyl-sulfoxide (DMSO) and was diluted with saline (1:10) to administer 0.1 mg/kg i.p. in 10% DMSO to mice. A subgroup of controls were treated with 10% DMSO in saline vehicle, they did not differ significantly from saline-treated mice in social and marble burying tests (F_1,7 <1.25; p > 0.3), so these groups were subsequently pooled. Injections were given 30 min prior to introduction into the testing arena, and 50 min prior to testing.

2.3 Behavioral testing and tissue collection

The three-chamber sociability testing procedure for mice was performed as described inGould et al. (2011). Briefly, pre-conditioning was performed under low red light (16 lux) first for 10 min with the subject confined to the center chamber, then with chamber doors opened so it could explore the whole arena for another 10 min. Just prior to testing, an empty wire cup cage was placed at one end of the arena, and a stranger mouse of the same strain was placed under a cup cage at the opposite end. Stranger mice were pre-conditioned to cup cage confinement in 3 separate 30 min sessions prior to testing, and were neither litter- nor cage-mates of the subjects. Cup cages were topped with weighted jars (9 cm high × 7 cm diameter) to prevent mice from climbing on top of them. Digital video cameras (Photosmart R742, Hewlett-Packard, Palo Alto, CA) positioned on tripods over the arenas were turned on, the doors were removed and social approach behavior was recorded for 10 min under low red light.

The social approach test ended with subject confinement in the center chamber by closing the doors. A new stranger mouse was placed under the empty cup cage, the doors were re-opened, and behavior was recorded for another 10 min under low red light to assess preference for social novelty. After this test, stranger mice were returned to their home cages for use in subsequent tests, and subjects were removed from the arenas and placed in marble burying tests. Between animal subjects the arena was cleaned with 70% ethanol and dried. Digital videos were analyzed for chamber entries, dwelling and social sniff times by treatment-blind observers.

Marble burying was assessed in a dark room (<16 lux) by placing 15 or 20 blue marbles on top of fresh wood chip bedding filled to a depth of 4–5 cm in a 22 × 42 cm clear acrylic rat cage covered with a filter top. Mice were placed in the cages to bury marbles for 30 min. Marbles that were at least 2/3 covered by bedding were considered buried, as described previously (Gould et al., 2011).

Following behavior tests, mice were sacrificed by cervical dislocation and decapitation. Trunk blood was collected into tubes containing 25 μl of 20 mM ethylenediaminetetraacetic acid and centrifuged for 10 min at 5000 rpm, serum was stored at −20°C. Whole brains were frozen on dry ice and stored at −80°C.

2.4 HPLC measurements of acetaminophen in serum and 5-HT in brain

To quantify serum acetaminophen and para-aminophenol, a 100 μL aliquot of thawed serum was mixed with 200 ng of the internal standard 3-acetamidophenol in 0.05 M aqueous sodium acetate solution (100 μL). The mixture was extracted with ethyl acetate and the organic extract was dried under nitrogen. The residue from each sample separately was taken again in 0.05 M aqueous sodium acetate solution (200 μL). An aliquot of 20 μL from this mixture was injected on a μ-Bondapak, reverse phase C-18, 3.9 × 300 mm HPLC column (Waters Corporation, Milford, MA) and eluted with 0.05 M aqueous sodium acetate mobile phase. The peaks of acetaminophen and para-aminophenol in biological samples were recognized by co-injection with standard acetaminophen and para-aminophenol separately. Peaks were quantified by a combination of the methods of Wang et al. (1985) and Campanero et al. (1999).

5-HT levels in frontal cortex were measured by HPLC with electrochemical detection as previously reported (O’Connor, 2009), with modifications. Briefly, frozen tissue (20 ± 2 mg) was homogenized and deproteinated in 100 μl methanol at 4° C, followed by centrifugation through a 0.2 μm filter column. The resulting supernatants were further diluted in .435mM perchlorate in 13% methanol. Mobile phase consisted of 17.2% acetonitrile, .018mM perchlorate, 0.7 μM EDTA and 2.54 mg/ml 1-octanesulfonic acid. Peaks were detected and integrated using a BAS 502 isocratic liquid chromatographic system (Bioanalytical Systems, West Lafayette, IN) with a Ag/AgCl reference electrode and 3mm glassy carbon electrode at +950mV.

2.5 GC-MS measurement of brain endocannabinoids

To measure levels of the fatty acid amides 2 arachidonyl glycerol (2-AG), anandamide (AEA), and oleoylethanolamide (OEA), frontal cortex samples were cut from frozen brain at −20°C, weighed and spiked with 50 pmol of [²H₄]anandamide, [²H₄]oleoylethanolamine and [²H₅]-2-arachidonyl glycerol (internal standards) and processed as in Hardison et al. (2006). Briefly, lipids were extracted by adding methanol/chloroform/water (1:2:1, v/v/v) and the chloroform layer was further purified by solid phase extraction using C18 Bond Elut cartridges (100 mg; Varian, Palo Alto, CA). Endocannabinoid-containing fractions were analyzed by gas chromatography/chemical ionization mass spectrometry (GC/MS) using isotope dilution as in Seillier et al. (2010).

2.6 Saturation Binding in Frontal Cortex Membrane Preparations

Frontal cortices from 3–4 month old naïve male mice (2 per sample) were homogenized in 40 ml of ice-cold 50 mM Tris-HCl buffer (pH 7.4 at 25°C) at 29,000 RPM for 10 sec on a Polytron (Brinkmann-Kinematica, Bohemia, NY). These homogenates were centrifuged at 30,600 × G for 10 min. The supernatants were discarded and pellets re-suspended in 25 ml buffer, and centrifuged at 30,600 × G. The final pellets were suspended in 10 ml of buffer. Protein concentration was determined with Bradford reagent (Sigma) by linear regression using bovine serum albumen (BSA) standards on a microplate reader (Synergy HT, Biotek, Winooski, VT).

Saturation binding to 0.003 – 12 nM [³H] CP 55,940 (Perkin-Elmer, Boston, MA) was carried out in triplicate at 25°C for 2 hours in 50mM Tris buffer supplemented with 5% BSA as in Herkenham et al. (1991). Non-specific binding was defined with 1 μM WIN 55,212-2. Incubation was terminated with 4 ml of 4°C buffer, and labeled homogenates were captured by filtration under vacuum onto glass fiber filters soaked in 0.5% polyethyleneimine with a tissue harvester (Brandel, Gaithersberg, MD). Filters were washed 3 times with 4 ml of 4°C buffer. Bound [³H] CP55,940 was measured on a liquid scintillation counter (Beckman, Brea, CA) with 61% efficiency. Data were analyzed by non-linear regression using DeltaGraph (Red Rock, Salt Lake City, UT).

2.7 Quantitative Autoradiography

Naïve 3–4 month old male mice were sacrificed by decapitation, their brains were frozen on dry ice, and stored at −80°C. Brains were sectioned 20 μm coronally from 1.3 to 1.6 mm pre-Bregma in a −18°C cryostat (Leica, Bannockburn, IL). Sections were thawed onto gelatin coated slides, desiccated at 4°C for 18–24h, and stored at −80°C.

For CB₁ receptor binding, brain sections on slides were pre-incubated in 50 mM Tris-HCl, 1% bovine serum albumin (BSA) buffer, pH 7.4 at 25°C for 30 min. They were then incubated in that same buffer containing 5 nM [³H] CP55,940 for 2 h. Nonspecific binding was determined using 0.2 mM WIN 55,212-2 (Ascent Scientific, Princeton, NJ) in the incubation solution for a subgroup of slides. Brain sections were washed in 50 mM Tris-HCl buffer, pH 7.4, containing 0.3 % BSA at 4°C for 4 h. This procedure was adapted from Herkenham et al., (1991), except lower concentrations of BSA were used.

To measure 5-HT_1A receptor density, [³H] 8-Hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, 2 nM, Perkin-Elmer) binding was performed exactly as described in Gould et al. (2011). Labeled sections were opposed to film (Biomax MR, Kodak, Rochester, NY) with [³H] standards (American Radiolabeled Chemicals (ARC), St. Louis, MO) for 6 weeks. Autoradiograms were captured as digital images an calibrated using NIH Image-J (NIH, Bethesda, MD) as described previously (Gould et al., 2011).

The methods of Seillier et al. (2010) were used for agonist-stimulated GTPγS binding. Briefly, sections on slides were equilibrated in buffer containing dithiothreitol (2 mM), pre-incubated in buffer containing 2mM GDP, and incubated in buffer containing 40 pM [³⁵S] GTPγS in the absence (basal) or presence (agonist-stimulated) of 1μM CP 55,940, 8-OH-DPAT or para-aminophenol. Nonspecific binding was defined under basal conditions with 10 μM GTPγS. Labeled sections were opposed to film (Biomax MR, Kodak) along with [¹⁴C] standards (ARC) for 48 hours. All autoradiograms were digitally captured and calibrated as described in Gould et al. (2011). Specific agonist-stimulated [³H]GTPγS binding was expressed as % above basal binding.

2.8 Statistical Analyses

Social interaction behavior was compared among treatment groups, first by global repeated-measures analysis of variance (ANOVA) to compare behavioral tendency shifts between the social interaction and social novelty phases of the test, then by mixed model ANOVA, with significant main effects or interactions (defined as p <0.05) examined post-hoc using Fisher’s LSD test. Mean times spent by treated BTBR mice in the chamber with the stranger mouse, as well as acetaminophen, endocannabinoid or 5-HT levels were compared using a two-tailed t-test, and saturation binding parameters were compared using one or two-way ANOVA, with significant effects examined post-hoc using Fisher’s LSD test. For autoradiography, mean densities were compared by one-way ANOVA, with significant main effects evaluated by Newman Keul’s test. Analyses were performed using Statistica (StatSoft, Tulsa, OK).

3.1 Behavior tests: Social approach and marble burying

 

3.1.1 Acute effects of acetaminophen in BTBR mice

The dose-response relationship for acetaminophen (1–400 mg/kg i.p.) to promote dwelling near a stranger mouse in the three chamber social approach test was initially determined in adult male BTBR mice. The lowest dose of acetaminophen to significantly increase time spent in the chamber with a stranger mouse above that of saline-injected controls was 100 mg/kg (F_4,25 = 4.6, p < 0.01, LSD p ≤ 0.05), as illustrated in Figure 1.

Figure 1

Acetaminophen doses of 100 mg/kg and 400 mg/kg increased dwelling near confined stranger mice by adult male BTBR mice

In subsequent three-chamber sociability tests, global repeated-measured ANOVA revealed significant interactions among acute drug treatments, test phase (social approach vs. social novelty) and duration of time spent in each side chamber (F_{INTERACTION 2,31} = 7.21, p < 0.005). In the social approach test, there was a significant interaction between drug treatment and chamber preference of BTBR mice as determined by mixed-model ANOVA (F_{INTERACTION 2,31} = 5.3, p ≤ 0.01). Acetaminophen (100mg/kg) treated mice spent more time in the chamber with the stranger mouse and than either vehicle control (saline ± 10% DMSO) or WIN 55,212-2 (0.1 mg/kg) treated mice (F_2,31 ≥ 4.8, p ≤ 0.015, LSD p < 0.025), and less time less time by the empty cage than controls (LSD p< 0.01), as shown in Figure 2a. However, there were no differences among treatment groups in time engaged in social sniff of the stranger mouse, or investigation of the empty cage, during the social approach test (F_2,31 < 1.1, p = 0.35 for both comparisons, see Figure 2b). Chamber entries did not differ among treated BTBR mice, and were on average 41 ± 5 during the 10 min social approach test. In the social novelty test, mixed model ANOVA revealed a significant interaction between drug treatment and chamber preference (F_{INTERACTION 2,31} = 3.4, p < 0.05). WIN 55,212-2 treated mice spent less time than others in the arena center (F_2,31 = 3.4, p < 0.05, LSD p <0.025), as shown in Figure 2c, tended to spend more time near the old stranger mice (F_2,31 = 1.9, p = 0.16, LSD p = 0.06), and made fewer chamber entries (19 ± 3) than either vehicle (45 ± 5) or acetaminophen (50 ± 7) treated mice (F_2,31 = 5.4, p < 0.01, LSD p<0.01) during social novelty testing. Number of marbles buried did not differ significantly among groups, but a trend toward WIN 55,212-2 treated mice burying less than controls was evident (F_2,17=2.4, p = 0.12, Figure 2d).

Figure 2

Acetaminophen increased BTBR mouse social interaction but not marble burying

 

3.1.2 Acute effects of acetaminophen in 129S1/SvImJ mice

Adult male 129S mice exhibited global differences in chamber dwelling patterns among drug treatments (F_2,24 =7.4, p < 0.003) and across the two sociability test phases (F_1,24 = 5.0, p < 0.03), without interaction. In the social approach test, all groups spent essentially equal time in chambers with a stranger and in chambers with an empty cage (Figure 3a). However, 129S mice treated with WIN 55,212-2 (0.1 mg/kg) spent more time than acetaminophen (100 mg/kg) or vehicle (saline ± 10% DMSO) treated mice in the center (F_2,24 = 5.0, p < 0.02, LSD p <0.05). WIN 55,212-2 treated mice also made fewer chamber entries on average (9 ± 4), than control mice (27 ± 5) but not acetaminophen treated mice (21 ± 5) during the social approach test (F_2,24 = 3.6, p < 0.05, LSD p <0.025). Time spent sniffing strangers or investigating empty cages did not differ among drug treatment groups (Figure 3b), although there was a trend toward acetaminophen-treated 129S mice spending more time investigating strangers that did not reach significance (F_2,24 = 2.5, p = 0.1). In the social novelty phase (Figure 3c), acetaminophen-treated 129S mice spent significantly more time than controls in the arena chamber with a new stranger mouse, while WIN 55,212-2 treatment increased dwelling in the center chamber (F_2,24 > 3.0, p < 0.05, LSD p <0.05 for both). The number of chamber entries in the social novelty test was similar across 129S treatment groups, and averaged 21 ± 4 over 10 min. Marble-burying in acetaminophen-treated mice was greater than in vehicle-control or WIN 55,212-2 treated 129S mice (F_2,24= 4.0, p < 0.05, LSD p<0.05), as shown in Figure 3d.

Figure 3

Acetaminophen increased 129S1/SvImJ social novelty preference and marble burying

 

3.1.3 Cannabinoid receptor 1 (CB₁) deficient mouse behavior

CB₁ receptor heterozygous knock-out mice exhibited similar behavior to wild-type mice in social approach and novelty preference tests, and in the marble burying task, as illustrated in figure 4. Between the social approach and social novelty tests there was a significant interaction between phase of test (approach vs. novelty) and time spent in the end chambers (F _{INTERACTION 1,26} = 41, P < 0.00001). This was due to a shift in preference from the side containing the stranger mouse in the initial social approach phase (Figure 4a), to the side containing a new stranger in the subsequent social novelty phase of the test (Figure 4c). There was no significant difference in any other parameter measured in the CB₁ deficient mouse sociability tests (F_1,26 < 2.0, P ≥ 0.1). CB₁ wild-type mice made 37 ± 2.7 chamber entries per test, and CB₁ deficient mice made 34 ± 3.3 entries per test during each phase of sociability testing. Marble burying was also similar among CB₁ genotypes, as shown in figure 4d.

Figure 4

Performance of cannabinoid CB1 receptor-deficient and wild-type mice is similar in sociability and marble-burying tasks

3.2 HPLC Analyses

3.3 Endocannabinoid levels in frontal cortex

Measures of endogenous cannabinoids in the frontal cortex from BTBR and 129S mice are summarized in table 1. BTBR mice that were either treated with acetaminophen and returned to home cages, or were saline-treated subjects in sociability tests had higher anandamide (AEA) levels in frontal cortex (drug effect and interaction F_1,19 > 4.11, p≤0.05, LSD p< 0.05, N = 5–7) than saline-treated controls, but these effects were not additive. Levels of 2 arachidonyl glycerol (2-AG) and oleoylethanolamide (OEA) did not differ among treatment groups (effects and interaction F_1,8 < 2, p > 0.18). However, there was a trend (behavior effect F_1,20 = 3.66, p = 0.07) toward BTBR mice that performed sociability tests having slightly higher 2-AG levels (4.7 ± 0.3 nmol/g) than those that did not (3.9 ± 0.2 nmol/g).

Table 1

Endogenous cannabinoid levels in mouse frontal cortex

In contrast, neither behavior testing nor acetaminophen treatment increased AEA in the 129S frontal cortex. Instead, among behavior naïve mice, acetaminophen reduced AEA levels relative to saline controls (drug effect F_1,25 = 4.29, p <0.05; LSD p<0.05). OEA levels were similar among 129S groups (F_1,25 < 1.0, p = 0.33). However, 2-AG levels were lower (F_1,25 = 15, p <0.001, LSD p < 0.001) in drug treated groups than in vehicle-control behavior test subjects, as shown in table 1.

3.4 CB₁ Receptor saturation binding in frontal cortex

[³H]CP55,940 saturation binding in the frontal cortex revealed similar maximal binding (Bmax, F_2,16 = 1.03, p = 0.37) and dissociation constant values (Kd, F_2,16 = 0.72, p = 0.49) for BTBR, C57 and 129S mice. Non-linear regression was used to fit these data to a single-site curve with Hill coefficients = 1. For each mouse strain, CB₁ receptor binding properties are summarized in table 2.

Table 2

Strain comparison of [³H] CP 55,940 binding in frontal cortex

3.5 Quantitative autoradiography: CB₁ receptor density and G-protein coupling capacity

CB₁ receptor density in cingulate cortex, labeled by [³H] CP 55,940 specific binding, did not differ among BTBR, 129S or C57BL/6 mice (F_2,21 = 0.78, p = 0.47, Figure 5a). Specific binding of [³H] 8-OH-DPAT to 5-HT_1A receptors in this brain region was higher in 129S mice than in other strains (F_2,21= 5.0, p < 0.02, Newman Keul’s p < 0.025, Figure 5b). When [³⁵S] GTPγS binding was stimulated by 1μM of the CB₁ receptor agonist CP 55,940, C57 mice had lower specific binding than either BTBR or 129S mice (F_2,21= 9.5, p < 0.002, Newman Keul’s p < 0.005, Figure 5c). In contrast, neither 1μM of para-aminophenol nor 1μM of 8-OH-DPAT were able to produce any [³⁵S] GTPγS binding in excess of 30% over basal binding. Basal binding did not differ among mouse strains (F_2,21= 2.5, p = 0.1) and was on average 225 ± 9 nCi/mg.

Figure 5

Comparison of CB₁ and 5-HT_1A receptor density and agonist-stimulated G-protein coupling in the cingulate cortex indicate higher CP55,940 sensitivity in socially impaired mice, and lack of para-aminophenol CB₁ agonist activity

4.1 Sociability promoting effects of acetaminophen

The neural circuitry involved in perception of physical pain and the experience of “social pain” or hurt feelings has been shown to overlap to some extent (Eisenberger et al., 2003;2006). Magnetic resonance imaging and cerebral blood-flow studies indicate that activity in the anterior cingulate region of the frontal cortex is involved in both pain perception and emotional response to social rejection, and that this activity is suppressed by acetaminophen administration (Baliki et al., 2006; DeWall et al., 2010; Newberg et al., 2011). Consistent with the idea of shared circuitry, acetaminophen administration is reported to reduce social pain associated with rejection (DeWall et al., 2010), and to promote social interaction (Chibnall et al., 2005) in clinical studies.

Umathe et al. (2009) were the first to describe that acetaminophen and andandamide could dose-dependently enhance social interaction behavior in Swiss mice, and that this effect could be blocked by the CB₁ antagonist AM251. In the present study, we found that acetaminophen also enhanced social behavior in BTBR T+/tf and 129S1/SvImJ mice, two inbred strains with inherently low sociability in three chambered tests. BTBR mice typically spend equal time investigating stranger mice and novel objects in social approach tests, but show a preference for social novelty (Moy et al., 2007). We observed that acetaminophen, but not the CB₁ agonist WIN 55,212-2, increased social interaction by BTBR mice in the social approach test, while WIN 55,212-2 treatment tended to diminish social novelty preference (Figure 2). In contrast, 129S mice normally exhibit both impaired social interaction, and a lack of preference for social novelty (Moy et al., 2007). Acetaminophen and WIN55,212-2 failed to enhance 129S social interaction in the social approach test, however, acetaminophen produced a preference for social novelty in 129S mice (Figure 3). Hence sociability was enhanced by acetaminophen in different ways in each strain.

4.2 Effects of acetaminophen or CB1 expression on marble burying

Marble burying by mice was initially thought to be an index of anxiety and obsessive compulsive behavior (e.g. Ichimaru et al., 1995), but a more recent study has characterized this genetically-variable but inherent behavior more properly as an index of restrictive-repetitive digging behavior, unrelated to novelty, and not correlated with other measures of anxiety (Thomas et al., 2009). In our study, vehicle-treated BTBR mice buried more marbles (13 ± 2 per 30 min) than 129S mice (3 ± 1.2 per 30 min), and acetaminophen only increased marble burying in 129S mice (Figures 2d and ​and3d).3d). WIN 55,212-treated BTBR mice tended to bury fewer marbles than controls, this trend, which did not reach significance, is consistent with a recent study in which mice treated with a higher dose of WIN 55,212 (1mg/kg) buried significantly fewer marbles than controls (Gomes et al., 2011). Since marble burying is a behavior that is sensitive to various pharmacological effects (e.g. Gould et al., 2011), its use may aid discovery of effective interventions for repetitive behaviors found in various psychiatric disorders.

4.3. Region-specific endocannabinoid induction vs. global full-agonist effects

The acetaminophen-induced increase in BTBR cortical AEA (Table 1) may have contributed to their enhanced social behavior. Paralleling this finding, in fatty acid amide hydrolase (FAAH) knock-out mice AEA tone, 5-HT tone and social behavior are all enhanced (Cassano et al., 2011). In contrast, WIN55,212-2 failed to improve sociability, but instead tended to worsen it in our tests. This second finding is consistent with a study in rats in which inhibition of AEA breakdown increased social play, while WIN55,212-2 reduced it (Trezza and Vanderschuren, 2008). The inconsistent behavioral effects of this full CB₁ agonist and AEA could be due to WIN55,212-2 having a greater affinity for, and agonist effect at CB₁ and/or CB₂ receptors then endogenous cannabinoids (Ashton et al., 2008). Further, WIN55,212-2 may not mimic the regional specificity of endogenous cannabinoids, and may instead recruit areas with deleterious effects on social interaction, or it may have longer-lasting effects (Wiley et al., 2005). Alternatively, the different behavioral effects of these drugs might be attributed to initial variability in cannabinoid or 5-HT tone. For example, withdrawal in phencyclidine-treated rats impairs social behavior, but treatment with the FAAH inhibitor URB597 restores sociability; yet alone it suppressed sociability in naïve rats (Seillier et al., 2010).

It is of interest that the full cannabinoid agonist, WIN 55,212-2, reduced locomotor activity in all of our behavior tests. We selected a 0.1 mg/kg dose through a combination of prior literature indicating a dose 1 mg/kg increased open-arm entries in the elevated plus maze without impairing locomotion (Haller et al., 2004; Patel and Hillard, 2006). However, we found in our own pilot studies that 1 mg/kg WIN 55,212-2 completely inhibited locomotor activity in BTBR and 129S mice, resulting in no chamber entries in sociability tests. Based on this, we reduced the dose to 0.1 mg/kg for these experiments. An earlier study also found that WIN55,212-2 suppressed mouse locomotor activity at low receptor occupancy, with an effective dose of 0.3 mg/kg (Gifford et al., 1999). Yet WIN 55,212-2 had demonstrated sedative effects only at higher doses in other mouse studies (e.g. Cosenza et al., 2000; Rutkowska et al., 2006). Sensitivity to WIN55,212-2 may be strain-specific, such that BTBR and 129S mice are more responsive to it than C57BL/6 mice. This would be consistent with our CP55,940 stimulated GTPγS binding results in the cingulate cortex (Figure 5c). We did not find any differences in the density of CB₁receptors in the frontal or cingulate cortex among BTBR, 129S or C57BL/6 mice (Table 2and Figure 5a), yet we found increased capacity for CB₁ agonist stimulated G-protein coupling in BTBR and 129S mice (Figure 5c). We also found no evidence of para-aminophenol stimulated G-protein coupling activity in the cingulate cortex among the strains we examined.

4.4. Acetaminophen, cannabinoid and 5-HT system interactions

The pain-relieving properties of acetaminophen appear to be mediated, in part, through both cannabinoid CB₁ receptor activation and 5-HT system modulation. Acetaminophen’s analgesic effects are blocked by CB₁ receptor antagonists or by complete depletion of CB₁receptors (Ottani et al., 2006; Dani et al., 2007; Mallet et al., 2008). However, acetaminophen is unlikely to act as a direct CB₁ receptor agonist. Instead the endocannabinoid transporter inhibitor AM404 is produced through its metabolism. AM404 itself has little affinity for CB₁ receptors (Beltramo et al., 1997), but it increases extracellular levels of endogenous cannabinoids such as AEA and 2-AG that activate CB1 receptors (Högestätt et al., 2005; Bertolini et al., 2006; Schultz, 2010). However, other receptors are implicated in acetaminophen’s effects, for example, its analgesic effects are lost in transient receptor potential vanilloid 1 (TRPV1) knock-out mice, and AM404 is a TRPV1 agonist (Mallet et al., 2010). Also, CB₂ receptors in the central nervous system may also modulate such responses (Onaivi et al., 2012).

Endocannabinoids also regulate glutamate signaling and plasticity in the frontal cortex (Lafourcade et al., 2007). Administration of the CB₁ agonists CP55,940, AEA, or WIN55,212-2 suppressed 5-HT release, perhaps by blocking glutamate transmission in the dorsal raphe, which projects neurons into the frontal cortex (Nakazi et al., 2000; Haj-Dahmane and Shen, 2009). Consistent with this, in CB₁ knock-out mice 5-HT negative feedback is lost, and 5-HT neurotransmission is facilitated (Aso et al., 2009).

Yet acetaminophen metabolites such as AM404 may also act as agonists at 5-HT receptors. For example, in a clinical study, antagonists of 5-HT₃ receptors effectively blocked acetaminophen’s antinociceptive properties (Pickering et al., 2006). In rats, 5-HT_1A and 5-HT_3/4 antagonists, as well as 5,7-dihydroxytryptamine-induced lesions of bulbospinal 5-HT pathways blocked acetaminophen-induced analgesia (Mallet et al., 2008). When the roles of 5-HT receptors in the analgesic actions of AM404 and acetaminophen were compared, the 5-HT₃ antagonist ondanseteron blocked both, while the 5-HT₂ antagonist ketanserin blocked only acetaminophen’s effects (Ruggieri et al., 2008). This latter result suggests that acetaminophen metabolites other than AM404 may activate 5-HT₂ receptors. Taken together, these findings suggest that enhancement, not suppression of serotonergic transmission is also involved in acetaminophen’s analgesic actions.

Further evidence for this comes from discovery that both acute and sub-chronic acetaminophen administration reduced 5-HT₂ receptor density by 30% in rat frontal cortex (Sandrini et al., 2007), an effect blocked by p-chlorophenylalanine-induced 5-HT depletion in the brain (Pini et al., 1996); and that the 5-HT_1&2 receptor antagonist methysergide blocked acetaminophen-induced changes in mouse hippocampal long term potentiation (Chen and Bazan, 2003). These findings suggest that acetaminophen is promoting 5-HT neurotransmission, instead of suppressing it, as might be expected to occur through CB₁ receptor activation. These outcomes may stem from high dose-acetaminophen (100 mg/kg) inhibiting the hepatic apoenzyme of tryptophan-2,3-dioxygenase (TDO), thereby increasing conversion of tryptophan to 5-HT in the brain, as was found in rats (Daya and Anoopkumar-Dukie, 2000). Another study concluded that Inhibition of 5-HT breakdown is unlikely to account for increased brain 5-HT levels 45 min after high dose (100–400 mg/kg) acetaminophen administration to rats, since monoamine oxygenase A activity and 5-hydroxyindoleacetic acid levels were unchanged (Courade et al., 2001). 5-HT levels in the frontal cortex of BTBR mice were not in excess of saline treated BTBR controls in frontal cortex 100 min after acetaminophen administration (100 mg/kg) in our study. However, this measurement was made in frozen frontal cortex collected at a later time than in either rat study, and acetaminophen and para-aminophenol may have been substantially cleared by then. It would be of interest to measure 5-HT turnover in the mouse frontal cortex, or extracellular 5-HT levels by microdialysis sooner after acetaminophen is given to see if 5-HT neurotransmission is enhanced. Nevertheless, social behaviors 50–70 min after acetaminophen administration were consistent with enhanced 5-HT neurotransmission, as occurs with 5-HT uptake blockade or buspirone treatment (Gould et al., 2011).

We also demonstrated that the potential for CB₁ agonist stimulated G-protein coupling in frontal cortex is much greater than for 5-HT_1A agonists, since we saw no evidence of strong 8-OH-DPAT (5-HT_1A agonist) stimulated G-protein activity in the cingulate cortex of any strain (Figure 5c). Yet acetaminophen could be acting at other types of 5-HT receptors, such as 5-HT_2/3 and/or in other brain regions. For example, in the dorsal hippocampus of BTBR mice, 8-OH-DPAT produces significant increases over basal [³⁵S]GTPγS binding, and busprione, a 5-HT_1A partial-agonist improved their sociability (Gould et al., 2011). Other key brain regions to social behavior that have yet to be identified might also respond similarly to acetaminophen treatment.

4.5. CB₁ deficient mice: Are they on the edge of impaired sociability?

We did not observe differences in sociability or marble burying between CB₁ heterozygous and wild-type mice (Figure 4). This was not surprising, since CB₁ knock-out sociability is similar to wild-type mice under dim lighting (Haller et al., 2004). However with bright lighting or in unfamiliar cages, Haller et al. (2004) found impaired sociability in CB₁knock-outs. Four common and many additional rare functional CB₁ receptor gene promoter polymorphisms occur in human populations, and some are associated with susceptibility to substance abuse, anxiety disorders and social reward (Zhang et al., 2004;Chakrabarti et al., 2006; Lazary et al., 2009). Subtle differences in CB₁ affinity for endocannabinoids from such polymorphisms may underlie different responses to social cues (Chakrabarti et al., 2006). It is of interest that in CB₁ deficient mice, WIN55,212-2 stimulated [³⁵S]GTPγS binding was only 25% lower than wild-types, despite 50% lower CB₁ receptor density, and region-dependent differences were found in the efficacy/potency of partial-agonists that the authors thought may result from either differential G-protein availability, or activation of different subtypes (Selley et al., 2001). There has been little follow-up on the role CB₁ polymorphisms may play in the impaired social behaviors of autism and schizophrenia, but given the involvement of CB₁ receptors in brain development (Trezza et al., 2008), CB₁ deficient mice may be a useful model in which to study these effects.

We thank Ken Hargreaves, DDS, PhD and Chair of Endodontics, Claudia Miller, MD and Raymond Palmer, PhD, Professors of Family and Community Medicine, and Bettie Sue Masters, PhD, Professor of Biochemistry at the University of Texas Health Science Center at San Antonio for their helpful suggestions for this project. We also thank Lynette Daws PhD, Professor of Physiology for use of her laboratory resources, and Irene Chapa PhD, Director of Science Outreach for making high school student involvement possible through the Mentor/Shadow Program. This study was supported by U.S. Navy Medical Research Unit contracts, and by grants from the National Institute of Mental Health (R03MH086708, R01MH090127 and P30MH089868), the Morrison Trust, Lindow Stephens Treat, and sub-awards from the Institute for Integration of Medicine and Science CTSA grant (UL1RR025767), and the South Texas Advanced Research Training Undergraduate Program grant (R25GM097632). Jason O’Connor has received an honorarium from Lundbeck Research, USA.

2-AG

2-arachidonyl glycerol

8-OH-DPAT

8-Hydroxy-2-(di-n-propylamino)tetralin

ACM

acetaminophen

AEA

anandamide

AM404

N-arachidonyl-phenolamine

ANOVA

analysis of variance

CB

cannabinoid

DMSO

dimethyl-sulfoxide

FAAH

fatty acid amide hydrolase

GDP

guanosine diphosphate

GTP

guanosine triphosphate

HPLC

high performance liquid chromatography

OEA

oleoylethanolamide

PCR

polymerase chain reaction

TRPV1

transient receptor potential vanilloid 1 cation channels

Tris

Tris(hydroxymethyl) aminomethane

WIN

WIN55,212-2

The other authors have no conflicts of interest to disclose.

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• Ashton JC, Wright JL, McPartland JM, Tyndall JD. Cannabinoid CB₁ and CB₂receptor ligand specificity and the development of CB2-selective agonists. Curr Med Chem. 2008;15:1428–43. [PubMed]
• Aso E, Renoir T, Mengod G, Ledent C, Hamon M, Maldonado R, Lanfumey L, Valverde O. Lack of CB₁ receptor activity impairs serotonergic negative feedback.J Neurochem. 2009;109:935–44. [PubMed]
• Baliki MN, Chialvo DR, Geha PY, Levy RM, Harden RN, Parrish TB, Apkarian AV. Chronic pain and the emotional brain: specific brain activity associated with spontaneous fluctuations of intensity of chronic back pain. J Neurosci.2006;26:12165–73. [PubMed]
• Bartolomucci A, Carola V, Pascucci T, Puglisi-Allegra S, Cabib S, Lesch KP, Parmigiani S, Palanza P, Gross C. Increased vulnerability to psychosocial stress in heterozygous serotonin transporter knockout mice. Dis Model Mech. 2010;3:459–70. [PubMed]
• 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. [PubMed]
• Bertolini A, Ferrari A, Ottani A, Guerzoni S, Tacchi R, Leone S. Paracetamol: new vistas of an old drug. CNS Drug Rev. 2006;12:250–75. [PubMed]
• Boylan CB, Blue ME, Hohmann CF. Modeling early cortical serotonergic deficits in autism. Behav Brain Res. 2007;176:94–108. [PMC free article] [PubMed]
• Campanero MA, Calahorra B, García-Quétglas E, López-Ocáriz A, Honorato J. Rapid liquid chromatographic assay for the determination of acetaminophen in plasma after propacetamol administration: application to pharmacokinetic studies. J Pharm Biomed Anal. 1999;20:327–34. [PubMed]
• Cassano T, Gaetani S, Macheda T, Laconca L, Romano A, Morgese MG, Cimmino CS, Chiarotti F, Bambico FR, Gobbi G, Cuomo V, Piomelli D. Evaluation of the emotional phenotype and serotonergic neurotransmission of fatty acid amide hydrolase-deficient mice. Psychopharmacology. 2011;214:465–76.[PMC free article] [PubMed]
• Chakrabarti B, Kent L, Suckling J, Bullmore E, Baron-Cohen S. Variations in the human cannabinoid receptor (CNR1) gene modulate striatal responses to happy faces. Eur J Neurosci. 2006;23:1944–8. [PubMed]
• Chibnall JT, Tait RC, Harman B, Luebbert RA. Effect of acetaminophen on behavior, well-being, and psychotropic medication use in nursing home residents with moderate-to-severe dementia. J Am Geriatr Soc. 2005;53:1921–9. [PubMed]
• Chen C, Bazan NG. Acetaminophen modifies hippocampal synaptic plasticity via a presynaptic 5-HT₂ receptor. Neuroreport. 2003;14:743–7. [PubMed]
• Cosenza M, Gifford AN, Gatley SJ, Pyatt B, Liu Q, Makriyannis A, Volkow ND. Locomotor activity and occupancy of brain cannabinoid CB₁ receptors by the antagonist/inverse agonist AM281. Synapse. 2000;38:477–82. [PubMed]
• Courade JP, Caussade F, Martin K, Besse D, Delchambre C, Hanoun N, Hamon M, Eschalier A, Cloarec A. Effects of acetaminophen on monoaminergic systems in the rat central nervous system. Naunyn Schmiedebergs Arch Pharmacol.2001;364:534–7. [PubMed]
• Dani M, Guindon J, Lambert C, Beaulieu P. The local antinociceptive effects of paracetamol in neuropathic pain are mediated by cannabinoid receptors. Eur J Pharmacol. 2007;573:214–5. [PubMed]
• Daya S, Anoopkumar-Dukie S. Acetaminophen inhibits liver trytophan-2,3-dioxygenase activity with a concomitant rise in brain serotonin levels and a reduction in urinary 5-hydroxyindole acetic acid. Life Sci. 2000;67:235–40.[PubMed]
• Derntl B, Seidel EM, Eickhoff SB, Kellermann T, Gur RC, Schneider F, Habel U. Neural correlates of social approach and withdrawal in patients with major depression. Soc Neurosci. 2011;6:482–501. [PMC free article] [PubMed]
• Dewall CN, Macdonald G, Webster GD, Masten CL, Baumeister RF, Powell C, Combs D, Schurtz DR, Stillman TF, Tice DM, Eisenberger NI. Acetaminophen reduces social pain: behavioral and neural evidence. Psychol Sci. 2010;21:931–7.[PubMed]
• Defensor EB, Pearson BL, Pobbe RL, Bolivar VJ, Blanchard DC, Blanchard RJ. A novel social proximity test suggests patterns of social avoidance and gaze aversion-like behavior in BTBR T+ tf/J mice. Behav Brain Res. 2010;217:302–8.[PMC free article] [PubMed]
• Eisenberger NI, Lieberman MD, Williams KD. Does rejection hurt? An FMRI study of social exclusion. Science. 2003;302:290–2. [PubMed]
• Eisenberger NI, Jarcho JM, Lieberman MD, Naliboff BD. An experimental study of shared sensitivity to physical pain and social rejection. Pain. 2006;126:132–8.[PubMed]
• Feldman R, Vengrober A. Posttraumatic stress disorder in infants and young children exposed to war-related trauma. J Am Acad Child Adolesc Psychiatry.2011;50:645–58. [PubMed]
• File SE, Seth P. A review of 25 years of the social interaction test. Eur J Pharmacol. 2003;463:35–53. [PubMed]
• Filipenko ML, Beilina AG, Alekseyenko OV, Dolgov VV, Kudryavtseva NN. Increase in expression of brain serotonin transporter and monoamine oxidase a genes induced by repeated experience of social defeats in male mice.Biochemistry. 2002;67:451–5. [PubMed]
• Gerretsen P, Graff-Guerrero A, Menon M, Pollock BG, Kapur S, Vasdev N, Houle S, Mamo D. Is desire for social relationships mediated by the serotonergic system in the prefrontal cortex? An [(18)F]setoperone PET study. Soc Neurosci.2010;5:375–83. [PubMed]
• Gifford AN, Bruneus M, Gatley SJ, Lan R, Makriyannis A, Volkow ND. Large receptor reserve for cannabinoid actions in the central nervous system. J Pharmacol Exp Ther. 1999;288:478–83. [PubMed]
• Gomes FV, Casarotto PC, Resstel LB, Guimarães FS. Facilitation of CB₁ receptor-mediated neurotransmission decreases marble burying behavior in mice. Prog Neuropsychopharmacol Biol Psychiatry. 2011;35:434–8. [PubMed]
• Gould GG, Hensler JG, Burke TF, Benno RH, Onaivi ES, Daws LC. Density and function of central serotonin (5-HT) transporters, 5-HT_1A and 5-HT_2A receptors, and effects of their targeting on BTBR T+tf/J mouse social behavior. J Neurochem. 2011;116:291–303. [PMC free article] [PubMed]
• Haj-Dahmane S, Shen RY. Endocannabinoids suppress excitatory synaptic transmission to dorsal raphe serotonin neurons through the activation of presynaptic CB₁ receptors. J Pharmacol Exp Ther. 2009;331:186–96.[PMC free article] [PubMed]
• Haller J, Varga B, Ledent C, Barna I, Freund TF. Context-dependent effects of CB₁ cannabinoid gene disruption on anxiety-like and social behaviour in mice.Eur J Neurosci. 2004;19:1906–12. [PubMed]
• Hardison S, Weintraub ST, Giuffrida A. Quantification of endocannabinoids in rat biological samples by GC/MS: technical and theoretical considerations.Prostaglandins Other Lipid Mediat. 2006;81:106–12. [PubMed]
• Herkenham M, Lynn AB, Johnson MR, Melvin LS, de Costa BR, Rice KC. Characterization and localization of cannabinoid receptors in rat brain: a quantitative in vitro autoradiographic study. J Neurosci. 1991;11:563–83.[PubMed]
• Högestätt ED, Jönsson BA, Ermund A, Andersson DA, Björk H, Alexander JP, Cravatt BF, Basbaum AI, Zygmunt PM. Conversion of acetaminophen to the bioactive N-acylphenolamine AM404 via fatty acid amide hydrolase-dependent arachidonic acid conjugation in the nervous system. J Biol Chem.2005;280:31405–12. [PubMed]
• Hoyle D, Juhasz G, Aso E, Chase D, del Rio J, Fabre V, Hamon M, Lanfumey L, Lesch KP, Maldonado R, Serra MA, Sharp T, Tordera R, Toro C, Deakin JF. Shared changes in gene expression in frontal cortex of four genetically modified mouse models of depression. Eur Neuropsychopharmacol. 2011;21:3–10.[PubMed]
• Ichimaru Y, Egawa T, Sawa A. 5-HT_1A-receptor subtype mediates the effect of fluvoxamine, a selective serotonin reuptake inhibitor, on marble-burying behavior in mice. Jpn J Pharmacol. 1995;68:65–70. [PubMed]
• Lafourcade M, Elezgarai I, Mato S, Bakiri Y, Grandes P, Manzoni OJ. Molecular components and functions of the endocannabinoid system in mouse prefrontal cortex. PLoS One. 2007;2:e709. [PMC free article] [PubMed]
• Lazary J, Lazary A, Gonda X, Benko A, Molnar E, Hunyady L, Juhasz G, Bagdy G. Promoter variants of the cannabinoid receptor 1 gene (CNR1) in interaction with 5-HTTLPR affect the anxious phenotype. Am J Med Genet B Neuropsychiatr Genet. 2009;150B:1118–27. [PubMed]
• Lau T, Schloss P. The cannabinoid CB₁ receptor is expressed on serotonergic and dopaminergic neurons. Eur J Pharmacol. 2008;578:137–41. [PubMed]
• Mallet C, Daulhac L, Bonnefont J, Ledent C, Etienne M, Chapuy E, Libert F, Eschalier A. Endocannabinoid and serotonergic systems are needed for acetaminophen-induced analgesia. Pain. 2008;139:190–200. [PubMed]
• Mallet C, Barrière DA, Ermund A, Jönsson BA, Eschalier A, Zygmunt PM, Högestätt ED. TRPV1 in brain is involved in acetaminophen-induced antinociception. PLoS One. 2010;5:e12748. [PMC free article] [PubMed]
• Moy SS, Nadler JJ, Young NB, Perez A, Holloway LP, Barbaro RP, Barbaro JR, Wilson LM, Threadgill DW, Lauder JM, Magnuson TR, Crawley JN. Mouse behavioral tasks relevant to autism: phenotypes of 10 inbred strains. Behav Brain Res. 2007;176:4–20. [PMC free article] [PubMed]
• Nakazi M, Bauer U, Nickel T, Kathmann M, Schlicker E. Inhibition of serotonin release in the mouse brain via presynaptic cannabinoid CB₁ receptors. Naunyn Schmiedebergs Arch Pharmacol. 2000;361:19–24. [PubMed]
• Newberg AB, Hersh EV, Levin LM, Giannakopoulos H, Secreto SA, Wintering NA, Farrar JT. Double-blind, placebo-controlled, randomized pilot study of cerebral blood flow patterns employing SPECT imaging in dental postsurgical pain patients with and without pain relief. Clin Ther. 2011;33:1894–1903. [PubMed]
• O’Connor JC, André C, Wang Y, Lawson MA, Szegedi SS, Lestage J, Castanon N, Kelley KW, Dantzer R. Interferon-gamma and tumor necrosis factor-alpha mediate the upregulation of indoleamine 2,3-dioxygenase and the induction of depressive-like behavior in mice in response to bacillus Calmette-Guerin. J Neurosci. 2009;29:4200–4209. [PMC free article] [PubMed]
• Onaivi ES, Ishiguro H, Gu S, Liu QR. CNS effects of CB2 cannabinoid receptors:beyond neuro-immuno-cannabinoid activity. J Psychopharmacol.2012;26:92–103. [PMC free article] [PubMed]
• Ottani A, Leone S, Sandrini M, Ferrari A, Bertolini A. The analgesic activity of paracetamol is prevented by the blockade of cannabinoid CB₁ receptors. Eur J Pharmacol. 2006;531:280–1. [PubMed]
• Patel S, Hillard CJ. Pharmacological evaluation of cannabinoid receptor ligands in a mouse model of anxiety: further evidence for an anxiolytic role for endogenous cannabinoid signaling. J Pharmacol Exp Ther. 2006;318:304–11.[PubMed]
• Pickering G, Loriot MA, Libert F, Eschalier A, Beaune P, Dubray C. Analgesic effect of acetaminophen in humans: first evidence of a central serotonergic mechanism. Clin Pharmacol Ther. 2006;79:371–8. [PubMed]
• Pini LA, Sandrini M, Vitale G. The antinociceptive action of paracetamol is associated with changes in the serotonergic system in the rat brain. Eur J Pharmacol. 1996;308:31–40. [PubMed]
• Pinkham AE, Hopfinger JB, Pelphrey KA, Piven J, Penn DL. Neural bases for impaired social cognition in schizophrenia and autism spectrum disorders.Schizophr Res. 2008;99:164–75. [PMC free article] [PubMed]
• Ruggieri V, Vitale G, Pini LA, Sandrini M. Differential involvement of opioidergic and serotonergic systems in the antinociceptive activity of N-arachidonoyl-phenolamine (AM404) in the rat: comparison with paracetamol. Naunyn Schmiedebergs Arch Pharmacol. 2008;377:219–29. [PubMed]
• Rutkowska M, Jamontt J, Gliniak H. Effects of cannabinoids on the anxiety-like response in mice. Pharmacol Rep. 2006;58:200–6. [PubMed]
• Sandrini M, Vitale G, Ruggieri V, Pini LA. Effect of acute and repeated administration of paracetamol on opioidergic and serotonergic systems in rats.Inflamm Res. 2007;56:139–42. [PubMed]
• Schultz ST. Can autism be triggered by acetaminophen activation of the endocannabinoid system? Acta Neurobiol Exp. 2010;70:227–31. [PubMed]
• Seillier A, Advani T, Cassano T, Hensler JG, Giuffrida A. Inhibition of fatty-acid amide hydrolase and CB1 receptor antagonism differentially affect behavioural responses in normal and PCP-treated rats. Int J Neuropsychopharmacol.2010;3:373–86. [PubMed]
• Selley DE, Rorrer WK, Breivogel CS, Zimmer AM, Zimmer A, Martin BR, Sim-Selley LJ. Agonist efficacy and receptor efficiency in heterozygous CB₁ knockout mice: relationship of reduced CB1 receptor density to G-protein activation. J Neurochem. 2001;77:1048–57. [PubMed]
• Sinning C, Watzer B, Coste O, Nüsing RM, Ott I, Ligresti A, Di Marzo V, Imming P. New analgesics synthetically derived from the paracetamol metabolite N-(4-hydroxyphenyl)-(5Z,8Z,11Z,14Z)-icosatetra-5,8,11,14-enamide. J Med Chem.2008;51:7800–5. [PubMed]
• Spencer CM, Alekseyenko O, Hamilton SM, Thomas AM, Serysheva E, Yuva-Paylor LA, Paylor R. Modifying behavioral phenotypes in Fmr1KO mice: genetic background differences reveal autistic-like responses. Autism Res. 2011;4:40–56.[PMC free article] [PubMed]
• Thomas A, Burant A, Bui N, Graham D, Yuva-Paylor LA, Paylor R. Marble burying reflects a repetitive and perseverative behavior more than novelty-induced anxiety. Psychopharmacology (Berl) 2009;204:361–73. [PMC free article][PubMed]
• Trezza V, Cuomo V, Vanderschuren LJ. Cannabis and the developing brain: insights from behavior. Eur J Pharmacol. 2008;585:441–52. [PubMed]
• Trezza V, Vanderschuren LJ. Bidirectional cannabinoid modulation of social behavior in adolescent rats. Psychopharmacology (Berl) 2008;197:217–27.[PubMed]
• Umathe SN, Manna SS, Utturwar KS, Jain NS. Endocannabinoids mediate anxiolytic-like effect of acetaminophen via CB1 receptors. Prog Neuropsychopharmacol Biol Psychiatry. 2009;33:1191–9. [PubMed]
• Wang LH, Rudolph AM, Benet LZ. Distribution and fate of acetaminophen conjugates in fetal lambs in utero. J Pharmacol Experimental Therap.1985;235:302–307. [PubMed]
• Wiley JL, Smith FL, Razdan RK, Dewey WL. Task specificity of cross-tolerance between Delta9-tetrahydrocannabinol and anandamide analogs in mice. Eur JPharmacol. 2005;510:59–68. [PubMed]
• Zhang PW, Ishiguro H, Ohtsuki T, Hess J, Carillo F, Walther D, Onaivi ES, Arinami T, Uhl GR. Human cannabinoid receptor 1: 5′ exons, candidate regulatory regions, polymorphisms, haplotypes and association with poly substance abuse. Mol Psychiatry. 2004;9:916–31. [PubMed]