Sparse metal bars allowed for paw access to the smooth acrylic floor, whereas dense-wire mesh did not. For high-fat diet CPP, animals were given one pellet of standard chow and an isocaloric amount of high-fat food . As high-fat pellets have a different color and consistency, they were also given to home cages the day before pre-conditioning to prevent neophobia. Statistical analyses. Results are expressed as means ± SEM. Significance was determined using two-tailed Student’s t-test, One-way or Two-way analysis of variance with Tukey’s post-hoc test and differences were considered significant if P<0.05. Analyses were conducted using GraphPad Prism .The use of marijuana is reinforced through activation of the mesolimbic reward circuit . In a related but distinct modulatory process, the neurotransmitter system mediating the effects of marijuana in the brain – the endocannabinoid system – also facilitates the reward of other stimuli, such as food or drugs of abuse . The endocannabinoid system has three main components: two lipid-derived local messengers – 2- arachidonoyl-sn-glycerol and anandamide , enzymes and transporters that mediate their formation and elimination, and receptors that are activated by endocannabinoids and regulate neuronal activity . Genetic and pharmacological studies have unveiled key roles of the CB1 receptor in the modulation of reward signaling. Less is known about the functions served by individual endocannabinoid messengers. In particular, an emerging question is whether endocannabinoids might also regulate the reward of social interactions. We recently demonstrated that anandamide regulates social reward via cooperative signaling with oxytocin, 4×4 grow table a neuropeptide that is crucial for social bonding . The role of 2-AG remains unknown, however. One way to assess the specific contribution of individual endocannabinoids is to manipulate the enzymes responsible for their formation and deactivation.

For example, pharmacological inhibition or genetic deletion of the enzyme that hydrolyzes anandamide, fatty acid amide hydrolase , markedly increases anandamide activity at CB1 cannabinoid receptors . Analogous strategies exist for 2-AG. Indeed, MGL-/- mice, in which the 2-AG-hydrolyzing enzyme monoacylglycerol lipase is deleted, andthus 2-AG levels are elevated, as well as DGL-α-/- mice, in which the 2-AG-synthesizing enzyme diacylglycerol lipase is deleted, and thus 2-AG levels are very low . However, the effects of these radical modifications are often difficult to interpret because of the emergence of profound compensatory changes in the brain, such as desensitization of CB1 receptors and elevation in anandamide and arachidonic acid levels . We have recently generated a novel transgenic mouse model – MGL-Tg mice – which selectively overexpress MGL in forebrain neurons under the control of the CaMKIIα promoter . These mutant mice display a forebrain-selective accrual in MGL hydrolyzing activity and a 50-75% decrement in 2-AG content. This reduction in 2-AG is not accompanied by overt changes in levels of other endocannabinoid-related lipids , cannabinoid receptors, or other endocannabinoid-related proteins . To investigate the role of 2-AG in reward-related behaviors, we tested MGL-Tg mice in conditioned place preference paradigms for high-fat food, social, or cocaine stimuli. Based on a rich theoretical framework, CPP assesses the rewarding value of test stimuli by pairing them with neutral environments . Because less is known about endocannabinoid signaling and social behavior, we also investigated the effects of social interaction on 2-AG signaling in reward-related regions of the brain. We hypothesized that MGL-Tg mice are deficient in reward signaling and that rewarding social stimuli drive 2-AG signaling in normal mice.Socially conditioned place preference . Procedures were previously described . Briefly, mice were placed in a two-chambered acrylic box .

A 30-min preconditioning session was used to establish baseline neutral preference to two types of autoclaved, novel bedding . These differed in texture and shade . Individual mice with strong baseline preference for either type of bedding were excluded – typically, those that spent more than 1.5x time on one bedding over the other. The next day, animals were randomly assigned to a social cage with cage-mates to be conditioned to one type of novel bedding for 24 h , then moved to an isolated cage with the other type of bedding for 24 h. On the next day, animals were tested alone for 30 min in the two-chambered box to determine post-conditioning preference for either type of bedding. Bedding volumes were 300 mL in each side of the two-chambered box and 550 mL in the home-cage. Familiar animals from the same cage were tested concurrently in four adjacent, opaque CPP boxes. Between trials, boxes were thoroughly cleaned with SCOE 10X odor eliminator . Scoring was automated using a validated image analysis script in ImageJ .Cocaine and high-fat diet CPP. Procedures were previously described . Briefly, these paradigms were similar to social CPP, including unbiased and counterbalanced design, cleaning and habituation, exclusion criteria, and scoring, except for the following main differences, which followed reported methods . Mice were conditioned and tested in a two-chambered acrylic box . Pre- and post conditioning tests allowed free access to both chambers and each had durations of 15 min and 20 min . For conditioning, animals underwent 30-min sessions alternating each day between saline/cocaine or standard chow pellet/high-fat pellet . The two chambers offered conditioning environments that differed in floor texture and wall pattern – sparse metal bars on the floor and solid black walls vs. dense-wire-mesh floors and striped walls. Sparse metal bars allowed for paw access to the smooth acrylic floor, whereas dense-wire mesh did not. For high-fat diet CPP, animals were given one pellet of standard chow and an isocaloric amount of high-fat food . As high-fat pellets have a different color and consistency, they were also given to home cages the day before pre-conditioning to prevent neophobia.Intake of high-fat pellets was recorded in free feeding mice using an automated monitoring system , as described previously . Food intake was measured for two days, and the average of intake was normalized to the body weight at the start of feeding.The test was conducted according to established methods .

To mimic the conditions of the social CPP task, mice were first isolated for 30 min and tested in dim-light conditions . Pairs of mice were tested in an open field arena for 5 min. Scoring for social interaction time included behaviors such as sniffing, following, grooming, mounting and crawling over or under. Passive interaction, in which mice were in close proximity but without these interactions, was not included in the scoring.The procedure was previously described , which was based on an established protocol . Briefly, test mice were first habituated to an empty three-chambered acrylic box , including to the center chamber for 10 min, cannabis drying system and then to all chambers for 10 additional min. Mice were then tested for 10 min. 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 used because they are relatively inert. They were trained to prevent erratic or aggressive behaviors, such as biting the cup. Weighted cups were placed on top of the pencil cups to prevent climbing. Low lighting was used. The apparatus was thoroughly cleaned with SCOE 10X odor eliminator between trials to preclude olfactory confounders. Chamber time scoring was automated using image analysis.Sniffing time was scored by trained assistants who were unaware of treatment conditions. Outliers in inactivity or side preference were excluded.The procedure was previously described . Briefly, whole brains were collected and flash-frozen in isopentane at -50 to -60 °C. Frozen brains were transferred to -20°C in a cryostat and kept for 1 h to attain local temperature. The brain was then cut to the desired coronal depth and micropunches from bilateral regions of interest were collected using a 1×1.5-mm puncher . The micropunches weighed approximately 1.75 mg. A reference micropunch was taken to normalize each punch to the brain’s weight. Bilateral punches were combined for lipid analyses.Procedures were previously described . 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 for15 min, then increased to 100% B in 1 min and kept at 100% B for 14 min. The column temperature was 15°C. Under these conditions, Na+ adducts of anandamide/H2 -anandamide had retention times of 6.9/6.8 min and m/z of 348/352, OEA/H2 -OEA had Rt 12.7/12.6 min and m/z 326/330, and 2-AG/2 H8-2-AG had Rt 12.4/12.0 min and m/z 401/409. An isotopedilution method was used for quantification.MGL-Tg mice eat less chow than do their wild-type littermates . The food intake phenotype, however, does not dissociate the effects of 2-AG signaling on metabolic and reinforcement processes.

Furthermore, altered feeding can be interpreted as either decreased or increased reward to the food stimulus. To isolate the effects of reduced 2-AG signaling on reward, we tested MGL-Tg mice and their wild-type littermates in a CPP task for high-fat food. In a standard CPP box, mice were conditioned for 30-min sessions to either standard chow or isocaloric high-fat food for 6 sessions each, alternating over 12 days total . In WT mice, we found that this conditioning protocol was sufficient to elicit a preference for the high-fat-paired chamber during post-conditioning testing. Animals spent 137 seconds more in the high-fat chamber compared to the standard-chow chamber . In contrast, MGL-Tg mice did not develop a preference for either chamber . This result suggests that 2-AG signaling is involved in conditioned reward processes of high-fat food. We then asked whether this role for 2-AG signaling could translate to the reward produced by social interaction . We conditioned mice for 24 h with cage-mates in their home-cage to one type of bedding, then we conditioned them for 24 h isolated to another bedding . In the post conditioning test in a standard CPP box, we found that this conditioning was sufficient to elicit a preference in WT mice for the social bedding . In contrast, MGL-Tg mice did not develop a preference for either bedding . Together with the high-fat-food CPP results, these results suggest that 2-AG signaling may underlie aspects of reward processes common to both natural stimuli.The lack of CPP can be attributed to impairments in the generation and processing of the reward, the consolidation of the memory for the reward, or a combination of these processes. To evaluate whether high-fat food stimuli are generated and processed properly, we measured initial intake of high-fat pellets over 2 days. MGL-Tg mice show a 16% reduction in normalized intake compared to WT littermates over this period . The combined phenotype of MGL-Tg mice showing a lack of CPP and decreased intake suggests that 2-AG plays a role in the generation and processing of high-fat food reward. Strict interpretation of these results, however, may be complicated by the role of 2-AG in energy metabolism . For the same reason, we also examined the direct social activity and the social approach interest of MGL-Tg mice using the social interaction test and the three chambered social approach test , respectively. These tests differ in two key ways: the social interaction test evaluates interactions that are reciprocal and direct, whereas the social approach test measures approach activity to a stimulus mouse that is sequestered in an inverted wire cup; the social interaction test uses familiar cage-mates, whereas the social approach test uses a novel mouse as a stimulus. In the social interaction test, we observed that MGL-Tg mice trend toward less interaction time, but this result was not significant . In the social approach test, we found that both MGL-Tg and WT mice preferred the social chamber over the object chamber and sniffed the stimulus mouse more than the object . MGL-Tg mice were similar to WT mice in the amount of time spent in the social chamber and sniffing the stimulus mouse .