In humans, marijuana can either facilitate or impair social interactions and social saliency, possibly depending on dose and context . Analogously, in animal models, cannabinoid receptor activation with direct-acting agonist drugs disrupts social interactions, whereas FAAH inhibition enhances them, which is suggestive of a role for anandamide in socialization . While important, these data leave unanswered two key questions. The first is whether the anandamide, whose functions in the modulation of stress-coping responses are well recognized , might influence social behavior by modulating stress reactivity . We addressed this question with two complementary sets of experiments. In one, we used a model of socially conditioned place preference that focuses specifically on the acquisition of incentive salience . Mice were conditioned to social contact with familiar cage-mates for 24 h, an intervention that does not cause stress. When this conditioning procedure was paired with FAAH inhibition, or faah-/- mice were used instead of wild-type mice, the animals displayed a markedly increased preference for the social context. In separate studies, we evaluated the impact of genetic FAAH deletion or pharmacological FAAH blockade in the social approach task, in which mice are given the option of interacting with a novel conspecific or a novel object for a relatively short period of time . Socially normal mice spend more time with their conspecific than with the object. We found that neither pharmacological nor genetic FAAH blockade had any effect in this model.
Collectively, hemp drying racks our findings support the conclusion that anandamide signaling at CB1 receptors specifically regulates the incentive salience of social interactions, and that this effect is independent of anandamide’s ability to modulate anxiety. A second question addressed by the present work pertains to the neural circuits responsible for recruiting endocannabinoid neurotransmission during socialization. We used convergent experimental approaches to show that OTR activation by endogenously released oxytocin triggers anandamide mobilization in the NAc. This result is consistent with evidence indicating that oxytocin acts as a social reinforcement signal within this limbic region, where it elicits a presynaptic form of long-term depression in medium spiny neurons . Thus, a plausible interpretation of our findings is that oxytocin triggers an anandamide-mediated paracrine signal in the NAc, which influences synaptic plasticity through activation of local CB1receptors. Activation of these receptors is known to induce presynaptic LTD at corticostriatal synapses . While providing an economical explanation for our results, the hypothesis formulated above also raises several new questions. Particularly important among them are questions about the roles that other modulatory neurotransmitters may play in regulating the interaction between oxytocin and anandamide. Previous studies point toward serotonin, which is needed for the expression of oxytocin-dependent plasticity in the NAc , and dopamine, which has been implicated in striatal anandamide signaling . Defining such roles will require, however, further investigation. In conclusion, our results illuminate a novel mechanism underlying the prosocial actions of oxytocin, and provide unexpected insights on possible neural substrates involved in the social facilitation caused by marijuana.
Pharmacological modulation of oxytocin-driven anandamide signaling – by utilizing, for example, FAAH inhibitors – might open new avenues to treat social impairment in autism spectrum disorders.We dissolved URB597 and AM251 in a vehicle of saline/propylene glycol/Tween-80 . L-368,899 was dissolved in saline for intraperitoneal injections or in DMSO for intracerebroventricular injections. WAY-267464 was dissolved in DMSO. Clozapine-N-oxide and cocaine were dissolved in saline. For lipid analyses, L-368,899 was administered 0.5 h before the start of socialization , WAY-267464 was administered by i.c.v. injection to 24-h isolated animals 0.5 h prior to sacrifice, and CNO to 24-h isolated animals 1 h before sacrifice. For the socially conditioned place preference test, animals were habituated to injections for 3 days leading up to the experiment. URB597, L-368,899 and AM251 were administered twice a day during social conditioning . Balancing vehicle treatments were given during isolation conditioning . For the social approach task, URB597 was administered i.p. 3 h before starting the test.We anesthetized mice with ketamine-xylazine and stereotaxically implanted a 22-gauge guide cannula positioned 1 mm above the right lateral ventricle at coordinates from bregma: AP -0.2, ML -1.0 and DV -1.3 mm . Animals were allowed to recover 10 days after surgery, during which they were maintained in social housing . Infusions were made in wake animals through a 33-gauge infusion cannula that extended 1 mm beyond the end of the guide cannula. The injector was connected to a 10- µL Hamilton syringe by PE-20 polyethylene tubing. The syringe was driven by an automated pump at a rate of 0.66 µL/min to provide a total infusion volume of 0.5 µL. Cannula placements were verified histologically.We used a serotype 2 AAV vector that was previously constructed and validated to express a modified muscarinic receptor and the fluorescent protein mCherry .
The hM3Dq receptor is exclusively activated by the otherwise inert compound clozapine-N-oxide. Expression of the virus is restricted by the oxytocin promoter, which directs oxytocin cell-specific expression of hM3Dq receptors and mCherry. mCherry expression in the paraventricular nucleus was verified using a Leica 6000B epifluorescence microscope.We bilaterally injected the AAV2-oxt-hM3Dq-mCherry construct into the PVN using the following coordinates: AP -0.70, ML ±0.30 and DV – 5.20 mm. We used an adaptor with 33-G needle connected via polyethylene tubing to a 10-µL Hamilton syringe driven by an automated pump . We waited for 5 min prior to infusion in order for tissue to seal around the needle, infused a total volume of 0.5 µL over 5 min and waited 10 min after infusion before removing the needle. Experiments were conducted 3 weeks after viral injections to allow for recovery and adequate expression. During this period, animals were maintained in social housing .Whole brains were collected and flash-frozen in isopentane at -50 to – 60 °C. Frozen brains were maintained in liquid nitrogen on the day of sacrifice until they were transferred to storage at -80 °C. To take micropunches of brain tissue, we first transferred frozen brains to -20°C in a cryostat and waited 1 h for brains to attain local temperature. We then cut to the desired coronal depth and collected micropunches from bilateral regions of interest 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 described previously . 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. 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 .Ketamine-xylazine anesthetized mice were perfused through the heart left ventricle, industrial rolling racks first with ice-cold saline solution and then with a fixation solution containing 4% paraformaldehyde in 0.1 M phosphate buffered saline . Brains were collected, post-fixed for 1.5 h and cryoprotected using 30% sucrose in PBS. Coronal sections were cut using a microtome and mounted on Superfrost plus slides . For cFos immunostaining, sections were washed in 0.1 M glycine solution to quench excess PFA. Sections were incubated for 1 h in blocking solution . Washed sections were incubated for 48 h at 4°C with anti-cFos antibody .
After washing with 0.1M PBS to remove unbound primary antibody, sections were then incubated for 1 h at room temperature with donkey anti-rabbit IgG conjugated to Alexa Fluor 594. Slides were cover-slipped with Vectashield plus DAPI . Images were captured using a 10x objective on a Leica 6000B epifluorescence microscope with PCO Scientific CMOS camera and Metamorph acquisition software.cFos quantification. Image montages were stitched together using FIJI . Variability in background fluorescence was standardized by subtracting a gaussian-blurred image of each image from itself. Objects of cellular size and shape were then detected using Python 2.7 and FIJI. Brain regions were traced by hand in FIJI using an atlas reference , and resulting coordinates were used to restrict cell counts. Because immunostaining varies across animals and experiments, values were normalized as a ratio to the dorsal striatum of the same animal. The dStr was selected as an internal control because it did not vary across compared groups . Socially conditioned place preference . Following previously described procedures , mice were placed in an opaque acrylic box , divided into two chambers by a clear acrylic wall with a small opening. In the box, a 30-min pre-conditioning test was used to establish baseline non-preference to two types of autoclaved, novel bedding , which differed in texture and shade . Individual mice with strong 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 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. Bedding assignments were counterbalanced for an unbiased design. Animals were then tested alone for 30 min in the two-chambered box to determine post conditioning preference for either type of bedding. Fresh bedding was used at each step and chambers were thoroughly cleaned between trials with SCOE 10X odor eliminator to avoid olfactory confounders. Volumes of bedding were measured to beconsistent – 300 mL in each side of the two-chambered box and 550 mL in the home-cage. Animals from the same cage were run concurrently in four adjacent, opaque CPP boxes. Scoring of chamber time and locomotion were automated using a validated image analysis script in ImageJ – the static background image was subtracted, moving objects of mouse shape and size were thresholded out and frames were counted in a position restricted manner.Three-chambered social approach task. Test mice were habituated to an empty three-chambered acrylic box , as previously described . Habituation consisted of a 10-min trial in the center chamber with doors closed, and then a 10-min trial in all chambers with doors open. Then, during the 10-min testing phase, 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, and they were trained to prevent aggressive or abnormal behaviors. 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. 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 as in social CPP. Subjects with outlying inactivity or side preference were excluded.Cocaine and high-fat diet CPP. These paradigms were largely similar to social CPP, including unbiased and counterbalanced design, cleaning and habituation, exclusion criteria, and scoring, except for the following key differences which followed established methods . Mice were conditioned and tested in a two-chambered opaque acrylic box with a small opening. 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.