Sex-dependent effects of in utero cannabinoid exposure on cortical function

1With 181.8 million estimated regular users aged 15 – 64, cannabis is the most commonly consumed illicit drug around the world (UNODC, 2015). The majority of users are young, and exposure largely occurs during their reproductive years (SAMSHA, 2015). In fact, data collected from 2002 to 2014 in the U.S. indicates that 7.5% of pregnant women between 18 and 25 years of age use cannabis, while the rate of use in all pregnant women is approximately 4%. A longitudinal prospective study on drug use in pregnancy showed that 48% of women who used cannabis in the year prior to their pregnancy continued to smoke cannabis throughout pregnancy. Moreover, 70% of pregnant and non-pregnant women believe that there is little or no harm in using cannabis once or twice per week. Thus, cannabis consumption during pregnancy is a major public health concern worthy of study as thousands of infants are prenatally exposed to cannabis.

2Worldwide legalization and/or decriminalization initiatives emphasize the urgent need for scientific evidence regarding possible harms and benefits of cannabis use. Epidemiological studies of children with a history of in utero cannabis exposure reveal normal intelligence scores but altered cognition and executive functions (attention, memory, concentration, inhibitory control) and higher levels of depression and anxiety during adolescence have been reported. In animal studies, prenatal exposure to various regimens of cannabinoids has been linked to alterations in locomotor, learning, memory, emotional functions and social interaction. A major caveat is that most studies investigated effects using male progeny only.

3Δ9-Tetrahydrocannabinol (THC), the major psychoactive ingredient in marijuana and other cannabis-based preparations, efficiently crosses the placental barrier and reaches the fetus. In the brain, cannabinoid receptor type 1 (CB1R) is the main target of THC. The endocannabinoid system, mainly through CB1R, has been implicated in neuronal proliferation, migration, morphogenesis and synaptogenesis. Consequently, it is crucial to understand the long-term consequences of cannabinoid exposure at this critical stage of development in both sexes.

4Here, we examined how prenatal cannabinoid exposure (PCE) (to synthetic and plant-derived cannabinoids) influences the synaptic and behavioral functions of the medial prefrontal cortex, a brain region implicated in neuropsychiatric disorders. We observed marked sexual divergence in the protracted effects of PCE. Following PCE, social interaction was altered in adult male rats and this alteration was paralleled by sex-specific deficits in synaptic plasticity, neuronal excitability and relevant mRNA levels in the mPFC. We discovered that pharmacological potentiation of mGlu5 or blockade of anandamide degradation both normalized behavioral and synaptic deficits. The data reveal new endophenotypes of PCE and identify new therapeutic strategies.

5In rodent models, the consumption of synthetic or plant-derived cannabinoids during gestation has multiple deleterious consequences on the progeny’s behavior (reviewed in;; ). In these earlier studies, different drug and treatment regimen were used and multiple behaviors studied in the progeny at various ages. Here, to simplify the analysis/maximize the significance of our comparisons of molecular, synaptic and behavioral measures we first decided to generate a coherent new dataset. Thus, gestating dams were exposed a single daily low dose of the synthetic cannabinoid WIN55,212 – 2 (WIN, 0.5 mg/kg, s.c. or vehicle or SHAM) between GD5 and GD20. Key findings were reproduced with the phytocannabinoid Δ9-Tetrahydrocannabinol, THC (5 mg/kg). This protocol of PCE is clinically relevant, since the doses of WIN and THC used here correspond to a moderate exposure of cannabis in humans, correcting for the differences in route of administration and body weight surface area. We compared male and female SHAM and cannabinoid-exposed progeny in a series of behavioral tests to explore social behavior, anxiety, locomotion and cognition at the adult stage (PND >90). In accord with international ethical guidelines to reduce the number of animal used and their treatment/manipulations, once we had established that there was no difference between sham and naive animals (Table 1), data from animals in both groups were pooled and naive rat dams were included in the SHAM group.

6We found that PCE impaired specific components of social interactions in adult male rats. Thus, PCE males had less contact and spent less time interacting with their congeners than those exposed in-utero to vehicle (Figure 1A–B). Detailed exploration of the various parameters of social interaction revealed that sniffing (Figure 1C–D) and playing (Figure 1E) behaviors were impaired in PCE males while the number of attacks remained unchanged (Figure 1F). The low socialization in PCE males was unlikely due to impaired motor locomotion, as we found no significant change in the distance traveled between WIN and SHAM groups during testing (3758 ± 117.8 cm, n = 10; and 3779 ± 228.8 cm, n = 8; t(7) = 0.02, p>0.05, t-test; SHAM- and WIN-exposed rats, respectively).

7Interestingly, we discovered that the deleterious effects of PCE on social behavior were specific to male offspring. In PCE adult female rats, social behavior was indistinguishable from that of the SHAM group; there was no difference in the total frequency and time (Figure 1) of social interaction in either group.

8Social behaviors require emotional control and cognitive abilities. To test for the generalization of PCE-induced deficits across these behavioral dimensions we estimated anxiety and cognition in our model. No differences between WIN- and SHAM prenatally-exposed rats were found in the elevated plus maze. Specifically, there was no change in the percentage of time spent in the open arms (Figure 2A), percentage of open arm entries (Figure 2B), in the time spent in the open and close arms (Figure 2C–D), number of closed-arm entries ([F(treat x sex)1,29 = 0.195 p=0.662]; data not shown), stretch-attended posture ([F(treat x sex)1,29 = 0.167 p=0.685]; data not shown) and frequency of head-dippings ([F(treat x sex)1,29 = 0.338 p=0.565; data not shown).

9Regarding their cognitive abilities, adult SHAM and WIN-exposed animals of both sexes displayed identical discrimination index and exploration time in both the temporal order memory task (Figure 2E–F) and the novel object recognition test (Figure 2G–H).

10Thus, while social interaction was specifically impaired in PCE males, locomotion, anxiety and cognition were spared in both sexes. These data reveal discrete and sex-specific behavioral consequences of PCE at adulthood.

11In theory, cannabinoid exposure during in-utero neurodevelopment may perturb synaptic functions in most brain areas. The finding of selective impairments of social interaction in the absence of emotional and cognitive deficits does not favor the hypothesis of widespread synaptic deficits. The prefrontal cortex and nucleus accumbens play prominent roles in social behaviors and prefronto-accumbens glutamatergic circuits modulate reward-related behaviors. Furthermore, the endogenous cannabinoid (eCB)-system of the accumbens core is instrumental to social interaction behavior. In-vivo cannabinoid exposure desensitizes CB1R and ablates eCB-mediated synaptic plasticity in the accumbens. Thus, we first compared eCB-mediated LTD (eCB-LTD) in our experimental groups. The data indicated that eCB-LTD in the accumbens is not affected by PCE (Figure 3).

12The extensive repertoire of synaptic plasticity displayed by medial prefrontal synapses is a consistent target of environmental and genetic insults. We first established that tetanic stimulation induced a robust eCB-LTD of excitatory synapses onto layer 5 mPFC pyramidal neurons in mPFC slices prepared from adult SHAM male and female rats (Figure 4A–B–C–D). In marked contrast to the accumbens, eCB-LTD in the mPFC was absent in WIN-exposed males (Figure 4A–B). Strikingly, LTD was normal in PCE female littermates (Figure 4C–D). In a second series of experiments a similar sex-specific ablation of eCB-LTD in the progeny of dams exposed to THC, the main psychoactive component of cannabis, was observed (5 mg/kg, s.c. GD5-20; Figure 5).

13To test if the effects of PCE extend to other forms of plasticity expressed at excitatory mPFC synapses we assessed two critical actors of bidirectional plasticity and reliable prefrontal endophenotypes of neuropsychiatric diseases: mGlu2/3-dependent LTD and NMDAR-dependent LTP. In contrast to the lack of eCB-LTD, mGlu2/3-mediated LTD was indistinguishable in SHAM- and WIN-exposed male and female (Figure 4—figure supplement 1 A-B ). Similarly, theta-burst stimulation (TBS) induces similar levels of NMDAR-dependent LTP at mPFC pyramidal synapses independent of sex and prenatal treatment (Figure 4—figure supplement 1 C-D-E-F). We also confirmed that prenatal THC does not affect prefrontal LTP at adulthood (Figure 4—figure supplement 2).

14Together the data reveal eCB-LTD in the mPFC as sex-specific target of in-utero cannabinoid exposure.

15Activity-induced modulation of intrinsic excitability is a powerful mean to control cortical circuits postulated to contribute to disorders of the CNS. To test the hypothesis that PCE altered prefrontal excitability, intrinsic firing properties of pyramidal neurons in our animal groups were compared. Sexual differences to the effects of prenatal exposure to cannabinoids were visible at this cellular level. Specifically, mPFC neurons in WIN-exposed rats showed impaired membrane reaction profiles in response to a series of somatic current steps (Figure 6A and E). Independent of the sex and treatment, the resting potential was similar (Figure 6B and F). While the rheobase (Figure 6C and G) was decreased in males prenatally exposed to WIN, females were spared. Moreover, this hyper-excitability in exposed-males was accompanied by an increased number of action potentials in response to somatic currents steps (Figure 6D). Similar results have been obtained in the rat progeny of dams treated with THC during gestation (Figure 7). Thus, PCE leads to an increased excitability of deep layers mPFC pyramidal cells in male progeny only.

16Motivated by the eCB-LTD deficiency in layer 5 mPFC of the male PCE progeny, we surveyed mRNA levels of key components of the endocannabinoid system in adult PCE rats using real time quantitative PCR (Figure 8 and Table 2). Key differences were decreased levels of mRNA for TRPV1, mGlu5, and DAGLα in the female PCE offspring and decreased levels of mGlu5 mRNA in the male offspring (Figure 8). A mild increase was seen in mGlu1 in the PCE males (Table 2). No differences were seen in other endocannabinoid system mRNAs after PCE, including CB1, CB2, DAGLβ, NAPE-PLD, FAAH, ABHD6, or CRIP1a (Table 2).

17The present data reveal a down-regulation of mGlu5 RNA expression levels in the mPFC of PCE males. Previous work from our group underlies the efficiency of mGlu5 positive allosteric modulation in correcting synaptic and behavioral deficits.

18Thus, we tested whether amplification of mGlu5, an effector of eCB-signaling complex in the PFC, could normalize LTD in PCE male. To this aim, we used CDPPB (10 μM), a well-described positive allosteric modulator (PAM) of mGlu5. We found that CDPPB fully restored the ability of excitatory mPFC synapses to express LTD in the prenatally WIN-exposed male group (Figure 9A–B). To verify that the curative properties of CDPPB were indeed due to eCB production, we tested the effects of CB1R and TRPV1 antagonists on the CDPPB rescue. Interestingly, preincubation with either SR141716A or capsazepine prevented the CDPPB rescue of synaptic plasticity (Figure 9C–D). Thus, the data showed that both receptors are engaged in the presence of the mGlu5 PAM.

19Strikingly, we found that systemic CDPPB normalized social cognition in the PCE group but remained without effect in the SHAM group, indicating selectivity of the drug's effects to the disease-state (Figure 9E–F). In agreement with the synaptic plasticity experiments, pre-treatment with either SR141716A (Figure 9G–H) or capsazepine prevented the ameliorative actions of CDPPB (Figure 9G–H). Thus, potentiating mGlu5 signaling normalized synaptic and behavioral deficits induced by prenatal exposure to cannabinoids via CB1R and TRPV1 activation.

20The observation that PCE did not alter CB1R RNA levels in either sex but lowered TRPV1R in the female PCE progeny (Figure 8, Table 2) drew our attention to the role of these two receptors at PFC synapses. Comparing full dose-response curves for the CB1 agonist CP55,940 in male SHAM vs PCE revealed no differences (Figure 4—figure supplement 3). Thus, the lack of LTD in PCE males cannot readily be attributed to altered sensitivity of presynaptic CB1R. We and others have shown that central excitatory synapses can engage TRPV1R and/or CB1R to trigger LTD. In PCE females, LTD was fully expressed in the presence of a specific CB1R antagonist (SR141716A 5 μM, Figure 10A–B–C) but completely prevented by TRPV1R antagonists (capsazepine 10 µM, Figure 10A–B–C). Thus, TRPV1R rather than CB1R mediates LTD in female PCE. This contribution of TRPV1R to LTD in female PCE could be part of the adaptive response to PCE or a previously unnoticed sexual difference in the mechanism of prefrontal synaptic plasticity. We next investigated the substrate of LTD in naive male and female rats (Figure 11). As expected from over a decade of studies in male rodents the CB1R antagonist prevented LTD induction in rat male slices (Figure 11D,E,F and Figure 11— figure supplement 1)). Unexpectedly, the same treatment was without effect in female slices (Figure 11A–B–C). In contrast, antagonism of TRPV1R was without effect in males (Figure 11A–C) but prevented the induction of eCB-LTD in females (Figure 11D–E–F).

21We confirmed and extended these findings with two other antagonists: the CB1R selective neutral antagonist NESS0327 (1 μM, Cayman Chemical, Figure 11—figure supplement 1A-B) but not the selective TRPV1R antagonist AMG9810 (3 µM, Tocris, Figure 11—figure supplement 1A-B) prevented LTD induction in naive male rat slices. Conversely, AMG9810 but not NESS0327 prevented the induction of LTD in naive female adult animals (Figure 11—figure supplement 1 C-D ).

22We thought of an additional strategy to reverse PCE-induced deficits based on our finding that TRPV1R mediates LTD in PCE females in spite of a down-regulation of TRPV1R RNA expression levels. Anandamide is an endogenous ligand of TRPV1 and we hypothesized that inhibition of anandamide’s main degrading enzyme, fatty acid amid hydrolase (FAAH), would increase anandamide levels to the threshold for LTD induction in the PCE males. Indeed, incubation of mPFC slices prepared from PCE male rats with the FAAH inhibitor URB597 effectively restored eCB-LTD (Figure 12A–B). Interestingly, we found that pre-incubation with the specific TRPV1 antagonist capsazepine did not prevent the curative effect of URB597. Rather, the CB1R antagonist SR141716 blocked the URB597 rescue (Figure 12C–D). Previous studies have shown that endocannabinoids, including anandamide, mediate social behavior in rodents. We directly tested the hypothesis that elevating circulating levels of a main eCB, anandamide, could normalize social deficits. We found that in vivo treatment with the FAAH inhibitor (URB597, 0.1 mg/kg) normalized social deficits in adult male PCE rats (Figure 12E–F). In line with our mPFC slice results, SR141716A but not capsazepine prevented the curative actions of URB597 (Figure 12G–H). Thus, blocking anandamide degradation normalized social deficits via CB1R.

23We discovered that fetal exposure to cannabinoids causes sex-specific alterations of behavioral and synaptic functions in the adult progeny. Specifically, we found that prenatal cannabinoid exposure (PCE) reduces social interactions in males but not females, while other behaviors were spared. In parallel, PCE specifically altered neuronal excitability and synaptic plasticity in the prefrontal cortex of male rats. We also identify a pharmacological strategy to normalize synaptic functions and socialization in adult prenatally exposed males.

24PFC malfunctions are a common denominator in several neuropsychiatric diseases (;;;;;; Martin et al.). Our data indicate that in the PCE group, plasticity deficits were circumscribed to eCB-LTD as two other types of synaptic plasticity, which act as biomarkers for prefrontal deficits in other model of neuropsychiatric diseases, were spared. Namely neither NMDAR-mediated LTP nor mGluR2/3-LTD were affected in PCE males. Furthermore, while excitatory synapses of the nucleus accumbens display reduced eCB-mediated plasticity in models of depression, autism, adult cannabis exposure or nutritional deficits, accumbal eCB-LTD was normal in both PCE males and females.

25Our behavioral exploration reveals that social interactions were reduced in males after PCE while female social behavior and other behavioral domains were spared in both sexes. Our results greatly extend that of Vargish and collaborators, who observed reduced social interaction in PCE mice. In the latter study, female progeny was unfortunately not tested. The current results add to previous work showing covariation between defects in PFC synaptic plasticity and social interaction behaviors. Together, these data suggest a certain level of regional PFC specificity to the effects of PCE. The fact that the PFC develops late in to postnatal life (i.e. late adolescence/early adulthood) may explain this sensitivity.

26In the hippocampus, PCE impaired eCB-dependent short-term depression at GABAergic synapses and NMDAR-dependent LTD. In contrast with the latter hippocampal study, we found no evidence that PCE modified release properties of PFC synapses, reinforcing the idea that region-specific mechanisms engage to alter PFC synapses in response to PCE. Our qRT-PCR data showed similar levels of CB1R mRNA in the PFC of male and female progeny exposed to cannabinoids during fetal life, in line with previous observations in the hippocampus and neocortex.

27While our work clearly shows previously undisclosed sexual divergence in the consequences of fetal cannabinoids, they also reveal sex differences in the main elements involved in eCB signaling: CB1R was similar in both sexes while lower mRNA levels of the 2-arachidonoylglycerol (2-AG) synthesizing (DAGLα) enzyme were found in naive females compared to naive males. Previous work highlighted sexual differences at the molecular level. CB1R affinity is higher in males while receptor density is similar in both sexes in the limbic forebrain; however, in the mesencephalon both parameters are higher in males. In the PFC, CB1R density is lower in males than in females. At the structural level, the medial PFC of rats is also sexually different: female prelimbic cortex pyramidal neurons have smaller/less complex apical dendritic arbors than in males. Thus, it is likely that both nanoscale differences in the relative position of the molecular elements of the eCB system and dissimilarities of mRNA/protein expression underlie these sex differences. At the behavioral level, sexual differences in fear-conditioning have also been identified. Sexual differences are unlikely exclusive to the eCB-LTD/PFC/fear conditioning. For instance, active avoidance, spatial memory and hippocampal LTP display sex differences. The data provide new impetus for the urgent need to investigate the functional and behavioral substrates of neuropsychiatric diseases in both sexes.

28Reduced bioavailability of endocannabinoids during LTD induction likely underlies the lack of synaptic plasticity found in PCE males. In support of this idea, basal levels of anandamide are reduced in limbic areas of prenatally WIN-exposed rats. We hypothesized that enhancing anandamide levels may lower the LTD-induction threshold and indeed found that FAAH inhibition with URB597 restored eCB-LTD in PCE males. Furthermore, based on the well-known, prominent role of mGlu5 in synaptic eCB-signaling, we found that positive allosteric modulation of mGlu5 with CDPPB restored LTD in PCE males. Strikingly, this finding translated well from the ex vivo slice preparations to the behavioral level, and in vivo treatments with either the FAAH inhibitor or the mGlu5 PAM normalized social interactions in PCE exposed adult males.

29In conclusion, these results provide compelling evidences for sexual divergence in the long-term functional and behavioral consequences of PCE and introduce strategies for reversing its detrimental effects.

30The experiments were performed following the ARRIVE (Animals in Research: Reporting In Vivo Experiments) guidelines. Wistar female rats (Charles River, France) weighing 250 – 280 g were housed at constant room temperature (20 ± 1°C) and humidity (60%), and exposed to a light cycle of 12 hr/day (08:00 a.m. to 08:00 p.m.), with food and water available ad libitum. For mating, pairs of females were placed with single male rat in the late afternoon. Vaginal smears were taken the following morning at 09:00 a.m. The day on which sperm was present was designated as the day 0 of gestation (GD 0).

31Sub-cutaneous (s.c.) injections were performed daily with the synthetic cannabinoid WIN55,212 – 2 (WIN, 0.5 mg/kg), the phytocannabinoid Delta9-Tetrahydrocannabinol, THC (5 mg/kg) or their respective vehicles from GD 5 to GD 20. WIN was suspended in 5% DMSO, 5% cremophor and saline, at a volume of 1 ml/kg. Control dams (SHAM group) received a similar volume injection of vehicle solution. THC was suspended in 5% Ethanol, 5% cremophor and saline, and injected s.c. at a volume of 1 ml/kg. Control dams (SHAM group) received a similar volume injection of vehicle solution. Newborn litters found up to 05:00 p.m. were considered to be born on that day (postnatal day (PND) 0). On PND 21, the pups were weaned and housed in groups of three. The experiments were carried out on the male and female offspring at adulthood (PNDs 90 – 130). One pup per litter from different litters per treatment group was used in the behavioral experiments. Sample size (n and scatter dot plot) is indicated in the figure legends.

32Animals were treated in compliance with the European Communities Council Directive (86/609/EEC) and the United States National Institutes of Health Guide for the care and use of laboratory animals.

33Body weights of the dams were taken daily throughout pregnancy and the length of pregnancy was determined. Litter size, weight gain of pups and postnatal vitality were also measured, see Table 3.

34Social interaction: Social behavior was assessed as previously described. The test was performed in a sound attenuated chamber under dim light conditions. The testing arena consisted of a Plexiglas cage measuring 45 × 45 cm, with 2 cm of wood shavings covering the floor. Rats were individually habituated to the test cage for 5 min on each of the 2 days prior to testing. Before testing, animals were socially isolated for 24 hr to enhance their social motivation and thus facilitate the expression of social behaviors during testing.

35We verified that the short period of social isolation before testing does not modify social interaction between males and females (Figure 1—figure supplement 1). Naive male and female rats not isolated before testing showed similar frequency and time of social interaction to rats isolated 24 hr prior social interaction testing (Figure 1—figure supplement 1A-B). Detailed exploration of the various parameters of social interaction revealed that sniffing (Figure 1—figure supplement 1C-D) was unchanged and the play events were reduced only in female animals (Figure 1—figure supplement 1E), while the number of attacks remained unchanged (Figure 1—figure supplement 1F).

36Drug treatments were counterbalanced so that cage mates were allocated to different treatment groups. The animals of each pair were similarly treated, did not differ more than 10 g in body weight and were not cage mates. Behavior was assessed per pair of animals and analyzed by a trained observer who was unaware of treatment condition using the Ethovision XT 13.0 video tracking software (Noldus, Wageningen, The Netherlands).

37The test consisted of placing two similarly treated animals into the test cage for 10 min. The total frequency of active social interactions was obtained as the sum of the frequency of the following behavioral elements scored per 10 min:

38Play-related behaviors: pouncing (on rat solicits another rat to play by attempting to nose or rub the nape of its neck), pinning (the rat that is pounced upon can respond in different ways – ‘pinning’ is the result when the animal lies with its dorsal surface on the floor with the other rat standing over it) and boxing (rats push or paw at each other with their forepaws, usually around the head, neck, shoulders of their opponent).

39Social behaviors unrelated to play: social exploration (sniffing any part of the body of the test partner), social grooming (one rat licks and chews the fur of the conspecific, while placing its forepaws on the back or the neck of the other rat), following/chasing (walking or running in the direction of the partner which stays where it is or moves away), crawling under/over (one animal crawls underneath or over the partner's body, crossing it transversely from one side to the other), kicking (the rat kicks backwards at the conspecific with one or both hind paws).

40Aggressive behavior: attack.

41The test was performed as we previously published. The apparatus consisted of a Plexiglas arena 45 × 45 cm, illuminated by fluorescent bulbs at a height of 2 m above the floor of the open field apparatus (light intensity of 30 Lux). The floor was cleaned between each trial to avoid olfactory clues. Each animal was transferred to the open-field facing a corner and was allowed to freely explore the experimental area for 10 min. A video tracking system (Ethovision XT, Noldus Information Technology) recorded the exact track of each rat as well as total distance traveled.

42The elevated plus-maze apparatus comprised two open (50 × 10 × 40 cm) and two closed arms (50 × 10 × 40 cm) that extended from a common central platform (10 × 10 cm). The test was performed as previously described. Rats were individually placed on the central platform of the maze for 5 min. Each 5 min session was recorded with a camera positioned above the apparatus for subsequent behavioral analysis carried out an observer, unaware of animal treatment, using the Observer 3.0 software (Noldus, The Netherlands). The following parameters were analyzed:

43% time spent in the open arms (% Time Open): (seconds spent on the open arms of the maze/seconds spent on the open +closed arms)×100;

44% open arm entries (% Open Entries): (the number of entries into the open arms of the maze/number of entries into open +closed arms)×100;

45Animals were habituated to the experimental arena (40 × 40 cm) without objects for 10 min daily for 2 days before testing. This task consisted of two sample phases and one test trial. In each sample phase, rats were allowed to explore two copies of an identical object for a total of 4 min. Different objects were used for sample Phases 1 and 2, with a delay between the sample phases of 1 hr. After 3 hr from sample Phase 2, rats performed the test trial (4 min duration) where a third copy of the objects from sample Phase 1 and a third copy of the objects from sample Phase 2 were used. The positions of the objects in the test and the objects used in sample Phase 1 and sample Phase 2 were counterbalanced between the animals. An intact temporal order memory requested the subjects to spend more time exploring the object from Sample 1 (i.e. the object presented less recently) compared with the object from Sample 2 (i.e. the ‘new’ object). The discrimination ratio was calculated as the difference in time spent by each animal exploring the object from sample Phase 1 compared with the object from sample Phase 2 divided by the total time spent exploring both objects in the test phase.

46After 2 days of 10 min habituation to the experimental arena, rats were exposed to the procedure of an acquisition or sample phase, followed by a preference test after a delay of 30 min. In the sample phase, duplicate copies (A1 and A2) of an object were placed near the two corners at either end of one side of the arena (8 cm from each adjacent wall). Rats were placed into the arena facing the center of the opposite wall and allowed a total of 4 min in the arena. At test (4 min duration), animals were placed in the arena, presented with two objects in the same positions: one object (A3) was a third copy of the set of the objects used in the sample phase, and the other object was a novel object (B). The positions of the objects in the test and the objects used as novel or familiar were counterbalanced between the rats. The discrimination ratio was calculated as the difference in time spent by each animal exploring the novel compared with the familiar object divided by the total time spent exploring both objects. Exploration was scored when the animal was observed sniffing or touching the object with the nose and/or forepaws. Sitting on objects was not considered to indicate exploratory behavior.

47The CB1 cannabinoid receptor antagonist SR141716A [5-(4-chloro-phenyl)−1-(2,4-dichlorophenyl)−4-methyl-N-1-piperidinyl-1H-pyrazole-3-carboxamide] (National Institute of Mental Health, USA), the positive allosteric modulator of mGlu5 receptors CDPPB (3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl) benzamide) (National Institute of Mental Health, USA), the vanilloid TRPV1 antagonist capsazepine (N-[2-(4-Chlorophenyl)ethyl]−1,3,4,5-tetrahydro-7,8-dihydroxy-2H-2-benzazepine-2-carbothioamide) (Tocris) and the FAAH inhibitor URB597 (Cyclohexylcarbamic acid 3'-(Aminocarbonyl)-[1,1'-biphenyl]−3-yl ester) (Tocris) were dissolved in 5% Tween 80/5% polyethylene glycol/saline and given intraperitoneally (i.p.). CDPPB (0.75 mg/kg) or its vehicle were administered 30 min before testing; URB597 (0.1 mg/kg) or its vehicle were administered 2 hr before testing; SR141716A was administered 30 min before CDPPB or URB597 at a dose that did not induce effects by itself (1 mg/kg) in adult rats. Capsazepine (5 mg/kg) was injected 30 min before CDPPB and URB597. Drug doses and pre-treatment intervals were based on the literature and on pilot experiments. Solutions were freshly prepared on the day of the experiment and were administered in a volume of 1 ml/kg.

48Adult male and female rats were anesthetized with isoflurane and killed as previously described. The brain was sliced (300 μm) in the coronal plane with a vibratome (Integraslice, Campden Instruments) in a sucrose-based solution at 4°C (in mm as follows: 87 NaCl, 75 sucrose, 25 glucose, 2.5 KCl, 4 MgCl2, 0.5 CaCl2, 23 NaHCO3 and 1.25 NaH2PO4). Immediately after cutting, slices containing the medial prefrontal cortex (PFC) or accumbens were stored for 1 hr at 32°C in a low-calcium ACSF that contained (in mm) as follows: 130 NaCl, 11 glucose, 2.5 KCl, 2.4 MgCl2, 1.2 CaCl2, 23 NaHCO3, 1.2 NaH2PO4, and were equilibrated with 95% O2/5% CO2 and then at room temperature until the time of recording. During the recording, slices were placed in the recording chamber and superfused at 2 ml/min with either low Ca2+ ACSF for mPFC or normal ACSF for the accumbens. All experiments were done at 32°C or room temperature for mPFC and accumbens respectively. The superfusion medium contained picrotoxin (100 µM) to block gamma-aminobutyric acid types A (GABA-A) receptors. All drugs were added at the final concentration to the superfusion medium.

49Whole cell patch-clamp of visualized layer five pyramidal neurons mPFC and field potential recordings were made in coronal slices containing the mPFC or the accumbens as previously described. Neurons were visualized using an upright microscope with infrared illumination. The intracellular solution was based on K+ gluconate (in mM: 145 K+ gluconate, 3 NaCl, 1 MgCl2, 1 EGTA, 0.3 CaCl2, 2 Na2+ATP, and 0.3 Na+ GTP, 0.2 cAMP, buffered with 10 HEPES). To quantify the AMPA/NMDA ratio we used a CH3O3SCs-based solution (in mM: 128 CH3O3SCs, 20 NaCl, 1 MgCl2, 1 EGTA, 0.3 CaCl2, 2 Na2+ATP, and 0.3 Na+ GTP, 0.2 cAMP, buffered with 10 HEPES, pH 7.2, osmolarity 290 – 300 mOsm). The pH was adjusted to 7.2 and osmolarity to 290 – 300 mOsm. Electrode resistance was 4 – 6 MOhms.

50Whole cell patch-clamp recordings were performed with an Axopatch-200B amplifier as previously described. Data were low-pass filtered at 2 kHz, digitized (10 kHz, DigiData 1440A, Axon Instrument), collected using Clampex 10.2 and analyzed using Clampfit 10.2 (all from Molecular Device, Sunnyvale, USA).

51A −2 mV hyperpolarizing pulse was applied before each evoked EPSC in order to evaluate the access resistance and those experiments in which this parameter changed >25% were rejected. Access resistance compensation was not used, and acceptable access resistance was <30 MOhms. The potential reference of the amplifier was adjusted to zero prior to breaking into the cell. Cells were held at −76 mV.

52Current-voltage (I-V) curves were made by a series of hyperpolarizing to depolarizing current steps immediately after breaking into the cell. Membrane resistance was estimated from the I–V curve around resting membrane potential.

53Field potential recordings were made in coronal slices containing the mPFC or the accumbens as previously described. During the recording, slices were placed in the recording chamber and superfused at 2 ml/min with either low Ca2+ ACSF for mPFC or normal ACSF for the accumbens. All experiments were done at 32°C or room temperature for mPFC and accumbens, respectively. The superfusion medium contained picrotoxin (100 μM) to block GABA Type A (GABA-A) receptors. All drugs were added at the final concentration to the superfusion medium. The glutamatergic nature of the field EPSP (fEPSP) was systematically confirmed at the end of the experiments using the ionotropic glutamate receptor antagonist CNQX (20 μM), which specifically blocked the synaptic component without altering the non-synaptic component (data not shown).

54Both fEPSP area and amplitude were analyzed. Stimulation was performed with a glass electrode filled with ACSF and the stimulus intensity was adjusted ∼60% of maximal intensity after performing an input–output curve (baseline EPSC amplitudes ranged between 50 and 150 pA). Stimulation frequency was set at 0.1 Hz.

55The magnitude of plasticity was calculated 35–40 min after induction as percentage of baseline responses. Statistical analysis of data was performed with Prism (GraphPad Software) using tests indicated in the main text after outlier subtraction. All values are given as mean ±SEM, and statistical significance was set at p<0.05.

56Frozen rat brains were coronally sectioned in a cutting block (Braintree Scientific, Inc., Braintree, MA, Cat.# BS-SS 605C) that had been pre-chilled to −20°C. One-millimeter sections were kept frozen throughout dissection with brain regions stored at −80°C until use. Total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen, Hilden, Germany, Cat.# 74034). RNA was reverse-transcribed using the RevertAid Kit (Thermo Fisher Scientific, Waltham, MA Cat.# K1621) as per manufacturer’s instructions. Taqman primers and probes were obtained through Applied Biosystems. Sequences used in qRT-PCR are as follows:

57TaqMan Gene Expression Master Mix from Applied Biosystems (Foster City, CA Cat.# 4369016) was used in generating expression data on a QuantStudio7 thermal cycler. Duplicates were run for each sample, and changes in gene expression were determined using the ΔΔCt method. Data was analyzed using Excel (Microsoft, Redmond, WA) and Prism 7 (Graphpad, La Jolla, CA) software.

58All the behavioral parameters were expressed as mean ± SEM. Group comparisons used t-test and two-way repeated measures (RM) ANOVA (for different sex and pharmacological treatments), followed by Student-Newman-Keuls post-hoc tests when appropriate.