Changes in endocannabinoid and N-acylethanolamine levels in rat brain structures following cocaine self-administration and extinction training

Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

Contents lists available at ScienceDirect

Progress in Neuro-Psychopharmacology & Biological Psychiatry journal homepage: www.elsevier.com/locate/pnp

Changes in endocannabinoid and N-acylethanolamine levels in rat brain structures following cocaine self-administration and extinction training Beata Bystrowska a,⁎, Irena Smaga a, Małgorzata Frankowska b, Małgorzata Filip a,b a b

Department of Toxicology, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland Laboratory of Drug Addiction Pharmacology, Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smętna 12, PL 31-343 Kraków, Poland

a r t i c l e

i n f o

Article history: Received 2 September 2013 Received in revised form 25 November 2013 Accepted 5 December 2013 Available online 12 December 2013 Keywords: “Yoked” procedure Cocaine self-administration Endocannabinoid N-acylethanolamine

a b s t r a c t Preclinical investigations have demonstrated that drugs of abuse alter the levels of lipid-based signalling molecules, including endocannabinoids (eCBs) and N-acylethanolamines (NAEs), in the rodent brain. In addition, several drugs targeting eCBs and/or NAEs are implicated in reward and/or seeking behaviours related to the stimulation of dopamine systems in the brain. In our study, the brain levels of eCBs (anandamide (AEA) and 2-arachidonoylglycerol (2-AG)) and NAEs (oleoylethanolamide (OEA) and palmitoylethanolamide (PEA)) were analyzed via an LC-MS/MS method in selected brain structures of rats during cocaine self-administration and after extinction training according to the “yoked” control procedure. Repeated (14 days) cocaine (0.5 mg/kg/infusion) self-administration and yoked drug delivery resulted in a significant decrease (ca. 52%) in AEA levels in the cerebellum, whereas levels of 2-AG increased in the frontal cortex, the hippocampus and the cerebellum and decreased in the hippocampus and the dorsal striatum. In addition, we detected increases (N 150%) in the levels of OEA and PEA in the limbic areas in both cocaine treated groups, as well as an increase in the tissue levels of OEA in the dorsal striatum in only the yoked cocaine group and increases in the tissue levels of PEA in the dorsal striatum (both cocaine groups) and the nucleus accumbens (yoked cocaine group only). Compared to the yoked saline control group, extinction training (10 days) resulted in a potent reduction in AEA levels in the frontal cortex, the hippocampus and the nucleus accumbens and in 2-AG levels in the hippocampus, the dorsal striatum and the cerebellum. The decreases in the limbic and subcortical areas were more apparent for rats that self-administered cocaine. Following extinction, there was a region-specific change in the levels of NAEs in rats previously injected with cocaine; a potent increase (ca. 100%) in the levels of OEA and PEA was detected in the prefrontal cortex and the hippocampus, whilst a drop was noted in the striatal areas versus yoked saline yoked animals. Our findings support the previous pharmacological evidence that the eCB system and NAEs are involved in reinforcement and extinction of positively reinforced behaviours and that these lipid-derived molecules may represent promising targets for the development of new treatments for drug addiction. © 2014 Elsevier Inc. All rights reserved.

1. Introduction Drug addiction is an extremely serious problem, both in terms of the proper functioning of the body and in terms of specific social behaviour that is often in conflict with the law. Amongst addictive drugs, cocaine belongs to the group of psychostimulants with a high abuse potential due to its induction of euphoric feelings, friendliness, empathy and hyperactivity (Kreek et al., 2012). The mechanism of action of cocaine

Abbreviations: AEA, anandamide; 2-AG, 2-arachidonoylglycerol; eCBs, endocannabinoids; LC-MS/MS, liquid chromatography tandem mass spectrometry; NAEs, N-acylethanolamines; OEA, oleoylethanolamide; PEA, palmitoylethanolamide. ⁎ Corresponding author at: Department of Toxicology, Faculty of Pharmacy, Jagiellonian University, Medical College, 9, Medyczna Street, 30-688 Krakow, Poland. Tel.: +48 12 6205630; fax: +48 12 6205643. E-mail address: [email protected] (B. Bystrowska). 0278-5846/$ – see front matter © 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.pnpbp.2013.12.002

includes interaction with monoaminergic (dopamine, noradrenalin and serotonin) neurotransmitter systems via inhibition of monoamine reuptake (Bossert et al., 2005; Nestler, 2004). The acute reinforcing effects of cocaine are associated with a marked increase in dopaminergic neurotransmission and an indirect activation of both D1 and D2 receptors at the synaptic termini of mesolimbic dopaminergic neurons (Di Chiara, 1995; Thomsen et al., 2009). Recent results indicate that cocaine – in addition to monoamine transporter binding – induces direct and/or indirect allosteric stimulation of D2 receptors. In fact, cocaine enhanced the ability of the D2-like receptor agonist quinpirole to reduce K+-evoked [3H]DA efflux from rat striatal synaptosomes (Ferraro et al., 2010). Cocaine also increased membrane-associated D2 receptor immunoreactivity in CHO cell lines lacking the dopamine transporter (Genedani et al., 2010) and the efficacy of dopamine to stimulate the binding of GTPγS to striatal D2-like receptors (Ferraro et al., 2012). In vivo studies also revealed that low concentrations of cocaine

2

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

amplified the quinpirole-induced reduction in accumbal extracellular glutamate levels and hyperlocomotion (Ferraro et al., 2012). In the late 20th century, a novel inhibitory feedback mechanism counteracting the dopamine-induced facilitation of behavioural and neurochemical functions was discovered. For instance, an in vivo microdialysis study indicated that the striatal administration of quinpirole evoked a marked local increase in the level of anandamide (AEA, N-arachidonoylethanolamine), a lipid-based molecule (Giuffrida et al., 1999), whilst an in vitro study revealed that cocaine or quinpirole perfusion into striatal slices evoked an increase in the AEA level (Centonze et al., 2004). These effects of cocaine were blocked by a D2 receptor antagonist both in vivo and in vitro. This evidence indicates that endocannabinoids (eCBs) are the downstream effectors of the action of cocaine in the dorsal striatum. These studies also suggest functional interactions between eCBs and dopaminergic systems during striatal signalling. Further preclinical reports have confirmed that the eCB system may play a role in cocaine addiction (Arnold, 2005), especially in the reinstatement of drug-seeking behaviours (Adamczyk et al., 2009; Budzyńska et al., 2009; Shoaib, 2008; Xi et al., 2006). Aside from AEA, the eCB system includes other endogenous lipid molecules, such as 2-arachidonoylglycerol (2-AG) (Onaivi et al., 2002). Via cleavage from the plasma membrane, synthesis of lipid precursors, including both AEA and 2-AG, is specifically regulated by neuronal activity. Once generated, eCBs primarily act via two receptors, CB1 and CB2, to regulate synaptic communication, membrane depolarisation and neurotransmitter release (Di Marzo, 2011; Onaivi et al., 2002; Piomelli, 2003). It has also been discovered that non-cannabinoid fatty acid ethanolamides may participate in the control of reward-related behaviours (Fu et al., 2008; Hansen and Diep, 2009; Melis et al., 2008). These compounds include oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), which act indirectly on CB receptors and may influence eCB function by competing for catabolic enzymes (Giuffrida et al., 2000). Both ethanolamides also possess neuromodulatory properties as endogenous ligands of the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR-α) and the capsaicin receptor transient receptor potential cation channel subfamily V member 1 (TRPV1) (TRPV1) (Melis et al., 2008). Investigations of cannabinoid and non-cannabinoid fatty acid ethanolamides (also known as N-acylethanolamines or acylethanolamides; NAEs) in rat brain structures following repeated cocaine administration in in vivo models are very limited, and the evaluation of active cocaine exposure via self-administration has not yet been studied. We sought to identify the magnitude of AEA, 2-AG, OEA and PEA levels in several rat brain structures via liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis during maintenance of cocaine self-administration and after drug withdrawal using a yoked-triad procedure. The latter procedure (in which each animal was paired with two rats that served as “yoked” controls, of which one received an injection of saline each time the paired rat self-administered a response-contingent injection of cocaine, and the second received an injection of cocaine in the same manner) allowed us to distinguish between the pharmacological and motivational effects of psychostimulant intake. 2. Materials and methods 2.1. Animals A group of 48 male Wistar rats (280–300 g) delivered by a licensed breeder (Charles River, Germany) were housed individually in standard plastic rodent cages in a colony room maintained at 20 ± 1 °C and at 40–50% humidity under a 12-h light–dark cycle (lights on at 06:00). The animals had free access to standard animal food and water during the 7-day habituation period. Then, the animals were divided into three groups (active, yoked and control, see Table 1) and the rats used

Table 1 Experimental protocol for behavioural studies. Group

1a 1b 1c 2a 2b 2c

Maintenance of self-administration

Extinction

14 days

10 days

Yoked saline Yoked cocaine Cocaine self-administration Yoked saline Yoked cocaine Cocaine self-administration

Saline Saline Saline

Rats (groups 1c and 2c) were trained to self-administered cocaine (0.5 mg/kg/infusion) during 2-h daily sessions. Groups 1a, 1b and 1c were sacrificed immediately following the last self-administration session, whilst groups 2a, 2b and 2c underwent 10-day extinction and were sacrificed immediately following the last 2-h extinction session.

in the cocaine self-administration procedures were maintained on limited water during initial training sessions (see below). All of the experiments were conducted during the light phase of the light–dark cycle (between 08:00 and 15:00) and were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with approval of the Bioethics Commission, as compliant with Polish Law (21 August 1997). The animals were experimentally naive. 2.2. Drugs Cocaine hydrochloride (Sigma-Aldrich, St. Louis, USA), dissolved in sterile 0.9% NaCl and given iv (0.1 ml/infusion). 2.3. Behavioural procedures 2.3.1. Cocaine self-administration and extinction training Rats were trained to press the lever of standard operant conditioning chambers (Med-Associates, USA) under a fixed ratio 5 schedule of water reinforcement. Two days following “lever-press” training and free access to water, the rats were chronically implanted with a silastic catheter in the external right jugular vein, as described previously (Frankowska et al., 2010). The catheters were flushed every day with 0.1 ml of saline solution containing heparin (70 U/ml, Biochemie GmbH, Austria) and 0.1 ml of solution of cephazolin (10 mg/ml; Biochemie GmbH, Austria). There was no problem with catheter patency. After a 10-day recovery period, all of the animals were water deprived for 18 h and trained to lever press to a fixed ratio 5 schedule of water reinforcement over a 2-h session. The subjects were then given access to cocaine during 2-h daily sessions performed 6 days/week (maintenance) and from that time they were given ad libitum water. The house light was illuminated throughout each session. Each completion of five presses on the active lever complex (the fixed ratio 5 schedule) resulted in a 5-s infusion of cocaine (0.5 mg/kg per 0.1 ml) and a 5-s presentation of a stimulus complex (activation of the white stimulus light directly above the active lever and the tone generator, 2000 Hz; 15 dB above ambient noise levels). Following each injection, there was a 20-s time-out period during which responding was recorded but had no programmed consequences. Response on the inactive lever never resulted in cocaine delivery. Acquisition of the conditioned operant response lasted a minimum of 10 days until the subjects met the following criteria: minimum requirement of 22 reinforcements with an average of 6 days, and active lever presses with an average of 6 consecutive days and a standard deviation within those 6 days of b10% of the average; this selected criterion was based on our prior experiments (Filip, 2005). After the last (2 h) self-administration session, the animals were decapitated. After 14 days of self-administration (once the rats met the maintenance criterion), a separate group of rats (n = 24) underwent 10-day extinction trials. During extinction, the animals experienced 2-h daily

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

training sessions; although active lever presses now resulted in neither the delivery of cocaine (saline was substituted for cocaine) nor the presentation of the conditioned stimulus. The rats remained in extinction until responding on the active lever fell to 10% of the level during maintenance. On the 10th day of extinction, the animals were sacrificed immediately following the last (2 h) experimental session. 2.3.2. Yoked self-administration procedure The rats were tested simultaneously in groups of three with two rats serving as “yoked” controls that received an injection of saline or cocaine which was not contingent on responding, and each time a response-contingent injection of 0.5 mg/kg cocaine was self-administered by the paired rat. Unlike self-administering rats, lever pressing by the yoked rats was recorded but had no programmed consequences (Frankowska et al., 2008). 2.4. eCB and NAE tissue level measurements 2.4.1. Brain structure isolation The rats were sacrificed by decapitation. Selected brain structures (i.e., the prefrontal cortex, frontal cortex, hippocampus, dorsal striatum, nucleus accumbens and cerebellum) were isolated, immediately frozen in dry ice and stored at − 80 °C. Tissues were dissected according to The Rat Brain Atlas (Paxinos and Watson, 1998) and to the relevant paragraph in Neuroproteomics (Spijker, 2011). 2.4.2. Liquid chromatography mass spectrometry analysis 2.4.2.1. Reagents. All chemical solvents and standards were of analytical grade. Standards of AEA, 2-AG, OEA and PEA were obtained from Tocris (Bristol, United Kingdom), AEA-d4, 2-AG-d5, OEA-d4 and PEA-d4 from Cayman Chemical (USA), acetonitrile and chloroform from Merck (Darmstadt, Germany), and methanol and formic acid from POCh (Katowice, Poland). The standard stock solutions were prepared in ethanol, except for 2-AG and 2-AG-d5 which were prepared in acetonitrile. All stock solutions were stored at −80 °C. Further dilutions were carried out in acetonitrile. 2.4.2.2. Lipid extraction from brain tissue. The brain tissues were weighted and subjected to eCB and NAE extraction. The extraction was carried out by the modified methods of isolation of lipid compounds developed by Folch et al. (1957). Tissues were homogenized using sonificator (UP50H, Hielscher) in an ice-cold mixture of methanol and chloroform (1:2; v/v) in a proportionate 10 mg of wet tissue to 150 μl of solvent to quench any possible enzymatic reaction that may interfere with the analysis. Next, 150 μl of homogenate was mixed with 2 μl of internal standard (AEA-d4, concentration 10 μg/ml; 2-AG-d5, concentration 100 μg/ml; PEA-d4, OEA-d4, concentration 5 μg/ml), 250 μl of formic acid (pH 3.0; 0.2 M) and 1500 μl of extraction mixture (methanol:chloroform; 1:2, v/v). The internal standard indicates analyte loss during a sample work-up. Afterwards, the samples were vortexed for 30 s and centrifuged for 10 min at 2000 rpm. Organic phases were collected and dried under a stream of nitrogen at 40 °C (this condition allows for the prevention of lipid oxidation). The residue was dissolved in 40 μl of acetonitrile, and 10 μl of the reconstituted extract was injected into the LC-MS/MS system for quantitative analysis.

3

The gradient began initially at 0% A during 1 min, increasing linearly to 90% at 2 min, and this was maintained for 2 min and then decreased to 0% at 6 min. Finally, the last 4 min of analysis was kept at 100% B. The sample temperature was maintained at 4 °C in the autosampler prior to analysis. A sample volume of 10 μl was injected into the analytical column for compound analysis. MS/MS analyses were accomplished on an Applied Biosystems MDS Sciex (Concord, Ontario, Canada) API 2000 triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) interface. ESI ionization was performed in the positive ionization mode. A standard polypropylene glycols solution (PPG standard) was used for instrument tuning and mass calibration at unit mass resolution according to the Applied Biosystems manual. The mass spectrometer was operated with a dwell time of 200 ms. To find the optimal parameters of the ion path and ion source of the studied compound, the quantitative optimization was done by direct infusion of the standards using a Hamilton syringe pump (Hamilton, Reno, Nevada). The multiple reaction monitoring (MRM) mode of the dominant product ion for each eCB/NAE was realized using optimal conditions. The ion source parameters were as follows: ion spray voltage (IS): 5500 V; nebulizer gas (gas 1): 30 psi; turbo gas (gas 2): 10 psi; temperature of the heated nebulizer (TEM): 400 °C; curtain gas (CUR): 25 psi. A comparison of the paired ion (precursor and product ion m/z values) and LC retention times with standards served to confirm the identification of eCBs/NAEs in the samples investigated. An ion pair was 348/62 for AEA, 379/287 for 2-AG, 326/62 for OEA, 300/62 for PEA, 352/66 for AEA-d4, 384/292 for 2-AG-d5, 330/66 for OEA-d4 and 304/66 for PEA-d4. Data acquisition and processing were accomplished using the Applied Biosystems Analyst version 1.4.2 software. 2.4.2.4. Calibration curve and quantification. eCB and NAE concentrations in the samples were calculated using the calibration curve that was prepared on the same day and analyzed in the same analytical run. Calibration curves were constructed after analyzing the samples of the brain tissues collected from naive rats. The homogenates were spiked with AEA, OEA and PEA to the following concentrations: blank, 0.1, 1, 10, 25, 50, and 100 ng/g. The solutions used for 2-AG were: blank, 0.4, 1, 5, 10, 25, and 50 μg/g. AEA-d4 2-AG-d5, PEA-d4, OEA-d4 were used as the internal standard. These samples were analyzed according to the procedure described for sample preparation (Section 2.4.2.2). 2.5. Statistical analyses All data were expressed as means ± SEM. For cocaine selfadministration and extinction training procedures, the number of responses on the active and on the inactive lever (including time-out responding), as well as cocaine infusions, was analyzed by a two-way analysis of variance (ANOVA) for repeated measures; and a post hoc Duncan's test was used to analyze differences between the group means. Additionally, Student's t-test was used to analyze differences between active vs. inactive lever presses. In biochemical assays, statistical analyses were performed with either a one-way ANOVA with post hoc Bonferroni's Multiple Comparison test or with Student's t-test. P b 0.05 was considered statistically significant. 3. Results 3.1. Cocaine self-administration and extinction training

2.4.2.3. LC-MS/MS conditions. LC was performed using an Agilent 1100 (Agilent Technologies, Waldbronn, Germany) LC system. Chromatographic separation was carried out with a Thermo Scientific BDS HYPERSIL C18 column (100 × 3 mm I.D., 3 μm particle size). The advance column, with its precolumn (100 × 3 mm I.D., 3 μm particle size), is set at 40 °C with a mobile phase flow rate of 0.3 ml/min. The gradient elution mobile phases consisted of formic acid (0.02 M) in acetonitrile (solvent A) and formic acid (0.02 M) in water (solvent B).

For rats sacrificed after the 14 cocaine self-administration sessions, more presses of the active lever than the inactive lever from the 3rd to the 14th experimental sessions were recorded (F(13,234) = 12.66, p b 0.001). During the last 3 self-administration sessions, the mean number of cocaine infusions per day varied from 20 to 24, with a mean of cocaine intake of 11.38 ± 0.18 mg/kg/day. During the 14 experimental sessions, the animals received a mean of 146.8 ± 17.4 mg/kg cocaine.

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10 yoked saline yoked cocaine active cocaine

25 20 x

*

15

*** ***

10 5

20

ER C

C N A

H

ST R

IP

FC

0 PF C

For rats sacrificed after the 10 extinction training sessions, neither drug nor drug-paired stimuli were provided in response to lever pressing during extinction, which resulted in a gradual decrease in presses of the active lever compared to previous responding during cocaine selfadministration. The rats pressed on the active lever significantly more frequently than the inactive lever from the 1st to the 18th experimental sessions (F(23,322) = 7.46, p b 0.001; Fig. 1). The amount of cocaine that these animals received during the drug self-administration sessions was 138.9 (±12.3) mg/kg. In the yoked cocaine and yoked saline groups, the difference between pressing the active versus the inactive lever failed to reach significance (data not shown). The yoked cocaine animals passively received exactly the same amount of cocaine (146.8 ± 17.4 mg/kg and 138.9 ± 12.3 mg/kg, respectively) at the same time as the rats that had learned to actively administer cocaine.

AEA [ng/g]

4

yoked saline yoked cocaine active cocaine

2-AG [ug/g]

15

A

lever presses/120 min

Self-administration (cocaine 0.5 mg/kg/inf)

Extinction (saline 0.1 ml/inf)

active lever inactive lever

200

150

100

50

0

2

4

6

8

10

12

14

16

18

20

22

24

session

lever presses/120 min

160

C

140

Active lever

120

Inactive lever

100 80 60 40

***

20 0

SA

xxx

**

***

*** xx

5

xx

xxx

***

*

C ER

C N A

ST R

H

IP

0

Fig. 2. Levels of eCBs in rat brain structures following the 14 days of cocaine selfadministration. AEA—anandamide, 2-AG—2-arachidonoylglycerol, PFC—prefrontal cortex, FC—frontal cortex, HIP—hippocampus, STR—dorsal striatum, NAC—nucleus accumbens, CER—cerebellum. All data are expressed as the means ± SEM. N = 8 rats/group. *p b 0.05; **p b 0.01; ***p b 0.001 vs. yoked saline. xp b 0.05; xxp b 0.01; xxxp b 0.001 active cocaine vs. yoked cocaine.

B

250

***

FC

3.2.1.1. Cocaine self-administration. In the yoked saline group, the AEA levels ranged from 8.70 to 22.55 ng/g, with the highest concentration observed in the cerebellum and the lowest in the hippocampus. As shown in Fig. 2, cocaine treatment resulted in a change in the AEA levels only in the cerebellum (F(2,21) = 47.42; p b 0.0001) and

10

C

3.2.1. eCBs

PF

3.2. Concentration of eCB and NAE in rat brain structures

EXT

Fig. 1. The mean number (±SEM) of presses of the active and inactive levers for rats that received cocaine at a dose of 0.5 mg/kg/injection via self-administration (A) and underwent extinction training (B). For comparison, the number (mean ± SEM) of active (black bars) and inactive (white bars) lever presses during the last 3 cocaine selfadministration sessions and the last 3 extinction training sessions is shown (C). N = 8 rats/group. ***p b 0.001 vs. inactive lever, Student's t-test.

in the prefrontal cortex (F(2,20) = 5.034; p b 0.05), but not in the frontal cortex (F(2,21) = 2.277; p N 0.05), hippocampus (F(2,21) = 0.4869; p N 0.05), dorsal striatum (F(2,21) = 2.826; p N 0.05) and nucleus accumbens (F(2,20) = 3.481; p N 0.05). In the cerebellum, a significant decrease was observed in animals which self-administered (p b 0.001) or passively received (p b 0.001) cocaine. There was a difference between cocaine treated groups in the prefrontal cortex (p b 0.05) with a significant reduction in yoked cocaine animals (Fig. 2). The concentration of 2-AG in the control (yoked saline) group ranged from 1.50 to 5.33 μg/g, with the highest concentration in the dorsal striatum and the lowest in the prefrontal cortex. Cocaine influenced the 2-AG levels in the following structures: frontal cortex (F(2,21) = 14.85; p b 0.0005), hippocampus (F(2,21) = 63.50; p b 0.0001), dorsal striatum (F(2,21) = 11.58; p b 0.001), and cerebellum (F(2,21) = 18.37; p b 0.0001), but not in the prefrontal cortex (F(2,20) = 1.76; p N 0.05) or nucleus accumbens (F(2,20) = 4.951; p N 0.05) during maintenance of cocaine self-administration. A reduction in the 2-AG level was seen in the hippocampus (p b 0.05) and in the dorsal striatum (p b 0.001), whilst in the frontal cortex (p b 0.01) and cerebellum (p b 0.001), in contrast, an increase was reported. In the animals passively administered cocaine, a significant enhancement in the 2-AG level in the frontal cortex (p b 0.001) and hippocampus (p b 0.001) was observed. Cocaine self-administration or yoked cocaine injections resulted in differences in the levels of 2-AG seen in the hippocampus (p b 0.001), dorsal striatum (p b 0.01), nucleus accumbens (p b 0.01) and cerebellum (p b 0.001) (Fig. 2). The levels of AEA in both yoked saline groups (1a and 2a) were as follows: prefrontal cortex—17.77 ng/g and 18.69 ng/g; frontal cortex—14.65 ng/g and 13.66 ng/g; hippocampus—13.20 ng/g and 14.80 ng/g; dorsal striatum—15.93 ng/g and 13.28 ng/g; nucleus accumbens—18.28 ng/g and 16.35 ng/g; cerebellum—17.08 ng/g and 15.59 ng/g respectively. The Student's t-test revealed a

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

significant difference between yoked saline groups only in the dorsal striatum (t = 4.906; p b 0.01). At the same time, the level of 2-AG did not change in yoked saline groups. 3.2.1.2. Extinction training. Extinction training resulted in potent changes in the AEA concentration in the frontal cortex (F(2,21) = 140.6; p b 0.0001), hippocampus (F(2,21) = 35.49; p b 0.0001), nucleus accumbens (F(2,21) = 11.57; p b 0.0005) and cerebellum (F(2,21) = 4.873; p b 0.05), but not in the prefrontal cortex (F(2,21) = 3.035; p N 0.05) and dorsal striatum (F(2,21) = 2.478; p N 0.05). We found a potent decrease of the AEA level in the frontal cortex (p b 0.001), hippocampus (p b 0.001) and nucleus accumbens (p b 0.001), whilst an increase in the cerebellum (p b 0.05) of rats that were previously self-administered cocaine was seen. In the yoked cocaine group, a drop in the AEA concentration in the frontal cortex (p b 0.001) and hippocampus (p b 0.001) was found. Cocaine active or inactive injections resulted in differences in the levels of AEA seen in the frontal cortex (p b 0.001), hippocampus (p b 0.05) and nucleus accumbens (p b 0.05) (Fig. 3). Withdrawal from cocaine resulted in a change of the 2-AG levels in all structures (the prefrontal cortex (F(2,21) = 8.025; p b 0.005), frontal cortex (F(2,21) = 11.14; p b 0.001), hippocampus (F(2,21) = 6.442; p b 0.01), dorsal striatum (F(2,21) = 9.569; p b 0.005), nucleus accumbens (F(2,21) = 243.1; p b 0.0001) and cerebellum (F(2,21) = 11.92; p b 0.0005)). During extinction, the level of 2-AG either decreased in the hippocampus (p b 0.01), dorsal striatum (p b 0.01) and cerebellum (p b 0.01), or increased in the prefrontal cortex (p b 0.01) and nucleus accumbens (p b 0.001) of rats previously self-administered cocaine. In the yoked cocaine group, there was an increase in the frontal cortex (p b 0.001), whilst a decrease of 2-AG level in the cerebellum (p b 0.001) was also reported. Cocaine active or inactive injections resulted in differences in the levels of 2-AG seen in the prefrontal cortex yoked saline

25

yoked cocaine active cocaine

3.2.2. NAEs 3.2.2.1. Cocaine self-administration. In the yoked saline group, the OEA levels ranged from 20.32 to 116.51 ng/g, with the highest concentration in the nucleus accumbens and the lowest in the prefrontal cortex. Cocaine treatment resulted in a change in the OEA level in the cortical structures (prefrontal cortex (F(2,20) = 139.4; p b 0.0001) and frontal cortex (F(2,21) = 20.12; p b 0.0001)), hippocampus (F(2,21) = 52.23; p b 0.0001) and dorsal striatum (F(2,21) = 10.41; p b 0.001), but not in the nucleus accumbens (F(2,20) = 0.5178; p N 0.05) and cerebellum (F(2,21) = 2.413; p N 0.05). Increases in the OEA levels in the prefrontal cortex (p b 0.001), frontal cortex (p b 0.01) and hippocampus (p b 0.001) in the animals self-administered cocaine were observed. In animals passively administered cocaine, significant rises in the OEA levels in the prefrontal cortex (p b 0.001), frontal cortex (p b 0.001), hippocampus (p b 0.001) and dorsal striatum (p b 0.001) were noticed. There was a difference between cocaine treated groups in the dorsal striatum (p b 0.05), with a significant increase in yoked cocaine animals (Fig. 4). The concentration of PEA ranged from 31.00 to 122.84 ng/g in the control (yoked saline) group, with the highest concentration in the cerebellum and the lowest in the frontal cortex. Cocaine administration induced changes in the PEA levels in almost all structures (the prefrontal cortex (F(2,20) = 86.82; p b 0.0001), frontal cortex (F(2,21) = 62.66; p b 0.0001), hippocampus (F(2,21) = 56.18; p b 0.0001), dorsal striatum (F(2,21) = 23.06; p b 0.0001) and nucleus accumbens (F(2,20) = 6.729; p b 0.01)), except for the cerebellum (F(2,21) = 2.79; p N 0.05). A potent increase in PEA levels was observed in the prefrontal cortex (p b 0.001), frontal cortex (p b 0.001), hippocampus (p b 0.001) and dorsal striatum (p b 0.001) during maintenance of cocaine self-administration in rats. In rats passively administered cocaine, increases of PEA levels in the prefrontal cortex (p b 0.001), frontal yoked saline yoked cocaine active cocaine

250 x

15

200

x

***

xxx

10

***

***

OEA [ng/g]

*** ***

5

*** ***

150 100

*** ***

***

x

***

**

50 C ER

PEA [ng/g]

10 xx

5

x

**

***

x

**

***

300 xxx

200

***

***

xxx

**

***

xx

***

*** ***

***

100

xxx

ER yoked saline yoked cocaine active cocaine

400

***

15

C

N A C

ST

IP H

FC

PF

yoked saline yoked cocaine active cocaine

20

R

0 C

N

A

C

ST R

IP H

PF

FC

C

0

xx

**

*** ** ER C

A C N

ST R

IP H

PF

ER C

N A C

R ST

H IP

FC

C PF

Fig. 3. Levels of eCBs in rat brain structures during the 10 days of extinction training after cocaine self-administration. AEA—anandamide, 2-AG—2-arachidonoylglycerol, PFC—prefrontal cortex, FC—frontal cortex, HIP—hippocampus, STR—dorsal striatum, NAC—nucleus accumbens, CER—cerebellum. All data are expressed as the means ± SEM. N = 8 rats/group. *p b 0.05; **p b 0.01; ***p b 0.001 vs. yoked saline. xp b 0.05; xx p b 0.01; xxxp b 0.001 active cocaine vs. yoked cocaine.

C

0

0

FC

AEA [ng/g]

(p b 0.05), frontal cortex (p b 0.01), dorsal striatum (p b 0.05) and nucleus accumbens (p b 0.001) (Fig. 3).

*

20

2-AG [ug/g]

5

Fig. 4. Levels of NAEs in rat brain structures following the 14 days of cocaine selfadministration. OEA—oleoylethanolamide, PEA—palmitoylethanolamide, PFC—prefrontal cortex, FC—frontal cortex, HIP—hippocampus, STR—dorsal striatum, NAC—nucleus accumbens, CER—cerebellum. All data are expressed as the means ± SEM. N = 8 rats/group. **p b 0.01; ***p b 0.001 vs. yoked saline. xp b 0.05; xxp b 0.01; xxxp b 0.001 active cocaine vs. yoked cocaine.

6

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

cortex (p b 0.001), hippocampus (p b 0.001), dorsal striatum (p b 0.001), and nucleus accumbens (p b 0.01) were reported. Cocaine active or inactive injections resulted in differences in the levels of PEA seen in the prefrontal cortex (p b 0.001), frontal cortex (p b 0.01) and hippocampus (p b 0.001) and nucleus accumbens (p b 0.01) (Fig. 4). The levels of OEA in both yoked saline groups (1a and 2a) were as follows: prefrontal cortex—30.77 ng/g and 46.86 ng/g; frontal cortex— 52.66 ng/g and 54.37 ng/g; hippocampus—49.10 ng/g and 62.96 ng/g; dorsal striatum—83.09 ng/g and 68.02 ng/g; nucleus accumbens— 84.81 ng/g and 80.53 ng/g; cerebellum—55.31 ng/g and 60.38 ng/g respectively. The Student's t-test revealed a significant difference between yoked saline groups only in the prefrontal cortex (t = 3.915; p b 0.01). In the yoked saline groups the levels of PEA were as follows: prefrontal cortex—53.03 ng/g and 63.77 ng/g; frontal cortex—52.97 ng/g and 67.13 ng/g; hippocampus—72.78 ng/g and 89.49 ng/g; dorsal striatum —68.97 ng/g and 96.03 ng/g; nucleus accumbens—75.68 ng/g and 67.89 ng/g; cerebellum—96.97 ng/g and 82.65 ng/g. The Student's t-test revealed a significant difference between yoked saline groups only in the prefrontal cortex (t = 3.411; p b 0.01). 3.2.2.2. Extinction training. In the extinction phase, the changes in the OEA levels were seen in the prefrontal in almost all structures (prefrontal cortex (F(2,21) = 68.04; p b 0.0001), hippocampus (F(2,21) = 37.32; p b 0.0001), dorsal striatum (F(2,21) = 6.459; p b 0.01), nucleus accumbens (F(2,21) = 15.85; p b 0.0001) and cerebellum (F(2,21) = 9.867; p b 0.005)), except for the frontal cortex (F(2,21) = 2.612; p N 0.05). Following the 10-day extinction, an increase of OEA concentration was noted in the prefrontal cortex (p b 0.001), hippocampus (p b 0.001) and nucleus accumbens (p b 0.05), whilst a decrease (p b 0.01) was reported in the dorsal striatum in rats previously self-administered cocaine. In the yoked cocaine group, only reductions in OEA levels were noticed for the dorsal striatum (p b 0.05), nucleus accumbens (p b 0.05) and cerebellum (p b 0.01). Cocaine active or inactive injections resulted in differences in the levels of OEA seen in the hippocampus (p b 0.001), nucleus accumbens (p b 0.001) and cerebellum (p b 0.01) (Fig. 5). yoked saline yoked cocaine active cocaine

250

OEA [ng/g]

200 150

*** 100

***

***

* xxx

xxx

xx

*

* **

50

**

ER

N

C

A C

R ST

IP H

FC

PF C

0

yoked saline

400

yoked cocaine

PEA [ng/g]

active cocaine

300 200

*** ***

x

***

* 100

x

******

xx

**

C ER

C A N

ST R

IP H

FC

PF C

0

Fig. 5. Levels of NAEs in rat brain structures during the 10 days of extinction training after cocaine self-administration. OEA—oleoylethanolamide, PEA—palmitoylethanolamide, PFC—prefrontal cortex, FC—frontal cortex, HIP—hippocampus, STR—dorsal striatum, NAC— nucleus accumbens, CER—cerebellum. All data are expressed as the means ± SEM. N = 8 rats/group. *p b 0.05; **p b 0.01; ***p b 0.001 vs. yoked saline. xp b 0.05; xxp b 0.01; xxx p b 0.001 active cocaine vs. yoked cocaine.

During extinction, we observed changes in the PEA levels in the prefrontal cortex (F(2,21) = 46.42; p b 0.0001), hippocampus (F(2,21) = 16.45; p b 0.0001), dorsal striatum (F(2,21) = 17.86; p b 0.0001) and cerebellum (F(2,21) = 7.666; p b 0.005), but not in the frontal cortex (F(2,21) = 2.589; p N 0.05) and nucleus accumbens (F(2,21) = 3,805; p b 0.05). A significant increase of PEA concentration was noted in the prefrontal cortex (p b 0.001) and hippocampus (p b 0.001), however, in the dorsal striatum (p b 0.001) a decrease was observed for the animals previously self-administered cocaine. In the yoked cocaine group, increases (in the prefrontal cortex (p b 0.001) and hippocampus (p b 0.05)) or reduction (in the dorsal striatum (p b 0.001) and cerebellum (p b 0.01)) were reported. There was a difference between cocaine treated groups in the hippocampus (p b 0.05), nucleus accumbens (p b 0.05) and cerebellum (p b 0.01), with a significant reduction in yoked cocaine animals (Fig. 5). 3.2.3. Comparison of eCB and NAE concentrations in rat brain structures between phases (maintenance vs. extinction training) Because the two groups of rats consumed similar amounts of cocaine during cocaine self-administration (see Section 3.1), we compared the tissue levels of eCBs and NAEs between the maintenance and extinction training phases. As shown in Table 2, significant decreases (35–56%) in AEA levels were found in the frontal cortex, the hippocampus, the striatum and the nucleus accumbens, whilst ca. 120% increases were detected in the cerebellums of both cocaine-receiving groups. Extinction training also resulted in a potent increase (N 200%) in accumbal levels of 2-AG limited to rats with previous cocaine self-administration. For those, as well as for their yoked cocaine controls, the levels of 2-AG decreased in the frontal cortex and the cerebellum. Relative to cocaine self-administration, extinction resulted in a decrease in the concentrations of OEA and PEA in the frontal cortex, the hippocampus and the striatum. Extinction training increased the OEA levels in the nucleus accumbens and the cerebellum in cocaine self-administering rats but not in the yoked cocaine controls (Table 2). 4. Discussion The present paper revealed the effects of cocaine self-administration and withdrawal on the ex vivo tissue levels of eCBs and NAEs using a yoked-triad procedure. Numerous brain regions were examined, particularly focussing on the structures involved in the rewarding properties and motivational aspects of cocaine intake (Fuchs et al., 2007; Gonzalez et al., 2002; Kalivas and McFarland, 2003). Several investigators have demonstrated that chronic treatment of animals with drugs such as nicotine, cocaine, morphine, cannabinoids and ethanol resulted in an increase or decrease in the level of 2-AG in the whole brain or in certain brain structures (Gonzalez et al., 2002, 2004; Sugiura et al., 2006; Vigano et al., 2003). Our results indicate that repeated cocaine injections for 14 days resulted in changes in the 2-AG levels, which differed depending on the brain structure. Specifically, increases in the 2-AG levels were detected in the frontal cortex and the cerebellum compared to the yoked saline groups, whilst decreases were detected in the hippocampus and the dorsal striatum. In addition, we found a significant reduction in the AEA levels in the cerebellum, but no alterations in other brain structures. As the changes in the eCB levels in the frontal cortex (2-AG) and the cerebellum (AEA) parallel those also found in yoked cocaine animals, these results associate the alterations in eCBs with the pharmacological properties of injected cocaine. Interestingly, alterations in the 2-AG levels were also detected in the prefrontal cortex, the frontal cortex, the hippocampus, the dorsal striatum, the nucleus accumbens and the cerebellum of rats during 10-day cocaine withdrawal with extinction training. The increases found in the prefrontal cortex and the nucleus accumbens and the decreases found in the hippocampus and the striatum occurred only in rats that had previously self-administered cocaine. Such a long-lasting

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

7

Table 2 Alterations in the tissue levels of eCBs and NAEs during cocaine self-administration and extinction training. Group

Cocaine self-administration

Brain structure

PFC

Yoked cocaine Cocaine self-administration

FC

Yoked cocaine Cocaine self-administration

HIP

Yoked cocaine Cocaine self-administration

STR

Yoked cocaine Cocaine self-administration

NAC

Yoked cocaine

Student's t-test

AEA

PEA

AEA

2-AG

OEA

PEA

−1.87

−29.86⁎⁎⁎

t = 1.768 df = 13 t = 1.109 df = 14 t = 11.20 df = 13 t = 4.733 df = 14 t = 5.687 df = 13 t = 3.397 df = 14 t = 4.487 df = 13 t = 6.328 df = 14 t = 7.410 df = 13 t = 2.479 df = 14 t = 10.25 df = 13 t = 6.386 df = 14

t = 5.009 df = 13 t = 1.592 df = 14 t = 4.155 df = 13 t = 4.296 df = 14 t = 1.887 df = 13 t = 6.995 df = 14 t = 0.6281 df = 13 t = 0.7801 df = 14 t = 14.29 df = 13 t = 0.6342 df = 14 t = 9.002 df = 13 t = 4.873 df = 14

t = 0.3724 df = 13 t = 2.048 df = 14 t = 7.410 df = 13 t = 5.873 df = 14 t = 3.845 df = 13 t = 8.307 df = 14 t = 6.155 df = 13 t = 10.55 df = 14 t = 3.060 df = 13 t = 4.459 df = 14 t = 2.387 df = 13 t = 0.2887 df = 14

t = 4.688 df = 13 t = 1.737 df = 14 t = 18.77 df = 13 t = 6.553 df = 14 t = 7.264 df = 13 t = 3.699 df = 14 t = 9.740 df = 13 t = 5.815 df = 14 t = 0.8334 df = 13 t = 4.131 df = 14 t = 0.2847 df = 13 t = 2.361 df = 14

−13.26

CER

2-AG

OEA

32.56⁎⁎⁎

9.00

19.58

15.29

16.49

−55.98⁎⁎⁎

−42.13⁎⁎

−61.76⁎⁎⁎

−51.20⁎⁎⁎

−44.94⁎⁎⁎

−32.99⁎⁎⁎

−57.94⁎⁎⁎

−52.26⁎⁎⁎

−35.45⁎⁎⁎

26.93

−24.19⁎⁎

−44.43⁎⁎⁎

−25.88⁎⁎

−42.13⁎⁎⁎

−61.21⁎⁎⁎

−35.06⁎⁎

−34.8⁎⁎⁎

12.92

−58.38⁎⁎⁎

−60.29⁎⁎⁎

−47.22⁎⁎⁎

−9.75

−64.00⁎⁎⁎

−52.44⁎⁎⁎

−48.55⁎⁎⁎

206.79⁎⁎⁎

37.34⁎⁎

6.36

6.88

−36.62⁎⁎⁎

−36.56⁎⁎

122.55⁎⁎⁎

−60.30⁎⁎⁎

45.73⁎

−3.59

75.75⁎⁎⁎

−49.04⁎⁎⁎

3.34

−22.40⁎

−16.16⁎

Yoked cocaine Cocaine self-administration

Extinction training [% change vs. maintenance of cocaine self-administration]

Rats self-administered cocaine (0.5 mg/kg/infusion) for 14 days. Another group of rats also underwent extinction training, in which animals self-administered saline (0.1 ml/infusion). Yoked cocaine animals were administered exactly the same amount of cocaine as the paired animals self-administered. N = 8 rats/group; *p b 0.05; **p b 0.01; ***p b 0.001 vs. maintenance. Statistically significant changes are marked in italics.

adaptation may encode behaviours involving drug availability and/or drug craving. To support such a hypothesis, the brain structures in which the eCB levels were altered mediate habit formation and automatism establishment (e.g., compulsive drug seeking). The observed parallel decreases in cerebellar AEA levels between rats self-administering and those passively cocaine receiving cannot be associated with motor adaptation during navigation (the classical role of the cerebellum), as only the rats actively working for cocaine were required to emit operant responses. As recently demonstrated, the cerebellum links sensory information with limbic brain structures, and there are functional connections from the cerebellum to the hippocampus, as well as from the cerebellum to the parietal and/or retrosplenial cortex, that are neural pathways for spatial cognition (Miquel et al., 2009; Ranganath and Ritchey, 2012). Moreover, Suarez et al. (2008) reported the presence of CB1 and CB2 receptors in the cerebellar tissue, as well as enzymes involved in the synthesis and degradation of eCBs, indicating possible participation of eCBs in the development of cerebellar synaptic plasticity. The cerebellar reduction in AEA levels did not result from the initial training procedures (learning to press the lever) in both cocaine groups, and AEA does not appear to play an important role in transferring information regarding the spatial distribution of the relevant stimuli (at the operant level) to improve detection at locations of high target probability, as yoked saline controls underwent the same training and injection procedures as yoked cocaine animals. In another study in which AEA concentrations were measured, there was a non-significant trend toward a reduction in the AEA levels in the striatum after experimenter-mediated administration of cocaine to mice (Kurtuncu et al., 2008) or rats (Gonzalez et al., 2002) via intraperitoneal (i.p.) drug injection for 5 days. Interestingly, an in vivo microdialysis study revealed decreases in accumbal AEA levels in short and low-dose (5 mg/kg/session) cocaine self-administered rats, but no changes were observed in animals administering 11–20 mg/kg per session (the present study; Caillé et al., 2007; Orio et al., 2009). In the present study, we also found that extinction training resulted in potent decreases in the AEA levels in animals previously receiving

cocaine either voluntarily or passively (compared to the yoked saline control groups). Such alterations were detected in limbic structures (the prefrontal and frontal cortices, the hippocampus and the nucleus accumbens) and reflect the pharmacological properties of chronic cocaine exposure but not the motivational and cognitive processes associated with reinforcement. Simultaneously, significant increases in the tissue levels of 2-AG in some brain areas (i.e., the prefrontal cortex, frontal cortex and nucleus accumbens) were detected. Our recent report (Adamczyk et al., 2012a) demonstrated that cocaine administration induced an increase in CB1 receptor expression in cortical and subcortical brain regions and that these changes persisted during withdrawal. As was the case for the 2-AG tissue levels (the present study), only active cocaine administration altered the levels of CB1 receptor expression in the dorsal striatum and the hippocampus, thus providing evidence for a direct link between these structures and the motivational and cognitive aspects of cocaine exposure. Interestingly, we report area- and phase-specific changes in eCB levels in the rat cortex. Thus, during the maintenance phase of cocaine self-administration, the 2-AG level increased in the frontal cortex in both cocaine groups, and this enhancement was preserved for up to 10 days of extinction in the yoked cocaine group. On the other hand, following extinction training, the levels of 2AG increased in the prefrontal cortex, and the levels of AEA in the frontal cortex significantly decreased, especially in those animals previously self-administering cocaine. Because different cortical regions mediate distinct functional responses (the prefrontal cortex is involved in working memory, impulsivity, motivation and seeking behaviour, whilst the frontal cortex is engaged in control of motor responses; Lafourcade et al., 2007; Gass and Chandler, 2013), further studies are required to address the specific role of eCBs in cocaine-evoked disruptions of cortical processes. The opposing regulation of AEA and 2-AG is consistent with previously published functional data from mouse striatal slices, in which the elevation of AEA reduced the levels, metabolism and physiological effects of 2-AG (Maccarrone et al., 2008). Those authors also revealed the involvement of TRPV1 receptors, through which endogenous AEA

8

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

reduced 2-AG metabolism (Maccarrone et al., 2008). Another ex vivo study in monkey brain found that increased AEA levels inhibited striatal 2-AG signalling via down-regulation of 2-AG production (Justinova et al., 2008b), which is a mechanism that may also have occurred in the present study. Up to now, there has not been general agreement regarding the role of eCBs in cocaine addiction and in the drug-induced alterations in AEA and 2-AG signalling of striatal plasticity. In fact, fatty acid amide hydrolase (FAAH) inhibition did not reduce the rewarding or sensitizing locomotor effects of cocaine (Adamczyk et al., 2009; Justinova et al., 2008a; Luque-Rojas et al., 2013), but did attenuate both cocaineand cue-induced drug-seeking behaviours (Adamczyk et al., 2009). Inhibition of monoacylglycerol lipase (MAGL) affected neither the acute psychomotor nor the conditioned locomotor activities of cocaine (Hnasko et al., 2007). Instead, MAGL inhibition only attenuated the induction of an already-acquired cocaine-induced behavioural sensitisation (Luque-Rojas et al., 2013). In contrast, cocaine self-administration and withdrawal resulted in long-lasting CB1 receptor up-regulation in many brain structures (Adamczyk et al., 2012a). Acute pharmacological blockade of CB1 receptors either reduced (Li et al., 2009; Soria et al., 2005; Xi et al., 2008) or did not change the rewarding properties of cocaine (Adamczyk et al., 2012b; Lesscher et al., 2005; Orio et al., 2009). The constitutive activation of CB1 receptors was associated with the external cues associated with cocaine that appeared during conditioned locomotion or drug-seeking behaviour (Adamczyk et al., 2012b; de Vries et al., 2001; Gerdeman et al., 2008). On the other hand, cocaine acts as a positive allosteric modulator in vitro, as cocaine exposure elevated the Emax of CP55940 (by 11%) on human CB1 receptors. The latter findings are, however, difficult to interpret in the context of our present data regarding the tissue levels of eCBs. There are also some recent data indicating that tonic activation of brain CB2 receptors facilitates the rewarding properties and locomotion-stimulating effects of cocaine (Xi et al., 2011). In parallel with the changes in AEA or 2-AG levels, repeated cocaine treatment via voluntary or passive exposure evoked potent, long-lasting changes (even detected after a 10-day drug-free period). These changes correlated with the up-regulation of OEA and PEA in limbic regions (i.e., the prefrontal cortex and the hippocampus). There was also an enhancement of accumbal OEA levels during the extinction training in animals that previously self-administered cocaine, and there was bi-directional modulation of the striatal levels of OEA and PEA. The latter changes reflected the state of addiction, with increases in NAEs detected during cocaine exposure and decreases detected during the drug-free period. Both OEA and PEA activate the PPAR-α receptors, which are engaged in lipid metabolism and energy balance. PEA also acts as an antinociceptive molecule and displays anti-inflammatory properties (Calignano et al., 1998; Jaggar et al., 1998; Rodríguez de Fonseca et al., 2001). In a PPAR-α receptor-dependent manner, OEA can enhance memory consolidation in rats (Mazzola et al., 2009) via regulation of the expression of genes involved in cholinergic neurotransmission and learning and memory processes (Cimini et al., 2005; Moreno et al., 2004). Recently, it was also demonstrated that PEA and OEA stimulate tyrosine kinase in a non-genomic manner, followed by phosphorylation and negative regulation of neuronal nicotinic acetylcholine receptors. It was shown that through this pathway, PEA and OEA participate in the rapid inhibition of nicotine-induced reward-related behaviours (Melis et al., 2008). However, it should be noted that OEA and PEA can serve as noncompetitive nicotinic acetylcholine receptor antagonists (Barrantes, 2004; Butt et al., 2002), and enhance dopamine levels in the hypothalamus (Serrano et al., 2011) or the nucleus accumbens (Murillo-Rodríguez et al., 2011). These properties of OEA and PEA do not support either the enhancement of memory consolidation or the inhibitory actions of OEA and PEA in the brain reward pathway. In fact, OEA bi-directionally influenced MDMA-induced deficits in learning and task recall in mice; at a low dose, OEA ameliorated these MDMA-

induced deficits, whilst at a high dose, OEA exacerbated these deficits (Plaza-Zabala et al., 2010). At present, it is also unclear which functions of OEA and PEA predominate or how each function predominates to propagate signals during cocaine self-administration and extinction training (as in this present study). Additionally, acute i.p. administration of OEA only weakly influenced cocaine behavioural sensitization (Bilbao et al., 2013), and direct stimulation of PPAR-α receptors did not affect the rewarding and drug-seeking responses to cocaine (Bilbao et al., 2013; Mascia et al., 2011) in rodents. It should also be noted that OEA and PEA also target several different receptors, ion channels and enzymes, e.g., vanilloid receptors, K+ channels (Kv4.3, Kv1.5); GPR119 receptors, ceramidases (Hansen, 2010; Syed et al., 2012). However, the relevance of these multi-targeted interactions to the reward and drug-seeking behaviours of cocaine has not been established. Similar to AEA, both OEA and PEA are primarily inactivated by FAAH (Cravatt and Lichtman, 2002; Fegley et al., 2005) via the so-called “entourage effects” (Di Marzo et al., 2001). However, when the levels of OEA and PEA were increased, we did not simultaneously detect differences in the tissue concentration of AEA during cocaine selfadministration (see above). There are several possible mechanisms by which cocaine altered the endogenous fatty acid levels: (i) intracellular degradation of AEA might occur via routes unrelated to AEA metabolism, such as the choline-specific phosphodiesterase NPP6 (Mulder and Cravatt, 2006), or alternative hydrolases (Tsuboi et al., 2005), lipoxygenases (Ueda et al., 1995) or cyclooxygenases (Kozak et al., 2002); (ii) OEA and PEA decomposition might be catalyzed by a distinct but as-yet-uncharacterised NAE amidase that operates at acidic pH values (Ueda et al., 1999, 2001). Another difference in the degradation process between AEA and both PEA and OEA is their interaction with a high-affinity cellular transporter. The first step in the AEA decomposition process requires its high-affinity transport into cells, but it has been found that OEA and PEA are poor substrates for this AEA transporter (Beltramo et al., 1997; Di Marzo et al., 1994). Because OEA shares properties with inhibitors of FAAH and the AEA transporter (Jonsson et al., 2001), the latter property of OEA might impede AEA transport into the cell, thereby maintaining the tissue levels of this eCB. The influence of cocaine on the metabolic pathways mentioned above remains unknown. Finally, the observed changes in the levels of eCBs and NAEs might be related to the feeding (Fu et al., 2003; Rodríguez de Fonseca et al., 2001), operant lever press training or drug self-administration procedures (Rivera et al., 2013). Moreover, in contrast to Buczynski et al. (2013) our behavioural procedures minimised the influence of the operant lever press procedure or the initial food restriction on the levels of eCBs and NAEs, as all of the experimental groups (including the yoked saline controls) were subjected to the same behavioural and injection conditions during the lever press training, maintenance and extinction phases. Stress is an inherent confound in yoked animals (Hill et al., 2010a,b) that should also be mentioned. In fact, the yoking procedure has been found to be an aversive stimulus that reduced the motivational aspect of cocaine (Twining et al., 2009) and enhanced the levels of corticosterone. Because we did not detect nonparallel changes in the levels of eCBs or NAEs in general during cocaine self-administration and extinction training in yoked cocaine animals compared to active cocaine self-administering animals or yoked saline animals, the impact of stress appears to be limited. The parallel changes in the levels of eCBs and NAEs that occurred in both cocaine groups reflect the pharmacological aspects of drug intake, whilst some of the differences in the effects that occurred following cocaine self-administration and extinction training or following yoked cocaine delivery and extinction may be due to different types of learning or synaptic adaptations related to learning. In fact, rats that self-administered cocaine learned to press the lever to receive cocaine infusion, which was an association using a conditioned stimulus. In those animals, the operant response was extinguished when a previously delivered unconditioned response and a conditioned stimulus were lacking. In other words, extinction

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

training induced a new type of learning, during which the rats omitted the non-reinforced behaviour. Rats subjected to yoked cocaine during the maintenance phase incorporated a Pavlovian association between the effects of cocaine and the stimulus, but during the extinction phase, this association was not present. Another difference between the cocaine groups was that the rats self-administering cocaine associated the cue with their operant response, whilst the yoked cocaine animals did not. This study also indicates some region-specific differences in the levels of AEA (the dorsal striatum), OEA (the prefrontal cortex) and PEA (the dorsal striatum) between the yoked saline groups compared to cocaine self-administering rats or those yoked to animals subjected to extinction training. Although it is difficult to establish definitively, these changes may reflect the alterations in the levels of eCBs and NAEs in the presence or absence of conditional stimuli (tone and light linked to the saline injections) in the maintenance or extinction training phases, respectively. 5. Conclusion As presented in this study, alterations in the levels of eCBs and NAEs in different rat brain structures indicate the importance of these lipids in cocaine reward and drug-seeking behaviours. To define the role of these lipids in the mechanism of drug addiction and to determine whether pharmacological modulation of this system may be an effective therapy for drug addiction, further investigations are required. Acknowledgement The authors would like to thank Agata Suder and Ewa Nowak for technical support. This study was supported by the grant no. N N404 273040 (Ministry of Science and Higher Education, Warszawa, Poland), statutory funds from the Jagiellonian University (K/ZDS/001295) and from the Institute of Pharmacology Polish Academy of Sciences (Kraków, Poland). References Adamczyk P, McCreary AC, Przegalinski E, Mierzejewski P, Bienkowski P, Filip M. The effects of fatty acid amide hydrolase inhibitors on maintenance of cocaine and food self-administration and on reinstatement of cocaine-seeking and food-taking behavior in rats. J Physiol Pharmacol 2009;60:119–25. Adamczyk P, Faron-Górecka A, Kuśmider M, Dziedzicka-Wasylewska M, Papp M, Filip M. Long-lasting increase in [3H]CP55,940 binding to CB1 receptors following cocaine self-administration and its withdrawal in rats. Brain Res 2012a;1451:34–43. Adamczyk P, Miszkiel J, McCreary AC, Filip M, Papp M, Przegaliński E. The effects of cannabinoid CB1, CB2 and vanilloid TRPV1 receptor antagonists on cocaine addictive behavior in rats. Brain Res 2012b;1444:45–54. Arnold JC. The role of endocannabinoid transmission in cocaine addiction. Pharmacol Biochem Behav 2005;81:396–406. Barrantes FJ. Structural basis for lipid modulation of nicotinic acetylcholine receptor function. Brain Res Brain Res Rev 2004;47:71–95. 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. Bilbao A, Blanco E, Luque-Rojas MJ, Suárez J, Palomino A, Vida M, et al. Oleoylethanolamide dose-dependently attenuates cocaine-induced behaviours through a PPARα receptorindependent mechanism. Addict Biol 2013;18:78–87. Bossert JB, Ghitza UE, Lu L, Epstein DH, Shaham Y. Neurobiology of relapse to heroin and cocaine seeking: an update and clinical implications. Eur J Pharmacol 2005;526: 36–50. Buczynski M, Polis I, Parsons L. The volitional nature of nicotine exposure alters anandamide and oleoylethanolamide levels in the ventral tegmental area. Neuropsychopharmacology 2013;38:574–84. Budzyńska B, Kruk M, Biała G. Effects of the cannabinoid CB1 receptor antagonist AM 251 on the reinstatement of nicotine-conditioned place preference by drug priming in rats. Pharmacol Rep 2009;61:304–10. Butt CM, Hutton SR, Marks MJ, Collins AC. Bovine serum albumin enhances nicotinic acetylcholine receptor function in mouse thalamic synaptosomes. J Neurochem 2002;83:48–56. Caillé S, Alvarez-Jaimes L, Polis I, Stouffer D, Parsons L. Specific alterations of extracellular endocannabinoid levels in the nucleus accumbens by ethanol, heroin, and cocaine self-administration. J Neurosci 2007;27:3695–702.

9

Calignano A, La Rana G, Giuffrida A, Piomelli D. Control of pain initiation by endogenous cannabinoids. Nature 1998;394:277–81. Centonze D, Battista N, Rossi S, Mercuri NB, Finazzi-Agrò A, Bernardi G, et al. A critical interaction between dopamine D2 receptors and endocannabinoids mediates the effects of cocaine on striatal GABAergic transmission. Neuropsychopharmacology 2004;29:1488–97. Cimini A, Benedetti E, Cristiano L, Sebastiani P, D'Amico MA, D'Angelo B, et al. Expression of peroxisome proliferator-activated receptors (PPARs) and retinoic acid receptors (RXRs) in rat cortical neurons. Neuroscience 2005;130:325–37. Cravatt BF, Lichtman AH. The enzymatic inactivation of the fatty acid amide class of signaling lipids. Chem Phys Lipids 2002;121:135–48. De Vries TJ, Shaham Y, Homberg JR, Crombag H, Schuurman K, Dieben J, et al. A cannabinoid mechanism in relapse to cocaine seeking. Nat Med 2001;7:1151–4. Di Chiara G. The role of dopamine in drug abuse viewed from the perspective of its role in motivation. Drug Alcohol Depend 1995;38:95–137. Di Marzo V. Endocannabinoid signaling in the brain: biosynthetic mechanisms in the limelight. Nat Neurosci 2011;14:9–15. Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz JC, et al. Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 1994;372:686–91. Di Marzo V, Melck D, Orlando P, Bisogno T, Zagoory O, Bifulco M, et al. Palmitoylethanolamide inhibits the expression of fatty acid amide hydrolase and enhances the anti-proliferative effect of anandamide in human breast cancer cells. Biochem J 2001;358:249–55. Fegley D, Gaetani S, Duranti A, Tontini A, Mor M, Tarzia G, et al. Characterization of the fatty acid amide hydrolase inhibitor cyclohexyl carbamic acid 3′-carbamoylbiphenyl-3-yl ester (URB597): effects on anandamide and oleoylethanolamide deactivation. J Pharmacol Exp Ther 2005;313:352–8. Ferraro L, Beggiato S, Marcellino D, Frankowska M, Filip M, Agnati LF, et al. Nanomolar concentrations of cocaine enhance D2-like agonist-induced inhibition of the K+-evoked [3H]-dopamine efflux from rat striatal synaptosomes: a novel action of cocaine. J Neural Transm 2010;117:593–7. Ferraro L, Frankowska M, Marcellino D, Zaniewska M, Beggiato S, Filip M, et al. A novel mechanism of cocaine to enhance dopamine d2-like receptor mediated neurochemical and behavioral effects. An in vivo and in vitro study. Neuropsychopharmacology 2012;37:1856–66. Filip M. Role of serotonin (5-HT)2 receptors in cocaine self-administration and seeking behavior in rats. Pharmacol Rep 2005;57:35–46. Folch J, Lees M, Sloane Stanley GH. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957;226:497–509. Frankowska M, Wydra K, Faron-Górecka A, Zaniewska M, Kuśmider M, Dziedzicka-Wasylewska M, et al. Neuroadaptive changes in the rat brain GABA B receptors after withdrawal from cocaine self-administration. Eur J Pharmacol 2008;599:58–64. Frankowska M, Gołda A, Wydra K, Gruca P, Papp M, Filip M. Effects of imipramine or GABA B receptor ligands on the immobility, swimming and climbing in the forced swim test in rats following discontinuation of cocaine self-administration. Eur J Pharmacol 2010;627:142–9. Fu J, Gaetani S, Oveisi F, Lo Verme J, Serreno A, Rodríguez De Fonseca F, et al. Oleylethanolamide regulates feeding and body weight through activation of the nuclear receptor PPAR-alpha. Nature 2003;425:90–3. Fu J, Kim J, Oveisi F, Astarita G, Piomelli D. Targeted enhancement of oleoylethanolamide production in proximal small intestine induces across-meal satiety in rats. Am J Physiol Regul Integr Comp Physiol 2008;295:45–50. Fuchs RA, Eaddy JL, Su Z, Bell GH. Interactions of the basolateral amygdala with the dorsal hippocampus and dorsomedial prefrontal cortex regulate drug context-induced reinstatement of cocaine-seeking in rats. Eur J Neurosci 2007;26:487–98. Gass JT, Chandler LJ. The plasticity of extinction: contribution of the prefrontal cortex in treating addiction through inhibitory learning. Front Psychiatry 2013;4(46):1–13. Genedani S, Carone C, Guidolin D, Filaferro M, Marcellino D, Fuxe K, et al. Differential sensitivity of A2A and especially D2 receptor trafficking to cocaine compared with lipid rafts in cotransfected CHO cell lines. Novel actions of cocaine independent of the DA transporter. J Mol Neurosci 2010;41:347–57. Gerdeman GL, Schechter JB, French ED. Context-specific reversal of cocaine sensitization by the CB1 cannabinoid receptor antagonist rimonabant. Neuropsychopharmacology 2008;33:2747–59. Giuffrida A, Parsons LH, Kerr TM, Rodriguez de Fonseca F, Navarro M, Piomelli D. Dopamine activation of endogenous cannabinoid signaling in dorsal striatum. Nat Neurosci 1999;2:358–63. Giuffrida A, Rodríguez de Fonseca F, Piomelli D. Quantification of bioactive acylethanolamides in rat plasma by electrospray mass spectrometry. Anal Biochem 2000;280:87–93. Gonzalez S, Cascio M, Fernandez-Ruiz J, Fezza F, Di Marzo V, Ramos JA. Changes in endocannabinoid contents in the brain of rats chronically exposed to nicotine, ethanol or cocaine. Brain Res 2002;954:73–81. Gonzalez S, Fernandez-Ruiz J, Di Marzo V, Hernandez M, Arevalo C, Nicanor C, et al. Behavioral and molecular changes elicited by acute administration of SR141716 to Δ9-tetrahydrocannabinol-tolerant rats: an experimental model of cannabinoid abstinence. Drug Alcohol Depend 2004;74:159–70. Hansen HS. Palmitoylethanolamide and other anandamide congeners. Proposed role in the diseased brain. Exp Neurol 2010;224:48–55. Hansen HS, Diep TA. N-acylethanolamines, anandamide and food intake. Biochem Pharmacol 2009;78:553–60. Hill M, McLaughlin R, Bingham B, Shrestha L, Lee T, Gray M, et al. Endogenous cannabinoid signaling is essential for stress adaptation. Proc Natl Acad Sci U S A 2010a;18(107):9406–11.

10

B. Bystrowska et al. / Progress in Neuro-Psychopharmacology & Biological Psychiatry 50 (2014) 1–10

Hill M, Patel S, Campolongo P, Tasker J, Wotjak C, Bains J. Functional interactions between stress and the endocannabinoid system: from synaptic signaling to behavioral output. J Neurosci 2010b;30:14980–6. Hnasko TS, Sotak BN, Palmiter RD. Cocaine-conditioned place preference by dopaminedeficient mice is mediated by serotonin. J Neurosci 2007;27:12484–8. Jaggar SI, Hasnie FS, Sellaturay S, Rice AS. The anti-hyperalgesic actions of the cannabinoid anandamide and the putative CB2 receptor agonist palmitoylethanolamide in visceral and somatic inflammatory pain. Pain 1998;76:189–99. Jonsson KO, Vandevoorde S, Lambert DM, Tiger G, Fowler CJ. Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide. Br J Pharmacol 2001;133:1263–75. Justinova Z, Mangieri RA, Bortolato M, Chefer SI, Mukhin AG, Clapper JR, et al. Fatty acid amide hydrolase inhibition heightens anandamide signaling without producing reinforcing effects in primates. Biol Psychiatry 2008a;64:930–7. Justinova Z, Munzar P, Panlilio LV, Yasar S, Redhi GH, Tanda G, et al. Blockade of THC-seeking behavior and relapse in monkeys by the cannabinoid CB(1)-receptor antagonist rimonabant. Neuropsychopharmacology 2008b;33:2870–7. Kalivas PW, McFarland K. Brain circuitry and the reinstatement of cocaine-seeking behavior. Psychopharmacology 2003;168:44–56. Kozak KR, Crews BC, Morrow JD, Wang LH, Ma YH, Weinander R. Metabolism of the endocannabinoids, 2-arachidonylglycerol and anandamide, into prostaglandin, thromboxane, and prostacyclin glycerol esters and ethanolamides. J Biol Chem 2002;277:44877–85. Kreek MJ, Levran O, Reed B, Schlussman SD, Zhou Y, Butelman ER. Opiate addiction and cocaine addiction: underlying molecular neurobiology and genetics. J Clin Invest 2012;122:3387–93. Kurtuncu M, Battista N, Uz T, D'Agostino A, Dimitrijevic N, Pasquariello N, et al. Effects of cocaine in 5-lipoxygenase-deficient mice. J Neural Transm 2008;115:389–95. 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. Lesscher H, Hoogveld E, Burbach JP, van Ree J, Gerrits M. Endogenous cannabinoids are not involved in cocaine reinforcement and development of cocaine-induced behavioural sensitization. Eur Neuropsychopharmacol 2005;15:31–7. Li X, Hoffman AF, Peng XQ, Lupica CR, Gardner EL, Xi ZX. Attenuation of basal and cocaine-enhanced locomotion and nucleus accumbens dopamine in cannabinoid CB1-receptor-knockout mice. Psychopharmacology (Berl) 2009;204:1–11. Luque-Rojas MJ, Galeano P, Suárez J, Araos P, Santín LJ, de Fonseca FR, et al. Hyperactivity induced by the dopamine D2/D3 receptor agonist quinpirole is attenuated by inhibitors of endocannabinoid degradation in mice. Int J Neuropsychopharmacol 2013;16:661–76. Maccarrone M, Rossi S, Bari M, De Chiara V, Fezza F, Musella A, et al. Anandamide inhibits metabolism and physiological actions of 2-arachidonoylglycerol in the striatum. Nat Neurosci 2008;11:152–9. Mascia P, Pistis M, Justinova Z, Panlilio LV, Luchicchi A, Lecca S, et al. Blockade of nicotine reward and reinstatement by activation of alpha-type peroxisome proliferatoractivated receptors. Biol Psychiatry 2011;69:633–41. Mazzola C, Medalie J, Scherma M, Panlilio LV, Solinas M, Tanda G, et al. Fatty acid amide hydrolase (FAAH) inhibition enhances memory acquisition through activation of PPAR-alpha nuclear receptors. Learn Mem 2009;16:332–7. Melis M, Pillolla G, Luchicchi A, Muntoni AL, Yasar S, Goldberg SR, et al. Endogenous fatty acid ethanolamides suppress -induced activation of mesolimbic dopamine neurons through nuclear receptors. J Neurosci 2008;28:13985–94. Miquel M, Toledo R, García L, Coria-Avila G, Manzo J. Why should we keep the cerebellum in mind when thinking about addiction? Curr Drug Abuse Rev 2009;2:26–40. Moreno S, Farioli-Vecchioli S, Cerù MP. Immunolocalization of peroxisome proliferatoractivated receptors and retinoid X receptors in the adult rat CNS. Neuroscience 2004;123:131–45. Mulder AM, Cravatt BF. Endocannabinoid metabolism in the absence of fatty acid amide hydrolase (FAAH): discovery of phosphorylcholine derivatives of N-acyl ethanolamines. Biochemistry 2006;45:11267–77. Murillo-Rodríguez E, Palomero-Rivero M, Millán-Aldaco D, Arias-Carrión O, Drucker-Colín R. Administration of URB597, oleoylethanolamide or palmitoylethanolamide increases waking and dopamine in rats. PLoS One 2011;6:e20766. Nestler EJ. Molecular mechanisms of drug addiction. Neuropharmacology 2004;47:24–32. Onaivi E, Leonard C, Ishiguro H, Wu Zhang P, Lin Z, Akinshola B, et al. Endocannabinoids and cannabinoid receptor genetics. Prog Neurobiol 2002;66:307–44.

Orio L, Edwards S, George O, Parsons LH, Koob GF. A role for the endocannabinoid system in the increased motivation for cocaine in extended-access conditions. J Neurosci 2009;29:4846–57. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 4th ed. San Diego: Academic Press; 1998. Piomelli D. The molecular logic of endocannabinoid signaling. Nat Rev Neurosci 2003;4: 873–84. Plaza-Zabala A, Berrendero F, Suarez J, Bermudez-Silva FJ, Fernandez-Espejo E, Serrano A, et al. Effects of the endogenous PPAR-alpha agonist, oleoylethanolamide on MDMA-induced cognitive deficits in mice. Synapse 2010;64:379–89. Ranganath C, Ritchey M. Two cortical systems for memory-guided behaviour. Nat Rev Neurosci 2012;13:713–26. Rivera P, Miguéns M, Coria SM, Rubio L, Higuera-Matas A, Bermúdez-Silva FJ, et al. Cocaine self-administration differentially modulates the expression of endogenous cannabinoid system-related proteins in the hippocampus of Lewis vs. Fischer 344 rats. Int J Neuropsychopharmacol 2013;16:1277–93. Rodríguez De Fonseca F, Gorriti MA, Bilbao A, Escuredo L, García-Segura LM, Piomelli D, et al. Role of the endogenous cannabinoid system as a modulator of dopamine transmission: implications for Parkinson's disease and schizophrenia. Neurotox Res 2001;3:23–35. Serrano A, Pavón FJ, Tovar S, Casanueva F, Señarís R, Diéguez C, et al. Oleoylethanolamide: effects on hypothalamic transmitters and gut peptides regulating food intake. Neuropharmacology 2011;60:593–601. Shoaib M. The cannabinoid antagonist AM251 attenuates nicotine self-administration and nicotine-seeking behaviour in rats. Neuropharmacology 2008;54:438–44. Soria G, Mendizábal V, Touriño C, Robledo P, Ledent C, Parmentier M, et al. Lack of CB1 cannabinoid receptor impairs cocaine self-administration. Neuropsychopharmacology 2005;30:1670–80. Spijker S. Dissection of rodent brain regions. In: Li KW, editor. Neuroproteomics, Neuromethods. Springer; 2011. p. 13–26. Suarez J, Bermudez-Silva F, Mackie K, Ledent C, Zimmer A, Cravatt B, et al. Immunohistochemical description of the endogenous cannabinoid system in the rat cerebellum and functionally related nuclei. J Comp Neurol 2008;509:400–21. Sugiura T, Kishimoto S, Oka S, Gokoh M. Biochemistry, pharmacology and physiology of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand. Prog Lipid Res 2006;45:405–46. Syed SK, Bui HH, Beavers LS, Farb TB, Ficorilli J, Chesterfield AK, et al. Regulation of GPR119 receptor activity with endocannabinoid-like lipids. Am J Physiol Endocrinol Metab 2012;303:1469–78. Thomsen M, Han D, Gu H, Caine B. Lack of cocaine self-administration in mice expressing a cocaine-insensitive dopamine transporter. J Pharmacol Exp Ther 2009;331:204–11. Tsuboi K, Sun YX, Okamoto Y, Araki N, Tonai T, Ueda N. Molecular characterization of N-acylethanolamine-hydrolyzing acid amidase, a novel member of the choloylglycine hydrolase family with structural and functional similarity to acid ceramidase. J Biol Chem 2005;280:11082–92. Twining R, Bolan M, Grigson P. Yoked delivery of cocaine is aversive and protects against the motivation for drug in rats. Behav Neurosci 2009;123:913–25. Ueda N, Yamamoto K, Yamamoto S, Tokunaga T, Shirakawa E, Shinkai H, et al. Lipoxygenase-catalyzed oxygenation of arachidonylethanolamide, a cannabinoid receptor agonist. Biochim Biophys Acta 1995;1254:127–34. Ueda N, Yamanaka K, Terasawa Y, Yamamoto S. An acid amidase hydrolyzing anandamide as an endogenous ligand for cannabinoid receptors. FEBS Lett 1999;454:267–70. Ueda N, Liu Q, Yamanaka K. Marked activation of the N-acylphosphatidylethanolaminehydrolyzing phosphodiesterase by divalent cations. Biochim Biophys Acta 2001;1532:121–7. Vigano D, Grazia Cascio M, Rubino T, Fezza F, Vaccani A, Di Marzo V, et al. Chronic morphine modulates the contents of the endocannabinoid, 2-arachidonoyl glycerol, in rat brain. Neuropsychopharmacology 2003;28:1160–7. Xi ZX, Newman AH, Gilbert JG, Pak AC, Peng XQ, Ashby Jr CR, et al. The novel dopamine D3 receptor antagonist NGB 2904 inhibits cocaine's rewarding effects and cocaine-induced reinstatement of drug-seeking behavior in rats. Neuropsychopharmacology 2006;31: 1393–405. Xi ZX, Spiller K, Pak AC, Gilbert J, Dillon C, Li X, et al. Cannabinoid CB1 receptor antagonists attenuate cocaine's rewarding effects: experiments with self-administration and brain-stimulation reward in rats. Neuropsychopharmacology 2008;33:1735–45. Xi ZX, Peng XQ, Li X, Song R, Zhang HY, Liu QR, et al. Brain cannabinoid CB receptors modulate cocaine's actions in mice. Nat Neurosci 2011;14:1160–6.