Selective Inhibition of PDE4B Reduces Binge Drinking in Two C57BL/6 Substrains

Cyclic AMP (cAMP)-dependent signaling is highly implicated in the pathophysiology of alcohol use disorder (AUD), with evidence supporting the efficacy of inhibiting the cAMP hydrolyzing enzyme phosphodiesterase 4 (PDE4) as a therapeutic strategy for drinking reduction. Off-target emetic effects associated with non-selective PDE4 inhibitors has prompted the development of selective PDE4 isozyme inhibitors for treating neuropsychiatric conditions. Herein, we examined the effect of a selective PDE4B inhibitor A33 (0–1.0 mg/kg) on alcohol drinking in both female and male mice from two genetically distinct C57BL/6 substrains. Under two different binge-drinking procedures, A33 pretreatment reduced alcohol intake in male and female mice of both substrains. In both drinking studies, there was no evidence for carry-over effects the next day; however, we did observe some sign of tolerance to A33’s effect on alcohol intake upon repeated, intermittent, treatment (5 injections of 1.0 mg/kg, every other day). Pretreatment with 1.0 mg/kg of A33 augmented sucrose intake by C57BL/6NJ, but not C57BL/6J, mice. In mice with a prior history of A33 pretreatment during alcohol-drinking, A33 (1.0 mg/kg) did not alter spontaneous locomotor activity or basal motor coordination, nor did it alter alcohol’s effects on motor activity, coordination or sedation. In a distinct cohort of alcohol-naïve mice, acute pretreatment with 1.0 mg/kg of A33 did not alter motor performance on a rotarod and reduced sensitivity to the acute intoxicating effects of alcohol. These data provide the first evidence that selective PDE4B inhibition is an effective strategy for reducing excessive alcohol intake in murine models of binge drinking, with minimal off-target effects. Despite reducing sensitivity to acute alcohol intoxication, PDE4B inhibition reduces binge alcohol drinking, without influencing behavioral sensitivity to alcohol in alcohol-experienced mice. Furthermore, A33 is equally effective in males and females and exerts a quantitatively similar reduction in alcohol intake in mice with a genetic predisposition for high versus moderate alcohol preference. Such findings further support the safety and potential clinical utility of targeting PDE4 for treating AUD.     

Dose and Radioadaptive Response Analysis of Micronucleus Induction in Mouse Bone Marrow

Enhanced cellular DNA repair efficiency and suppression of genomic instability have been proposed as mechanisms underlying radio-adaptive responses following low-dose radiation exposures. We previously showed that low-dose γ irradiation does not generate radio-adaptation by lowering radiation-induced cytogenetic damage in mouse spleen. Since radiation may exert tissue-specific effects, we extended these results here by examining the effects of γ radiation on cytogenetic damage and proliferative index in bone marrow erythrocytes of C57BL/6 and BALB/c mice. In C57BL/6 mice, the induction of micronuclei in polychromatic erythrocytes (MN-PCE) was observed at radiation doses of 100 mGy and greater, and suppression of erythroblast maturation occurred at doses of >500 mGy. A linear dose–response relationship for MN-PCE frequencies in C57BL/6 mice was established for radiation doses between 100 mGy and 1 Gy, with departure from linearity at doses of >1 Gy. BALB/c mice exhibited increased MN-PCE frequencies above baseline following a 20 mGy radiation exposure but did not exhibit radio-sensitivity relative to C57BL/6 mice following 2 Gy exposure. Radio-adaptation of bone marrow erythrocytes was not observed in either strain of mice exposed to low-dose priming γ irradiation (single doses of 20 mGy or 100 mGy or multiple 20 mGy doses) administered at various times prior to acute 2 Gy irradiation, confirming the lack of radio-adaptive response for induction of cytogenetic damage or suppression or erythrocyte proliferation/maturation in bone marrow of these mouse strains.     

Phosphodiesterases Expression during Murine Cardiac Development

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes playing a fundamental role in the control of intracellular levels of cAMP and cGMP. Emerging evidence suggested an important role of phosphodiesterases in heart formation, but little is known about the expression of phosphodiesterases during cardiac development. In the present study, the pattern of expression and enzymatic activity of phosphodiesterases was investigated at different stages of heart formation. C57BL/6 mice were mated and embryos were collected from 14.5 to 18.5 days of development. Data obtained by qRT-PCR and Western blot analysis showed that seven different isoforms are expressed during heart development, and PDE1C, PDE2A, PDE4D, PDE5A and PDE8A are modulated from E14.5 to E18.5. In heart homogenates, the total cAMP and cGMP hydrolytic activity is constant at the evaluated times, and PDE4 accounts for the majority of the cAMP hydrolyzing ability and PDE2A accounts for cGMP hydrolysis. This study showed that a subset of PDEs is expressed in developing mice heart and some of them are modulated to maintain constant nucleotide phosphodiesterase activity in embryonic and fetal heart.     

Effect of Central Administration of Brain-Derived Neurotrophic Factor (BDNF) on Behavior and Brain Monoamine Metabolism in New Recombinant Mouse Lines Differing by 5-HT1A Receptor Functionality

Experiments were carried out on recombinant B6.CBA-D13Mit76C (B6-M76C) and B6.CBA-D13Mit76B (B6-M76B) mouse lines created by transferring a 102.73–118.83 Mbp fragment of chromosome 13, containing the 5-HT_1A receptor gene, from CBA or C57BL/6 strains to a C57BL/6 genetic background, correspondingly. We have recently shown different levels of 5-HT1A receptor functionality in these mouse lines. The administration of BDNF (300 ng/mouse, i.c.v.) increased the levels of exploratory activity and intermale aggression only in B6-M76B mice, without affecting depressive-like behavior in both lines. In B6-M76B mice the behavioral alterations were accompanied by a decrease in the 5-HT_2A receptor functional activity and the augmentation of levels of serotonin and its main metabolite, 5-HIAA (5-hydroxyindoleacetic acid), in the midbrain. Moreover, the levels of dopamine and its main metabolites, HVA (homovanillic acid) and DOPAC (3,4-dihydroxyphenylacetic acid), were also elevated in the striatum of B6-M76B mice after BDNF treatment. In B6-M76C mice, central BDNF administration led only to a reduction in the functional activity of the 5-HT_1A receptor and a rise in DOPAC levels in the midbrain. The obtained data suggest the importance of the 102.73–118.83 Mbp fragment of mouse chromosome 13, which contains the 5-HT_1A receptor gene, for BDNF-induced alterations in behavior and the brain monoamine system.     

Effects of a Fully Humanized Type II Anti-CD20 Monoclonal Antibody on Peripheral and CNS B Cells in a Transgenic Mouse Model of Multiple Sclerosis

Successful therapy with anti-CD20 monoclonal antibodies (mAbs) has reinforced the key role of B cells in the immunopathology of multiple sclerosis (MS). This study aimed to determine the effects of a novel class of anti-CD20 mAbs on vascular and extravascular central nervous system (CNS)-infiltrating B cells in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Male hCD20xhIgR3 mice and wild-type C57BL/6 (B6) mice were immunized with human myelin oligodendrocyte glycoprotein (MOG)_1–125 to induce EAE. While hCD20xhIgR3 mice were injected intravenously with an anti-human CD20 mAb (5 mg/kg) (rituximab (a type I anti-CD20 mAb) or obinutuzumab (a type II anti-CD20 mAb), B6 mice received the anti-mouse CD20 antibody 18B12. Neither mAb affected clinical disease or serum antibody levels. Obinutuzumab and rituximab had an impact on splenic and CNS-infiltrated B cells with slightly differential depletion efficacy. Additionally, obinutuzumab had beneficial effects on spinal cord myelination. B cell depletion rates in the 18B12/B6 model were comparable with those observed in obinutuzumab-treated hCD20xhIgR3 mice. Our results demonstrate the usefulness of anti-CD20 mAbs for the modulation of B cell-driven peripheral immune response and CNS pathology, with type II antibodies potentially being superior to type I in the depletion of tissue-infiltrating B cells.     

Phosphodiesterase-4 Inhibition Reduces Cutaneous Inflammation and IL-1β Expression in a Psoriasiform Mouse Model but Does Not Inhibit Inflammasome Activation

Apremilast (Otezla^®) is an oral small molecule phosphodiesterase 4 (PDE4) inhibitor approved for the treatment of psoriasis, psoriatic arthritis, and oral ulcers associated with Behçet’s disease. While PDE4 inhibition overall is mechanistically understood, the effect of apremilast on the innate immune response, particularly inflammasome activation, remains unknown. Here, we assessed the effect of apremilast in a psoriasis mouse model and primary human cells. Psoriatic lesion development in vivo was studied in K5.Stat3C transgenic mice treated with apremilast for 2 weeks, resulting in a moderate (2 mg/kg/day) to significant (6 mg/kg/day) resolution of inflamed plaques after 2-week treatment. Concomitantly, epidermal thickness dramatically decreased, the cutaneous immune cell infiltrate was reduced, and proinflammatory cytokines were significantly downregulated. Additionally, apremilast significantly inhibited lipopolysaccharide- or anti-CD3-induced expression of proinflammatory cytokines in peripheral mononuclear cells (PBMCs). Notably, inflammasome activation and secretion of IL-1β were not inhibited by apremilast in PBMCs and in human primary keratinocytes. Collectively, apremilast effectively alleviated the psoriatic phenotype of K5.Stat3 transgenic mice, further substantiating PDE4 inhibitor-efficiency in targeting key clinical, histopathological and inflammatory features of psoriasis. Despite lacking direct effect on inflammasome activation, reduced priming of inflammasome components upon apremilast treatment reflected the indirect benefit of PDE4 inhibition in reducing inflammation.     

Altered Accumbal Dopamine Terminal Dynamics Following Chronic Heroin Self-Administration

Administration of heroin results in the engagement of multiple brain regions and the rewarding and addictive effects are mediated, at least partially, through activation of the mesolimbic dopamine system. However, less is known about dopamine system function following chronic exposure to heroin. Withdrawal from chronic heroin exposure is likely to drive a state of low dopamine in the nucleus accumbens (NAc), as previously observed during withdrawal from other drug classes. Thus, we aimed to investigate alterations in NAc dopamine terminal function following chronic heroin self-administration to identify a mechanism for dopaminergic adaptations. Adult male Long Evans rats were trained to self-administer heroin (0.05 mg/kg/inf, IV) and then placed on a long access (FR1, 6-h, unlimited inf, 0.05 mg/kg/inf) protocol to induce escalation of intake. Following heroin self-administration, rats had decreased basal extracellular levels of dopamine and blunted dopamine response following a heroin challenge (0.1 mg/kg/inf, IV) in the NAc compared to saline controls. FSCV revealed that heroin-exposed rats exhibited reduced stimulated dopamine release during tonic-like, single-pulse stimulations, but increased phasic-like dopamine release during multi-pulse stimulation trains (5 pulses, 5–100 Hz) in addition to an altered dynamic range of release stimulation intensities when compared to controls. Further, we found that presynaptic D3 autoreceptor and kappa-opioid receptor agonist responsivity were increased following heroin self-administration. These results reveal a marked low dopamine state following heroin exposure and suggest the combination of altered dopamine release dynamics may contribute to increased heroin seeking.     

Effects of β-Phenylethylamine on Psychomotor,Rewarding, and Reinforcing Behaviors and Affective State: The Role of Dopamine D1 Receptors

Beta-phenylethylamine (β-PEA) is a well-known and widespread endogenous neuroactive trace amine found throughout the central nervous system in humans. In this study, we demonstrated the effects of β-PEA on psychomotor, rewarding, and reinforcing behaviors and affective state using the open-field test, conditioned place preference (CPP), self-administration, and ultrasonic vocalizations (USVs) paradigms. We also investigated the role of the dopamine (DA) D1 receptor in the behavioral effects of β-PEA in rodents. Using enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting, we also determined the DA concentration and the DA-related protein levels in the dorsal striatum of mice administered with acute β-PEA. The results showed that acute β-PEA increased stereotypic behaviors such as circling and head-twitching responses in mice. In the CPP experiment, β-PEA increased place preference in mice. In the self-administration test, β-PEA significantly enhanced self-administration during a 2 h session under fixed ratio (FR) schedules (FR1 and FR3) and produced a higher breakpoint during a 6 h session under progressive ratio schedules of reinforcement in rats. In addition, acute β-PEA increased 50-kHz USV calls in rats. Furthermore, acute β-PEA administration increased DA concentration and p-DAT and TH expression in the dorsal striatum of mice. Finally, pretreatment with SCH23390, a DA D1 receptor antagonist, attenuated β-PEA-induced circling behavior and β-PEA-taking behavior in rodents. Taken together, these findings suggest that β-PEA has rewarding and reinforcing effects and psychoactive properties, which induce psychomotor behaviors and a positive affective state by activating the DA D1 receptor in the dorsal striatum.     

Sex- and Genotype-Dependent Nicotine-Induced Behaviors in Adolescent Rats with a Human Polymorphism (rs2304297) in the 3′-UTR of the CHRNA6 Gene

In human adolescents, a single nucleotide polymorphism (SNP), rs2304297, in the 3′-UTR of the nicotinic receptor subunit gene, CHRNA6, has been associated with increased smoking. To study the effects of the human CHRNA6 3′-UTR SNP, our lab generated knock-in rodent lines with either C or G SNP alleles. The objective of this study was to determine if the CHRNA6 3′-UTR SNP is functional in the knock-in rat lines. We hypothesized that the human CHRNA6 3′-UTR SNP knock-in does not impact baseline but enhances nicotine-induced behaviors. For baseline behaviors, rats underwent food self-administration at escalating schedules of reinforcement followed by a locomotor assay and a series of anxiety tests (postnatal day (PN) 25-39). In separate cohorts, adolescent rats underwent 1- or 4-day nicotine pretreatment (2×, 30 μg/kg/0.1 mL, i.v.). After the last nicotine injection (PN 31), animals were assessed behaviorally in an open-field chamber, and brain tissue was collected. We show the human CHRNA6 3′-UTR SNP knock-in does not affect food reinforcement, locomotor activity, or anxiety. Further, 4-day, but not 1-day, nicotine exposure enhances locomotion and anxiolytic behavior in a genotype- and sex-specific manner. These findings demonstrate that the human CHRNA6 3′-UTR SNP is functional in our in vivo model.     

Istradefylline Mitigates Age-Related Hearing Loss in C57BL/6J Mice

Age-related hearing loss (ARHL) is the most common sensory disorder among older people, and yet, the treatment options are limited to medical devices such as hearing aids and cochlear implants. The high prevalence of ARHL mandates the development of treatment strategies that can prevent or rescue age-related cochlear degeneration. In this study, we investigated a novel pharmacological strategy based on inhibition of the adenosine A_2A receptor (A_2AR) in middle aged C57BL/6 mice prone to early onset ARHL. C57BL/6J mice were treated with weekly istradefylline (A_2AR antagonist; 1 mg/kg) injections from 6 to 12 months of age. Auditory function was assessed using auditory brainstem responses (ABR) to tone pips (4–32 kHz). ABR thresholds and suprathreshold responses (wave I amplitudes and latencies) were evaluated at 6, 9, and 12 months of age. Functional outcomes were correlated with quantitative histological assessments of sensory hair cells. Cognitive function was assessed using the Morris water maze and the novel object recognition test, and the zero maze test was used to assess anxiety-like behaviour. Weekly injections of istradefylline attenuated ABR threshold shifts by approximately 20 dB at mid to high frequencies (16–32 kHz) but did not improve ABR suprathreshold responses. Istradefylline treatment improved hair cell survival in a turn-dependent manner, whilst the cognitive function was unaffected by istradefylline treatment. This study presents the first evidence for the rescue potential of istradefylline in ARHL and highlights the role of A_2AR in development of age-related cochlear degeneration.     

B6 Mouse Strain: The Best Fit for LPS-Induced Interstitial Cystitis Model

Interstitial cystitis (IC) is a chronic inflammatory disease characterized by bladder pain and increased urinary frequency. Although the C57BL/6J (B6) and FVB/NJ (FVB) mouse strains are commonly used as animal models for studies involving the urinary system, few reports have compared their lower urinary tract anatomy, despite the importance of such data. Our study aimed to characterize bladder function changes in FVB and B6 mouse strains with lipopolysaccharide (LPS)-induced IC, to understand mouse model-based bladder research. The bladder function parameters were measured by cystometrogram. Histological assay was examined by hematoxylin and eosin stain, Masson’s trichrome stain, and immunofluorescence staining. Results indicated that the two strains in the control group exhibited different bladder structures and functions, with significant anatomical differences, including a larger bladder size in the FVB than in the B6 strain. Furthermore, cystometry tests revealed differences in bladder function pressure. LPS-treated B6 mice presented significant changes in peak pressure, with decreased intercontraction intervals; these results were similar to symptoms of IC in humans. Each strain displayed distinct characteristics, emphasizing the care required in choosing the appropriate strain for bladder-model studies. The results suggested that the B6 mouse strain is more suitable for IC models.     

A20 Attenuates the Fibrotic Response in the Trabecular Meshwork

Although the extracellular matrix (ECM) in trabecular meshwork (TM) cells is known to be important in intraocular pressure (IOP) regulation, the molecular mechanisms involved in generating a glaucomatous environment in the TM are not completely understood. Recently we identified a molecular pathway, transforming growth factor beta 2 (TGFβ2)–toll-like receptor 4 (TLR4) signaling crosstalk, as an important regulator of glaucomatous damage in the TM, which contributes to fibrosis. Here we evaluate a novel molecular target, A20, also known as tumor necrosis factor alpha-induced protein 3 (TNFAIP3), which may help to block pathological TGFβ2–TLR4 signaling. Primary human TM cells were analyzed for A20 message and for A20 and fibronectin protein expression after treatment with TGFβ2. A20 message increased when the TLR4 pathway was inhibited in TM cells. In addition, TGFβ2, a known inducer of fibrosis, increased fibronectin expression, while at the same time decreasing the expression of A20. We then overexpressed A20 in TM cells in order to test the effect on treatment with TGFβ2, lipopolysaccharide (LPS), or cellular fibronectin extra domain A (cFN-EDA). Importantly, overexpression of A20 rescued the fibrotic response when TM cells were treated with TGFβ2, LPS, or cFN-EDA. In situ hybridization was used to probe for A20 RNA expression in age-matched control (C57BL/6J) mice and mice that constitutively express the EDA isoform of fibronectin (B6.EDA^+/+). In this novel mouse model of glaucoma, A20 RNA was increased versus age-matched control mice in a cyclic manner at 6 weeks and 1 year of age, but not at 8 months. Overall, these data suggest that A20 may work through a negative feedback mechanism attenuating the ability of TGFβ2–TLR4 signaling to induce fibrosis.     

The Anti-Atherosclerosis Effect of Anakinra,a Recombinant Human Interleukin-1 Receptor Antagonist,in Apolipoprotein E Knockout Mice

Interleukin (IL)-1β plays an important role in atherosclerosis pathogenesis. We aimed to investigate the effect of anakinra, a recombinant human IL-1 receptor antagonist, on the progression of atherosclerosis in apolipoprotein E knockout (ApoE^–/–) mice. ApoE^–/– mice (8-week male) were treated with saline (control), anakinra 10, 25, and 50 mg/kg, respectively (n = 10 in each group). Mice were fed a standard chow (4 weeks) followed by an atherogenic diet (35kcal% fat, 1.25% cholesterol, 12 weeks). Atheromatous plaques in ApoE^–/– mice and the expression of inflammatory genes and signaling pathways in human umbilical vein endothelial cells (HUVECs), rat aortic smooth muscle cells (RAOSMCs), and 3T3-L1 adipocytes were assessed. Anakinra reduced the plaque size of the aortic arch (30.6% and 25.2% at the 25 mg/kg and 50 mg/kg doses, both p < 0.05) and serum triglyceride in ApoE^–/– mice and suppressed inflammatory genes (IL-1β and IL-6) expressions in HUVECs and RAOSMCs (all p < 0.05). In RAOSMCs, anakinra reduced metalloproteinase-9 expression in a dose-dependent manner and inhibited cell migration. Anakinra-treated mice exhibited trends of lower CD68+ macrophage infiltration in visceral fat and monocyte chemoattractant protein-1 expression was reduced in 3T3-L1 adipocytes. Anakinra could be a useful component for complementary treatment with a standard regimen to reduce the residual cardiovascular risk.     

Cannabidiol Modulates the Motivational and Anxiety-Like Effects of 3,4-Methylenedioxypyrovalerone (MDPV) in Mice

3,4-Methylenedioxypyrovalerone (MDPV) is a new psychoactive substance (NPS) and the most widespread and life-threatening synthetic cathinone of the “bath salts”. Preclinical research has proven the cocaine-like psychostimulant effects of MDPV and its potential for abuse. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid that has emerged as a new potential treatment for drug addiction. Here, we tested the effects of CBD (20 mg/kg) on MDPV (2 mg/kg)-induced conditioned place preference and MDPV (0.05 and 0.075 mg/kg/infusion) self-administration paradigms. In addition, we assessed the effects of the co-administration of CBD and MDPV (3 and 4 mg/kg) on anxiety-like behaviour using the elevated plus maze (EPM). CBD mitigated the MDPV-induced conditioned place preference. On the contrary, CBD administration throughout the MDPV (0.075 mg/kg/infusion) self-administration increased drug-seeking and taking behaviours, but only in the high-responders group of mice. Furthermore, CBD exerted anxiolytic-like effects, exclusively in MDPV-treated mice. Taken together, our results indicate that CBD modulation of MDPV-induced motivational responses in mice varies depending on the requirements of the learning task, resulting in a complex response. Therefore, further research attempting to decipher the behavioural and molecular interactions between CBD and MDPV is needed.     

Concomitant Antihyperalgesic and Antitumor Effects of Gabapentin in a Murine Cancer Pain Model

Cancer pain may be the consequence of physical nerve compression by a growing tumor. We employed a murine model to study whether gabapentin was able to regulate tumor growth, in addition to controlling hyperalgesic symptoms. A fluorescent melanoma cell line (B16–BL6/Zs green) was inoculated into the proximity of the sciatic nerve in male C57BL/6 mice. The tumor gradually compressed the nerve, causing hypersensitivity. Tumor growth was characterized via in vivo imaging techniques. Every other day, gabapentin (100 mg/Kg) or saline was IP administered to each animal. In the therapeutic protocol, gabapentin was administered once the tumor had induced increased nociception. In the preventive protocol, gabapentin was administered before the appearance of the positive signs. Additionally, in vitro experiments were performed to determine gabapentin’s effects on cell-line proliferation, the secretion of the chemokine CCL2, and calcium influx. In the therapeutically treated animals, baseline responses to noxious stimuli were recovered, and tumors were significantly reduced. Similarly, gabapentin reduced tumor growth during the preventive treatment, but a relapse was noticed when the administration stopped. Gabapentin also inhibited cell proliferation, the secretion of CCL2, and calcium influx. These results suggest that gabapentin might represent a multivalent strategy to control cancer-associated events in painful tumors.     

Alterations in Rat Accumbens Dopamine,Endocannabinoids and GABA Content During WIN55,212-2 Treatment: The Role of Ghrelin

The endocannabinoid/CB1R system as well as the central ghrelin signalling with its growth hormone secretagogoue receptors (GHS-R1A) are importantly involved in food intake and reward/reinforcement processing and show distinct overlaps in distribution within the relevant brain regions including the hypothalamus (food intake), the ventral tegmental area (VTA) and the nucleus accumbens (NAC) (reward/reinforcement). The significant mutual interaction between these systems in food intake has been documented; however, the possible role of ghrelin/GHS-R1A in the cannabinoid reinforcement effects and addiction remain unclear. Therefore, the principal aim of the present study was to investigate whether pretreatment with GHS-R1A antagonist/JMV2959 could reduce the CB1R agonist/WIN55,212-2–induced dopamine efflux in the nucleus accumbens shell (NACSh), which is considered a crucial trigger impulse of the addiction process. The synthetic aminoalklylindol cannabinoid WIN55,212-2 administration into the posterior VTA induced significant accumbens dopamine release, which was significantly reduced by the 3 mg/kg i.p. JMV2959 pretreatment. Simultaneously, the cannabinoid-increased accumbens dopamine metabolic turnover was significantly augmented by the JMV2959 pretreament. The intracerebral WIN55,212-2 administration also increased the endocannabinoid arachidonoylethanolamide/anandamide and the 2-arachidonoylglycerol/2-AG extracellular levels in the NACSh, which was moderately but significantly attenuated by the JMV2959 pretreatment. Moreover, the cannabinoid-induced decrease in accumbens γ-aminobutyric acid/gamma-aminobutyric acid levels was reversed by the JMV2959 pretreatment. The behavioural study in the LABORAS cage showed that 3 mg/kg JMV2959 pretreatment also significantly reduced the systemic WIN55,212-2-induced behavioural stimulation. Our results demonstrate that the ghrelin/GHS-R1A system significantly participates in the rewarding/reinforcing effects of the cannabinoid/CB1 agonist that are involved in cannabinoid addiction processing.