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Background and Purpose Spice/K2 herbal mixtures, containing synthetic cannabinoids such as JWH-018, have been marketed as marijuana surrogates since 2004. JWH-018 has cannabinoid CB1 receptor-dependent reinforcing properties and acutely increases dopaminergic transmission selectively in the NAc shell. Here, we tested the hypothesis that repeated administration of JWH-018 (i) modulates behaviour, (ii) affects dopaminergic transmission and its responsiveness to motivational stimuli, and (iii) is associated with a neuroinflammatory phenotype. Experimental Approach Rats were administered with JWH-018 once a day for 14 consecutive days. We then performed behavioural, electrophysiological, and neurochemical evaluation at multiple time points after drug discontinuation. Key Results Repeated JWH-018 exposure (i) induced anxious and aversive behaviours, transitory attentional deficits, and withdrawal signs; (ii) decreased spontaneous activity and number of dopamine neurons in the VTA; and (iii) reduced stimulation of dopaminergic transmission in the NAc shell while potentiating that in the NAc core, in response to acute JWH-018 challenge. Moreover, (iv) we observed a decreased dopamine sensitivity in the NAc shell and core, but not in the mPFC, to a first chocolate exposure; conversely, after a second exposure, dialysate dopamine fully increased in the NAc shell and core but not in the mPFC. Finally, selected dopamine brain areas showed (v) astrogliosis (mPFC, NAc shell and core, VTA), microgliosis (NAc shell and core), and downregulation of CB1 receptors (mPFC, NAc shell and core). Conclusion and Implications Repeated exposure to JWH-018 may provide a useful model to clarify the detrimental effects of recurring use of Spice/K2 drugs. Drug Policies Department, Presidency of the Council of Ministers, Italy, project: "INSIDE-018"; Drug Policies Department, Presidency of the Council of Ministers, Italy, project: "Effects of NPS: development of a multicentre research for the information enhancement of the Early Warning System"; Drug Policies Department, Presidency of the Council of Ministers, Italy, project: RAS-FSC 2018 [RC_CRP_034, CUP RASSR03071]; Dipartimento Salute Mentale e Dipendenze (DSMD)-zona Sud-ATS Sardegna within the Convenzione sanitaria in materia di studio e ricerca tossicologica con il DiSB (UniCa) in oggetto al "PROGRAMMA REGIONALE PER L'ASSISTENZA SANITARIA DELLE PERSONE TOSSICODIPEN [121] Published version This research has been funded by the Drug Policies Department, Presidency of the Council of Ministers, Italy, projects: "INSIDE-018" (PI: Prof. De Luca, University of Cagliari) and "Effects of NPS: development of a multicentre research for the information enhancement of the Early Warning System" (PI: Prof. Marti, University of Ferrara) to M.A.D.L., and RAS-FSC 2018 (Codice intervento: RC_CRP_034; CUP RASSR03071; project: "Multidisciplinary preclinical study on NPS and evaluation of their behavioral and neurophysiological effects related to age and sex") to M.A.D.L., N.S., and L.F. Prof. De Luca would gratefully like to thank the Dipartimento Salute Mentale e Dipendenze (DSMD)-zona Sud-ATS Sardegna within the Convenzione sanitaria in materia di studio e ricerca tossicologica con il DiSB (UniCa) in oggetto al "PROGRAMMA REGIONALE PER L'ASSISTENZA SANITARIA DELLE PERSONE TOSSICODIPENDENTI NEGLI ISTITUTI PENITENZIARI DELLA SARDEGNA" (Resolution of the Special Commissioner ATS n. 121 of 21-02-2020).

Preclinical and clinical studies indicate that synthetic psychoactive substances, in addition to having abuse potential, may elicit toxic effects of varying severity at the peripheral and central levels. Nowadays, toxicity induced by synthetic psychoactive substances poses a serious harm for health, since recreational use of these substances is on the rise among young and adult people. The present review summarizes recent findings on the peripheral and central toxicity elicited by “old” and “new” synthetic psychoactive substances in humans and experimental animals, focusing on amphetamine derivatives, hallucinogen and dissociative drugs and synthetic cannabinoids.

Methoxetamine (MXE) is a dissociative substance of the arylcyclohexylamine class that has been present on the designer drug market as a ketamine-substitute since 2010. We have previously shown that MXE (i) possesses ketamine-like discriminative and positive rewarding effects in rats, (ii) affects brain processing involved in cognition and emotional responses, (iii) causes long-lasting behavioral abnormalities and neurotoxicity in rats and (iv) induces neurological, sensorimotor and cardiorespiratory alterations in mice. To shed light on the mechanisms through which MXE exerts its effects, we conducted a multidisciplinary study to evaluate the various neurotransmitter systems presumably involved in its actions on the brain. In vivo microdialysis study first showed that a single administration of MXE (0.25 and 0.5 mg/kg, i.v.) is able to significantly alter serotonin levels in the rat medial prefrontal cortex (mPFC) and nucleus accumbens. Then, we observed that blockade of the serotonin 5-HT

Substance Use Disorder (SUD) is a chronic and relapsing disease characterized by craving, loss of control, tolerance and physical dependence. At present, the combination of pharmacotherapy and psychosocial intervention is the most effective management strategy in preventing relapse to reduce dropout rates and promote abstinence in SUD patients. However, only few effective medications are available. Transcranial Magnetic Stimulation (TMS) is a non-invasive brain stimulation technique that modulates the cellular activity of the cerebral cortex through a magnetic pulse applied on selected brain areas. Recently, the efficacy of TMS has been investigated in various categories of SUD patients. The present review analyzes the application of repetitive TMS in patients with alcohol, tobacco, and cocaine use disorder. Although the number of clinical studies is still limited, repetitive TMS yields encouraging results in these patients, suggesting a possible role of TMS in the treatment of SUD.

In both animals and human beings, males and females differ in their genetic background and hormonally driven behaviour and show sex-related differences in brain activity and response to internal and external stimuli. Gender-specific medicine has been a neglected dimension of medicine for long time, and only in the last three decades it is receiving the due scientific and clinical attention. Research has recently begun to identify factors that could provide a neurobiological basis for gender-based differences in health and disease and to point to gonadal hormones as important determinants of male-female differences. Animal studies have been of great help in understanding factors contributing to sex-dependent differences and sex hormones action. Here we review and discuss evidence provided by clinical and animal studies in the last two decades showing gender (in humans) and sex (in animals) differences in selected psychiatric disorders, namely eating disorders (anorexia nervosa, bulimia nervosa, binge eating disorder), schizophrenia, mood disorders (anxiety, depression, obsessive-compulsive disorder) and neurodevelopmental disorders (autism spectrum disorders, attention-deficit/hyperactivity disorder).

The inability to maintain drug abstinence is often referred to as relapse and consists of a process by which an abstaining individual slips back into old behavioral patterns and substance use. Animal models of relapse have been developed over the last decades and significantly contributed to shed light on the neurobiological mechanisms underlying vulnerability to relapse. The most common procedure to study drug-seeking and relapse-like behavior in animals is the "extinction-reinstatement model." Originally elaborated by Pavlov and Skinner, the concepts of reinforced operant responding were applied to addiction research not before 1971 (Stretch et al., Can J Physiol Pharmacol 49:581-589, 1971), and the first report of a reinstatement animal model as it is now used worldwide was published only 10 years later (De Wit and Stewart, Psychopharmacology 75:134-143, 1981). According to the proposed model, opioids are typically self-administered intravenously, as humans do, and although rodents are most often employed in these studies, a variety of species including nonhuman primates, dogs, cats, and pigeons can be used. Several operant responses are available, depending on the species studied. For example, a lever press or a nose poke response typically is used for rodents, whereas a panel press response typically is used for nonhuman primates. In this chapter we describe a simple and easily reproducible protocol of heroin-seeking reinstatement in rats, which proved useful to study the neurobiological mechanisms underlying relapse to heroin and vulnerability factors enhancing the resumption of heroin-seeking behavior.

Opioid addiction in humans is a chronically relapsing disorder characterized by discontinuous periods of drug use and abstinence resulting in dependence. With time, the probability of falling into renewed drug consumption becomes particularly high and constitutes a considerable problem in the management of opioid addicts. Opioid addiction represents an important health concern and animal models have been crucial in understanding the neurobiology and pathophysiology of this complex disease. Although animal models of addiction do not fully reproduce the human condition, they do permit investigation of specific elements of the process as well as identification of potential therapeutic targets. In this chapter, we provide a step-by-step description of the morphine-conditioned place preference (CPP) model that represents a useful preclinical animal model extensively used to study the rewarding/aversive effect of drugs.

Opioid addiction in humans is a chronically relapsing disorder characterized by discontinuous periods of drug use and abstinence resulting in dependence. With time, the probability of falling into renewed drug consumption becomes particularly high and constitutes a considerable problem in the management of opioid addicts. Opioid addiction represents an important health concern and animal models have been crucial in understanding the neurobiology and pathophysiology of this complex disease. Although animal models of addiction do not fully reproduce the human condition, they do permit investigation of specific elements of the process as well as identification of potential therapeutic targets. In this chapter, we provide a step-by-step description of the morphine-conditioned place preference (CPP) model that represents a useful preclinical animal model extensively used to study the rewarding/aversive effect of drugs.

The inability to maintain drug abstinence is often referred to as relapse and consists of a process by which an abstaining individual slips back into old behavioral patterns and substance use. Animal models of relapse have been developed over the last decades and significantly contributed to shed light on the neurobiological mechanisms underlying vulnerability to relapse. The most common procedure to study drug-seeking and relapse-like behavior in animals is the "extinction-reinstatement model." Originally elaborated by Pavlov and Skinner, the concepts of reinforced operant responding were applied to addiction research not before 1971 (Stretch et al., Can J Physiol Pharmacol 49:581-589, 1971), and the first report of a reinstatement animal model as it is now used worldwide was published only 10 years later (De Wit and Stewart, Psychopharmacology 75:134-143, 1981). According to the proposed model, opioids are typically self-administered intravenously, as humans do, and although rodents are most often employed in these studies, a variety of species including nonhuman primates, dogs, cats, and pigeons can be used. Several operant responses are available, depending on the species studied. For example, a lever press or a nose poke response typically is used for rodents, whereas a panel press response typically is used for nonhuman primates. In this chapter we describe a simple and easily reproducible protocol of heroin-seeking reinstatement in rats, which proved useful to study the neurobiological mechanisms underlying relapse to heroin and vulnerability factors enhancing the resumption of heroin-seeking behavior.

An altered dopamine transmission has been described for different types of addiction for a long time. Preclinical and clinical evidence support the hypodopaminergic hypothesis and underpin the need to increase dopamine transmission to obtain therapeutic benefit. Repetitive transcranial magnetic stimulation (rTMS) of prefrontal cortex shows efficacy in treating some forms of addiction. Recent imaging studies confirmed that the therapeutic effect of rTMS is correlated with an enhancement of dopamine transmission. Novel targets for rTMS are under evaluation to increase its effectiveness in treating addiction, and research is ongoing to find the optimal protocol to boost dopaminergic transmission in the addicted brain. TMS can thus be considered a useful tool to test the dopamine hypothesis of drug addiction and instrumental in the search for addiction therapeutics.

Synthetic cannabinoids have long been studied for their therapeutic potentials. However, during the last decade, new generations of synthetic cannabinoid agonists appeared on the drug market. These new psychoactive substances are currently sold as 'marijuana-like' products as they claim to mimic the effects of the psychoactive component of cannabis, delta-9-tetrahydrocannabinol (THC). Yet, their effects are more intense and potent than THC, typically last longer and are often associated to serious psychiatric consequences. Animal models of drug addiction are frequently used in preclinical research to assess the abuse potential of new compounds, evaluate drug positive reinforcing effects and analyze drug-induced behaviors. Some of these protocols have been used recently to study the newly synthesized cannabinoid agonists and have started elucidating their pharmacology and actions in the brain. The aim of this review is to summarize the major findings reported by animal studies that tested synthetic cannabinoids of first, second, and third generation by using self-administration and reinstatement models, drug discrimination and conditioned place preference procedures. Altogether, behavioral studies clearly indicate that synthetic cannabinoids possess abuse liability, are likely to activate the brain reward circuit and induce positive subjective and reinforcing effects.

Methoxetamine (MXE) is a dissociative substance of the arylcyclohexylamine class that has been present on the designer drug market as a ketamine-substitute since 2010. We have previously shown that MXE (i) possesses ketamine-like discriminative and positive rewarding effects in rats, (ii) affects brain processing involved in cognition and emotional responses, (iii) causes long-lasting behavioral abnormalities and neurotoxicity in rats and (iv) induces neurological, sensorimotor and cardiorespiratory alterations in mice. To shed light on the mechanisms through which MXE exerts its effects, we conducted a multidisciplinary study to evaluate the various neurotransmitter systems presumably involved in its actions on the brain. In vivo microdialysis study first showed that a single administration of MXE (0.25 and 0.5 mg/kg, i.v.) is able to significantly alter serotonin levels in the rat medial prefrontal cortex (mPFC) and nucleus accumbens. Then, we observed that blockade of the serotonin 5-HT2 receptors through two selective antagonists, ketanserin (0.1 mg/kg, i.p.) and MDL 100907 (0.03 mg/kg, i.p.), at doses not affecting animals behavior per se, attenuated the facilitatory motor effect and the inhibition on visual sensory responses induced by MXE (3 mg/kg, i.p.) and ketamine (3 mg/kg, i.p.), and prevented MXE-induced reduction of the prepulse inhibition in rats, pointing to the 5-HT2 receptors as a key target for the recently described MXE-induced sensorimotor effects. Finally, in-vitro electrophysiological studies revealed that the GABAergic and glutamatergic systems are also likely involved in the mechanisms through which MXE exerts its central effects since MXE inhibits, in a concentration-dependent manner, NMDA-mediated field postsynaptic potentials and GABA-mediated spontaneous currents. Conversely, MXE failed to alter both the AMPA component of field potentials and presynaptic glutamate release, and seems not to interfere with the endocannabinoid-mediated effects on mPFC GABAergic synapses. Altogether, our results support the notion of MXE as a NMDA receptor antagonist and shed further lights into the central mechanisms of action of this ketamine-substitute by pointing to serotonin 5-HT2 receptors as crucial players in the expression of its sensorimotor altering effects and to the NMDA and GABA receptors as potential further important targets of action.

BACKGROUND AND PURPOSE Methoxetamine (MXE) is a novel psychoactive substance that is emerging on the Internet and induces dissociative effects and acute toxicity. Yet, its pharmacological effects are still poorly investigated. EXPERIMENTAL APPROACH We examined a range of behavioural effects induced by acute intraperitoneal administration of MXE (0.5-5 mg/kg) in rats, and whether it causes rapid neuroadaptive molecular changes. KEY RESULTS MXE (0.5-5 mg/kg) significantly affected motor activity in a dose- and time-dependent manner, inducing hypermotility and hypomotility at low and high doses, respectively. At low/intermediate doses (0.5 and 1 mg/kg) MXE induced anxious and/or obsessive-compulsive traits (marble burying test), slightly (but not significantly) increased sociability (social interaction test) but did not induce spatial anxiety (elevated plus maze test). At a high dose (5 mg/kg), MXE induced transient analgesia (tail flick and hot plate test), significantly decreased social interaction time (social interaction test) and significantly reduced immobility time while increasing swimming activity (forced swim test), suggesting an antidepressant effect. Acute MXE administration did not affect self-grooming behaviour at any dose tested. Immunohistochemistry analysis showed that behaviourally active doses of MXE (1 and 5 mg/kg) increased the phosphorylation of ribosomal protein S6 (rpS6) in the medial prefrontal cortex and hippocampus. CONCLUSIONS AND IMPLICATIONS Findings indicate that MXE differentially affects motor activity, behaviour and emotional states in rats depending on the dose tested. As recently reported for ketamine, rpS6 phosphorylation was increased in MXE-treated animals thus providing a ‘molecular snapshot’ of rapid neuroadaptive molecular changes induced by behaviourally active doses of MXE.

Dopamine agonists have been proposed as therapeutic tools for cocaine addiction. We have recently demonstrated that indirect dopamine agonists, including levodopa (L-DOPA), markedly increase cocaine-induced dopamine release in the medial prefrontal cortex (mPFC) of rats leading to the suppression of cocaine-seeking behavior. This study was aimed to understand the behavioral and neurochemical effects of L-DOPA on cocaine-taking and cocaine-seeking in rats. After reaching a stable pattern of intravenous cocaine self-administration under a continuous fixed ratio (FR-1) schedule of reinforcement, male rats were treated with L-DOPA at different steps of the self-administration protocol. We found that L-DOPA reduced cocaine self-administration under FR-1 schedule of reinforcement and decreased the breaking points and the amount of cocaine self-administered under the progressive ratio schedule of reinforcement. Levodopa also decreased cocaine-seeking behavior both in a saline substitution test and in the cue priming-induced reinstatement test, without affecting general motor activity. Importantly, L-DOPA greatly potentiated cocaine-induced dopamine release in the mPFC of self-administering rats while reducing their cocaine intake. In the same brain area, L-DOPA also increased dopamine levels during cue priming-induced reinstatement of cocaine-seeking behavior. The potentiating effect was also evident in the mPFC but not nucleus accumbens core of drug-naive rats passively administered with cocaine. Altogether, these findings demonstrate that L-DOPA efficaciously reduces the reinforcing and motivational effects of cocaine likely potentiating dopamine transmission in the mPFC. Its ability to prevent cue priming-induced reinstatement of cocaine-seeking suggests that it might be effective in reducing the risk to relapse to cocaine in abstinent patients.

Men and women affected by schizophrenia display different age of onset, symptom profile and course of the disease. Similarly, men and women differ in the prevalence and frequency of drug use, pattern and reasons of use, and vulnerability to develop drug addiction. An understanding of the role of sex in modulating brain processes and behavior in patients with substance use disorder and/or schizophrenia-like symptoms has broad implications for gender-tailored treatment approaches. Cognizant of the considerable recent evidence for sex and gender differences in drug addiction and schizophrenia, we focused this review on the sex-dependent differences in drug-induced psychosis and on factors that may contribute to such male–female differences.

Transcranial Magnetic Stimulation (TMS) is earning a role in the therapeutic arsenal of cocaine use disorder. A widespread and still growing number of studies have reported beneficial use of repeated TMS (rTMS) in reduction of craving, intake and cue-induced craving in cocaine addicts. In spite of these encouraging findings, many issues are still unresolved such as brain area to be stimulated, laterality of the effects, coil geometry and stimulation protocols/parameters. Intermittent theta burst stimulation (iTBS) is a more tolerable protocol administered at lower intensities and shorter intervals than conventional repeated TMS (rTMS) protocols. Yet, its effects on cocaine craving and length of abstinence in comparison with standard high frequency (10-15Hz) protocols have never been evaluated so far. In the present paper we describe the effect of the bilateral iTBS of the prefrontal cortex (PFC) in a population (n=25) of treatment-seeking cocaine addicts, in an outpatient setting, and compare them with 15 Hz stimulation of the same brain area (n=22). The results indicate that iTBS produces effects on cocaine consumption and cocaine craving virtually superimposable to the 15 Hz rTMS group. Both treatments had low numbers of dropouts and similar side-effects, safety and tolerability profiles. While larger studies are warranted to confirm these observations, iTBS appears to be a valid approach to be considered in treatment-seeking cocaine addicts, especially in light of its brief duration (3 mins) vs 15 Hz stimulation (15 mins). The use of iTBS would allow increasing the number of patients treated per day with current rTMS devices, thus reducing patient discomfort and hopefully reducing drop-out rates without compromising clinical effectiveness.

JWH-018-Cl, JWH-018-Br and AM-2201 (JWH-018 halogenated-derivatives; JWH-018-R compounds) are synthetic cannabinoid agonists illegally marketed as “Spice”, “K2”, “herbal blend” and research chemicals for their cannabis-like psychoactive effects. In rodents, JWH-018 and its halogenated derivatives reproduce the typical effects of Δ9-tetrahydrocannabinol (Δ9-THC), i.e. hypothermia, analgesia, hypolocomotion and akinesia. Yet, the effects of JWH-018-R compounds on sensorimotor functions are still unknown. This study was designed to investigate the effect of an acute intraperitoneal (i.p.) administration of JWH-018-R compounds (0.01–6 mg/kg) on sensorimotor functions in mice and to compare them to those caused by the reference compound JWH-018 and Δ9-THC. A well validated battery of behavioral tests was used to investigate the effects of these synthetic cannabinoids on the visual, auditory and tactile responses in mice, while the pre-pulse inhibition (PPI) test was used to investigate their effect on sensorimotor gating. The effect of the synthetic cannabinoids on spontaneous locomotion was also measured by a video tracking analysis to assess potential cannabinoid-induced motor impairment. Results showed that, similarly to JWH-018, systemic administration of JWH-018-R compounds inhibits sensorimotor and PPI responses at lower doses (0.01-0.1 mg/kg) and reduced spontaneous locomotion at intermediate/high doses (1–6 mg/kg). All effects were prevented by the administration of the selective cannabinoid CB1 receptor antagonist/inverse agonist AM-251 thus confirming a CB1 receptor-mediated action. Finding that lower doses of JWH-018-R compounds selectively impair sensorimotor and PPI responses without affecting locomotion should be carefully considered to better understand the potential danger that halogenated-derivatives of JWH-018 may pose to public health, with particular reference to decreased performance in driving and hazardous works.

Methoxetamine (MXE) is a novel psychoactive substance that can induce several short-term effects on emotional states and behavior. However, little is known about the persistent emotional and behavioral effects of MXE. Moreover, neurotoxic effects of MXE have been hypothesized, but never demonstrated in vivo. To clarify these issues, rats received repeated treatment with MXE every other day (0.1–0.5 mg/kg, i.p., × 5), and 7 days later they were challenged with MXE (0.1–0.5 mg/kg, i.p.). Behavioral effects of MXE were first evaluated by measuring emission of ultrasonic vocalizations and locomotor activity after each administration. Thereafter, persistent behavioral effects of MXE were evaluated, starting 8 days after challenge, through elevated plus maze, spontaneous alternation, novel object recognition, and marble burying tests. After completion of behavioral analysis, neurotoxic effects of MXE were evaluated by measuring densities of dopamine transporter, tyrosine hydroxylase, and serotonin transporter in various brain regions. Repeated treatment and challenge with MXE affected neither calling behavior nor locomotor activity of rats. Conversely, rats previously treated with MXE exhibited behavioral alterations in the elevated plus maze, marble burying and novel object recognition tests, suggestive of increased anxiety and impaired non-spatial memory. Noteworthy, the same rats displayed dopaminergic damage in the medial prefrontal cortex, nucleus accumbens, caudate-putamen, substantia nigra pars compacta, and ventral tegmental area, along with accumbal serotonergic damage. Our findings show for the first time that repeated administration of MXE induces persistent behavioral abnormalities and neurotoxicity in rats, which can help elucidating the risks associated with human MXE consumption.