Neuropharmacology of New Psychoactive Substances (NPS): Focus on the Rewarding and Reinforcing Properties of Cannabimimetics and Amphetamine-Like Stimulants

New psychoactive substances (NPS) are a heterogeneous and rapidly evolving class of molecules available on the global illicit drug market (e.g smart shops, internet, “dark net”) as a substitute for controlled substances. The use of NPS, mainly consumed along with other drugs of abuse and/or alcohol, has resulted in a significantly growing number of mortality and emergency admissions for overdoses, as reported by several poison centers from all over the world. The fact that the number of NPS have more than doubled over the last 10 years, is a critical challenge to governments, the scientific community, and civil society [EMCDDA (European Drug Report), 2014; UNODC, 2014b; Trends and developments]. The chemical structure (phenethylamines, piperazines, cathinones, tryptamines, synthetic cannabinoids) of NPS and their pharmacological and clinical effects (hallucinogenic, anesthetic, dissociative, depressant) help classify them into different categories. In the recent past, 50% of newly identified NPS have been classified as synthetic cannabinoids followed by new phenethylamines (17%) (UNODC, 2014b). Besides peripheral toxicological effects, many NPS seem to have addictive properties. Behavioral, neurochemical, and electrophysiological evidence can help in detecting them. This manuscript will review existing literature about the addictive and rewarding properties of the most popular NPS classes: cannabimimetics (JWH, HU, CP series) and amphetamine-like stimulants (amphetamine, methamphetamine, methcathinone, and MDMA analogs). Moreover, the review will include recent data from our lab which links JWH-018, a CB1 and CB2 agonist more potent than Δ9-THC, to other cannabinoids with known abuse potential, and to other classes of abused drugs that increase dopamine signaling in the Nucleus Accumbens (NAc) shell. Thus the neurochemical mechanisms that produce the rewarding properties of JWH-018, which most likely contributes to the greater incidence of dependence associated with “Spice” use, will be described (De Luca et al., 2015a). Considering the growing evidence of a widespread use of NPS, this review will be useful to understand the new trends in the field of drug reward and drug addiction by revealing the rewarding properties of NPS, and will be helpful to gather reliable data regarding the abuse potential of these compounds.

New psychoactive substances (NPS) are a heterogeneous and rapidly evolving class of molecules available on the global illicit drug market (e.g smart shops, internet, "dark net") as a substitute for controlled substances. The use of NPS, mainly consumed along with other drugs of abuse and/or alcohol, has resulted in a significantly growing number of mortality and emergency admissions for overdoses, as reported by several poison centers from all over the world. The fact that the number of NPS have more than doubled over the last 10 years, is a critical challenge to governments, the scientific community, and civil society [EMCDDA (European Drug Report), 2014; UNODC, 2014b; Trends and developments]. The chemical structure (phenethylamines, piperazines, cathinones, tryptamines, synthetic cannabinoids) of NPS and their pharmacological and clinical effects (hallucinogenic, anesthetic, dissociative, depressant) help classify them into different categories. In the recent past, 50% of newly identified NPS have been classified as synthetic cannabinoids followed by new phenethylamines (17%) (UNODC, 2014b). Besides peripheral toxicological effects, many NPS seem to have addictive properties. Behavioral, neurochemical, and electrophysiological evidence can help in detecting them. This manuscript will review existing literature about the addictive and rewarding properties of the most popular NPS classes: cannabimimetics (JWH, HU, CP series) and amphetamine-like stimulants (amphetamine, methamphetamine, methcathinone, and MDMA analogs). Moreover, the review will include recent data from our lab which links JWH-018, a CB1 and CB2 agonist more potent than 9 -THC, to other cannabinoids with known abuse potential, and to other classes of abused drugs that increase dopamine signaling in the Nucleus Accumbens (NAc) shell. Thus the neurochemical mechanisms that produce the rewarding properties of JWH-018, which most likely contributes to the greater incidence of dependence associated with "Spice"

INTRODUCTION
Over the last decade, New Psychoactive Substances (NPS) have become a global phenomenon. The emergence of these substances have been reported in almost 100 countries and territories, and more than 500 NPS have been identified worldwide based on reports by national governments, as well as the EU, and international institutions (UNODC, 2014a, 2015) (Figure 1). In 2014, in Europe alone, 101 NPS have been detected showing an increase of 25%, as compared to 2013 [EMCDDA (New psychoactive substances in Europe), 2015b]. NPS are able to mimic the effects of controlled substances and are mainly synthetic cannabinoids, stimulants, hallucinogens, and opioids.
Previous studies show that the use of NPS occurs among different subject groups: school students, partygoers, psychonauts, prisoners, and injecting drug users. Motivations for use include factors such as legal status, availability, and cost, as well as the desire to avoid detection and user preferences for particular pharmacological properties [González et al., 2013;Helander et al., 2013Helander et al., , 2014; EMCDDA (European Drug Report), 2015a; EMCDDA (New psychoactive substances in Europe), 2015b]. Although global research is not available yet about NPS, prevalence of use among the population, single national surveys (with respect to substances and subpopulations) show that the use of NPS amongst the general adult population is relatively low compared with the use of other illicit drugs. However, adolescents use more NPS than illicit drugs mostly because many of them are legal and easily available on the web [Drug Policy Department Italian Presidency of the Council of Ministers, 2014;Fraser, 2014;Kikura-Hanajiri et al., 2014;EMCDDA (New FIGURE 1 | Number of NPS reported worldwide (2009. Adapted from UNODC (2014a).
Similar to many known illicit drugs, NPS can cause severe physical and psychological symptoms that can even result in death [Drug Policy Department Italian Presidency of the Council of Ministers, 2013b;Fraser, 2014;EMCDDA (European Drug Report), 2015a;UNODC, 2015]. A recent analysis by the European Drug Emergencies Network, monitoring emergency admissions in the last 5 years in 10 EU countries, found that 9% of all drug-related emergencies involved NPS, primarily synthetic cannabinoids and cathinones (Wood et al., 2014). Consequently, there is increasing evidence that NPS play a great role in hospital emergencies and some drug-induced deaths [EMCDDA (European Drug Report), 2015a]. However, the analytical detection of NPS for emergency services is not technically available so far; their recognition by means of second level analysis requires standards solution, methodologies and analytical equipment not accessible to every laboratory yet [Drug Policy Department Italian Presidency of the Council of Ministers, 2013b; UNODC, 2014a; EMCDDA (European Drug Report), 2015a]. Therefore, their identification in biological samples, as well as in seized or collected samples, represents one major difficulty.
Notably, the internet is an important marketplace for the sale of NPS. Evidence is emerging of so-called "gray marketplaces"online sites selling NPS which operate on both the surface as well as the deep web (Deluca et al., 2012; Drug Policy Department Italian Presidency of the Council of Ministers, 2013b;Burns et al., 2014;Corazza et al., 2014). Therefore, NPS can be sold via the internet to everyone, including young, underage teenagers, with complete anonymity and an easy avoidance of law enforcement and health controls [Drug Policy Department Italian Presidency of the Council of Ministers, 2013b; UNODC, 2014a; EMCDDA (European Drug Report), 2015a]. The growth of online and virtual drug markets strongly contributes to the uncontrolled widespread use of these substances, increasing health risks for consumers, and challenging drug control policies.
The largest increase in terms of newly reported NPS involves synthetic cannabinoids, synthetic cathinones, and phenethylamines [EMCDDA (European Drug Report), 2015a; EMCDDA (New psychoactive substances in Europe), 2015b]. The first synthetic cannabinoids were identified in 2008 in preparations called "herbal mixtures" or "herbal blends" (i.e., Spice) and sold as incense or air fresheners. Their effects are similar, if not superior, to those caused by cannabis consumption (Hohmann et al., 2014;Khullar et al., 2014;Mills et al., 2015). Recently, a survey of the use of synthetic cannabinoids among US students showed that Spice products were the second most used drug after marijuana, with a prevalence of 7.4-7.9% in those aged between 15 and 18 years (Johnston et al., 2013). Adolescence, a critical developmental period commonly associated with an increase in drug abuse in the human population, may be a stage of particular vulnerability to the effects of the new psychoactive drugs (Johnston et al., 2013). In fact, most of the brain receptor systems have been shown to mature slowly, reaching maximal levels around age 20. Indeed, the use of these drugs might influence neurodevelopment inducing psychiatric disorders or other mental deficits (Paus, 2005;Sussman et al., 2008). Several NPS such as mephedrone, pentedrone and MDPV which mimic the effects of amphetamine-like stimulants (ATS), can be classified as synthetic cathinones with stimulant and empathogenic properties, or as phenethylamines which can induce stimulant and hallucinogenic effects (UNODC, 2014a(UNODC, , 2015. Similar to other NPS, synthetic cannabinoids and ATS are largely available online and are often sold as research chemical components. They are mainly produced in Eastern Europe, Central Asia and China, and then shipped and sold to Europe and the USA (UNODC, 2015 The forensic identification of NPS is very difficult. These may concern the lack of knowledge on NPS available to the professionals performing analytical analysis. In addition, analytical methodologies are still not sufficient to detect the presence of all of the NPS in the analyzed samples and many laboratories lack appropriate analytical equipment for their recognition (Drug Policy Department Italian Presidency of the Council of Ministers, 2013a). These are all important aspects to take into account when considering the legal, health, and social consequences related to NPS.
To date, several behavioral, neurochemical, and electrophysiological studies have helped us to understand the pharmacological mechanisms of action of NPS. However, many of them have been focused on the acute toxicological consequences of NPS use. As they are relatively new and novel, there are no epidemiological studies to show the long-term effects of these psychoactive compounds. Also, there is not a lot of evidence on the addictive properties of NPS. This work has been divided into two main parts based on pharmacological classification of the most popular and public health-concerning NPS classes: amphetamine-like stimulants and cannabimimetic drugs. Moreover, specific references to recent papers by the authors have been presented. A thorough analysis of the rewarding and reinforcing properties of NPS and their abuse liability will hopefully, provide to be useful for understanding the new disturbing trends in the field of drug addiction and provide strategies to tackle this growing problem.
Phenethylamines, piperazines, tryptamines, and synthetic catinones exhibit stimulant and hallucinogenic effects, making up the distinct class of entactogens, which are described as psychoactive substances that enhance feelings of empathy, love, and emotional closeness to others (Schifano et al., 2007). Entactogens can be chemically divided into phenethylamines, amphetamines, synthetic cathinones, piperazines, pipradrols/piperidines, aminoindanes, benzofurans, and tryptamines (see Table 2). Stimulant drugs usually inhibit monoamine reuptake, increasing the quantity of noradrenaline, dopamine and serotonin in the synaptic cleft leading to sympathomimetic effects (Schifano, 2013). Phenethylamines are synthetic compounds commercially known as "party pills" (e.g., tablets of different colors/shapes, capsules, powder/crystal). They act on serotoninergic receptors leading to psychedelic effects and some of them inhibit the monoamine reuptake as well (Nelson et al., 2014); 3,4-methylenedioxy-methamphetamine (MDMA), widely known as "ecstasy, " is one of the most popular drugs among young people because of its stimulant effects. But, recently a growing use of new dangerous molecules on the recreational drug scene, such as 2C and its derivatives (e.g., "N-Bomb, " "B-Fly, " and "Dr. Death"), 2-D series drugs, 3C-bromo-Dragonfly, 4-MTA, 6-APB, 4,4 ′ -DMAR and MPA, that are novel derivatives of classic psychedelic phenethylamines/MDMAlike drugs (Nelson et al., 2014) has been reported; several cases of intoxications have been reported with symptoms such as hypertension, vomiting, hyperthermia, convulsions, dissociation, hallucinations, respiratory deficits, liver, and kidney failure and death in case of overdose (Winstock and Schifano, 2009;Schifano et al., 2010;Corazza et al., 2011;Dean et al., 2013;Bersani et al., 2014;Le Roux et al., 2015;Maas et al., 2015). The lead compound in piperazines, N-Benzylpiperazin (BZP), has a typical central nervous system stimulant structure so it triggers the release of dopamine and norepinephrine and inhibits the uptake of dopamine, norepinephrine and serotonin (Smith et al., 2015). Although BZP is structurally similar to amphetamine, it is reported to have only one-tenth

HUMAN AND ANIMAL STUDIES ON AMPHETAMINE-LIKE STIMULANTS EFFECTS: PSYCHOACTIVE EFFECTS, COGNITIVE DEFICITS, EMOTIONAL ALTERATIONS, AND DEPENDENCE
In the second part of the 90s, a global trend of escalating amphetamine-like stimulant use was observed and synthetic tryptamines appeared on illicit drug markets. Instead of replacing or displacing MDMA and cocaine, mephedrone, and other NPS from this group appear to have been added to the established repertoire of psychostimulant narcotics (Sanders et al., 2008;Zawilska, 2015).
In animal models of addiction, cathinones have displayed potential rewarding and reinforcing effects. For example, mephedrone produces conditioned place preference (CPP), facilitates intracranial self-stimulation and is self-administered in rats (Hadlock et al., 2011;Lisek et al., 2012;Motbey et al., 2013;Bonano et al., 2014;Gregg et al., 2015). Prior studies demonstrated that MDPV and methylone, another synthetic cathinone, increase locomotor activity in rodents (López-Arnau et al., 2012;Marusich et al., 2012;Aarde et al., 2013;Gatch et al., 2013) and also enhance intracranial self-stimulation (Watterson et al., 2012(Watterson et al., , 2014Bonano et al., 2014) and engender conditioned place preference , effects that are indicators of high abuse potential (Schindler et al., 2015). This evidence suggests that each compound could produce behavioral effects consistent with psychostimulant drugs displaying high abuse liability, possibly higher than amphetamine. In fact, in rats trained to self-administer MDPV or methamphetamine, dose-substitution studies demonstrated that behavior was dose-sensitive for both drugs, but MDPV showed greater potency and efficacy than methamphetamine (Paillet-Loilier et al., 2014). Moreover, in mice models, mephedrone, methylone, and MDPV produce CPP equal or higher than amphetamine, strongly suggesting their addictive properties . In addition, MDMA, methylone, and mephedrone are self-administered in female rats with a higher intake in mephedrone-trained rats compared to methylonetrained animals. This seems to suggest that mephedrone might have greater reinforcing effects compared to methylone or MDMA , despite their shared mechanism of action. They are non-selective transporter substrates that increase the release of dopamine, norepinephrine and 5-HT in vitro (Baumann et al., 2012;Eshleman et al., 2013;Simmler et al., 2013). Importantly, the 5-HT-releasing ability of these drugs is more prevalent than their effects on dopamine in vivo (Baumann et al., 2008(Baumann et al., , 2012Kehr et al., 2011;Wright et al., 2012) suggesting empathogen-like effects. Such findings indicate that self-administration of MDMA-like drugs is influenced by 5-HT release, but also drug pharmacokinetics, effects on noradrenergic systems, or non-transporter sites of action (Schindler et al., 2015).
In humans, synthetic cathinones produce psychotropic effects similar to MDMA and cocaine (Simmler et al., 2013). The typical dose range varies according to the different cathinone derivatives. However, according to information released from users in drug forums, where people discuss their experiences with recreational drugs (e.g., "Drugs-Forum, " "Urban 75, " "Erowid"), they usually start with a small dose and gradually increase it. This is in line with scientific reports which show that an excessive increase of noradrenergic signals could promote the onset of adverse effects and that the potency of a substance to activate the noradrenergic system is inversely correlated to the doses typically used recreationally (Simmler et al., 2013). All cathinones exhibit higher dopaminergic activity when compared with their non βketo amphetamines analogs. Recent studies of the mechanisms by which b-ketoamphetamines interact with DAT, offer significant insight into why these drugs have such divergent effects on neurotoxicity. "Bath salts" have been classified as substrates and non-substrates based on whether or not they are transported by the DAT (Anneken et al., 2015). This increased dopaminergic property of the cathinones suggests higher stimulant-type effects and a greater risk for dependence (Aarde et al., 2013). Stimulant and entactogenic properties are typical of cathinones. In fact, desired or pleasant effects most often described by users include euphoria, intensification of sensory senses, increased sociability, increased energy, mental stimulation, empathy connection, openness, increased sensory perception, decreased inhibition, and sexual arousal; but side effects such as cognitive confusion, cognitive impairment, psychiatric irritability, aggression that sometimes progresses to violent or even criminal behavior, and self-destructive behavior have also been reported [IACP (International Association of Chiefs of Police), 2012]. MDPV and mephedrone have been directly implicated in a number of fatalities in medical literature. In one case involving MDPV, the cause of death was consistent with excited delirium syndrome, a condition associated with stimulant drug overdose and attributable to excessive dopaminergic transmission (Baumann et al., 2012).
However, amphetamine remains the prototype of psychostimulants causing agitation, insomnia, loss of appetite and, at higher doses, "amphetamines psychosis" characterized by paranoia, hallucinations and delusion (Iversen et al., 2014). In experimental animals, low doses of amphetamine cause hyperactivity and higher doses lead to stereotyped Frontiers in Neuroscience | www.frontiersin.org repetitive behaviors (Whelpton, 2007). The neurotoxic effects of amphetamines has been vastly studied and their ability to damage brain monoaminergic cells was shown by long-term deficits in dopaminergic and serotoninergic systems in several brain areas of animals (Teixeira-Gomes et al., 2014). One of the major neurotoxic actions of amphetamines observed in laboratory animals is the sustained depletion of monoamine brain levels. In addition to the damage to dopaminergic and serotoninergic neuronal systems, amphetamines can also induce neuronal death. For example, in several studies, MDMA administration in mice and rats produced neuronal death in several brain areas including the cortex, hippocampus, amygdala, ventromedial/ventrolateral thalamus, and teniatecta (Teixeira-Gomes et al., 2014). MDMA is still one of the most widely used recreational drugs and many NPS were designed to mimic its effects or as substitutes for MDMA in ecstasy pills.
The potency of abused psychostimulants to activate the brain reward circuitry increases the risk of potential for abuse and addiction in humans ( Table 3). In contrast, a relative activation of the serotonin system would be linked to a reduction in abuse potential. Thus, the DAT/SERT inhibition ratio and dopamine/serotonin release potency has been proposed to predict the effects of psychostimulants in humans (Paillet-Loilier et al., 2014). Data currently available has shown that the frequent consumption of high doses of synthetic cathinones induce tolerance, dependence, craving, and withdrawal syndrome after sudden suspension [NDIC (National Drug Intelligence Center), 2011]. Indeed, Kehr et al. (2011) showed that mephedrone induces a stimulation of the dopamine transmission in the nucleus accumbens, that might be a starting point for developing drug-addiction (Volkow et al., 2003;Di Chiara et al., 2004). Although the typical dose range of MDPV appears to be between 5 and 30 mg in a single administration, some users reported tolerance with the consumption of a single dose, higher than 200 mg (Coppola and Mondola, 2012). Several users have reported a withdrawal syndrome after abrupt cessation of long-term use of methcathinone, mephedrone and MDPV (Winstock et al., 2011). Moreover, Gatch et al. (2013) showed that all of the cathinone derivatives fully substituted for methamphetamine or cocaine in drug discrimination tests. Results suggest that these drugs are comparable to cocaine and methamphetamine, and are likely to induce dependence (Iversen et al., 2014).

Spice and CB1 "Super Agonists"
Synthetic Cannabimimetic agents (SC), also known as Cannabimimetics, are substances with pharmacological properties similar to delta-9-tetrahydrocannabinol ( 9 -THC) assessed by in vitro and in vivo animal studies such as binding studies and functional assays (Compton et al., 1992;EMCDDA, 2009b). SC have been detected in "Spice, " "K2, " and spice-like samples all over the world. Spice is a smokable herbal mixture marketed as a safe, legal alternative to Cannabis, composed by shredded plant material laced with a variety of SC compounds [NIDA (National Institute on Drug Abuse), 2012]. These compounds are "smokable" since they are small (typically 20-26 carbon atoms) and highly lipophilic molecules. A few hundred of SC of the JWH, HU, and CP series are currently available. They retain very high cannabinoid receptor binding affinity levels, with a dose-response efficacy significantly higher than 9 -THC itself (Brents et al., 2011;Fattore and Fratta, 2011;Schifano et al., 2015). New legal regulations have been enacted to control the global diffusion of Spice. As a consequence of that, three subsequent Several studies show that SC are remarkably different from and more dangerous than THC. Indeed, while THC is a partial CB1 agonist, in vitro studies have clearly shown that these compounds are full agonists with higher potency and efficacy as compared to 9 -THC (Atwood et al., 2010(Atwood et al., , 2011Marshell et al., 2014). More recent studies have been shown that selected third generation compounds, such as 5F-PB-22 and BB-22, retain greater CB1 receptor agonist potency (five-and seven-fold, respectively) and efficacy and a higher binding affinity (26-and 30-fold, respectively) at CB1 receptors compared to JWH-018 (De Luca et al., 2015b).
Moreover, studies performed in rats and mice showed that many SC displayed locomotor depressant effects and a characteristic tetrad profile at lower doses compared to 9 -THC (Chaperon and Thiébot, 1999;Wiley et al., 2012Wiley et al., , 2014Forster, 2014, 2015;Vigolo et al., 2015). In addition, JWH-018 and its congeners are readily metabolized to a series of cannabimimetics (Seely et al., 2012). That, together with the presence of several different SC in Spice/K2 products and their unpredictable dosing when consumed , might explain their acute severe toxicity and even lethal medical complications in humans (Brents et al., 2011;Papanti et al., 2013;Brents and Prather, 2014;Brewer and Collins, 2014;Santacroce et al., 2015), leading to severe withdrawal syndrome and dependence as well in some cases (Zimmermann et al., 2009;Gunderson et al., 2012;Macfarlane and Christie, 2015). In addition, clinical evidence indicates that JWH-018 can generate/cause psychosis in vulnerable individuals (Every-Palmer, 2011). Notably, SC misuse has been associated with anxiety, agitation/panic attacks, paranoid ideation, suicidal ideation, and hallucinations (Fattore and Fratta, 2011;Wells and Ott, 2011;Thomas et al., 2012;Besli et al., 2015), and also been related to mood, cognitive (i.e.,

Substance Dosage Regimen Studies References
Desoxypipradrol Rat brain slices from the nucleus accumbens core were exposed to desoxypipradrol (1, 3, or 10 µM) for 60 min Dopamine efflux was electrically evoked and recorded using fast cyclic voltammetry. Desoxypipradrol increased the peak dopamine efflux and also slowed dopamine re-uptake. Desoxypipradrol was more potent than cocaine causing a seven fold increase in peak dopamine levels and increasing dopamine re-uptake half-life 15-fold Davidson and Ramsey, 2011 5-APB Voltammetric studies in rat accumbens brain slices revealed that 5-APB slowed dopamine reuptake, and at high concentrations caused reverse transport of dopamine Dawson et al., 2014 Pentedrone Pentedrone at 3 and 10 mg/kg significantly increased conditioned place preference in mice, while pentedrone at 0.3 mg/kg/infusion significantly increased self-administration in rats Pentedrone produces CPP in mice and self-administration in rats. These results demonstrate the abuse liability of pentedrone in both models Results revealed that, repeated mephedrone injections cause a rapid decrease in striatal dopamine (DA) and hippocampal serotonin (5-hydroxytryptamine; 5HT) transporter function. Mephedrone also inhibited both synaptosomal DA and 5HT uptake. Like methylenedioxymethamphetamine, but unlike methamphetamine or methcathinone, repeated mephedrone administrations also caused persistent serotonergic, but not dopaminergic, deficits. However, mephedrone caused DA release from a striatal suspension approaching that of methamphetamine Hadlock et al., 2011 (Continued) Frontiers in Neuroscience | www.frontiersin.org This study support the hypothesis that elevations in extracellular 5-HT in the brain can dampen positive reinforcing effects of cathinone-type drugs. Nevertheless, MDPV and methylone are both self-administered by rats, suggesting these drugs possess significant abuse liability in humans Schindler et al., 2015 Methylone Rats were randomly assigned to one of four groups based upon methylone dose (0.05, 0.1, 0.2, or 0.5 mg/kg per infusion) This study reveal that methylone may possess an addiction potential similar to or greater than MDMA, yet patterns of self-administration and effects on brain reward function suggest that this drug may have a lower potential for abuse and compulsive use than prototypical psychostimulants Watterson et al., 2012 (Continued) Frontiers in Neuroscience | www.frontiersin.org Groups of female Wistar rats were trained to self-administer mephedrone, methylone or MDMA (0.5 mg/kg/inf) under a Fixed-Ratio (FR) 1 schedule of reinforcement for 14 sessions. Following the acquisition interval, animals were evaluated in FR (0.0, 0.125, 0.25, 0.5, 1.0, 2.5 mg/kg/inf) and Progressive-ratio-PR (0.125, 1.0 mg/kg/inf) dose-substitution procedures The results show that female rats acquired the self-administration of all three compounds with intakes in mephedrone-trained rats that were significantly higher than that of methylone-trained or MDMA-trained rats. In doses substitution under either FR or PR contingencies, however, the potencies of all three drugs were similar within the original training groups. The mephedrone-trained animals exhibited higher intakes of all drugs during dose-substitution, indicating lasting consequences of the training drug. Abuse liability of these three compounds is therefore predicted to be similar in established stimulant users but may differ in liability if they are primary drugs of initiation Creehan et al., 2015 Mephedrone Mephedrone (1 or 3 mg/kg) The neurochemical and functional properties of mephedrone resemble those of MDMA, but it also shows an amphetamine-like effect in that it evokes a rapid release and elimination of DA in the brain reward system, a feature that may contribute to its potent re-inforcing properties Kehr et al., 2011 (+)-amphetamine MDMA MDMA (3 mg/kg) (+)-amphetamine (1 mg/kg) memory impairment, attention difficulties), neurological (i.e., dizziness, sensation changes, seizures, tremor) and psychotic (i.e., agitation, aggression, catatonia, paranoia, hallucinations, depersonalization, dissociation, prolonged psychosis, perceptual alterations) episodes, with a higher incidence in comparison to those seen with 9 -THC use (Papanti et al., 2013;Spaderna et al., 2013;Van Amsterdam et al., 2015).

Rewarding and Reinforcing Properties of Cannabimimetics
Recent literature shows that SC have emerged as new drugs of abuse. As previously reported, an incredibly huge number of SC have been detected in Marijuana substitutes (Denooz et al., 2013;Brents and Prather, 2014;Maxwell, 2014). Being CB1 receptor agonists with extremely high affinity, SC probably act in brain regions where CB1 receptors are heavily expressed, such as the amygdala, cingulate cortex, prefrontal cortex (PFC), ventral pallidum, caudate putamen, nucleus accumbens (NAc), ventral tegmental area (VTA), and lateral hypothalamus (Glass et al., 1997;Wang et al., 2003). All these brain regions have a recognized involvement in reward, addiction and cognitive functions (Koob and Volkow, 2010). Furthermore, CB1 receptors are located in limbic regions, such as VTA, NAc, ventral pallidum, CeA, BNST, and PFC (Herkenham et al., 1991;Glass et al., 1997;Wang et al., 2003); the integration of excitatory and inhibitory inputs, coming from these structures, influence, and modulate reward processing (Sidhpura and Parsons, 2011;Panagis et al., 2014). Several studies in mice and rats showed that these compounds affect the mesolimbic dopaminergic transmission and influence conditioned behaviors (Table 4). Similar to other drugs of abuse, THC activates dopamine (DA) transmission in the ventral striatum in humans (Volkow et al., 2003;Bossong et al., 2009). In addition, animal studies showed that both 9 -THC and WIN 55.212-2, a CB1 and CB2 agonist, elicit dopamine release in the NAc (Chen et al., 1993;Cheer et al., 2004) with a specific activation of the NAc shell subregion (Tanda et al., 1997;Lecca et al., 2006;De Luca et al., 2012). The NAc plays a crucial role in brain reward circuits involved in motivational and cognitive functions (Heimer et al., 1991;Zahm and Brog, 1992). In particular, it has been shown that stimulation of DA transmission in the NAc shell is directly involved in the rewarding properties of both natural reward and addictive drugs (Di Chiara et al., 2004). Microdialysis studies on awake freely moving animals performed in our laboratories showed that JWH-018, at the dose of 0.25 mg/kg i.p., increases DA transmission in the NAc shell but not in the NAc core nor in mPFC (Figure 4). Surprisingly, DA transmission in the NAc shell was not stimulated after administration of lower (0.125 mg/kg ip) or higher (0.5 mg/kg ip) doses producing an inverted Ushape dose response curve for the effect of JWH-018 (De Luca et al., 2015a). Further studies in mice and rats showed a similar effect after the intraperitoneal administration of JWH-073 and JWH-250 as well (Ossato et al., 2016), and after the intravenous administration of BB-22 (De Luca et al., 2015b). Notably, as previously reported by De Luca et al. (2012), THC stimulated extracellular DA release in the NAc shell at a dose fourfold higher than JWH-018 when administered intraperitoneally. In addition, BB-22 stimulates NAc shell DA release at the dose of 0.01 mg/kg iv, while THC increases extracellular DA in the same area at dose of 0.15 mg/kg iv (Tanda et al., 1997). These results show that both JWH-018 and BB-22 are more potent than THC in inducing NAc shell DA release, suggesting a putative higher abuse liability of synthetic vs. natural cannabinoids. Electrophysiological studies show that the stimulation of DA extracellular levels in the NAc shell by JWH-018 is thought to be due to the activation of CB1 receptors located on presynaptic GABAergic afferents directed to VTA DA neurons, leading to a reduction of GABA A receptors mediated inhibition of DA neuronal activity in the VTA (Lupica and Riegel, 2005;Mátyás et al., 2008;Melis et al., 2014;  Adult male rats trained to discriminate 3 mg/kg (9)-THC or 0.3 mg/kg JWH-018 from vehicle JWH-018, JWH-073, and JWH-210 fully substituted in (9)-THC-trained rats and (9)-THC substituted in JWH-018-trained rats Wiley et al., 2014JWH-018 JWH-073 JWH-250 JWH-200 JWH-203 AM-2201 These compounds were then tested for substitution in rats trained to discriminate -THC (3 mg/kg, intraperitoneally) Each of the compounds fully substituted for the discriminative stimulus effects of -THC, mostly at doses that produced only marginal amounts of rate suppression. JWH-250 and CP 47,497-C8-homolog suppressed response rates at doses that fully substituted for -THC Gatch and Forster, 2014 CP 55940 Acute and repeated administration (7 days Luca et al., 2015a). Rewarding effects of cannabimimetics have also been assessed by different experimental paradigms such as intracranial self-stimulation (ICSS), place conditioning tests, drug-discrimination and intravenous self-administration (IVSA) studies. ICSS of the medial forebrain bundle is the operant conditioning method used in rodents to evaluate the role of the mesolimbic dopamine pathway in rewarding behavioral effects (Carlezon and Chartoff, 2007) and evaluating potential of abuse (Negus and Miller, 2014). Not surprisingly, to date no data on the effect of new SC on ICSS are available. 9 -THC does not facilitate ICSS, but has a dose-dependent inhibitory influence on ICSS (Vlachou et al., 2007). Similarly, a depression of ICSS is observed after the administration of WIN55212-2, CP55940, HU210 (Antoniou et al., 2005;Vlachou et al., 2005;Mavrikaki et al., 2010). Differences in developing tolerance to depression of ICSS after repeated exposure to cannabinoids have been reported. Tolerance is completely developed after repeated exposure to 9 -THC (Kwilasz and Negus, 2012) but partially developed after CP55940 (Grim et al., 2015), and not developed after WIN55212-2 administration (Mavrikaki et al., 2010), suggesting that the different affinity of 9 -THC vs. SC for the CB1 receptors could play a role in developing this tolerance (Grim et al., 2015). On the other hand, place conditioning tests in animals showed that WIN 55212-2 and HU210 established a robust place aversion (CPA), reversed by the CB1 receptor antagonist/inverse agonist SR 141716A, in a similar way as seen with 9 -THC (Chaperon et al., 1998;Cheer et al., 2000;Valjent and Maldonado, 2000). In addition, the CB1 antagonist/inverse agonist AM281 did not induce conditioned place preference (CPP). However, a pretreatment of 14 days with AM281 prior to the CPP test with the same drug, showed significant CPP (Botanas et al., 2015). Accordingly, it has been hypothesized that the endogenous cannabinoid system in the brain may act as a counter-reward system, and blocking or antagonizing this system would therefore produce the reward (Sañudo-Peña et al., 1997;Botanas et al., 2015). This could represent a limitation on the therapeutic use of CB1 antagonist/inverse agonists (Seely et al., 2011).
These studies typically serve as an integration of the results obtained by intravenous self-administration (SA) experiments. As for SA studies, while there is some disputable data concerning the reinforcing properties of 9 -THC based on its ability to be persistently self-administered in squirrel monkeys (Tanda et al., 2000) but not in rodents, there is still favorable evidence available about SC SA. Thus it has been reported that monkeys, mice and rats acquire and maintain WIN 55.212-2 SA (Martellotta et al., 1998;Fattore et al., 2001;Justinova et al., 2004;Lecca et al., 2006). Recently, it has been reported that JWH-018 is self-administered in rodents (Figure 5). In the study by De Luca et al. (2015a) both rats and mice readily acquired two different operant behaviors: nose-poking into an optical switch (rats) and lever-pressing (mice). Rats self-administered JWH-018 at the dose of 20 µg/kg/infusion in daily 1 h FR3 sessions ( Figure 5A). As expected, a reduction of SA after the injection of SR141617A (1 mg/kg ip, 30 min prior to the SA session) was observed, consistent with the lack of JWH-018 mediated reinforcement. Intriguingly, nose-poking for JWH-018 significantly increased from the first session (30th session, Figure 5A) performed after the administration of SR141617A for 2 consecutive days, confirming that these effects of JWH-018 are mediated through cannabinoid receptors. SA behavior did not decrease when JWH-018 was replaced by vehicle. A control group of rats trained for vehicle, failed to acquire SA behavior. It has been hypothesized that the absence of extinction-like response patterns was unrelated to responsecontingent training for JWH-018 because the vehicle failed to induce responding. We think that this probably occurred as a result of a habit learning conditioned by JWH-018, in fact contextual cues were sufficient to maintain responding (De Luca et al., 2015a). This confirmed previous evidence showing that endocannabinoid signaling through CB1 receptors is significant for the habit formation (Hilário et al., 2007). Indeed, in mice, CB1 receptor knockdown can enhance or blunt habit formation, whereas 9 -THC tolerance enhances habit formation; in humans, cannabis use enhances the stimulusresponse/habit memory (for review, see Goodman and Packard, 2015).
surprisingly, active lever-pressing did not decrease, while inactive lever-pressing increased becoming superimposable to the active ones. Unlike the SA experiments with rats, in these experiments, the drug associated cues were removed after the first three of a total of 12 sessions during the extinction phase. However, during the JWH-018 reinstatement, SA behavior was immediately reinstated and inactive lever-presses decreased since the SA behavior was specifically regulated by the drug infusion confirming the reinforcing properties of JWH-018. This may also prove that JWH-018 alters cortical processes important for the context updating and the automatic orientation of attention (D'Souza et al., 2012) with consequent disruption of cognitive functioning, emotional processing, and affective states as different SC make in humans (Zimmermann et al., 2009).
Further research is needed to evaluate the impact of chronic exposure to SC.

CONCLUDING REMARKS
The review of the biomedical data here presented, clearly demonstrates the unsafe nature of these new drugs of abuse. This is particularly alarming since adolescents seem to be the most exposed subjects to these dangerous NPS. Indeed, users are often unaware of the consequences of ingesting synthetic compounds that are sold as "legal alternative" to classical drugs, and their unexpected, sometimes fatal adverse effects. Only awareness can reduce NPS use since stopping their synthesis and diffusion seems to be an improbable task and, morbidity and mortality reports keep increasing as NPS gain popularity worldwide. Awareness campaigns about these substances and their devastating effects should be organized to inform everyone, including clinicians, who should be able to recognize symptoms of intoxication induced by NPS (Simonato et al., 2013;Papanti et al., 2013;Schifano et al., 2015).
Ultimately, this paper intends to be helpful to drive governments and civil society to not underestimate the NPS issue, and to encourage the scientific community to deeply evaluate the pharmacology and toxicological effects of NPS and to develop effective treatments for NPS intoxication. Additionally, this paper intends to be useful for advising law enforcement agencies, which need updated information for the prevention and fight against trafficking and sale of NPS, and in the long run, hopefully contribute to better protect public health and safety.

AUTHOR CONTRIBUTIONS
This is a review, different authors contributed as follows: CM: Section NPS: From Chemistry to Pharmacological Effects;