Background and Purpose: Fentanyl analogues have been implicated in many cases of intoxication and death with overdose worldwide. The aim of this study is to investigate the pharmaco-toxicology of two fentanyl analogues: butyrylfentanyl (BUF) and 4-fluorobutyrylfentanyl (4F-BUF). Experimental Approach: In vitro, we measured agonist opioid receptor efficacy, potency, and selectivity and ability to promote interaction of the mu receptor with G protein and beta-arrestin 2. In vivo, we evaluated thermal antinociception, stimulated motor activity and cardiorespiratory changes in female and male CD-1 mice injected with BUF or 4F-BUF (0.1-6 mgkg(-1)). Opioid receptor specificity was investigated using naloxone (6 mgkg(-1)). We investigated the possible role of stress in increasing cardiorespiratory toxicity using the corticotropin-releasing factor 1 (CRF1) antagonist antalarmin (10 mgkg(-1)). Key Results: Agonists displayed the following rank of potency at mu receptors: fentanyl > 4F-BUF > BUF. Fentanyl and BUF behaved as partial agonists for the beta-arrestin 2 pathway, whereas 4F-BUF did not promote beta-arrestin 2 recruitment. In vivo, we revealed sex differences in motor and cardiorespiratory impairments but not antinociception induced by BUF and 4F-BUF. Antalarmin alone was effective in blocking respiratory impairment induced by BUF in both sexes but not 4F-BUF. The combination of naloxone and antalarmin significantly enhanced naloxone reversal of the cardiorespiratory impairments induced by BUF and 4F-BUF in mice. Conclusion and Implications: In this study, we have uncovered a novel mechanism by which synthetic opioids induce respiratory depression, shedding new light on the role of CRF1 receptors in cardiorespiratory impairments by mu agonists.

Γ-valerolactone (GVL), marketed online as "Tranquilli-G" and "excellent Valium", is used as a legal substitute for γ-hydroxybutyric acid (GHB); however, until now, GVL has only been connected to one Drug-Facilitated Sexual Assault (DFSA) case. Moreover, the pharmaco-toxicological effects of GVL are poorly studied. The aim of this study was to investigate the 1) in vivo effects of gavage administration of GVL (100-3000 mg/kg) on neurological (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, motor activity (bar, drag, and accelerod test) and cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in CD-1 male mice and the 2) in silico ADMET profile of GVL in comparison to GHB and the open active form γ-hydroxyvaleric acid (GHV). The present study demonstrates that GVL inhibits, in a dose-dependent manner, sensorimotor and motor responses and induces cardiorespiratory depression (at a dose of 3000 mg/kg) in mice. The determination of the ED50 in sensorimotor and motor responses revealed that GVL is about 4-5 times less potent than GHB. In silico prediction of ADMET profiles revealed toxicokinetic similarities between GHB and GHV, and differences with GVL. These results suggest that GVL could be used as a substitute for GHB and should be added to forensic toxicology screenings.

JWH-073 is a synthetic cannabinoid (SCB) that is illegally marketed within an “herbal blend”, causing psychoactive effects more intense than those produced by Cannabis. Users report that JWH-073 causes less harmful effects than other SCBs, misrepresenting it as a “safe JWH-018 alternative”, which in turn prompts its recreational use. The present study is aimed to investigate the in vivo pharmacological activity on physiological and neurobehavioral parameters in male CD-1 mice after acute 1 mg/kg JWH-073 administration. To this aim we investigate its effect on sensorimotor (visual, acoustic, and tactile), motor (spontaneous motor activity and catalepsy), and memory functions (novel object recognition; NOR) in mice coupling behavioral and EEG data. Moreover, to clarify how memory function is affected by JWH-073, we performed in vitro electrophysiological studies in hippocampal preparations using a Long-Term Potentiation (LTP) stimulation paradigm. We demonstrated that acute administration of JWH-073 transiently decreased motor activity for up to 25 min and visual sensorimotor responses for up to 105 min, with the highest effects at 25 min (~48 and ~38%, respectively), while the memory function was altered up to 24 h (~33%) in treated-mice as compared to the vehicle. EEG in the somatosensory cortex showed a maximal decrease of α (~23%) and γ (~26%) bands at 15 min, β (~26%) band at 25 min, a maximal increase of θ (~14%) band at 25 min and δ (~35%) band at 2 h, and a significant decrease of θ (~18%), α (~26%), and β (~10%) bands during 24 h. On the other hand, EEG in the hippocampus showed a significant decrease of all bands from 10 min to 2 h, with the maximal effect at 30 min for θ (~34%) and γ (~26%) bands and 2 h for α (~36%), β (~29%), and δ (~15%) bands. Notably, the δ band significant increase both at 5 min (~12%) and 24 h (~19%). Moreover, in vitro results support cognitive function impairment (~60% of decrease) by interfering with hippocampal synaptic transmission and LTP generation. Our results suggest that JWH-073 deeply alters brain electrical responsiveness with minor behavioral symptoms. Thus, it poses a subtle threat to consumers who mistakenly consider it safer than other SCBs.

Fentanyl derivatives (FENS) belongs to the class of Novel Synthetic Opioids that emerged in the illegal drug market of New Psychoactive Substances (NPS). These substances have been implicated in many cases of intoxication and death with overdose worldwide. Therefore, the aim of this study is to investigate the pharmacodynamic profiles of three fentanyl (FENT) analogues: Acrylfentanyl (ACRYLF), Ocfentanyl (OCF) and Furanylfentanyl (FUF). In vitro, we measured FENS opioid receptor efficacy, potency, and selectivity in calcium mobilization studies performed in cells coexpressing opioid receptors and chimeric G proteins and their capability to promote the interaction of the mu receptor with G protein and beta-arrestin 2 in bioluminescence resonance energy transfer (BRET) studies. In vivo, we investigated the acute effects of the systemic administration of ACRYLF, OCF and FUF (0.01-15 mg/kg i.p.) on mechanical and thermal analgesia, motor impairment, grip strength and cardiorespiratory changes in CD-1 male mice. Opioid receptor specificity was investigated in vivo using naloxone (NLX; 6 mg/kg i.p) pre-treatment. In vitro, the three FENS were able to activate the mu opioid receptor in a concentration dependent manner with following rank order potency: FUF > FENT=OCF > ACRYLF. All compounds were able to elicit maximal effects similar to that of dermorphin, with the exception of FUF which displayed lower maximal effects thus behaving as a partial agonist. In the BRET G-protein assay, all compounds behaved as partial agonists for the beta-arrestin 2 pathway in comparison with dermorphin, whereas FUF did not promote beta-arrestin 2 recruitment, behaving as an antagonist. In vivo, all the compounds increased mechanical and thermal analgesia with following rank order potency ACRYLF = FENT > FUF > OCF and impaired motor and cardiorespiratory parameters. Among the substances tested, FUF showed lower potency for cardiorespiratory and motor effects. These findings reveal the risks associated with the use of FENS and the importance of studying the pharmaco-dynamic properties of these drugs to better understand possible therapeutic interventions in the case of toxicity.

In the last decade, the market for new psychoactive substances has been enriched by numerous psychedelic phenethylamines, which mimic the psychoactive effect of lysergic acid diethylamide (LSD). In particular, the -NBOMe series, which are more potent than their 2C compounds analogs, are considered worthy substitutes for LSD by users. The purpose of this study was to assess the effects of 25H-NBOMe and its halogenated derivatives (25I-NBOMe and 25B-NBOMe) in comparison to their 2C compounds analogs and LSD on the sensorimotor (visual, acoustic, and overall tactile), reaction time, spontaneous (total distance traveled) and stimulated (drag, accelerod test) motor activity, grip strength test, and prepulse inhibition (PPI) responses in mice. Systemic administration of -NBOMe, 2C compounds analogs, and LSD (0.001–10 mg/kg) differently impaired the sensorimotor, reaction time, motor, and PPI responses in mice. In particular, halogenated (25I and 25B)-NBOMe derivatives appear to be more effective than the entire class of 2C compounds analogs in altering visual and acoustic responses, affecting reaction time, and motor and sensory gating in PPI test. In fact, the specific rank order of compounds potency for nearly all of the experiments showed that (25I and 25B)-NBOMe were more potent than 2C compounds analogs and LSD. -NBOMe and 2C compounds analogs impaired not only the reception of incoming sensory stimuli (visual and acoustic), but their correct brain processing (PPI) in an equal and sometimes stronger way than LSD. This sensory impairment directly affected the spontaneous motor response and reaction time of mice, with no change in performance in stimulated motor activity tests. These aspects should be carefully considered to better understand the potential danger that psychedelic phenethylamines, in particular -NBOMe, may pose to public health, with particular reference to decreased performance in driving and hazardous works that require special sensorimotor skills.

Background: Reports concerning the causal link between aggressive behavior and use and abuse of different substances (i.e., alcohol, MDPV) can be found in the literature. Nonetheless, the topic concerning the effects of acute ethanol administration on MDPV and cocaine induced aggressive behavior has yet to be thoroughly investigated. The aim of this study was to investigate such synergistic effects. Materials and methods: A total of 360 male mice were employed in the study. Ethanol was diluted with saline solution and administered 10 min before MDPV or cocaine injection via oral gavage needles. Similarly, MDPV and cocaine were dissolved in saline solution and administered by intraperitoneal injection. Different associations of specific drug doses were then tested. To investigate the acute effects of MDPV and cocaine and their interaction with ethanol on aggression in mice, a resident-intruder test was used. Results: Ethanol alone was ineffective at dosages of 0.05 g/kg and 0.25 g/kg but increased the aggressiveness of the mice at 0.125 g/kg. Similarly, the injection of both cocaine alone and MDPV alone did not significantly increase the aggressiveness of the mice; conversely, the combination of ethanol and cocaine and ethanol and MDPV enhanced aggression at specific ethanol dosages (0.05 g/kg and 0.125 g/kg). Conclusion: This study demonstrated that acute ethanol administration enhances MDPV-and cocaine-induced aggressive behavior in mice. This aggressive response is particularly enhanced when MDVP and cocaine are coupled with specific ethanol dosages, proving that psychostimulant drugs may act synergistically under certain conditions.

Recreational use of illicit methiopropamine (MPA) is a public health concern because it produces neurochemical effects comparable with those induced by methamphetamine (METH). The present study investigated the effects of MPA on the expression of an aggressive behaviour. Eighty CD-1 male mice, after receiving intraperitoneal injection of saline, MPA (0.01–10 mg/kg), METH (0.01–10 mg/kg), or AMPH (0.01–10 mg/kg), once a week over a 5-week period, underwent the resident–intruder test and spontaneous locomotor activity measurement. Results showed that all psychostimulants induce aggressive behaviour even at low doses, with a dose-dependent increase and a time-dependent sensitisation. MPA potency was similar to METH and superior to AMPH. Therefore, MPA-induced aggressive behaviour may appear even at MPA dosages free of cardiovascular or other behavioural adverse effects and could become a non-intentional side effect that users experience after increasing and repeating MPA consumption.

4,4’-Dimethylaminorex (4,4’-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±)cis (0.1–60 mg/kg) and (±)trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±)cis at 1, 10 or 60 mg/kg + (±)trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4′-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±)cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±)trans stereoisomer was ineffective whilst the stereoisomers’ co-administration resulted in a worsening of the toxic (±)cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±)trans stereoisomer reduced the metabolism of the (±)cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.

N-methyl-2-pyrrolidone (NMP) and γ-hydroxybutyrate acid (GHB) are synthetic solvents detected in the recreational drug market. GHB has sedative/hypnotic properties and is used for criminal purposes to compromise reaction ability and commit drug-facilitated sexual assaults and other crimes. NMP is a strong solubilizing solvent that has been used alone or mixed with GHB in case of abuse and robberies. The aim of this experimental study is to compare the acute pharmaco-toxicological effects of NMP and GHB on neurological signs (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, and motor activity (bar, drag, and accelerod test) in CD-1 male mice. Moreover, since cardiorespiratory depression is one of the main adverse effects related to GHB intake, we investigated the effect of NMP and GHB on cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in mice. The present study demonstrates that NMP inhibited sensorimotor and motor responses and induced cardiorespiratory depression, with a lower potency and efficacy compared to GHB. These results suggest that NMP can hardly be used alone as a substance to perpetrate sexual assault or robberies.

INTRODUCTION:The anterior cingulate cortex (ACC) has shown to play a role in impulsivity, fear, and anxiety. Considering, its high glutamate receptor density, it was chosen as a region of interest to investigate the role of glutamate transmission in drug dependance. We investigated the correlations between personality trait scores and glutamate-to-glutamine (Glx) ratio concentrations in the ACC in order to evaluate if (1) personality traits may increase the probability of drug use and (2) drug use can modify cerebral metabolic pattern contributing to addictive behaviors.MATERIALS AND METHODS:Glx ratio concentrations in the ACC region were measured with high-resolution multivoxel proton magnetic resonance spectroscopy (1H-MRS). Personality traits were evaluated utilizing Cloninger's TCI-revised test. Bivariate correlations between personality scores of 28 teens cannabis users (males, mean age = 18.54 ± 2.80) were evaluated.RESULTS:In the ACC, we observed negative correlation between GG concentrations (r = -0.44, P = 0.05) and co-operativeness values (CO), choline (cho), and novelty seeking (NS) values (r = -0,45, P = 0.05). Low levels of glutamate and high levels of cho in the ACC were closely related to the CO and NS personality traits.CONCLUSIONS:Metabolic and personality patterns seems to be related to the risk of substance predisposition in adolescents. Our data contribute a possible support to the "top-down" control of the ACC on brain metabolism, due to the particular cerebral metabolic pattern found in "drug-using" adolescents.

MT-45 is a synthetic opioid that was developed in the 1970s as an analgesic compound. However, in recent years MT-45 has been associated with multiple deaths in Europe and has been included in the class of novel psychoactive substances known as novel synthetic opioids (NSOs). Little is known about the pharmaco-toxicological effects of MT-45. Therefore, we used a dynamic mass redistribution (DMR) assay to investigate the pharmacodynamic profile of this NSO in vitro compared with morphine. We then used in vivo studies to investigate the effect of the acute systemic administration of MT-45 (0.01-15 mg/kg i.p.) on motor and sensorimotor (visual, acoustic and tactile) responses, mechanical and thermal analgesia, muscle strength and body temperature in CD-1 male mice. Higher doses of MT-45 (6-30 mg/kg i.p.) were used to investigate cardiorespiratory changes (heart rate, respiratory rate, SpO(2) saturation and pulse distention). All effects of MT-45 were compared with those of morphine. In vitro DMR assay results demonstrated that at human recombinant opioid receptors MT-45 behaves as a potent selective mu agonist with a slightly higher efficacy than morphine. In vivo results showed that MT-45 progressively induces tail elevation at the lowest dose tested (0.01 mg/kg), increased mechanical and thermal antinociception (starting from 1 to 6 mg/kg), decreased visual sensorimotor responses (starting from 3 to 6 mg/kg) and reduced tactile responses, modulated motor performance and induced muscle rigidity at higher doses (15 mg/kg). In addition, at higher doses (15-30 mg/kg) MT-45 impaired the cardiorespiratory functions. All effects were prevented by the administration of the opioid receptor antagonist naloxone. These findings reveal the risks associated with the ingestion of opioids and the importance of studying these drugs and undertaking more clinical studies of the current molecules to better understand possible therapeutic interventions in the case of toxicity.

AKB48 is a designer drug belonging to the indazole synthetic cannabinoids class, illegally sold as herbal blend, incense, or research chemicals for their psychoactive cannabis-like effects. In the present study, we investigated the in vivo pharmacological and behavioral effects of AKB48 in male rats and measured the pharmacodynamic effects of AKB48 and simultaneously determined its plasma pharmacokinetic. AKB48 at low doses preferentially stimulated dopamine release in the nucleus accumbens shell (0.25 mg/kg) and impaired visual sensorimotor responses (0.3 mg/kg) without affecting acoustic and tactile reflexes, which are reduced only to the highest dose tested (3 mg/kg). Increasing doses (0.5 mg/kg) of AKB48 impaired place preference and induced hypolocomotion in rats. At the highest dose (3 mg/kg), AKB48 induced hypothermia, analgesia, and catalepsy; inhibited the startle/pre-pulse inhibition test; and caused cardiorespiratory changes characterized by bradycardia and mild bradipnea and SpO2 reduction. All behavioral and neurochemical effects were fully prevented by the selective CB1 receptor antagonist/inverse agonist AM251. AKB48 plasma concentrations rose linearly with increasing dose and were correlated with changes in the somatosensory, hypothermic, analgesic, and cataleptic responses in rats. For the first time, this study shows the pharmacological and behavioral effects of AKB48 in rats, correlating them to the plasma levels of the synthetic cannabinoid.Chemical Compound Studied in This Article: AKB48 (PubChem CID: 57404063); AM251 (PubChem CID: 2125).

JWH-018-C1, 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 Delta(9)-tetrahydrocannabinol (Delta(9)-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 Delta(9)-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.

MDPV is a synthetic cathinone illegally marketed and consumed for its psychostimulant effects, which are similar to those produced by cocaine, amphetamines, and MDMA. Clinical reports indicate that MDPV produces euphoria, increases alertness, and at high doses causes agitation, psychosis, tachycardia and hypertension, hallucinations, delirium, hyperthermia, rhabdomyolysis, and even death. In rodents, MDPV reproduces the typical physiological effects of psychostimulant drugs, demonstrating greater potency than cocaine. Nevertheless, its role in aggressive behavior has been reported but not yet experimentally confirmed. Therefore, the aim of this study was to evaluate the effects of acute and repeated MDPV (0.01–10 mg/kg i.p.) administration on aggressive behavior in mice and to compare them with those of cocaine (0.01–10 mg/kg i.p.) administration. To this purpose, the resident–intruder test in isolated mice and the spontaneous and stimulated aggressiveness tests for group-housed mice were employed. The present study shows for the first time that MDPV enhances aggressive behavior and locomotion in mice with greater potency and efficacy than cocaine treatment. Moreover, the aggressive and locomotor responses are enhanced after repeated administration, indicating that a sensitization mechanism comes into play. These results, although from preclinical investigation, are suggestive that human MDPV intake could be a problem for public health and the criminal justice system. Thus, investigation by police officers and medical staff is needed to prevent interpersonal violence induced by the consumption of synthetic cathinones.

The phenomenon of Drug-Facilitated Sexual Assaults (DFSA), has dramatically raised in the past decades, also due to the high number of drugs that can potentially be used. Analytical methods able to detect a large number of drugs with high sensitivity are sought for the diagnosis of DFC. The aim of the present study was the development of an analytical protocol for the identification of a wide number of possible incapacitating substance in biological specimens for its application to alleged victims of DFC. The procedure involves the combination of ten analytical methods, performed by LC-MS/MS, GC-MS, GC-FID. The procedure allows the screening of about 200 drugs/metabolites in urine, blood and hair. The protocol is generally applied to urine samples. Hair analysis is performed in cases where more than 48 h elapsed from the suspected administration; a blood sample is analysed only when less than 12 h elapsed and urine is positive for one or more substance. For its preliminary evaluation, the protocol was applied to analyse biological samples collected on 120 victims of alleged DFSA. Ethanol and/or its metabolite ethylglucuronide was detected in 66% of cases; benzodiazepines and analogues (zolpidem) in 18%; other substances detected were cocaine (in 15 cases), cannabis (in 10 cases) MDMA (8 cases), antipsychotics, antihystaminics, antidepressants, antiepileptics and opiates. GHB was detected at a concentration greater than 10 mu g/ml in urine only in one case. The results demonstrate the suitability of the protocol to detect substances potentially used to commit or to facilitate a DFC.

JWH-018 and AKB48 are two synthetic cannabinoids (SCBs) belonging to different structural classes and illegally marketed as incense, herbal preparations, or chemical supply for theirs psychoactive cannabis-like effects. Clinical reports from emergency room reported psychomotor agitation as one of the most frequent effects in people assuming SCBs. This study aimed to investigate the psychostimulant properties of JWH-018 and AKB48 in male CD-1 mice and to compare their behavioral and biochemical effects with those caused by cocaine and amphetamine. In vivo studies showed that JWH-018 and AKB48, as cocaine and amphetamine, facilitated spontaneous locomotion in mice. These effects were prevented by CB1 receptor blockade and dopamine (DA) D1/5 and D2/3 receptors inhibition. SPECT-CT studies on dopamine transporter (DAT) revealed that, as cocaine and amphetamine, JWH-018 and AKB48 decreased the [123I]-FP-CIT binding in the mouse striatum. Conversely, in vitro competition binding studies revealed that, unlike cocaine and amphetamine, JWH-018 and AKB48 did not bind to mouse or human DAT. Moreover, microdialysis studies showed that the systemic administration of JWH-018, AKB48, cocaine, and amphetamine stimulated DA release in the nucleus accumbens (NAc) shell of freely moving mice. Finally, unlike amphetamine and cocaine, JWH-018 and AKB48 did not induce any changes on spontaneous [3H]-DA efflux from murine striatal synaptosomes. The present results suggest that SCBs facilitate striatal DA release possibly with different mechanisms than cocaine and amphetamine. Furthermore, they demonstrate, for the first time, that JWH-018 and AKB48 induce a psychostimulant effect in mice possibly by increasing NAc DA release. These data, according to clinical reports, outline the potential psychostimulant action of SCBs highlighting their possible danger to human health.

It is well known that an impairment of learning and memory function is one of the major physiological effects caused by natural or synthetic cannabinoid consumption in rodents, nonhuman primates and in humans. JWH-018 and its halogenated derivatives (JWH-018-Cl and JWH-018-Br) are synthetic CB1/CB2 cannabinoid agonists, illegally marketed as "Spice" and "herbal blend" for their Cannabis-like psychoactive effects. In the present study the effects of acute exposure to JWH-018, JWH-018-Cl, JWH-018-Br (JWH-018-R compounds) and Δ(9)-THC (for comparison) on Novel Object Recognition test (NOR) has been investigated in mice. Moreover, to better characterize the effects of JWH-018-R compounds on memory function, in vitro electrophysiological and neurochemical studies in hippocampal preparations have been performed. JWH-018, JWH-018-Cl and JWH-018-Br dose-dependently impaired both short- and long-memory retention in mice (respectively 2 and 24 h after training session). Their effects resulted more potent respect to that evoked by Δ(9)-THC. Moreover, in vitro studies showed as JWH-018-R compounds negatively affected electrically evoked synaptic transmission, LTP and aminoacid (glutamate and GABA) release in hippocampal slices. Behavioral, electrophysiological and neurochemical effects were fully prevented by CB1 receptor antagonist AM251 pretreatment, suggesting a CB1 receptor involvement. These data support the hypothesis that synthetic JWH-018-R compounds, as Δ(9)-THC, impair cognitive function in mice by interfering with hippocampal synaptic transmission and memory mechanisms. This data outline the danger that the use and/or abuse of these synthetic cannabinoids may represent for the cognitive process in human consumer.

BACKGROUND:Clinical reliability of self-reported data for alcohol, caffeine, and nicotine consumptions is lacking, particularly in adolescents. OBJECTIVES:To compare a self-report questionnaire and hair analysis to assess the reliability and effectiveness of the self-report. METHODS:A cross-sectional study on 14-15-year-old Italian students (n = 874, 38% males, 62% females) was performed comparing self-reported data to hair analysis. The latter quantified hair concentrations of caffeine, nicotine, cotinine, ethyl glucuronide (EtG), and fatty acid ethyl esters (FAEEs) using mass spectrometry. RESULTS:Concordance between self-report and hair testing ranged from good to poor across substances and levels of use: poor for heavy alcohol intake (EtG: k = 0.36, 20 positive cases by hair analysis, false negative by self-report, 2.3% of total sample; FAEE k = 0.31, 25 positive cases, 2.9% of total sample); fair to poor for active smokers (k = 0.40, 125 positive cases, 14.3% of total sample); and moderate for caffeine (k = 0.57, 56 positive cases, 6.4% of total sample). CONCLUSIONS:Epidemiological studies on alcohol, caffeine, and nicotine consumption in adolescents may benefit from the inclusion of toxicological analysis on hair samples to overcome the under-reporting phenomenon of questionnaires and detect more cases of problematic substance use.

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 Δ(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" 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.

gamma-Hydroxybutyric acid (GHB) spread for recreational purposes or as "rape drug" represents a hard issue for forensic toxicologists due to its endogenous nature. It is clear that an actual and reliable discrimination between basal and exogenous levels is mandatory to achieve a correct evaluation of conscious/unconscious administration. This research aimed to study the GHB baseline in hair samples, collected from 150 volunteers, non-consumers of any drugs of abuse, in order to evaluate if a generic cut-off value could be accepted, also focusing on potential influences of gender and age. The analysis consisted of an overnight incubation with NaOH at 56 degrees C, liquid-liquid extraction with ethylacetate and trimethylsylil derivatization. Detection was carried out through gas chromatography-mass spectrometry in single ion monitoring (m/z 233, 234, 147 for GHB; m/z 239, 240 and 147 for GHB-d6). The endogenous amount in "blank" hair was estimated by the standard addition method. Concentration range was 0.279-2.839 ng/mg. In males, the average GHB levels were higher than in females (0.829 vs 0.596 ng/mg, respectively), especially in the first age category (<30 years, 1.008 vs 0.606 ng/mg, respectively). Age influences on GHB levels seemed to be different among the two sexes: in male population concentrations were higher <30 (1.008 ng/mg) and similar in the other age ranges (0.762 ng/mg, 30-50; 0.763 ng/mg, >50); in female, quite similar levels were registered throughout all the age categories (0.606 ng/mg, <30; 0.536 ng/mg, 30-50; 0.691 ng/mg, >50). Further study should be performed on GHB physiology in order to better understand these differences among ages and genders. Moreover, we demonstrated that for hair analysis a cut-off reference value is not strictly mandatory, underlining the great interpretative valence of segmental analysis. (c) 2015 Elsevier B.V. All rights reserved.