Psychedelic-inspired drug discovery using an engineered biosensor.

Ligands can induce G protein-coupled receptors (GPCRs) to adopt a myriad of conformations, many of which play critical roles in determining the activation of specific signaling cascades associated with distinct functional and behavioral consequences. For example, the 5-hydroxytryptamine 2A receptor (5-HT2AR) is the target of classic hallucinogens, atypical antipsychotics, and psychoplastogens. However, currently available methods are inadequate for directly assessing 5-HT2AR conformation both in vitro and in vivo. Here, we developed psychLight, a genetically encoded fluorescent sensor based on the 5-HT2AR structure. PsychLight detects behaviorally relevant serotonin release and correctly predicts the hallucinogenic behavioral effects of structurally similar 5-HT2AR ligands. We further used psychLight to identify a non-hallucinogenic psychedelic analog, which produced rapid-onset and long-lasting antidepressant-like effects after a single administration. The advent of psychLight will enable in vivo detection of serotonin dynamics, early identification of designer drugs of abuse, and the development of 5-HT2AR-dependent non-hallucinogenic therapeutics.

The Effects of Psychedelics on Neuronal Physiology.

Psychedelics are quite unique among drugs that impact the central nervous system, as a single administration of a psychedelic can both rapidly alter subjective experience in profound ways and produce sustained effects on circuits relevant to mood, fear, reward, and cognitive flexibility. These remarkable properties are a direct result of psychedelics interacting with several key neuroreceptors distributed across the brain. Stimulation of these receptors activates a variety of signaling cascades that ultimately culminate in changes in neuronal structure and function. Here, we describe the effects of psychedelics on neuronal physiology, highlighting their acute effects on serotonergic and glutamatergic neurotransmission as well as their long-lasting effects on structural and functional neuroplasticity in the cortex. We propose that the neurobiological changes leading to the acute and sustained effects of psychedelics might be distinct, which could provide opportunities for engineering compounds with optimized safety and efficacy profiles.

From taboo to treatment: The emergence of psychedelics in the management of pain and opioid use disorder.

The rise of psychedelics in contemporary medicine has sparked interest in their potential therapeutic applications. While traditionally associated with countercultural movements and recreational use, recent research has shed light on the potential benefits of psychedelics in various mental health conditions. In this review, we explore the possible role of psychedelics in the management of chronic pain and opioid use disorder (OUD), 2 critical areas in need of innovative treatment options. Pain control remains a significant clinical challenge, particularly for individuals with OUD and those who receive long-term opioid therapy who develop marked tolerance to opioid-induced analgesia. Despite the magnitude of this problem, there is a scarcity of controlled studies investigating pain management alternatives for these populations. Drawing from preclinical and human evidence, we highlight the potential of psychedelics to act on shared neurobiological substrates of chronic pain and OUD, potentially reversing pain- and opioid-induced neuroadaptations, such as central sensitization. We elaborate on the multifaceted dimensions of the pain experience (sensory, affective and cognitive) and their intersections that overlap with opioid-related phenomena (opioid craving and withdrawal), hypothesizing how these processes can be modulated by psychedelics. After summarizing the available clinical research, we propose mechanistic insights and methodological considerations for the design of future translational studies and clinical trials, building on a shared clinical and neurobiological understanding of chronic pain and OUD. Our intention is to provide timely perspectives that accelerate the development and exploration of novel therapeutics for chronic pain and OUD amidst the escalating opioid crisis.

Harnessing psilocybin: antidepressant-like behavioral and synaptic actions of psilocybin are independent of 5-HT2R activation in mice.

Depression is a widespread and devastating mental illness and the search for rapid-acting antidepressants remains critical. There is now exciting evidence that the psychedelic compound psilocybin produces not only powerful alterations of consciousness, but also rapid and persistent antidepressant effects. How psilocybin exerts its therapeutic actions is not known, but it is widely presumed that these actions require altered consciousness, which is known to be dependent on serotonin 2A receptor (5-HT2AR) activation. This hypothesis has never been tested, however. We therefore asked whether psilocybin would exert antidepressant-like responses in mice and, if so, whether these responses required 5-HT2AR activation. Using chronically stressed male mice, we observed that a single injection of psilocybin reversed anhedonic responses assessed with the sucrose preference and female urine preference tests. The antianhedonic response to psilocybin was accompanied by a strengthening of excitatory synapses in the hippocampus-a characteristic of traditional and fast-acting antidepressants. Neither behavioral nor electrophysiological responses to psilocybin were prevented by pretreatment with the 5-HT2A/2C antagonist ketanserin, despite positive evidence of ketanserin's efficacy. We conclude that psilocybin's mechanism of antidepressant action can be studied in animal models and suggest that altered perception may not be required for its antidepressant effects. We further suggest that a 5-HT2AR-independent restoration of synaptic strength in cortico-mesolimbic reward circuits may contribute to its antidepressant action. The possibility of combining psychedelic compounds and a 5-HT2AR antagonist offers a potential means to increase their acceptance and clinical utility and should be studied in human depression.

25X-NBOMe compounds - chemistry, pharmacology and toxicology. A comprehensive review.

Recently, a growing number of reports have indicated a positive effect of hallucinogenic-based therapies in different neuropsychiatric disorders. However, hallucinogens belonging to the group of new psychoactive substances (NPS) may produce high toxicity. NPS, due to their multi-receptors affinity, are extremely dangerous for the human body and mental health. An example of hallucinogens that have been lately responsible for many severe intoxications and deaths are 25X-NBOMes - N-(2-methoxybenzyl)-2,5-dimethoxy-4-substituted phenethylamines, synthetic compounds with strong hallucinogenic properties. 25X-NBOMes exhibit a high binding affinity to serotonin receptors but also to dopamine, adrenergic and histamine receptors. Apart from their influence on perception, many case reports point out systemic and neurological poisoning with these compounds. In humans, the most frequent side effects are tachycardia, anxiety, hypertension and seizures. Moreover, preclinical studies confirm that 25X-NBOMes cause developmental impairments, cytotoxicity, cardiovascular toxicity and changes in behavior of animals. Metabolism of NBOMes seems to be very complex and involves many metabolic pathways. This fact may explain the observed high toxicity. In addition, many analytical methods have been applied in order to identify these compounds and their metabolites. The presented review summarized the current knowledge about 25X-NBOMes, especially in the context of toxicity.

IUPHAR - invited review - Ibogaine - A legacy within the current renaissance of psychedelic therapy.

Ibogaine is a powerful psychoactive substance that not only alters perception, mood and affect, but also stops addictive behaviors. Ibogaine has a very long history of ethnobotanical use in low doses to combat fatigue, hunger and thirst and, in high doses as a sacrament in African ritual contexts. In the 1960's, American and European self-help groups provided public testimonials that a single dose of ibogaine alleviated drug craving, opioid withdrawal symptoms, and prevented relapse for weeks, months and sometimes years. Ibogaine is rapidly demethylated by first-pass metabolism to a long-acting metabolite noribogaine. Ibogaine and its metabolite interact with two or more CNS targets simultaneously and both drugs have demonstrated predictive validity in animal models of addiction. Online forums endorse the benefits of ibogaine as an "addiction interrupter" and present-day estimates suggest that more than ten thousand people have sought treatment in countries where the drug is unregulated. Open label pilot studies of ibogaine-assisted drug detoxification have shown positive benefit in treating addiction. Ibogaine, granted regulatory approval for human testing in a Phase 1/2a clinical trial, joins the current landscape of psychedelic medicines in clinical development.

Behavioral and Pharmacokinetics Studies of N-Methyl-2-Aminoindane (NM2AI) in Mice: An Aminoindane Briefly Used in the Illicit Drug Market.

Drug forums are considered as the main platform sources that have contributed to the increase in NPS popularity, especially for those not yet known to law enforcement and therefore not yet illegal. An example is the new synthetic stimulant NM2AI, which has a very short history of human use and abuse. Little is known regarding this compound, but some information from internet forums and the scientific literature indicates NM2AI as a structural derivate of MDAI, which is known for its entactogenic activity. Indeed, the purpose of this study is to evaluate, for the first time, the in vivo acute effect induced by the intraperitoneal injection of NM2AI (1-10-30-100 mg/kg) in mice. We demonstrate the sensory (by visual placing and object tests) and physiological (core temperature measurement) function variations, nociceptor (by tail pinch test) and strength (grip test) alterations, and sensorimotor (time on rod and mobility) decrease. Moreover, we verify the mild hallucinogenic effect of NM2AI (by startle/prepulse inhibition test). Lastly, we perform a pharmacokinetic study on mice blood samples, highlighting that the main active metabolite of NM2AI is 2-aminoindane (2AI). Taken together, our data confirm the suspected entactogenic activity of NM2AI; however, these in vivo effects appear atypical and less intense with respect to those induced by the classic stimulants, in surprising analogy with what is reported by networked users.

Psilocybin Efficacy and Mechanisms of Action in Major Depressive Disorder: a Review.

PURPOSE OF THE REVIEW: We aim to provide an overview of the current state of knowledge about the efficacy of psilocybin in the treatment of depression, as well as its mechanisms of action. RECENT FINDINGS: Psilocybin has a large, rapid, and persistent clinical effect in the treatment of resistant or end-of-life depression. Tolerance is good, with mild side effects limited to a few hours after dosing. The studies conducted to date have had small sample sizes. One clinical trial has been conducted against a reference treatment (escitalopram) without showing a significant superiority of psilocybin in the main outcome. The neurobiological mechanisms, mostly unknown, differ from those of SSRI antidepressants. Psilocybin represents a promising alternative in the treatment of depression. Further research with larger sample sizes, particularly against reference treatments, is needed to better understand the neurobiological factors of its effects and to investigate its potential for use in everyday practice.

Synthesis of psychotropic alkaloids from Galbulimima.

Efficient syntheses of valuable natural products open gateways from kind learning environments to wicked worlds, where long-term, interdisciplinary research questions can be asked and answered. In this Perspective, we discuss the Galbulimima (GB) alkaloids, metabolites of a rainforest canopy tree that exhibit potent but poorly understood effects in humans, including accounts of hallucination. Recent syntheses from our group have opened up GB alkaloid chemical space for investigation by way of new cross-coupling reactions and gram-scale target production. Although natural product synthesis can be challenging, its objective is obvious. Realization of long-term, enabling goals will be a circuitous journey at the interface of chemistry, pharmacology and neuroscience-a potent mix to foster discovery in the coming century.

Chemical evidence for the use of multiple psychotropic plants in a 1,000-year-old ritual bundle from South America.

Over several millennia, various native plant species in South America have been used for their healing and psychoactive properties. Chemical analysis of archaeological artifacts provides an opportunity to study the use of psychoactive plants in the past and to better understand ancient botanical knowledge systems. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to analyze organic residues from a ritual bundle, radiocarbon dated to approximately 1,000 C.E., recovered from archaeological excavations in a rock shelter located in the Lípez Altiplano of southwestern Bolivia. The site is located at an elevation of ∼3,900 m above sea level and contains evidence of intermittent human occupations during the last 4,000 years. Chemical traces of bufotenine, dimethyltryptamine, harmine, and cocaine, including its degradation product benzoylecgonine, were identified, suggesting that at least three plants containing these compounds were part of the shamanic paraphernalia dating back 1,000 years ago, the largest number of compounds recovered from a single artifact from this area of the world, to date. This is also a documented case of a ritual bundle containing both harmine and dimethyltryptamine, the two primary ingredients of ayahuasca. The presence of multiple plants that come from disparate and distant ecological areas in South America suggests that hallucinogenic plants moved across significant distances and that an intricate botanical knowledge was intrinsic to pre-Columbian ritual practices.

Disparities in Use/Misuse of Specific Illicit and Prescription Drugs among Sexual Minority Adults in a National Sample.

Background: Compared to heterosexual adults, lesbian, gay, and bisexual (LGB) adults have higher rates of any illicit drug use and any prescription drug misuse, yet disparities regarding specific drugs remain poorly characterized. Methods: We examined disparities by sexual identity and sex for 8 illicit and prescription drugs using 2015-2019 National Survey on Drug Use and Health data. Outcomes included past-year use/misuse of cocaine/crack, hallucinogens, inhalants, methamphetamine, heroin, prescription opioids, prescription stimulants, prescription tranquilizers/sedatives, and level of polydrug use/misuse (2 substances; 3+ substances). For each outcome, odds ratios relative to heterosexual adults of same sex were estimated using logistic regression controlling for demographics; significant estimates were interpreted as a disparity. Results: Among gay men, significant disparities were present for all drugs except prescription stimulants and heroin; inhalant use was particularly elevated. Bisexual women exhibited significant disparities for every drug examined, as did bisexual men (except heroin). Among lesbian/gay women, disparities were only present for prescription opioids and stimulants. Relative to heterosexual peers, use of 3+ substances was 3 times higher among gay men and bisexual women and 2 times higher among bisexual men. Conclusions: Consistent with minority stress theory, prevalences of illicit and prescription drug use/misuse were 2-3 times higher among LGB adults than heterosexual adults. Illicit drug use should not be perceived as only impacting gay/bisexual men - bisexual women had similar - or higher - prevalences of hallucinogen, cocaine, methamphetamine, and heroin use. Yet, in contrast to bisexual women, lesbian/gay women did not exhibit disparities for any illicit drugs.

Migraine prevalence in visual snow with prior illicit drug use (hallucinogen persisting perception disorder) versus without.

BACKGROUND AND PURPOSE: This study was undertaken to investigate migraine prevalence in persons with hallucinogen persisting perception disorder (HPPD) presenting as visual snow syndrome (VSS). METHODS: Persons with visual snow as a persisting symptom after illicit drug use (HPPD) were recruited via a Dutch consulting clinic for recreational drug use. A structured interview on (visual) perceptual symptomatology, details of drugs use, and medical and headache history was taken. As a control group, persons with visual snow who had never used illicit drugs prior to onset were included. The primary outcome was lifetime prevalence of migraine. Symptom severity was evaluated by the Visual Snow Handicap Inventory (VHI), a 25-item questionnaire. RESULTS: None of the 24 HPPD participants had migraine, whereas 20 of 37 (54.1%) controls had migraine (p < 0.001). VHI scores did not differ significantly between the two groups; in both groups, the median score was 38 of 100. In most HPPD cases (17/24, 70.9%), visual snow had started after intake of ecstasy; other psychedelic drugs reported included cannabis, psilocybin mushrooms, amphetamine, 4-fluoroamphetamine, 3-methylmethcathinone, 4-Bromo-2,5-dimethoxypenethylamine, and nitrous oxide. CONCLUSIONS: Whereas none of the HPPD participants had migraine, more than half of the visual snow controls without prior use of illicit drugs had migraine. This suggests that at least partly different pathophysiological factors play a role in these disorders. Users of ecstasy and other hallucinogens should be warned of the risk of visual snow. Further studies are needed to enhance understanding of the underlying neurobiology of HPPD and VSS to enable better management of these conditions.

Solid-Contact Potentiometric Sensors Based on Main-Tailored Bio-Mimics for Trace Detection of Harmine Hallucinogen in Urine Specimens.

All-solid-state potentiometric sensors have attracted great attention over other types of potentiometric sensors due to their outstanding properties such as enhanced portability, simplicity of handling, affordability and flexibility. Herein, a novel solid-contact ion-selective electrode (SC-ISE) based on poly(3,4-ethylenedioxythiophene) (PEDOT) as the ion-to-electron transducer was designed and characterized for rapid detection of harmine. The harmine-sensing membrane was based on the use of synthesized imprinted bio-mimics as a selective material for this recognition. The imprinted receptors were synthesized using acrylamide (AA) and ethylene glycol dimethacrylate (EGDMA) as functional monomer and cross-linker, respectively. The polymerization process was carried out at 70 °C in the presence of dibenzoyl peroxide (DBO) as an initiator. The sensing membrane in addition to the solid-contact layer was applied to a glassy-carbon disc as an electronic conductor. All performance characteristics of the presented electrode in terms of linearity, detection limit, pH range, response time and selectivity were evaluated. The sensor revealed a wide linearity over the range 2.0 × 10-7-1.0 × 10-2 M, with a detection limit of 0.02 µg/mL and a sensitivity slope of 59.2 ± 0.8 mV/hamine concentration decade. A 40 mM Britton-Robinson (BR) buffer solution at pH of 6 was used for all harmine measurements. The electrode showed good selectivity towards harmine over other common interfering ions, and maintained a stable electrochemical response over two weeks. After applying the validation requirements, the proposed method revealed good performance characteristics. Method precision, accuracy, bias, trueness, repeatability, reproducibility, and uncertainty were also evaluated. These analytical capabilities support the fast and direct assessment of harmine in different urine specimens. The analytical results were compared with the standard liquid chromatographic method. The results obtained demonstrated that PEDOT/PSS was a promising solid-contact ion-to-electron transducer material in the development of harmine-ISE. The electrodes manifested enhanced stability and low cost, which provides a wide number of potential applications for pharmaceutical and forensic analysis.

Differential signaling signatures evoked by DOI versus lisuride stimulation of the 5-HT2A receptor.

The 5-HT2A receptor is a target for hallucinogenic and non-hallucinogenic ligands that evoke unique behavioral, electrophysiological and molecular consequences. Here, we explored the differential effects of distinct 5-HT2A receptor ligands on signaling pathways downstream to the 5-HT2A receptor. The hallucinogenic 5-HT2A receptor agonist DOI evoked an enhanced signaling response compared to the non-hallucinogenic 5-HT2A receptor agonist lisuride in human/rat 5-HT2AR-EGFP receptor expressing HEK293 cell lines and cortical neuronal cultures. We noted higher levels of phospho-PLC, pPKC, pERK, pCaMKII, pCREB, as well as higher levels of IP3 and DAG production following 5-HT2A receptor stimulation with DOI. Our study reveals distinct signaling signatures, differing in magnitude and kinetics at the 5-HT2A receptor in response to DOI versus lisuride.

Psychedelics as an emerging novel intervention in the treatment of substance use disorder: a review.

Classical psychedelics are a group of drugs characterized by their activation of the serotonin-2A (5-hydroxytryptamine-2A; 5-HT2A) receptor and the unique hallucinogenic and mystical-type experiences that result. After a substantial period of restrictions limiting investigations into the therapeutic potential of psychedelics, a relatively recent recommencement of interest has sparked the burgeoning possibility for these drugs to play a part in the treatment of a wide array of psychopathologies. One of the most promising is in the study of addiction. Evidence has emerged that psychedelic agents may provide a novel avenue for the clinical treatment of patients dealing with substance use disorders (SUD). These serotonergic hallucinogens have displayed remarkable and enduring positive outcomes in this area, even when administered as one or two doses. The neural targets for these psychedelics are varied and underlie a complex mechanism of action-modulating multiple neural networks. It is believed that these agents allow for the reorganization of disordered neural pathways in the default mode network and attenuate maladaptive signaling in mesolimbic reward circuitry. The aim of this review is to examine the current standing of evidence regarding psychedelic psychopharmacology and to provide an overview of the use and effectiveness of these drugs in the treatment of SUD, alcohol use disorder, and for smoking cessation.

In vitro structure-activity relationship determination of 30 psychedelic new psychoactive substances by means of ß-arrestin 2 recruitment to the serotonin 2A receptor.

Serotonergic psychedelics, substances exerting their effects primarily through the serotonin 2A receptor (5-HT2AR), continue to comprise a substantial portion of reported new psychoactive substances (NPS). The exact mechanisms of action of psychedelics still remain to be elucidated further, and certain pathways remain largely unexplored on a molecular level for this group of compounds. A systematic comparison of substances belonging to different subclasses, monitoring the receptor-proximal ß-arrestin 2 recruitment, is lacking. Based on a previously reported in vitro bioassay employing functional complementation of a split nanoluciferase to monitor ß-arrestin 2 recruitment to the 5-HT2AR, we here report on the setup of a stable HEK 293 T cell-based bioassay. Following verification of the performance of this new stable cell system as compared to a system based on transient transfection, the stable expression system was deemed suitable for the pharmacological characterization of psychedelic NPS. Subsequently, it was applied for the in vitro assessment of the structure-activity relationship of a set of 30 substances, representing different subclasses of phenylalkylamine psychedelics, among which 12 phenethylamine derivatives (2C-X), 7 phenylisopropylamines (DOx) and 11 N-benzylderivatives (25X-NB). The resulting potency and efficacy values provide insights into the structure-activity relationship of the tested compounds, overall confirm findings observed with other reported in vitro assays, and even show a significant correlation with estimated common doses. This approach, in which a large series of psychedelic NPS belonging to different subclasses is comparatively tested, using a same assay setup, monitoring a receptor-proximal event, not only gives pharmacological insights, but may also allow prioritization of legal actions related to the most potent -and potentially dangerous- compounds.

25B-NBOMe, a novel N-2-methoxybenzyl-phenethylamine (NBOMe) derivative, may induce rewarding and reinforcing effects via a dopaminergic mechanism: Evidence of abuse potential.

An increasing number of N-2-methoxybenzyl-phenethylamine (NBOMe) derivatives are being misused worldwide, including the potent hallucinogen 2-(4-bromo-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25B-NBOMe). However, the number of studies characterizing the abuse potential and psychopharmacological properties of 25B-NBOMe is limited; thus, we examined its rewarding and reinforcing effects using conditioned place preference (CPP) and self-administration (SA) tests. Pretreatment with SCH23390 (SCH), Haloperidol (HAL), and ketanserin (KS), antagonists of dopamine D1 (DRD1 ), dopamine D2 (DRD2 ), and serotonin 2A (5-HT2A receptor) receptors, respectively, was utilized during a CPP test to investigate the involvement of the dopaminergic and serotonergic systems in 25B-NBOMe-mediated effects. We also examined the effects of 25B-NBOMe on the expression of dopamine-related proteins in the nucleus accumbens (NAcc) and ventral tegmental area (VTA). Then, we measured the dopamine level, phosphorylated CREB (p-CREB), deltaFosB (ΔFosB), and brain-derived neurotrophic factor (BDNF) in the NAcc. In addition, we explored the involvement of 5-HT2A receptors in the 25B-NBOMe-induced head twitch response (HTR). We also examined the effects of 25B-NBOMe on brain wave activity using electroencephalography. 25B-NBOMe elicited CPP and SA. SCH and HAL blocked 25B-NBOMe-induced CPP, whereas KS did not. Moreover, 25B-NBOMe altered the DRD1 , DRD2 , and dopamine transporter expression and increased dopamine levels. It also induced changes in p-CREB, ΔFosB, and BDNF expression. 25B-NBOMe induced HTR and increased 5-HT2A receptor mRNA levels, effects inhibited by KS. Furthermore, 25B-NBOMe altered delta and gamma wave activity, which was normalized by SCH and HAL. These findings show that 25B-NBOMe may induce rewarding and reinforcing effects via a dopaminergic mechanism, suggesting its abuse potential.

Abuse and Effects of Salvia divinorum in a Sample of Patients Hospitalized for Substance Dependence.

The study goal is to document the prevalence of salvia use among patients admitted for detoxification of other illicit drug use and to determine its effect. This cross-sectional study included 47 heavy drug users who were admitted for detoxification of other illicit drug abuse at a psychiatric hospital in Lebanon. The prevalence of salvia use was 66%. The salvia effect started and dissipated rapidly (15 min). No significant difference was found between salvia and non-salvia users in terms of affect, cognition and somaesthesia subscales of the Hallucinogen Rating Scale. Ratings of intensity and volition subscales were higher in non-salvia users than salvia users, while perception score was higher in users. Salvia use was correlated with perceptual alteration and hallucinogenic effects.

Intravenous self-administration of benzydamine, a non-steroidal anti-inflammatory drug with a central cannabinoidergic mechanism of action.

Benzydamine (BZY) is a non-steroidal anti-inflammatory drug used for the topical treatment of inflammations of the oral and vaginal mucosae. Virtually nothing is known about the central pharmacological actions of BZY. Yet there are reports of voluntary systemic overdosage of BZY in drug addicts, resulting in a euphoric, hallucinatory state. In the present study, we investigated the reinforcing properties of BZY in a rat self-administration paradigm. We found that BZY has a powerful reinforcing effect and that this effect is greatly facilitated in animals that already had substance experience, having previously self-administered heroin and cocaine, indicating cross sensitization between BZY and other common drugs of abuse. We then assessed the effect of BZY on prelimbic cortex-to-nucleus accumbens glutamatergic transmission, using field recordings in rat parasagittal brain slices. BZY dose-dependently reduced both field excitatory post synaptic potential amplitude and paired pulse ratio, suggesting a presynaptic mechanism of action. Similarly to the in vivo paradigm, also the electrophysiological effects of BZY were potentiated in slices from animals that had undergone cocaine and heroin self-administration. Furthermore, BZY-induced Long Term Depression (LTD)-like responses in the prelimbic cortex-to-nucleus accumbens circuitry were significantly reduced in the presence of the CB1 receptor antagonist AM251. These findings provide firm evidence of the abuse liability of BZY and suggest a possible cannabinoidergic mechanism of action. Further research is needed in order to give insights into the molecular mechanism underlying BZY psychoactive and reinforcing effects, to better understand its abuse potential.