Psychedelics have emerged as potential therapeutics for substance use disorders, yet preclinical data validating their efficacy remain limited. Here, we investigated the effects of a clinically inspired dose-escalation protocol of psilocybin and ibogaine on extinction and cue-induced reinstatement in Wistar male rats following intravenous cocaine self-administration (IVSA). Rats were trained on a fixed ratio 1 (FR1) schedule with cocaine dose-escalation during the acquisition phase (0.25 mg/kg/infusion, followed by 0.5 mg/kg/infusion). Following acquisition, animals were randomised into treatment groups and then subjected to 10 days of extinction. Psilocybin (1.25 mg/kg and 5 mg/kg) or ibogaine (10 mg/kg and 40 mg/kg) was administered subcutaneously and intraperitoneally, respectively, on extinction days 1 and 5. A cue-induced reinstatement test was conducted 6 days after the last treatment. Both treatments significantly modulated behaviour during extinction; psilocybin reduced active lever pressing 1 day after the second dose, with a nonsignificant reduction already apparent after the first dose, while the effect of ibogaine was significant even after the first administration. However, neither compound significantly altered reinstatement behaviour, although psilocybin showed a trend toward attenuation. The applied treatment had no side effects on general locomotor activity or anxiety-like behaviour, as measured in the open field test 24 h after each administration. These findings support a role for psilocybin and ibogaine in facilitating extinction learning and suggest possible protective effects against relapse, warranting further research into their antiaddictive efficacy.

Psilocin (4-hydroxy-N, N-dimethyltryptamine) is a substituted tryptamine alkaloid and a nonselective serotonergic agonist acting predominantly at 5-HT2A/C receptors, with substantial binding to 5-HT1A and 5-HT2B receptors. Microdosing is the practice of taking a very small, sub-perceptual dose, typically 5% to 10% of a full recreational dose, to improve mood, creativity, and focus without hallucinogenic effects. However, rigorous preclinical evidence for its behavioral and neurobiological effects remains limited. We therefore examined whether chronic psilocin microdosing alters behavior and dentate gyrus (DG) cell proliferation in adult male Wistar rats. Psilocin was administered subcutaneously at 0.05 or 0.075 mg/kg. Animals received six doses of psilocin or saline on alternate days over 18 days prior to the first behavioral assessment, and microdosing on alternate days continued between behavioral tasks for five weeks. To minimize acute drug effects, all behavioral assessments were performed 48 h after the preceding dose. Animals were tested sequentially in the Elevated Plus Maze, Hole-Board, Open Field, Social Interaction, and modified Forced Swim Test, with six-day intervals between tests. DG cell proliferation was quantified by BrdU and Ki-67 immunohistochemistry. Across this regimen, psilocin microdosing did not measurably affect locomotor activity, depressive-like behavior, sociability, or novelty seeking, and it did not increase DG proliferation by either marker. A small anxiogenic effect was detected in the Elevated Plus Maze. These data indicate that, under the present dosing schedule and endpoints, chronic psilocin microdosing produces limited behavioral effects and does not enhance hippocampal progenitor proliferation in rats.

N-benzylphenethylamines are a class of new psychoactive substances (NPS) that are increasingly being used as recreational drugs with a wide range of adverse effects, possibly even death. Currently, the appearance of new N-benzylphenethylamines far outweighs the studies of their metabolism. One of such compounds, 25E-NBOH, has been on the market for the last seven years but its pharmacological and toxicological effects have not yet been thoroughly reported. To provide a basis for such studies, we investigated 25E-NBOH metabolism in three different systems: human liver microsomes, Wistar rat urine, and Cunninghamella elegans fungus, which contains enzymes similar to those found in mammals and serves as an environmentally sustainable and ethically favourable alternative to animal-based metabolic models. Untargeted LC-HRMS/MS was used to detect phase I and phase II 25E-NBOH metabolites in all three systems. A total of 56 metabolites were annotated, many of which occurred in multiple isomeric forms. Despite metabolic differences between the systems, several abundant metabolites were found in all of them. The primary metabolic pathways detected were hydroxylation at various positions, O-demethylation, and N-debenzylation, followed by conjugation with glucuronic acid, sulphate, or glucose. To confirm the presence of metabolites, we synthesised and measured ten substances under the same LC-HRMS/MS conditions as the real samples. Seven of these were successfully matched to detected metabolites based on retention time and MS/MS spectra, enabling structural assignment and isomer distinction. Additionally, the identity of 2C-E, a known psychoactive substance, was confirmed as one of the metabolites using a commercial reference standard. Lastly, we report the structures of three main biomarkers, suggested by both this study and prior literature. This study provides the first comprehensive in vitro and in vivo metabolic profile of 25E-NBOH, identifying the structures of specific metabolites using in-house synthesised reference standards and proposing structures for the main biomarkers. These findings establish a solid foundation for future pharmacological and toxicological studies, supporting clinicians in the accurate diagnosis of intoxication cases.

Methoxphenidine (MXP) is classified as a new psychoactive substance that has recently emerged on the illicit drug market. Understanding the pharmacological and behavioural profiles of newly emerging drugs is essential for a better understanding of their psychotropic effects and potential toxicity. Therefore, in this study, we investigated a broad range of effects of acute MXP administration: pharmacokinetics in the brain and serum; behaviour (open field and prepulse inhibition), systemic toxicity (lethal dose; LD 50), and histopathology changes in parenchymal organs of Wistar rats. MXP rapidly crossed the blood-brain barrier, reaching peak median concentrations in both serum and brain 30 min post-administration, followed by an elimination phase with a half-life of 2.15 h. Locomotor activity in the open field test displayed a dose-response effect at low to moderate doses (10-20 mg/kg MXP). At higher doses (40 mg/kg), locomotor activity decreased. All doses of MXP significantly disrupted prepulse inhibition and the effect was present during the onset of its action as well as 60 min after treatment. Additionally, MXP demonstrated moderate acute toxicity, with an estimated LD50 of 500 mg/kg when administered subcutaneously. In summary, MXP exhibited a profile similar to typical dissociative anesthetics, producing stimulant and anxiogenic effects at lower doses, sedative effects at higher doses, and disrupting sensorimotor gating. The accumulation of MXP in brain tissue is likely to contribute to acute intoxication in humans, potentially leading to negative experiences. Our findings highlight the potentially dangerous effects of recreational MXP use and underscore the risks of inducing serious adverse health outcomes.

BACKGROUND:Hexahydrocannabinol (HHC) is a new psychoactive substance known for its mind-altering effects and temporary legal status. It is widely used in parts of the Europe and United Kingdom as a legal alternative to ∆9-tetrahydrocannabinol, yet little research has explored its effects and safety. This study examined how HHC is processed in the body, its toxicity, and its impact on behavior in male Wistar rats. METHODS:A 1:1 mixture of (9R)-HHC and (9S)-HHC was administered via intragastric gavage at doses of 1, 5, and 10 mg/kg. Behavioral effects were assessed using the open field test and the prepulse inhibition of acoustic startle response. RESULTS:Two hours after the highest dose (10 mg/kg), peak concentrations of HHC were detected in blood and brain tissue. The Organization for Economic Co-operation and Development 423 toxicity test classified HHC as a Category 4 substance, estimating a lethal dose of 1000 mg/kg. Compared to controls (administered by sunflower oil), 10 mg/kg HHC reduced movement, increased anxiety, and impaired sensory processing. CONCLUSIONS:Overall, HHC crosses the blood-brain barrier, exhibits mild toxicity, and induces behavioral effects similar to tetrahydrocannabinol. Its dose-dependent anxiogenic properties and impact on information processing highlight the importance of the appropriate dosing in any potential therapeutic use.

BACKGROUND:Recent evidence suggests that psychedelics can induce rapid and long-lasting antidepressant effects. The generally acknowledged explanation for these traits is the phenomenon of neuroplasticity, although the exact underlying molecular mechanisms remain unclear. AIMS:This study investigates the effects of psilocin, lysergic acid diethylamide (LSD) and N,N-dimethyltryptamine (DMT) on synaptogenesis and immediate early genes (IEGs) expression in direct comparison with ketamine, fluoxetine and lithium after acute (1 h) and/or prolonged (24 h) treatment in vitro. METHODS:Rat primary cortical cultures were treated with 10 µM psilocin, 1 µM LSD, 90 µM DMT, 1 µM ketamine, 10 µM fluoxetine and 5 mM lithium. Analysis of synaptic puncta was performed; puncta of presynaptic marker synapsin I/II, postsynaptic density protein 95 (PSD-95) and their co-localization (established synapse) were assessed 24 h after drug treatment. Next, expressions of IEGs encoding activity-regulated cytoskeleton-associated protein (Arc), early growth response 1 (Egr1), and neuronal PAS (Per-Arnt-Sim) domain protein 4 (Npas4) were analysed 1 and 24 h after drug treatments. RESULTS:Psilocin increased synaptic puncta count and induced Arc expression. The effect to promote synaptogenesis was comparable to ketamine and lithium; ketamine additionally increased PSD-95 puncta count. LSD and DMT did not induce any significant effects. Interestingly, fluoxetine had no effect on synaptic puncta count, but upregulated Egr1 and Npas4. CONCLUSIONS:Psilocin demonstrated synaptogenic effects comparable to those of ketamine and lithium, and acutely upregulated IEG Arc expression, adding another piece of evidence to its profile as a promising therapeutic agent.

Baeocystin is a naturally occurring tryptamine-based compound found in various psychoactive mushrooms, including in several species of Psilocybe genus. Due to its structural similarity to psilocybin, which has shown a therapeutic potential in treatment of psychiatric disorders, there is a growing interest in investigating whether baeocystin exhibits comparable effects. This study investigated the pharmacokinetic profile and acute behavioural effects of baeocystin in Wistar rats. Behavioural assessments including locomotor activity and its spatial characteristics (in the open field test) and sensorimotor gating measured by prepulse inhibition were evaluated after subcutaneous administration of 1.25 or 5 mg/kg baeocystin. Pharmacokinetics and brain-serum ratios were analysed after the 5 mg/kg sc. dose. Pharmacokinetics demonstrated that both baeocystin and its metabolite, norpsilocin, have a very limited ability to cross the blood-brain barrier. Consistent with the pharmacokinetic profile, baeocystin had no significant effects on locomotor activity, exploratory behaviour, anxiety-like responses, or sensorimotor gating at doses of either 1.25 or 5 mg/kg. In conclusion, our results suggest that baeocystin has minimal to no behavioural effects in rats, probably due to its poor permeability across the blood-brain barrier. This limited penetration may account for its negligible neurobiological and psychedelic activity.

Background: Psychedelics, particularly psilocin, are increasingly being studied for their mind-altering effects and potential therapeutic applications in psychiatry. Visual hallucinations, especially the illusion of motion in static images, are a hallmark of their action. Despite growing interest, the underlying mechanisms remain poorly understood, as their systematic evaluation in both humans and animals is challenging. Methods: To investigate psilocin-induced visual distortions, we designed a 2-choice visual discrimination task. Human participants and male rats indicated whether an image appeared static or moving while the image either actually moved or did not. In humans, performance was compared with self-reported hallucination intensity, Altered States of Consciousness scale scores, and psilocin plasma levels. Rats were tested in 2 distinct tasks, a luminance-based task and a motion-based task. Their performance was evaluated alongside decision time. Results: Both species exhibited significant impairment in distinguishing static from dynamic visual stimuli while under psilocin’s influence. In humans, this impairment followed the time course of psilocin plasma levels and hallucination intensity. In rats, psilocin selectively impaired performance in the motion-based task, while performance in the luminance-based task remained intact, indicating a specific effect on visual perception. Decision time was linked to discrimination impairment. Conclusions: Psilocin impaired static-dynamic discrimination in both species, providing the first evidence that rats experience visual distortions similar to those reported by humans. The correlations between discrimination impairment, psilocin levels, and hallucination intensity in humans reinforce psilocin’s effects on visual perception. This approach provides a valuable tool for investigating the neurobiology of altered visual perception in drug-induced states and psychiatric conditions.

Rationale: Mescaline is a classical psychedelic compound with a phenylethylamine structure that primarily acts on serotonin 5-HT2A/C receptors, but also binds to 5-HT1A and 5-HT2B receptors. Despite being the first psychedelic ever isolated and synthesized, the precise role of different serotonin receptor subtypes in its behavioral pharmacology is not fully understood. Objectives: In this study, we aimed to investigate how selective antagonists of 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT1A receptors affect the behavioral changes induced by subcutaneous administration of mescaline (at doses of 10, 20, and 100 mg/kg) in rats. Methods: We used adult male Wistar rats in all our experiments. We evaluated locomotor activity using the open field test, and assessed sensorimotor gating deficits by measuring prepulse inhibition (PPI) of acoustic startle reaction (ASR). Results: While the highest dose of mescaline induced hyperlocomotion, which almost all the other antagonists reversed, the PPI deficits were selectively normalized by the 5-HT2A antagonist. The 5-HT2C antagonist partially reversed the small decrease in locomotor activity induced by lower doses of mescaline. Conclusion: Our findings suggest that mescaline-induced changes in behavior are primarily mediated by the 5-HT2A receptor subtype, with less pronounced contributions from the 5-HT2C receptor. The other antagonists had limited effects. ### Competing Interest Statement TP and JH declare to have shares in Psyon s.r.o., and have founded PSYRES - Psychedelic Research Foundation. TP has shares in Spolecnost pro podporu neurovedniho vyzkumu s.r.o. and reports consulting fees from GH Research and CB21-Pharma outside the submitted work. TP and/or JH are/were involved in Compass Pathways, MAPS, GH-Research, Ketabon clinical trials with psilocybin, MDMA, 5-MeO-DMT, ketamine and MDMA outside the submitted work.

RATIONALE:Mescaline is a classical psychedelic compound with a phenylethylamine structure that primarily acts on serotonin 5-HT2A/C receptors, but also binds to 5-HT1A and 5-HT2B receptors. Despite being the first psychedelic ever isolated and synthesized, the precise role of different serotonin receptor subtypes in its behavioral pharmacology is not fully understood. OBJECTIVES:In this study, we aimed to investigate how selective antagonists of 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT1A receptors affect the behavioral changes induced by subcutaneous administration of mescaline (at doses of 10, 20, and 100 mg/kg) in rats. METHODS:We used adult male Wistar rats in all our experiments. We evaluated locomotor activity using the open field test, and assessed sensorimotor gating deficits by measuring prepulse inhibition (PPI) of acoustic startle reaction (ASR). RESULTS:While the highest dose of mescaline induced hyperlocomotion (p < 0.001), which almost all the other antagonists reversed (p < 0.05-0.001), the PPI deficits were selectively normalized by the 5-HT2A antagonist (p < 0.05-0.01). The 5-HT2C antagonist partially reversed the small PPI deficit induced by lower doses of mescaline (p = 0.0017). CONCLUSION:Our findings suggest that mescaline-induced changes in behavior are primarily mediated by the 5-HT2A receptor subtype, with less pronounced contributions from the 5-HT2C receptor. The other antagonists had limited effects.

Introduction: N-2-methoxy-benzylated (“NBOMe”) analogues of phenethylamine are a group of new psychoactive substances (NPS) with reported strong psychedelic effects in sub-milligram doses linked to a number of severe intoxications, including fatal ones. In our present work, we provide a detailed investigation of pharmacokinetics and acute behavioural effects of 2C-B-Fly-NBOMe (2-(8-bromo-2,3,6,7-tetrahydrobenzo [1,2-b:4,5-b′]difuran-4-yl)-N-[(2-methoxybenzyl]ethan-1-amine), an analogue of popular psychedelic entactogen 2C-B (4-Bromo-2,5-dimethoxyphenethylamine). Methods: All experiments were conducted on adult male Wistar rats. Pharmacokinetic parameters of 2C-B-Fly-NBOMe (1 mg/kg subcutaneously; s. c.) in blood serum and brain tissue were analysed over 24 h using liquid chromatography-mass spectrometry (LC/MS). For examination of behavioural parameters in open field test (OFT) and prepulse inhibition (PPI) of acoustic startle reaction (ASR), 2C-B-Fly-NBOMe (0.2, 1 and 5 mg/kg s. c.) was administered in two temporal onsets: 15 and 60 min after administration. Thermoregulatory changes were evaluated in individually and group-housed animals over 8 h following the highest dose used in behavioural experiments (5 mg/kg s. c.). Results: Peak drug concentrations were detected 30 and 60 min after the drug application in serum (28 ng/ml) and brain tissue (171 ng/g), respectively. The parental compound was still present in the brain 8 h after administration. Locomotor activity was dose-dependently reduced by the drug in both temporal testing onsets. ASR was also strongly disrupted in both temporal onsets, drug’s effect on PPI was weaker. 2C-B-Fly-NBOMe did not cause any significant thermoregulatory changes. Discussion: Our results suggest that 2C-B-Fly-NBOMe penetrates animal brain tissue in a relatively slow manner, induces significant inhibitory effects on motor performance, and attenuates sensorimotor gating. Its overall profile is similar to closely related analogue 2C-B and other NBOMe substances.

N-(2-methoxybenzyl)phenethylamines (NBOMes) are a family of potent 5-HT2A agonists containing substances emerging on the illicit drug market as a replacement for N,N-diethyllysergamide (LSD). Despite the increasing use of NBOMes for diagnostic, research and recreational purposes, only a limited number of studies have focussed on their in vivo effect. Here, we investigated pharmacokinetics, systemic toxicity, thermoregulation in individually and group-housed animals, and acute behavioural effects after subcutaneous administration of 2,5-dimethoxy-4-(2-((2-methoxybenzyl)amino)ethyl)benzonitrile (25CN-NBOMe; 0.2, 1, and 5 mg/kg) in Wistar rats. Drug concentration peaked 1 h after the administration of 5 mg/kg in both blood serum and brain tissue with a half-life of 1.88 and 2.28 h, respectively. According to Organisation for Economic Co-operation and Development 423 toxicity assay, the drug is classified into category 3 with a lethal dose of 300 mg/kg and an estimated LD50 value of 200 mg/kg. Histological examination of organs collected from rats injected with the lethal dose revealed subtle pathological changes, highly suggestive of acute cardiovascular arrest due to malignant arrhythmia. Altered thermoregulation after 5 mg/kg was demonstrated by reduced body temperature in individually housed rats (p < 0.01). Behavioural effects assessed by the Open Field test and Prepulse Inhibition of Startle Response revealed that the two lower doses (0.2 and 1 mg/kg) caused a reduction in locomotor activity (p < 0.01), increased anxiety (p < 0.05) and 5 mg/kg additionally impaired sensorimotor gating (p < 0.001). In summary, 25CN-NBOMe readily passes the blood-brain barrier and exhibits a moderate level of toxicity and behavioural effect comparable with other NBOMes.