Underlying pharmacological mechanisms of psilocin-induced broadband desynchronization and disconnection of EEG in rats.

Introduction: Psilocybin is one of the most extensively studied psychedelic drugs with a broad therapeutic potential. Despite the fact that its psychoactivity is mainly attributed to the agonism at 5-HT2A receptors, it has high binding affinity also to 5-HT2C and 5-HT1A receptors and indirectly modulates the dopaminergic system. Psilocybin and its active metabolite psilocin, as well as other serotonergic psychedelics, induce broadband desynchronization and disconnection in EEG in humans as well as in animals. The contribution of serotonergic and dopaminergic mechanisms underlying these changes is not clear. The present study thus aims to elucidate the pharmacological mechanisms underlying psilocin-induced broadband desynchronization and disconnection in an animal model. Methods: Selective antagonists of serotonin receptors (5-HT1A WAY100635, 5-HT2A MDL100907, 5-HT2C SB242084) and antipsychotics haloperidol, a D2 antagonist, and clozapine, a mixed D2 and 5-HT receptor antagonist, were used in order to clarify the underlying pharmacology. Results: Psilocin-induced broadband decrease in the mean absolute EEG power was normalized by all antagonists and antipsychotics used within the frequency range 1-25 Hz; however, decreases in 25-40 Hz were influenced only by clozapine. Psilocin-induced decrease in global functional connectivity and, specifically, fronto-temporal disconnection were reversed by the 5-HT2A antagonist while other drugs had no effect. Discussion: These findings suggest the involvement of all three serotonergic receptors studied as well as the role of dopaminergic mechanisms in power spectra/current density with only the 5-HT2A receptor being effective in both studied metrics. This opens an important discussion on the role of other than 5-HT2A-dependent mechanisms underlying the neurobiology of psychedelics.

Acute pharmacological profile of 2C-B-Fly-NBOMe in male Wistar rats-pharmacokinetics, effects on behaviour and thermoregulation.

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.

Effects of intra-hippocampal corticosterone and sleep on consolidation of memories of aversive experience in rats.

Formation and consolidation of memories for highly stressful (traumatic) events is a complex process that involves interplay between multiple memory systems and has implications for etiology and treatment of stress- and trauma-related disorders. Here we study effects of sleep/wake states and high intra-hippocampal corticosterone on consolidation of aversive contextual memories, as well as consolidation of association between auditory unpaired phasic background cues and fear response in rats. Animals were implanted with EEG and EMG electrodes for sleep assessment and cannulas for intra-hippocampal corticosterone application. They were familiarized to a "safe box" and then trained in a fear conditioning paradigm in a distinct "shock box" with a prominent unpaired phasic background auditory cue. Immediately after conditioning, animals received bilateral intra-hippocampal saline (1 µl) or corticosterone (10 ng in 1 µl) injection and were either allowed to sleep or were kept awake for a following two-hour consolidation period. Memory tests 24 h later revealed that the saline-injected animals that slept during consolidation had significantly stronger fear responses in the shock box compared to the safe box as well as increased fear response in response to the auditory cue. Lack of sleep during the consolidation period in saline injected animals led to generalization of the fear response to the safe context, while association between auditory cue and fear response was preserved. High intra-hippocampal corticosterone levels during memory consolidation led to generalization of fear response to the safe context, regardless of sleep/wake state, while enhancement of response to auditory cue was not observed. Our results show how manipulation of conditions during consolidation can lead to greatly variable memories for an aversive episode with distinct behavioral outcomes. Observed overgeneralization of fear to safe context and altered fear response to background phasic cue has implications for understanding etiology of pathological memory alternations in stress-related conditions e.g., in posttraumatic stress disorder in humans.

Pharmacokinetics, systemic toxicity, thermoregulation and acute behavioural effects of 25CN-NBOMe.

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.

Psilocin, LSD, mescaline, and DOB all induce broadband desynchronization of EEG and disconnection in rats with robust translational validity.

Serotonergic psychedelics are recently gaining a lot of attention as a potential treatment of several neuropsychiatric disorders. Broadband desynchronization of EEG activity and disconnection in humans have been repeatedly shown; however, translational data from animals are completely lacking. Therefore, the main aim of our study was to assess the effects of tryptamine and phenethylamine psychedelics (psilocin 4 mg/kg, LSD 0.2 mg/kg, mescaline 100 mg/kg, and DOB 5 mg/kg) on EEG in freely moving rats. A system consisting of 14 cortical EEG electrodes, co-registration of behavioral activity of animals with subsequent analysis only in segments corresponding to behavioral inactivity (resting-state-like EEG) was used in order to reach a high level of translational validity. Analyses of the mean power, topographic brain-mapping, and functional connectivity revealed that all of the psychedelics irrespective of the structural family induced overall and time-dependent global decrease/desynchronization of EEG activity and disconnection within 1-40 Hz. Major changes in activity were localized on the large areas of the frontal and sensorimotor cortex showing some subtle spatial patterns characterizing each substance. A rebound of occipital theta (4-8 Hz) activity was detected at later stages after treatment with mescaline and LSD. Connectivity analyses showed an overall decrease in global connectivity for both the components of cross-spectral and phase-lagged coherence. Since our results show almost identical effects to those known from human EEG/MEG studies, we conclude that our method has robust translational validity.

mTOR inhibitor improves autistic-like behaviors related to Tsc2 haploinsufficiency but not following developmental status epilepticus.

BACKGROUND: Tuberous sclerosis complex (TSC), a multi-system genetic disorder often associated with autism spectrum disorder (ASD), is caused by mutations of TSC1 or TSC2, which lead to constitutive overactivation of mammalian target of rapamycin (mTOR). In several Tsc1+/- and Tsc2+/- animal models, cognitive and social behavior deficits were reversed by mTOR inhibitors. However, phase II studies have not shown amelioration of ASD and cognitive deficits in individuals with TSC during mTOR inhibitor therapy. We asked here if developmental epilepsy, common in the majority of individuals with TSC but absent in most animal models, could explain the discrepancy. METHODS: At postnatal day P12, developmental status epilepticus (DSE) was induced in male Tsc2+/- (Eker) and wild-type rats, establishing four experimental groups including controls. In adult animals (n = 36), the behavior was assessed in the paradigms of social interaction test, elevated plus-maze, light-dark test, Y-maze, and novel object recognition. The testing was carried out before medication (T1), during a 2-week treatment with the mTOR inhibitor everolimus (T2) and after an 8-week washing-out (T3). Electroencephalographic (EEG) activity was recorded in a separate set of animals (n = 18). RESULTS: Both Tsc2+/- mutation and DSE caused social behavior deficits and epileptiform EEG abnormalities (T1). Everolimus led to a persistent improvement of the social deficit induced by Tsc2+/-, while deficits related to DSE did not respond to everolimus (T2, T3). CONCLUSIONS: These findings may contribute to an explanation why ASD symptoms in individuals with TSC, where comorbid early-onset epilepsy is common, were not reliably ameliorated by mTOR inhibitors in clinical studies.

Electrical Source Imaging in Freely Moving Rats: Evaluation of a 12-Electrode Cortical Electroencephalography System.

This work presents and evaluates a 12-electrode intracranial electroencephalography system developed at the National Institute of Mental Health (Klecany, Czech Republic) in terms of an electrical source imaging (ESI) technique in rats. The electrode system was originally designed for translational research purposes. This study demonstrates that it is also possible to use this well-established system for ESI, and estimates its precision, accuracy, and limitations. Furthermore, this paper sets a methodological basis for future implants. Source localization quality is evaluated using three approaches based on surrogate data, physical phantom measurements, and in vivo experiments. The forward model for source localization is obtained from the FieldTrip-SimBio pipeline using the finite-element method. Rat brain tissue extracted from a magnetic resonance imaging template is approximated by a single-compartment homogeneous tetrahedral head model. Four inverse solvers were tested: standardized low-resolution brain electromagnetic tomography, exact low-resolution brain electromagnetic tomography (eLORETA), linear constrained minimum variance (LCMV), and dynamic imaging of coherent sources. Based on surrogate data, this paper evaluates the accuracy and precision of all solvers within the brain volume using error distance and reliability maps. The mean error distance over the whole brain was found to be the lowest in the eLORETA solution through signal to noise ratios (SNRs) (0.2 mm for 25 dB SNR). The LCMV outperformed eLORETA under higher SNR conditions, and exhibiting higher spatial precision. Both of these inverse solvers provided accurate results in a phantom experiment (1.6 mm mean error distance across shallow and 2.6 mm across subcortical testing dipoles). Utilizing the developed technique in freely moving rats, an auditory steady-state response experiment provided results in line with previously reported findings. The obtained results support the idea of utilizing a 12-electrode system for ESI and using it as a solid basis for the development of future ESI dedicated implants.

Mephedrone (4-Methylmethcathinone): Acute Behavioral Effects, Hyperthermic, and Pharmacokinetic Profile in Rats.

Mephedrone (MEPH) is a synthetic cathinone derivative with effects that mimic MDMA and/or cocaine. Our study in male Wistar rats provides detailed investigations of MEPH's and its primary metabolite nor-mephedrone's (nor-MEPH) pharmacokinetics and bio-distribution to four different substrates (serum, brain, lungs, and liver), as well as comparative analysis of their effects on locomotion [open field test (OFT)] and sensorimotor gating [prepulse inhibition of acoustic startle reaction (PPI ASR)]. Furthermore, in order to mimic the crowded condition where MEPH is typically taken (e.g., clubs), the acute effect of MEPH on thermoregulation in singly- and group-housed rats was evaluated. Pharmacokinetics of MEPH and nor-MEPH after MEPH (5 mg/kg, sc.) were analyzed over 8 h using liquid chromatography with mass spectrometry. MEPH (2.5, 5, or 20 mg/kg, sc.) and nor-MEPH (5 mg/kg, sc.) were administered 5 or 40 min before the behavioral testing in the OFT and PPI ASR; locomotion and its spatial distribution, ASR, habituation and PPI itself were quantified. The effect of MEPH on rectal temperature was measured after 5 and 20 mg/kg, sc. Both MEPH and nor-MEPH were detected in all substrates, with the highest levels detected in lungs. Mean brain: serum ratios were 1:1.19 (MEPH) and 1:1.91 (nor-MEPH), maximum concentrations were observed at 30 min; at 2 and 4 h after administration, nor-MEPH concentrations were higher compared to the parent drug. While neither of the drugs disrupted PPI, both increased locomotion and affected its spatial distribution. The effects of MEPH were dose dependent, rapid, and short-lasting, and the intensity of locomotor stimulant effects was comparable between MEPH and nor-MEPH. Despite the disappearance of behavioral effects within 40 min after administration, MEPH induced rectal temperature elevations that persisted for 3 h even in singly housed rats. To conclude, we observed a robust, short-lasting, and most likely synergistic stimulatory effect of both drugs which corresponded to brain pharmacokinetics. The dissociation between the duration of behavioral and hyperthermic effects is indicative of the possible contribution of nor-MEPH or other biologically active metabolites. This temporal dissociation may be related to the risk of prolonged somatic toxicity when stimulatory effects are no longer present.