Behavioral, neurochemical and pharmaco-EEG profiles of the psychedelic drug 4-bromo-2, 5-dimethoxyphenethylamine (2C-B) in rats

Páleníček and colleagues describe 4-bromo-2,5-dimethoxyphenethylamine (2C-B), a recreational phenylethylamine with reported psychedelic and entactogenic effects, as one of a family of “2Cs” whose human use outstrips detailed preclinical characterisation. Earlier literature provided limited behavioural data (one older human study and a single avian study) and little information on monoamine neurotransmission; binding data suggested partial agonism at serotonin 5-HT2A/2C and 5-HT1A/B receptors but left gaps about dopaminergic effects and whole-brain functional consequences. The authors positioned 2C-B between classic psychedelics and entactogens such as MDMA, and noted the need to assess behavioral, neurochemical and electrophysiological actions in a rodent model to better understand its pharmacology and potential risks. The study set out to provide an integrated characterisation in rats, combining open-field locomotion and sensorimotor gating (prepulse inhibition of acoustic startle, PPI), microdialysis of dopamine and metabolites in nucleus accumbens (NAc), and quantitative EEG (QEEG) from 12 cortical electrodes. Amphetamine was included as a comparator stimulant to help dissociate entactogenic/serotonergic from stimulant/dopaminergic effects. The investigators emphasised time-dependent assessments to capture both onset and peak-brain-concentration phases after subcutaneous dosing, and they aimed to relate behavioural changes to neurochemical and EEG markers of brain functional connectivity.

Adult male Wistar rats (200-250 g for behavioural/EEG work; 280-330 g for microdialysis surgery) were used. Animals were housed in pairs on a 12-h light/dark cycle and acclimatised for 7–10 days. Typical group sizes were 9–11 per behavioural treatment, 7 animals planned per NAc compartment for microdialysis (though fewer were included in final analyses), and 12 animals per EEG group were introduced to surgery with variable final inclusion due to data quality. 2C-B hydrochloride (≥98% purity) was administered subcutaneously at 2.5, 10, 25 or 50 mg/kg depending on the experiment. Microdialysis used 25 mg/kg; EEG recordings used 10 and 50 mg/kg. D-amphetamine sulfate served as a comparator (behavioural doses 1 and 4 mg/kg, EEG 4 mg/kg). Behavioural testing occurred in two timing paradigms to capture early and later drug effects: testing began either 15 min or 60 min after dosing, with independent groups for each timing. Vehicle controls were saline administered s.c. in the same volumes. Locomotion and spatial behaviour were assessed in an open-field arena tracked by EthoVision for 30 min, with locomotor data parsed in 5-min bins. Spatial metrics included thigmotaxis (proportion of peripheral zone entries) and time spent in the central 9 zones. Sensorimotor gating was evaluated using prepulse inhibition (PPI) of acoustic startle reaction (ASR) in calibrated startle chambers; PPI was calculated as percentage inhibition of startle amplitude when a weak prepulse preceded the pulse. Animals with very low baseline startle (<10 arbitrary units) were excluded as non-responders. Microdialysis probes were stereotaxically implanted 2 mm above NAc core and shell, perfused with artificial CSF at 2 μl/min, and dialysates collected at 20-min intervals. After three baseline samples, 2C-B 25 mg/kg s.c. was given and sampling continued for a total of 5 h. Probe placements were histologically verified; due to misplacements or perfusion failures the final microdialysis sample comprised 4 animals for core and 5 for shell, which the authors subsequently pooled for analysis. Dopamine (DA) and metabolites (DOPAC, HVA, 3-MT) were quantified by LC–MS using a Varian HPLC coupled to a triple-quadrupole mass spectrometer with specific precursor→product transitions for each analyte. EEG methods involved stereotactic implantation of 14 silver electrodes (12 active cortical leads across frontal, motor, parietal and temporal sites, plus reference and ground). EEGs were recorded in freely moving rats with baseline 10-min pre-dose recording followed by post-dose recording (total 75 min for 2C-B/vehicle, 45 min for amphetamine). EEG preprocessing used FIR bandpass filtering (0.5–45 Hz), selection of artefact-free 10-min segments at defined post-dose intervals, and FFT spectral analysis at 0.5-Hz resolution. Spectral bands analysed included delta (1–4 Hz), theta (4–7 Hz), alpha (8–12 Hz), beta (12–25 Hz), high beta (25–30 Hz) and gamma. Coherence (a measure of functional connectivity between electrode pairs) was computed for multiple intra- and interhemispheric electrode pairs. Behavioural data were analysed by two-way ANOVA or repeated-measures ANOVA as appropriate, with Newman–Keuls post hoc tests; microdialysis used Friedman repeated-measures ANOVA on ranks with Wilcoxon post hoc tests. EEG power used one-way repeated-measures ANOVA and coherence used paired t tests with Familywise Error correction for multiple comparisons where stated; significance was set at p<0.05 (corrected thresholds applied for coherence).

Behaviour: Páleníček and colleagues observed a time- and dose-dependent, biphasic locomotor profile for 2C-B. When administered 15 min before testing, 2C-B 25 mg/kg reduced total locomotion (post hoc p<0.05) with a trend for 50 mg/kg (p=0.06). Conversely, when testing began 60 min after dosing, higher doses (10, 25 and 50 mg/kg) produced increased locomotion from the third 5-min interval onward compared with vehicle (treatment×time interactions reported, p<0.01). Thigmotaxis showed no treatment effects, while time spent in the centre increased markedly after 2C-B 50 mg/kg given 15 min prior (vehicle 68.8 s vs 2C-B 282.4 s, p<0.001). Acoustic startle and PPI: 2C-B reduced ASR at both 15 and 60 min (treatment effect p<0.001; effect more pronounced at 15 min). Prepulse inhibition was disrupted by multiple doses: at 15 min doses of 10, 25 and 50 mg/kg lowered PPI (p values ranged from <0.05 to <0.001), and at 60 min doses of 2.5, 10 and 50 mg/kg also reduced PPI (p<0.05-p<0.01), with 25 mg/kg showing only a trend (p=0.06). Amphetamine produced prototypical stimulant results: both 1 and 4 mg/kg increased locomotion (one-way ANOVA p<0.001) and amphetamine 4 mg/kg increased ASR and disrupted PPI (p<0.05). Microdialysis: 2C-B 25 mg/kg produced robust increases in extracellular dopamine in pooled NAc samples, reaching up to 4.5× baseline (significant by Friedman ANOVA and post hoc tests, p<0.05–0.001) and persisting for approximately 120 min. Metabolites HVA and 3-MT rose substantially as well (HVA up to 6× baseline, 3-MT up to 7× baseline; p<0.05–0.001). In contrast, DOPAC levels decreased, which the authors interpret as evidence for monoamine oxidase (MAO) inhibition. Statistical tests for DA and metabolites were significant (Friedman χ2 values reported, p<0.001). EEG absolute power spectra: Low-dose 2C-B (10 mg/kg) decreased mean power in higher frequency bands during both early (15–25 min) and later (55–65 min) windows: beta reduced to 88% of baseline, high beta to 77% and gamma to 74% (one-way RM ANOVAs significant, p values <0.05 to <0.001). High-dose 2C-B (50 mg/kg) produced an initial decrease in beta/high beta/gamma (to ~76% of baseline at 15–25 min) but later showed an increase in theta power to 135% of baseline at 55–65 min (p<0.05). Amphetamine 4 mg/kg increased theta (143.5% of baseline) and alpha (163.7% of baseline) power during its onset (10–20 min, p<0.01 and p<0.05 respectively), with these increases persisting into 25–35 min; amphetamine produced a modest beta decrease. EEG coherence: Coherence changes paralleled behavioural activity and dose. 2C-B 10 mg/kg induced a general decrease in coherence across bands, most notably interhemispheric temporal and intrahemispheric parieto‑temporal and fronto‑temporal pairings, at both 15–25 and 55–65 min. By contrast, 2C-B 50 mg/kg produced mixed effects: decreases in frontal/temporal/parietal interhemispheric coherence in lower bands during onset, alongside increases in intrahemispheric fronto‑temporal and fronto‑parietal coherence in beta and higher frequencies. Amphetamine elicited a broad increase in coherence in theta and alpha bands at 10–20 min, with some later reductions in beta/high beta coherence. Vehicle-treated animals showed minimal early EEG changes but developed decreases in high beta and gamma power and alterations in coherence at later timepoints, which the authors attribute partly to sleep-wake influences and handling. Links between measures: The authors report temporal correlations between increased locomotor activity, elevations in NAc dopamine and increases in theta/alpha power and coherence for both 2C-B (at later timepoints, especially higher dose) and amphetamine, while the initial 2C-B effects (hypolocomotion, reduced high-frequency power and decreased coherence) were temporally dissociated from the dopamine peak.

The authors interpret 2C-B as a centrally active compound whose behavioural, neurochemical and electrophysiological profile overlaps with psychedelics, entactogens and stimulants. They emphasise the biphasic time- and dose-dependent pattern: an early phase of locomotor suppression, reduced high-frequency EEG power and decreased coherence followed by a later phase of hyperlocomotion, increased theta/alpha EEG power and elevated coherence at higher doses. This later phase temporally coincided with a pronounced rise in nucleus accumbens dopamine, supporting a model in which early effects are driven more by serotonergic receptor actions while later effects increasingly reflect dopaminergic involvement. Páleníček and colleagues link the decreased DOPAC together with increased DA, HVA and 3-MT to possible inhibition of monoamine oxidase (MAO) by 2C-B, though they acknowledge inconsistencies in how HVA is produced given reduced DOPAC and indicate that further work is required to disentangle MAO-A vs MAO-B contributions and other metabolic pathways. The dopamine increase in both NAc shell and core is discussed as a substrate for the observed hyperlocomotion, PPI disruption and a theoretical contribution to addictive or psychotomimetic potential. Regarding EEG, the authors note that decreases in higher-frequency power and coherence seen at lower doses are consistent with other serotonergic psychedelics, whereas increases in theta/alpha power and coherence that align with motor activation are similar to amphetamine effects and may reflect dopaminergic-driven wakefulness and locomotion. They suggest that coherence changes, as a marker of functional connectivity, may map onto disturbed information processing underlying altered locomotion, stereotypy and disrupted sensorimotor gating. The discussion recognises experimental caveats: vehicle-treated controls showed later spectral/coherence shifts likely related to sleep–wake state and handling, and the microdialysis sample was reduced by probe misplacements and perfusion failures. The authors therefore call for additional studies to clarify mechanisms (MAO isoform specificity, transporter interactions, active metabolites) and to further relate receptor-level pharmacology to the multi-modal effects observed.

The study concludes that 2C-B is a potent centrally active drug producing time- and dose-dependent behavioural, neurochemical and electrophysiological changes in rats that resemble features of classic psychedelics, entactogens and stimulants. Biphasic effects appear linked to early serotonergic actions and later dopaminergic activation; alterations in dopamine levels and metabolite turnover in the nucleus accumbens suggest possible MAO inhibition and raise concerns about psychotomimetic and addictive potential. EEG measures revealed robust modifications in functional connectivity that the authors propose relate to disrupted sensorimotor processing and psychedelic-like effects.