Psilocybin lacks antidepressant-like effect in the Flinders Sensitive Line rat

Depression is a major global health problem with substantial unmet needs: current pharmacological and psychological treatments leave a sizeable minority of patients non-responsive, have delayed onset for many, and carry high relapse rates. Re-purposing older compounds with novel mechanisms has therefore attracted interest. Psilocybin, a serotonergic psychedelic found in 'magic mushrooms', has shown promising, rapid and sustained antidepressant effects in small human trials when administered alongside psychological support, but its neurobiological mechanisms remain poorly understood and require translational animal work to explore causal pathways. Jefsen and colleagues set out to test whether psilocybin or its active metabolite psilocin produce antidepressant-like effects in an established rat model of depression, the Flinders Sensitive Line (FSL). Using immobility in the forced swim test (FST) as the primary behavioural readout and the Flinders Resistant Line (FRL) as a control strain, the investigators conducted a series of experiments varying dose, treatment paradigm and timing to assess whether psilocybin/psilocin reduce immobility consistent with an antidepressant-like effect. They hypothesised that drug treatment would lower immobility in FSL rats compared with saline-treated FSL controls.

All procedures received prior approval from the Danish National Committee for Ethics in Animal Experimentation. The study comprised four consecutive experiments (labelled E1–E4) using male Flinders Line rats (FSL and FRL) from an in-house colony. Individual animals were randomly allocated to treatment groups using a random number generator and tested in a pseudo-random order; behavioural testing occurred between 08:00 and 14:00 with a maximum 2.5-hour window per testing session. Sample size calculations with G*Power assumed an effect size of Cohen's d = 1.6 (a ≥20% reduction in immobility judged clinically meaningful), SD = 12.5%, α = 0.05 and power = 0.80, yielding eight animals per group as the planned sample size. Animals were pair-housed under controlled temperature and light/dark conditions with ad libitum food and water; welfare was monitored daily. Psilocin and psilocybin (sourced from THC Pharm) were dissolved in acidified 0.9% saline to pH 5–6 and administered intraperitoneally at 1 ml per injection in the home cage. Dose ranges tested were psilocin 0.5 and 2.0 mg/kg, and psilocybin 2.0, 3.0 (given as 3 × 3 mg/kg in one paradigm) and 10 mg/kg; doses were chosen based on literature and an allometric conversion from human therapeutic doses. Psilocybin is a prodrug hydrolysed to psilocin, and the investigators note psilocin is ~1.4 times more potent per milligram than psilocybin. Based on rat pharmacokinetics, behavioural testing commenced no earlier than 3 hours after injection to avoid capturing acute intoxication. Locomotor activity was assessed in an open field test (OFT): animals were placed in the centre of a 100 × 100 × 30 cm arena for 5 minutes while infrared video-tracking quantified total distance travelled. Immediately after the OFT, rats underwent a modified forced swim test (FST) without a pre-swim session: Plexiglas cylinders (60 cm height, 20 cm diameter) were filled with 40 cm water at 24 ± 2°C and rats were video-recorded for 5 minutes. A single trained observer, blinded to strain and treatment, scored immobility, swimming and struggling every 5 s; each video was scored at least twice and an average of the two closest scores was used. Statistical analysis treated the individual animal as the unit. Outliers were identified by the ROUT test (Q = 0.01). Two-way ANOVA assessed main effects and interactions of strain and treatment, followed by Tukey's multiple comparisons; analyses were performed in GraphPad Prism 7 and significance set at p < 0.05. Investigators were blinded to strain during injections but not to treatment; the outcome assessor for behaviour was blinded.

Open field test: Across all four experiments, FSL rats exhibited greater locomotor activity than FRL controls. The two-way ANOVAs showed highly significant main effects of strain in E1–E4 (E1: F(1,42) = 22.17, p < 0.0001; E2: F(1,30) = 112.9, p < 0.0001; E3: F(1,31) = 49.04, p = 0.0001; E4: F(1,32) = 26.41, p < 0.0001). Treatment with either psilocin or psilocybin produced no statistically significant effect on locomotor activity in any experiment (E1: F(2,42) = 1.617, p = 0.2106; E2: F(1,30) = 0.1895, p = 0.6664; E3: F(1,31) = 1.284, p = 0.265; E4: F(1,32) = 1.663, p = 0.2064). The authors interpret these null treatment effects to indicate that stimulant or sedative confounding of FST performance is unlikely at the tested time points. Forced swim test: Treatment with psilocin or psilocybin did not alter immobility time, struggling or swimming behaviour in the FST in any of the four experiments. Two-way ANOVA results for treatment on immobility were non-significant across experiments (E1: F(2,42) = 0.196, p = 0.8228; E2: F(1,26) = 0.00462, p = 0.9463; E3: F(1,27) = 1.087, p = 0.3065; E4: F(1,32) = 0.4132, p = 0.5249). By contrast, the strain effect was robust in all experiments, with FSL rats showing markedly higher immobility than FRL rats (E1: F(1,42) = 93.5, p < 0.0001; E2: F(1,26) = 68.87, p < 0.0001; E3: F(1,27) = 38.33, p < 0.0001; E4: F(1,32) = 29.64, p < 0.0001). Sample sizes reported in figure text included mostly n = 8 per group in E1 and E4, with occasional exclusions: one animal was excluded as a statistical outlier from an E2 struggling outcome, and one animal died unexpectedly in E3. Overall, the measured data show no antidepressant-like effect of psilocin or psilocybin in FSL rats under the tested conditions.

Jefsen and colleagues interpret the principal result as a clear lack of antidepressant-like effect of psilocin and psilocybin in FSL rats when assessed using the FST and OFT. No changes were observed in immobility, swimming or struggling in the FST, and no locomotor effects were seen in the OFT, despite the model strain (FSL) displaying the expected increased immobility relative to FRL controls. This outcome contradicts the investigators' hypothesis and contrasts with reported antidepressant effects of psilocybin in humans. The authors discuss several possible explanations. Methodological considerations include the suitability of the FST as a translational assay and whether measuring a single behavioural endpoint is sufficient; immobility can be interpreted in multiple ways and may not equate to human depressive states. Biological explanations focus on serotonergic receptor differences: FSL rats display markedly lower 5-HT2A receptor mRNA expression in frontal cortex and hippocampus (35% and 37% of FRL levels), and psilocin/psilocybin effects in humans depend on 5-HT2A activation. In addition, species differences in 5-HT2A receptor sequence produce substantially different psilocin affinity (the authors cite a 15-fold higher affinity at the human receptor versus the rat receptor due to a Ser-242/Ala-242 substitution), which could lead to divergent pharmacodynamics between humans and rodents. Psychological and contextual factors are also emphasised: clinical psilocybin trials always combine drug administration with psychotherapy, supportive settings and integration, factors that are difficult to model in animals and may be necessary for therapeutic effects. Limitations acknowledged by the investigators include the restricted dose range (0.5–10 mg/kg), uncertainty about which dose is translationally relevant, the focus on a single behavioural measure (FST) rather than a broader behavioural phenotype, and partial blinding (investigators were not blinded to treatment during injections). They also note they did not present group-specific baseline characteristics or perform covariate adjustment. Despite these caveats, the authors consider the study low risk of bias in terms of randomisation, outcome assessor blinding and reporting of obtained results. Concluding, they suggest that the antidepressant actions of psilocybin in humans may not be straightforwardly back-translatable to this animal model and advocate testing psilocybin across additional behavioural assays and preclinical models to better capture its therapeutic mechanisms.