Blocking 5-HT2B receptors abolishes psilocybin’s efficacy in the rat forced swim test

Major depressive disorder is described as a highly burdensome condition, and the paper notes that conventional antidepressants such as SSRIs and serotonin-norepinephrine reuptake inhibitors are limited by delayed onset, side effects, and inadequate response in about 30% of patients. Psilocybin has attracted attention because earlier research and clinical studies suggested rapid and sometimes sustained antidepressant effects, but the receptor mechanisms behind these benefits remained unclear. Most work had focused on 5-HT2A receptors because they are strongly linked to psychedelic effects, yet the paper highlights emerging evidence that psilocybin’s antidepressant-like actions may be separable from its hallucinogenic effects and may involve other serotonin receptors. Seillier and colleagues set out to test whether blocking 5-HT2B receptors would prevent psilocybin’s behavioural effects in the rat forced swim test, a preclinical assay with predictive validity for antidepressant activity. They also aimed to see whether 5-HT2B blockade would alter psilocybin-induced head-twitch response, which is used in rodents as a proxy for psychedelic-like 5-HT2A-mediated effects, and to assess whether body temperature and locomotion were affected. The study was designed to clarify whether 5-HT2B receptors contribute to psilocybin’s antidepressant-like profile independently of its psychedelic actions.

The researchers used 64 male Wistar rats housed under standard conditions. Animals were allocated to eight groups, with eight rats per group, using a Cyclic Latin Square design to balance testing order across the 8-week experimental period. On day 0, rats received pretreatment with the selective 5-HT2B receptor antagonist RS-127445 at 0.32, 1.0, or 3.2 mg/kg, or its vehicle, followed 30 minutes later by saline or psilocybin at 0.32 mg/kg. Psilocybin and RS-127445 were given intraperitoneally. The study also assessed rectal temperature, open-field locomotor activity, head-twitch response, and forced swim test behaviour. Rectal temperature was measured before pretreatment, before the second injection, and then every 30 minutes for 90 minutes after treatment. Locomotor activity was recorded in an open field for 30 minutes immediately after saline or psilocybin administration, and head-twitch responses were manually scored by a blinded experimenter. The forced swim test was run twice to assess both rapid and sustained effects: a test on day 1 with a pre-test on day 0, and a second test on day 21 with a pre-test on day 20. The main forced swim outcomes were immobility, climbing, and swimming. Statistical analyses included two-way and three-way ANOVAs for normally distributed data, mixed ANOVA for repeated measures such as temperature and forced swim outcomes across time, and Kruskal-Wallis tests with Dunn’s post hoc comparisons for head-twitch counts and other nonparametric data. Outliers were handled using a predefined median-based rule, and significance was set at 0.05.

Neither psilocybin nor RS-127445 affected body weight gain over the 21-day observation period. Rectal temperature also did not differ between treatment groups; only time had a significant effect, while antagonist, agonist, and their interaction were not significant. Similarly, open-field measures of locomotor activity showed no significant treatment effects on mobility time, distance travelled, or velocity. Psilocybin significantly increased head-twitch responses, consistent with a psychedelic-like effect, and this was not blocked by RS-127445 at any dose. The Kruskal-Wallis test showed an overall treatment effect on head-twitch counts (H = 44.63, p < 0.0001), and all psilocybin-treated groups had higher head-twitch ranks than the vehicle-saline group, regardless of antagonist pretreatment. In the forced swim test, psilocybin produced both rapid and sustained antidepressant-like behavioural changes. On day 1 and day 21, it reduced immobility and increased climbing behaviour compared with saline, while swimming did not change significantly. RS-127445 dose-dependently reversed these psilocybin-induced effects. At 0.32 mg/kg, RS-127445 did not fully block the psilocybin effect, but at 1.0 and 3.2 mg/kg it reversed the reductions in immobility and the increases in climbing. The statistical tests showed significant antagonist, agonist, and interaction effects for immobility and climbing at both timepoints, supporting a treatment-by-treatment interaction. The paper reports no significant effect on swimming in either forced swim test session.

Seillier and colleagues interpret the findings as evidence that 5-HT2B receptor activation is required for psilocybin’s rapid and enduring behavioural effects in the forced swim test, whereas it is not needed for psilocybin’s head-twitch response. They argue that this produces a pharmacological dissociation between the compound’s psychedelic-like effects and its antidepressant-like behavioural effects, extending previous work suggesting that psilocybin’s therapeutic profile is not solely dependent on 5-HT2A receptor activation. In their view, the results add a previously underappreciated non-5-HT2A mechanism to the discussion of psilocybin’s actions. The authors place their findings in the context of earlier research showing rapid antidepressant effects of psilocybin in preclinical and clinical settings, including in treatment-resistant depression, and note that psilocybin can produce longer-lasting effects than ketamine in some reports. They also discuss the climbing-dominant pattern observed in the forced swim test, suggesting it may reflect recruitment of noradrenergic circuitry rather than a simple serotonergic pattern, although they present this as a possibility rather than a confirmed mechanism. They further note that psilocin has relatively high affinity for 5-HT2B receptors, making this receptor a plausible mediator that had been overlooked compared with 5-HT2A. The authors acknowledge several limitations. The study was limited to healthy Wistar rats and to the forced swim test, so the findings may not generalise to depressed animals or to other behavioural paradigms. They also emphasise that 5-HT2B blockade was shown to be necessary for the observed effects, but not sufficient on its own to prove that 5-HT2B agonism fully explains psilocybin’s antidepressant-like profile. They discuss possible involvement of non-neuronal 5-HT2B receptors, particularly in astrocytes and microglia, and suggest that future work should examine these pathways more directly. In terms of implications, they suggest that identifying non-psychedelic receptor mechanisms could help in developing psychedelic-inspired treatments that retain antidepressant potential while reducing hallucinogenic or other adverse effects.

The authors conclude that 5-HT2B receptors are a critical and previously underappreciated contributor to psilocybin’s rapid and sustained behavioural effects in the rat forced swim test. They state that RS-127445 abolished psilocybin’s reduction in immobility and increase in climbing, while leaving head-twitch responses intact, supporting a separation between therapeutic-like and psychedelic-like pathways. They suggest that this finding strengthens the case for multireceptor explanations of psilocybin’s actions and may inform the development of treatments that preserve antidepressant efficacy with fewer psychedelic effects.