Harnessing psilocybin: antidepressant-like behavioral and synaptic actions of psilocybin are independent of 5-HT2R activation in mice

Depression is common and often treatment-resistant, and there is growing clinical evidence that the psychedelic compound psilocybin produces rapid and sustained antidepressant effects in humans. Psilocin, the active metabolite of psilocybin, is a potent agonist at multiple serotonin receptors, and the intense alterations of perception caused by psilocybin in humans have been linked to activation of the serotonin 2A receptor (5-HT2AR). A widespread assumption in the field is that these perceptual or ‘‘psychedelic’’ effects are necessary for therapeutic benefit, but that hypothesis has not been directly tested in humans or preclinical models. Hesselgrave and colleagues therefore tested whether a single dose of psilocybin produces antidepressant-like effects in a validated mouse model of stress-induced anhedonia and, crucially, whether those effects require 5-HT2R activation. They used behavioural assays of hedonic state together with electrophysiological measures of synaptic strength, and included pharmacological blockade of 5-HT2A/2C receptors with ketanserin as a functional test of the necessity of 5-HT2R-mediated psychedelic actions for any observed antidepressant-like outcomes.

This was an experimental preclinical study using male C57BL/6J mice (8 weeks old). Two cohorts were bred in-house for the chronic stress and anhedonia experiments; separate cohorts were used for in vivo electrophysiology and locomotion assessments. Animals were single housed for the behavioural and stress protocols, kept on a 12 h light/dark cycle, and assigned to groups balanced on hedonic behaviour after stress. Chronic multimodal stress (CMMS) was used to induce an anhedonic phenotype: mice experienced 4 h per day of restraint stress combined with strobe lighting and white noise for 10 to 14 consecutive days, applied near the start of the light cycle. Hedonic behaviour was assessed at baseline, after CMMS, and 24 h after drug injection using two appetitive choice tasks: a two-bottle sucrose preference test (SPT; 1% sucrose versus water, measured across two nights) and a female urine sniffing test (FUST; time spent sniffing swabs soaked in female versus male urine). A priori inclusion criteria required baseline sucrose preference >65%; mice whose sucrose preference fell to <70% after CMMS were classified as stress-susceptible, while those remaining >70% were classified as resilient. Drug treatments were administered intraperitoneally. The primary behavioural experiments used psilocybin at 1 mg/kg given as a single injection; ketanserin (+)-tartrate (2 mg/kg) or saline was given 60 min before psilocybin or vehicle. Higher psilocybin doses (5 and 10 mg/kg) were used in locomotion and in vivo electrophysiology experiments respectively. Head-twitch responses were video scored for 0–15 min post-psilocybin by blinded raters in a subset of animals. Separate cohorts were used for open-field locomotion and for forced swim testing (FST; measured at 1, 3 and 7 days post-injection). For synaptic physiology, hippocampal slices (400 μm) were prepared from animals after behavioural testing. Extracellular field recordings quantified AMPA/NMDA ratios at the temporoammonic input to distal CA1 (TA–CA1), using DNQX and APV to isolate AMPAR and NMDAR components; AMPA and NMDA slopes were normalised to fiber volley amplitude and averaged across slices to obtain each animal's mean AMPA/NMDA and AMPA/FV and NMDA/FV. In vivo local field potentials (LFPs) were recorded from CA1 under isoflurane anaesthesia to measure low-frequency (delta, 0–4 Hz) power before and after a high psilocybin dose (10 mg/kg), with or without ketanserin pretreatment, as a positive control of 5-HT2AR blockade. Statistical analyses included t tests and one-, two- and three-way ANOVAs with multiple-comparison corrections; experimental blinding was applied for behavioural scoring and electrophysiological analysis where stated.

CMMS produced clear, reproducible reductions in hedonic responses. Mice showed strong baseline preferences for 1% sucrose and for female urine, and both preferences decreased significantly after 10–14 days of CMMS. Following a single i.p. injection of psilocybin (1 mg/kg) there was a rapid restoration of hedonic behaviour: sucrose and female urine preferences were significantly increased 24–48 h after psilocybin compared with post-stress values in vehicle-treated mice. Stress-resilient mice (those not losing sucrose preference after CMMS) did not change their behaviour after psilocybin. Group sizes reported in the main behavioural figure included vehicle–vehicle n = 12, ketanserin–vehicle n = 6, vehicle–psilocybin n = 13, and ketanserin–psilocybin n = 7. A three-way repeated-measures ANOVA showed a significant effect of stress on sucrose preference (F2,68 = 60.26, P < 0.0001) and a Stress × Psilocybin interaction (F2,68 = 4.50, P = 0.015), while the Stress × Psilocybin × Ketanserin interaction was not significant (F2,68 = 0.05917, P = 0.9426). For female urine preference, stress reduced preference (F2,30 = 43.41, P < 0.0001) and a Stress × Psilocybin interaction was observed (F2,30 = 4.26, P < 0.024), with no significant three-way interaction including ketanserin (F2,30 = 0.8677, P = 0.4302). To test whether 5-HT2R activation was necessary for the behavioural effect, ketanserin was given 60 min before psilocybin. Ketanserin pretreatment did not prevent psilocybin’s restoration of sucrose or female urine preference: psilocybin significantly increased both measures whether mice were pretreated with ketanserin or vehicle. As a positive control that ketanserin was active, vehicle-pretreated mice given psilocybin exhibited markedly increased head-twitch counts (behaviourally indicative of 5-HT2AR activation), whereas ketanserin largely blocked this head-twitch response. A post hoc separation of psilocybin-treated mice into high and low head-twitch groups (>7 head twitches/15 min versus <7/15 min) showed that both subgroups displayed significant improvements in sucrose and female urine preference, consistent with a behavioural effect independent of head-twitching (and therefore, 5-HT2AR activation). In vivo LFP recordings provided a second positive control of ketanserin efficacy. A relatively high psilocybin dose (10 mg/kg) produced a decrease in delta (0–4 Hz) power within 15 min that lasted >60 min; ketanserin pretreatment greatly attenuated this psilocybin-induced reduction in delta power, confirming effective blockade of 5-HT2AR-dependent electrophysiological effects under the pretreatment protocol. At the synaptic level, slices from stress-susceptible mice treated with psilocybin showed significantly higher AMPA/NMDA ratios at TA–CA1 synapses than slices from CMMS mice given vehicle or ketanserin alone. Normalisation of response components to fiber volley amplitude revealed that the increased AMPA/NMDA ratio reflected an increased AMPAR-mediated component, with no significant change in the NMDAR-mediated component. Pretreatment with ketanserin did not impair the ability of psilocybin to restore AMPA/NMDA ratios: AMPA/NMDA was elevated in psilocybin-treated animals regardless of ketanserin pretreatment. AMPA/NMDA ratios correlated positively with sucrose preference across animals. These synaptic changes persisted days after administration, consistent with persistent synaptic strengthening analogous to human reports of altered functional connectivity after psilocybin. The authors report no effect of psilocybin (1 mg/kg) on immobility in the forced swim test in unstressed male or female C57BL/6J mice at 1, 3, or 7 days post-injection. They also note ketanserin-induced hypolocomotion in some assays, consistent with reported activity at the dose used.

Hesselgrave and colleagues interpret their results as evidence that a single administration of psilocybin produces rapid antidepressant-like effects in mice exposed to chronic stress, and that these effects are accompanied by persistent strengthening of excitatory synapses in a hippocampal pathway implicated in reward and emotion. They underscore that these synaptic and behavioural changes outlast the pharmacokinetic presence of the drug, mirroring human observations of sustained changes in functional connectivity after psilocybin. Central to their interpretation is the finding that ketanserin—a 5-HT2A/2C antagonist—blocked canonical indices of 5-HT2AR activation (head twitches and decreases in low-frequency LFP power) but did not prevent the restoration of hedonic behaviour or the increase in AMPA/NMDA ratios. From this, the investigators conclude that 5-HT2R activation and the associated perceptual alterations may not be required for the antidepressant-like and synaptic actions of psilocybin in this mouse model. They note that this raises the possibility of combining psilocybin with a safe 5-HT2R antagonist to reduce psychedelic effects while preserving therapeutic benefit, or of developing non-hallucinogenic analogues biased toward synaptic strengthening. The authors acknowledge several limitations and uncertainties. Animal models cannot capture the subjective human psychedelic experience or its potential therapeutic interaction with psychotherapy. Their experiments used only male C57BL/6J mice and primarily examined outcomes at 24–48 h post-injection; they cannot exclude a role for 5-HT2AR activation at later time points. The lack of an effect in the forced swim test and the limited examination of female mice are noted as areas needing further work. Finally, the specific serotonin receptor(s) mediating the synaptic and behavioural antidepressant-like effects remain undefined; the authors suggest 5-HT1B receptors as a candidate based on prior work but emphasise that defining these mechanisms will be important for developing alternatives to psilocybin and for understanding whether psychedelic perceptual experiences contribute to therapeutic outcomes in humans.