Sex differences and serotonergic mechanisms in the behavioural effects of psilocin

Psilocin, the active metabolite of psilocybin, has high affinity for several serotonin receptor subtypes, notably 5-HT2 and 5-HT1A, and is used both as a pharmacological model of psychosis in preclinical work and as an experimental therapeutic agent in clinical research. Previous animal studies have shown that serotonergic hallucinogens, including psilocin/psilocybin, reliably alter spontaneous behaviour (for example decreased locomotion, reduced exploration, signs of behavioural serotonin syndrome) and can affect sensorimotor gating measured as prepulse inhibition (PPI), but the receptor mechanisms beyond 5-HT2A and possible sex-dependent differences have not been fully characterised. Tylš and colleagues designed experiments to fill two gaps: whether behavioural responses to psilocin differ by sex and by phase of the female oestrous cycle, and whether receptor subtypes other than 5-HT2A—specifically 5-HT1A, 5-HT2B and 5-HT2C—contribute to psilocin’s behavioural effects. The investigators assessed spontaneous behaviour in the open-field and sensorimotor gating (PPI) in adult Wistar rats, and probed receptor involvement using selective antagonists in male animals.

Adult Wistar rats were used, with males weighing 200–250 g and females 150–180 g. Animals were acclimatised for at least 7 days, housed under a 12 h light/dark cycle, and tested during the light phase. Group sizes were 8–12 animals and each subject was tested once. Experiments complied with relevant animal-care guidelines. The design had two parts. First, both sexes were tested in the open-field and PPI paradigms with psilocin at 0.25, 1 and 4 mg/kg to evaluate sex differences and the influence of the female oestrous cycle. Female rats were assigned to pro-oestrus+oestrus (PE) or metoestrus+dioestrus (MD) groups based on vaginal smears taken before and immediately after testing. Second, receptor mechanism experiments were performed in males only: selective antagonists for 5-HT1A (WAY100635, 1 mg/kg), 5-HT2A (MDL100907, 0.5 mg/kg), 5-HT2B (SB215505, 1 mg/kg) and 5-HT2C (SB242084, 1 mg/kg) were administered to probe contributions of these receptors. Behavioural endpoints in the open-field included automatically tracked locomotor activity (total distance moved, time in centre, thigmotaxis/peripheral time) recorded for 30 min, and a separate 5-min qualitative scoring of behaviours (rearing, grooming, sniffing, immobility, flat body posture, wet dog shakes). PPI testing used a standard startle chamber protocol with background noise (62 dB), startle pulses (120 dB) and prepulses (+13 dB) presented 100 ms before the pulse; PPI was calculated as percentage suppression [100 − (mean prepulse–pulse response / single-pulse startle) × 100]. Animals with baseline startle <10 were considered nonresponders and excluded; nonresponder counts did not differ by group. Drugs were given subcutaneously in 2 ml/kg volume, with psilocin and antagonists administered 15 min before testing in separate injections. Vehicles were tested against saline controls. Statistical analysis used two-way ANOVA with psilocin dose and sex (or antagonist) as factors, followed by Newman–Keuls post-hoc tests when appropriate. Outliers more than 2 SD from the mean were excluded (0.3% of data). Significance was set at P < 0.05.

Sex differences in locomotor activity: Two-way ANOVA showed significant main effects of psilocin treatment [F(3,107) = 5.84, P < 0.001] and sex [F(2,107) = 19.64, P < 0.001], with a sex × treatment interaction approaching significance [F(6,107) = 2.01, P = 0.071]. Psilocin produced a dose-dependent decrease in locomotion in male and MD females but not in PE females. In males, a significant reduction occurred at 1 mg/kg; both sexes showed reductions at 4 mg/kg. Overall, females were less sensitive to psilocin’s locomotor-inhibitory effects than males, with PE females showing the greatest resistance. Analysis of time spent in the centre revealed a main effect of sex [F(3,108) = 12.36, P < 0.001], with both female groups spending more time in the centre than males after psilocin; thigmotaxis did not change significantly. Sex differences in qualitative behaviour: Psilocin significantly affected all scored behaviours (rearing, grooming, sniffing, immobility, flat body posture, wet dog shakes) [e.g. rearing F(3,129) = 39.60, P < 0.001]. There was a sex effect only for rearing [F(3,129) = 3.23, P < 0.05], and no overall sex × treatment interactions. Post-hoc tests showed that especially the 4 mg/kg dose reduced rearing, grooming and sniffing and increased immobility and flat body posture across groups. Wet dog shakes increased after 0.25 mg/kg psilocin in both female groups but not in males. Isolated sex differences included greater sniffing in females after 1 mg/kg and more wet dog shakes in females after 0.25 mg/kg. Sex differences in PPI and ASR: Two-way ANOVA indicated a significant effect of psilocin on acoustic startle reaction (ASR) [F(3,121) = 2.58, P < 0.01] but no main effect of sex or interaction; post-hoc tests did not identify dose effects on ASR except that males had lower ASR than MD females (P = 0.05). For PPI the overall ANOVA showed a borderline effect of psilocin [F(3,121) = 2.58, P = 0.056] with no sex effect. A one-way ANOVA restricted to males revealed a significant treatment effect [F(3,38) = 3.14, P < 0.05], with 1 mg/kg psilocin disrupting PPI (post-hoc P < 0.05); the authors report that the PPI after 1 mg/kg in males was about 35% of control. Vehicle controls: Vehicles did not differ from saline in any test. Effects of serotonin antagonists on locomotion: In male-only antagonist experiments two-way ANOVA showed significant main effects of psilocin [F(3,176) = 11.62, P < 0.001] and antagonist treatment [F(4,176) = 17.06, P < 0.001] and a significant interaction [F(12,176) = 2.565, P < 0.01]. Post-hoc analyses found that the 5-HT1A antagonist WAY100635 and the 5-HT2C antagonist SB242084 restored locomotion suppressed by 1 and 4 mg/kg psilocin (P < 0.05–0.01). Co-administration of WAY100635 with 0.25 mg/kg psilocin produced hyperlocomotion (P < 0.01). The 5-HT2B antagonist SB215505 did not significantly reverse hypolocomotion, although activity was not different from controls; MDL100907 (5-HT2A antagonist) had no reversing effect and in some comparisons slightly potentiated locomotor inhibition at 1 and 4 mg/kg. Effects of antagonists on PPI and ASR: Two-way ANOVA showed significant effects of psilocin [F(3,185) = 5.32, P < 0.01] and antagonist [F(4,185) = 5.05, P < 0.001] on ASR and a significant interaction [F(12,185) = 2.03, P < 0.05], but post-hoc comparisons yielded a single clear finding: MDL100907 with 0.25 mg/kg psilocin produced a higher ASR than either 0.25 mg/kg psilocin alone or saline (P < 0.01–0.001). For PPI, ANOVA showed main effects of psilocin [F(3,185) = 5.54, P < 0.01] and antagonist [F(4,185) = 3.91, P < 0.01] but no interaction; post-hoc tests only indicated a statistical trend for a disruptive effect of 1 mg/kg psilocin (P = 0.08). The authors note that antagonists tended to increase PPI toward control values, most notably the 5-HT2A antagonist, though these normalising effects were not robustly significant in post-hoc testing.

Tylš and colleagues highlight two principal conclusions: first, psilocin’s behavioural effects are not solely mediated by 5-HT2A receptors—5-HT1A and 5-HT2B/C receptors make important contributions particularly to locomotor suppression; second, female rats, especially during pro-oestrus and oestrus, are generally less affected by psilocin than males. The investigators relate the locomotor inhibition, reduced exploration and signs of a behavioural serotonin syndrome to agonist actions at multiple serotonin receptors. Evidence that WAY100635 (5-HT1A antagonist) and SB242084 (5-HT2C antagonist) reversed hypolocomotion supports roles for 5-HT1A and 5-HT2C in motor inhibition, while a 5-HT2B antagonist produced a similar normalising effect and is hypothesised to act, perhaps via cerebellar (Purkinje cell) sites, on ataxia and motor control. A dopaminergic mechanism is offered to reconcile these receptor effects: 5-HT2A stimulation can increase phasic dopamine release, whereas 5-HT2C activation tends to decrease tonic dopaminergic activity, which may underlie locomotor suppression; accordingly, 5-HT2C blockade could restore dopamine tone and locomotion. The paradoxical hyperlocomotion observed when WAY100635 was combined with the lowest psilocin dose is discussed as potentially unmasking other serotonergic effects that increase dopamine release. Regarding sensorimotor gating, the authors report that disruption of PPI by 1 mg/kg psilocin in males is most consistent with a 5-HT2A-mediated mechanism, though the data suggest minor contributions from 5-HT1A and 5-HT2B/C as well. They note that antagonist treatments tended to shift PPI toward control values, particularly the 5-HT2A antagonist, but these effects were not robust in post-hoc testing. The investigators also discuss a biphasic dose response—loss of PPI disruption and wet dog shakes at the highest dose—which they attribute either to general motor inhibition at high doses or to a masking effect from strong 5-HT2C agonism. Sex differences are interpreted in the context of steroid modulation of serotonergic and dopaminergic systems. Female rats in pro-oestrus/oestrus have higher serotonin levels and reduced responsiveness to 5-HT1A agonists; oestrogens may downregulate 5-HT1A autoreceptors while altering 5-HT2A expression, and oestrogen/progesterone increase dopaminergic activity. These neuroendocrine differences could buffer females from psilocin-induced motor inhibition and account for their relative resistance in many behavioural measures. The authors acknowledge limitations: preclinical tests cannot capture subjective hallucinatory phenomena, testing during the light phase (when activity is lower) may influence results, selectivity and possible additive actions of antagonists cannot be fully excluded, and some antagonist effects did not reach significance in post-hoc comparisons. They suggest further experiments to clarify receptor selectivity, localisation and dose-related biphasic mechanisms.

Psilocin produced behavioural effects similar to other serotonergic hallucinogens, including locomotor inhibition, reduced exploration and signs of behavioural serotonin syndrome. The study indicates an important contribution of 5-HT1A and 5-HT2B/C receptors to these actions in addition to 5-HT2A. It also confirms that female rats, particularly during pro-oestrus and oestrus, are relatively protected from some psilocin effects. The authors note that while human studies have not consistently reported sex differences in responses to psilocybin, such differences might be subtle or underexplored.