Psilocin and ketamine microdosing: effects of subchronic intermittent microdoses in the elevated plus-maze in male Wistar rats

Ketamine and psilocin are psychoactive compounds with distinct pharmacologies but overlapping behavioural effects at moderate doses. Ketamine is primarily a noncompetitive NMDA receptor antagonist with additional actions at the serotonin transporter and on dopaminergic and opioid systems. Psilocin (the active metabolite of psilocybin) is a serotonin receptor agonist, particularly at 5-HT1A and 5-HT2A/C receptors. Both classes of drugs can produce psychomimetic effects in humans and rodents, including anxiety, and have more recently been investigated for therapeutic properties in mood and anxiety disorders. Against this background, an intermittent low-dose "microdosing" practice has emerged in humans, typically using about one-tenth of a full active dose on an intermittent schedule, with anecdotal reports of sustained reductions in anxiety and improved mood. Horsley and colleagues noted a lack of preclinical data testing microdosing regimens. The current study therefore tested whether subchronic, intermittent subcutaneous microdoses of ketamine (0.5 and 3 mg/kg) or psilocin (0.05 and 0.075 mg/kg) produce lasting anxiolytic or anxiogenic effects when animals are tested drug-free on the elevated plus-maze (EPM), a standard preclinical screen for anxiety-related behaviour.

The study used a between-subjects design in which ketamine and psilocin data were analysed separately; for each drug the factor was Drug with three levels (low dose, high dose, saline). Forty experimentally naive male Wistar rats were used, allocated in groups such that each experimental cell comprised eight animals. The same saline control animals were used in both drug analyses to minimise animal use. All procedures complied with relevant ethical guidelines and local approvals. Behavioural testing used a standard elevated plus-maze (two open arms, two closed arms, central hub), elevated 50 cm above the floor. After a 1 h habituation adjacent to the testing room, each rat was placed on the central square facing an open arm and allowed to explore the maze for 5 min while behaviour was video recorded and tracked. Testing occurred during the light phase and maze order was counterbalanced; behaviours were scored automatically and supplemented by ethological measures. Rats received three subcutaneous injections of their assigned treatment over 6 days (administrations occurred in the home cage between 12:00 and 16:00), with the final dose on day 6. Testing on the EPM was performed 48 h after the final injection (day 8), such that animals were behaviourally drug-free at test. Ketamine (racemic) was prepared in saline and given at 0.5 or 3 mg/kg (1 ml/kg). Psilocin was dissolved in a small volume of acidified saline then diluted to deliver 0.05 or 0.075 mg/kg (1 ml/kg); the authors note the psilocin vehicle contained a minute amount of acid while the saline control vehicle did not. Primary dependent variables comprised arm entry frequencies and percentage time spent in zones (open arms, closed arms, central square), total arm entries and total distance travelled, distance per visit to open and closed arms, and ethological measures (rears, stretch-attend postures, head dips). Arm entries were counted when the animal's body centre crossed into the arm. Each dependent variable was analysed separately using a one-way analysis of variance (ANOVA) per drug, with α = 0.05 (two-tailed) and planned independent t-tests to explore significant or marginal main effects. Levene's test was used to assess equality of variances and corrected statistics reported where appropriate.

General: Behavioural testing was performed 48 h after the final microdose administration so that animals were drug-free at the time of testing. The authors report modest or borderline statistical effects overall; patterns of means were similar across ketamine and psilocin conditions. Ketamine: One animal in the 3 mg/kg ketamine group was excluded from all analyses because its behaviour was an outlier (>2 SDs from the group mean). A marginal main effect of ketamine on frequency of open arm entries was reported [F(2,20) = 3.23, P = 0.06]. Planned comparisons indicated that the low dose (0.5 mg/kg) significantly reduced visits to the open arms versus saline [t(8) = 2.41, P < 0.05], whereas the higher dose (3 mg/kg) did not differ from saline [t(13) = 1.56, not significant]. Total arm entries, closed arm entries and distance measures showed no clear drug effects. Percentage time data suggested reduced time in the open arms and centre and increased time in the closed arms after ketamine, but these differences did not reach conventional significance; a marginal effect was reported for time in the centre [F(2,20) = 1.96, P = 0.08, one-tailed], with a marginal reduction at 3 mg/kg on one-tailed testing [t(13) = 1.68, P = 0.06, one-tailed]. Ethological measures showed no effect on stretch-attend postures or head dips, and a marginal main effect on rears [F(2,20) = 2.18, P = 0.07, one-tailed], with the low dose producing an increase in rearing [t(13) = 1.87, P < 0.05, one-tailed]. Psilocin: The extracted results text is incomplete, but the authors report that means suggested reduced open arm entries after psilocin. In the Discussion the investigators state that the lower psilocin dose (0.05 mg/kg) produced mild anxiogenesis, with the higher dose (0.075 mg/kg) showing a similar pattern though nonsignificant on the primary open-arm entry measure. No locomotor confound was evident for psilocin; ethological rearing was not increased. The authors also note that the vehicle for psilocin contained a small amount of acid while the saline control did not, representing a methodological caveat. Precise ANOVA and t-test statistics for psilocin are not clearly reported in the extracted text.

Horsley and colleagues interpret their principal finding as a mild anxiogenic effect of intermittent microdosing with ketamine and psilocin on the elevated plus-maze, most evident at the lower doses. Reduced open arm entries—the primary EPM measure of anxiety—drove this interpretation, and the investigators argue these changes are unlikely to be secondary to altered locomotion since closed arm entries and total entries were not reduced. The authors discuss the pattern of results in the context of possible shared mechanisms between serotonergic hallucinogens and dissociative anaesthetics, noting that both drug classes can engage forebrain circuits involving 5-HT2A receptors and frontocortical glutamatergic systems. They highlight that the behavioural effects were observed 48 h after dosing, when acute pharmacological levels of ketamine and psilocin are expected to have subsided in rats, and therefore suggest lasting neuroadaptive changes may underlie the observations. Pharmacokinetic considerations are presented: ketamine and its metabolites have relatively short elimination half-lives in rats, making residual drug unlikely at 48 h, whereas psilocin metabolites such as psilocin-O-glucuronide might theoretically persist longer; the authors do not exclude this possibility. Several limitations are acknowledged. Statistical effects were modest and in some cases only marginal; the study relied on a single behavioural assay, sample sizes were small per group, and the same saline controls were used for both drug analyses. The psilocin vehicle differed from the saline control by containing a tiny amount of acid, which could confound interpretation. The results derive from male Wistar rats only, limiting generalisability. The authors therefore counsel caution in extrapolating to clinical microdosing practices in humans. For future work, they recommend replication across additional animal models of anxiety and depression, investigation of neurobiological mechanisms (including 5-HT2A and NMDA-related processes), and the establishment of an appropriate preclinical screening paradigm for microdosing. Given the popularity of self-administered microdosing, the investigators emphasise the need for more rigorous evidence to inform harm-reduction advice and clinical practice.

Intermittent subchronic microdoses of ketamine or psilocin altered anxiety-related behaviour on the elevated plus-maze in male Wistar rats, producing a pattern consistent with mild anxiogenesis rather than anxiolysis. The statistical effects were modest but showed similar mean patterns across both compounds, which the authors view as suggestive of a small but potentially meaningful effect. They urge caution in interpretation because findings derive from a single test with small group sizes and some methodological caveats, and they call for further preclinical and mechanistic research to confirm these observations and to assess their translational relevance to human microdosing practices.