Effects of acute and repeated treatment with serotonin 5-HT2A receptor agonist hallucinogens on intracranial self-stimulation in rats

Sakloth and colleagues frame the study within renewed scientific and popular interest in prototype 5-HT2A receptor agonist hallucinogens—LSD, mescaline, and psilocybin—which are legally classified as Schedule I in the United States yet have shown potential in clinical contexts for disorders such as depression, anxiety, and substance-use problems. The authors note that, paradoxically, these compounds often fail to produce robust signals in standard preclinical assays of abuse liability (for example, drug self-administration), and that intracranial self-stimulation (ICSS) is a complementary preclinical procedure commonly used to detect abuse-related rewarding effects of drugs. The present study set out to characterise acute and repeated effects of LSD, mescaline, and psilocybin on ICSS in rats using a frequency-rate procedure. Initial aims were to determine dose–effect relationships and time courses for acute dosing. A secondary aim was to evaluate whether repeated daily LSD dosing (to model, in part, emerging human microdosing practices and to test whether chronic exposure could reveal latent abuse-related effects) altered LSD's own ICSS effects or modified ICSS effects produced by the psychostimulant methamphetamine or the kappa-opioid receptor agonist U69,593.

Male Sprague-Dawley rats were used; the paper reports 52 animals overall. For acute dose–effect and time-course studies, separate groups of six rats each received LSD (0.0032–0.1 mg/kg; N=6), mescaline (0.32–10 mg/kg; N=6), or psilocybin (0.032–1.0 mg/kg; N=6). For repeated-LSD studies, 27 drug‑naïve rats were allocated to four groups receiving daily IP injections of saline, 0.01, 0.1, or 0.32 mg/kg/day LSD (N=7 per group except N=6 for the 0.32 mg/kg/day group). All animals were implanted stereotaxically with a cathodal electrode targeting the left medial forebrain bundle (coordinates reported relative to bregma) and trained to lever press for brain stimulation in operant chambers. Training used an FR1 schedule and a frequency-rate ICSS procedure: sessions comprised three 10-min components, each component containing 10 one-minute trials in which stimulation frequency decreased across trials from 2.2 log Hz to 1.75 log Hz. After stable baseline frequency-rate curves were achieved, acute test sessions consisted of three baseline components followed by a time-out and two test components; saline or drug was administered at the start of the time-out and test order was randomised in Latin-square designs. Time-course tests sampled effects at 10, 30, 100, 180, and 300 minutes after injection. For repeated-LSD studies a nine-day protocol was used: daily dosing on Days 1–6, cumulative-dosing assessment of LSD on Day 7 (five injections at 30-min intervals increasing cumulative dose in 0.5 log-unit steps), and cumulative-dosing tests with methamphetamine and U69,593 on Days 8–9 (same cumulative procedure). Primary outcomes were reinforcement rate per trial normalised to percent maximum control rate (%MCR) and total stimulations per component expressed as percentage of baseline. Data analysis relied on two-way repeated-measures ANOVA (factors included dose or time and ICSS frequency), with Holm–Sidak post hoc tests and a significance threshold of P < 0.05. Predrug baselines for repeated-dosing experiments used the three days prior to any LSD administration. Drugs (LSD tartrate, mescaline HCl, psilocybin, methamphetamine HCl, U69,593 HCl) were dissolved in sterile water and administered intraperitoneally.

Baseline measures for the 25 rats used in the acute studies averaged MCR = 55 ± 6 stimulations/trial and ~253 ± 17 stimulations/component. Across acute dose–effect and time-course experiments, Sakloth and colleagues report that LSD, mescaline, and psilocybin produced weak and inconsistent ICSS facilitation at low doses and reliable ICSS depression at higher doses. LSD showed biphasic effects: a very low dose (0.0032 mg/kg) produced facilitation at a single brain-stimulation frequency (1.95 log Hz), and 0.01 mg/kg facilitated responding at two frequencies (2.00 and 2.05 log Hz) and significantly increased total stimulations per component. A 0.032 mg/kg dose had no significant effect, whereas 0.1 mg/kg produced statistically significant depression of ICSS across a broad frequency range (2.0–2.15 log Hz) and reduced total stimulations; a higher 0.32 mg/kg dose (tested in a subset of three rats) generally produced further depression. Time-course data showed no effect of 0.01 mg/kg LSD at any sampled time; 0.32 mg/kg depressed ICSS at 10 and 30 minutes, with effects dissipating by 100 minutes. Mescaline produced ICSS depression only at the highest tested dose (10 mg/kg), with decreases across 2.0–2.2 log Hz and a near-significant reduction in total stimulations (P = 0.052). In time-course testing, a 1 mg/kg low dose transiently facilitated ICSS at one frequency (2.05 log Hz) at 10 minutes, whereas 10 mg/kg depressed ICSS at 10 minutes. Psilocybin produced ICSS depression at 1.0 mg/kg across 2.0–2.2 log Hz and decreased total stimulations; in time-course testing, 0.1 mg/kg transiently facilitated at one frequency (2.05 log Hz) at 30 minutes, and 1.0 mg/kg depressed ICSS at 10 and 30 minutes. For all three drugs, experimentally observed effects had dissipated by 100 minutes and were absent at 180 and 300 minutes. In the repeated-LSD cohort (27 rats; mean MCR = 56 ± 1, mean stimulations/component = 232 ± 16), predose baselines did not differ from Day 7 baselines in any group, indicating that repeated daily saline or LSD (0.01–0.32 mg/kg/day) did not change baseline ICSS. Cumulative-dosing tests showed that cumulative LSD (0.0032–0.32 mg/kg) and cumulative U69,593 (0.056–0.56 mg/kg) produced dose-dependent ICSS depression across groups, while cumulative methamphetamine (0.032–0.32 mg/kg) produced dose-dependent ICSS facilitation in all groups. Between-group comparisons revealed a tendency for repeated LSD to attenuate effects of the highest cumulative doses of LSD, methamphetamine and U69,593, but the only statistically reliable between-group effect was that repeated 0.1 and 0.32 mg/kg/day LSD attenuated U69,593-induced ICSS depression. Repeated LSD did not alter methamphetamine-induced facilitation. The authors also report a post hoc power analysis: power to detect an interaction between frequency and LSD treatment was >0.99 for LSD and U69,593 data but only 0.51 for methamphetamine data; achieving 0.8 power for methamphetamine would require increasing group sizes from 6–7 to about 11 per group. No sex comparisons were made; all data derive from male rats.

Sakloth and colleagues interpret their findings as showing three principal results: first, acute administration of LSD, mescaline, and psilocybin produced weak and inconsistent evidence of abuse-related ICSS facilitation at low doses but consistent ICSS depression at higher doses. Second, repeated daily LSD dosing for one week did not produce tolerance to LSD-induced ICSS depression nor did it unmask or increase ICSS facilitation by LSD. Third, repeated LSD did not change baseline ICSS or methamphetamine-induced facilitation, but repeated higher-dose LSD (0.1 and 0.32 mg/kg/day) significantly attenuated U69,593-induced ICSS depression. The authors position these outcomes relative to earlier preclinical work by noting concordance with prior reports that 5-HT2A agonists tend not to maintain drug self-administration and that another 5-HT2A agonist (TCB-2) also failed to facilitate ICSS. They emphasise that ICSS has greater predictive validity for drugs that promote patterns of frequent, sustained human use (for example amphetamines, opioids, nicotine) and is less sensitive to drugs associated with transient or infrequent consumption (for example ketamine, salvinorin). Thus, the weak ICSS facilitation is consistent with the clinical observation that classic hallucinogens are not typically used in sustained, high-frequency patterns. Several limitations acknowledged by the authors temper generalisation. First, the study used only male rats and sex differences in serotonergic drug effects have been reported previously. Second, ICSS is better validated for predicting abuse potential than for predicting therapeutic effects against drug abuse or depression; therefore extrapolation to clinical efficacy for methamphetamine addiction or depression must be cautious. Third, the methamphetamine-related analyses may have been underpowered (post hoc power = 0.51), so failure to detect an effect of repeated LSD on methamphetamine-induced facilitation could reflect sample size constraints. Fourth, the repeated-dosing regimen employed here may not faithfully model human microdosing: the lowest repeated LSD dose used (0.01 mg/kg/day) approximates one tenth of discrimination doses in rats but, after allometric adjustment, remains higher than typical human microdose equivalents; moreover, attenuation of U69,593 effects was observed only at the higher repeated LSD doses, not at the lowest dose. The investigators conclude that their data do not support significant abuse-related effects of these 5-HT2A agonist hallucinogens in ICSS procedures but provide limited preclinical support for repeated LSD reducing kappa-opioid receptor agonist–mediated depressant effects, a finding the authors suggest merits further mechanistic and translational study. They recommend additional research with larger samples, inclusion of females, varied dosing regimens that more closely mimic human microdosing, and complementary behavioural paradigms to clarify therapeutic potential.