Regional specificity of the cingulate cortex thickness association with the intensity of psilocybin experience: a replication study

RESEARCH Psychopharmacology https://doi.org/10.1007/s00213-025-06983-9 Introduction Psychedelic substances, particularly psilocybin, have recently garnered significant scientific interest for their therapeutic potential and insights into human consciousness (Nichols 2016; Tyls et al. 2014). Psilocybin, the primary psychoactive component in mushrooms from the Psilocybe genus, primarily exerts its effects through serotonin 2 A receptor (5HT2A) agonism, leading to profound alterations in cognition, perception, and emotional states (Viktorin et al. 2022; Nichols 2016; Carhart-Harris et al. 2014; Vollenweider and Kometer 2010; Preller and Vollenweider 2018; Bravermanova et al. 2018). Individual variability in psilocybin responses is substantial and clinically significant, as David Greguš [email protected] Tomáš Páleníček [email protected] 1 National Institute of Mental Health, Topolová 748, Klecany 250 67, Czech Republic 2 3rd Faculty of Medicine, Charles University, Ruská 87, Prague 10, Prague 100 00, Czech Republic 3 Institute of Computer Science of the Czech Academy of Sciences, PraguePod Vodárenskou věží 2, Prague 8, 182 00, Czech Republic Abstract Rationale Individual variability in psilocybin response is a major challenge for psychedelic-assisted therapy, with structural brain features potentially serving as predictive biomarkers. (Lewis et al. Biomedicines 8(2):34 2020) reported that rostral anterior cingulate cortex thickness predicted emotional experiences under psilocybin, suggesting cortical morphometry as a marker of psychedelic responsivity. Objectives This study sought to replicate and extend these findings by examining associations between cingulate thickness and psilocybin-induced altered states of consciousness using comprehensive assessment and rigorous statistical control. Methods Twenty-five healthy participants underwent a double-blind, placebo-controlled crossover design with psilocybin (0.26 mg/kg) and placebo. High-resolution T1-weighted magnetic resonance imaging (MRI) measured cortical thickness across cingulate subregions. Subjective effects were assessed with the Altered States of Consciousness (ASC) questionnaire. Analyses applied false discovery rate (FDR) correction for multiple comparisons. Results The primary Lewis et al. finding—that rostral anterior cingulate cortex thickness predicts emotional psilocybin responses—showed a comparable effect size (β = 0.523 vs. their range 0.324–0.572) that did not achieve statistical significance (p = 0.297), likely reflecting limited statistical power given our smaller sample (N = 25 vs. N = 55). We identified an anterior–posterior gradient in cingulate thickness that significantly predicted psychedelic experience intensity (r = 0.676, FDR p = 0.0004). Conclusions Findings indicate that spatial organization within the cingulate cortex provides a neuroanatomical marker of variability in psychedelic response. Results highlight the importance of organizational patterns within the cingulate cortex, rather than focal regional measures, when predicting psychedelic effects. Keywords Psilocybin · Cingulate cortex · Cortical thickness · Psychedelic experience · Replication study Received: 5 September 2025 / Accepted: 28 November 2025 © The Author(s) 2025 Regional specificity of the cingulate cortex thickness association with the intensity of psilocybin experience: a replication study DavidGreguš1· JaroslavHlinka1,3· FilipTylš 1,2· VojtěchViktorin1· MichaelaViktorinová1· AnnaBravermanová1· Renáta Androvičová1· VeronikaAndrashko1,2· JakubKorčák1· Marek Nikolič1,2· Petr Adámek1,2· MichalBeneš1,2· TomášPáleníček1,2· JiříHoráček1,2 1 3 Psychopharmacology the intensity and quality of subjective experiences predict therapeutic outcomes in depression, anxiety, and substance use disorders (Yaden and Griffiths 2020). The cingulate cortex represents a critical neuroanatomical region related both to pathophysiology of depression and the effect of psilocybin. In depression, the cingulate cortex exhibits multiple structural and functional abnormalities that have been consistently documented across neuroimaging studies. The subgenual anterior cingulate cortex shows pronounced gray matter volume reductions and decreased metabolic activity (Drevets et al. 1997), while functional connectivity analyses have revealed increased connectivity between different cingulate subregions and areas involved in negative emotional processing, including increased coupling between the posterior cingulate cortex and lateral orbitofrontal cortex (Cheng et al. 2018) and altered connectivity patterns between anterior cingulate regions and orbitofrontal areas (Rolls et al. 2019). These connectivity alterations are thought to underlie core depressive symptoms such as rumination, anhedonia, and emotional dysregulation (Cheng et al. 2018; Berman et al. 2011). Given the central role of cingulate dysfunction in depression, it is particularly noteworthy that psychedelic compounds produce profound alterations in cingulate cortex activity and connectivity. Psilocybin consistently decreases cerebral blood flow and neural activity in both anterior and posterior cingulate regions (Carhart-Harris et al. 2012), while also causing broadband cortical desynchronization that disrupts normal rhythmic activity patterns throughout cingulate networks (Muthukumaraswamy et al. 2013). Similarly, conventional antidepressants such as selective serotonin reuptake inhibitors (SSRIs) have been shown to induce structural changes in the anterior cingulate cortex (ACC), including increases in rostral and caudal ACC thickness following treatment (Bartlett et al. 2018; Nemati and Abdallah 2020), highlighting the ACC as a common neuroplasticity target across different pharmacological interventions for mood disorders. Lewis et al. (2020) reported that rostral anterior cingulate cortex (rACC) thickness predicted emotional experiences induced by psilocybin in healthy adults, finding significant positive correlations between right rACC thickness and emotional subscales of the Five-Dimensional Altered States of Consciousness(5D-ASC) questionnaire. However, their analysis focused only on limited emotional dimensions of the 5D-ASC. This fact together with employed statistical approaches which did not fully address the problem of multiple comparison (inherent in neuroimaging research) call for a replication study to evaluate in more detail the role of cingulate cortex thickness parameters in the intensity and nature of the psilocybin experience. The present study aimed to investigate the relationship between cingulate cortex structure and psilocybin-induced altered states of consciousness. Our goals were to: (1) replicate Lewis et al.‘s analytical approach on our dataset under rigorous statistical control, (2) explore the broader dimensionality of psychedelic experiences using comprehensive ASC assessment, and (3) identify neuroanatomical predictors of psychedelic response. Methods Study approval and safety measures The study adhered to the Guidelines for Safety in Human Hallucinogen Research (Johnson et al. 2008). Participants abstained from drug use during the study period, including alcohol (one week before the experiment) and caffeine and tobacco (two hours before testing). The study was approved by the Ethical Committee of the National Institute of Mental Health/Psychiatrické centrum Praha (IGA MZČR NT13897, approved on 11 December 2013) and by the Czech State Institute for Drug Control. Written informed consent was obtained from all participants prior to study participation. Experimental design This study employed an exploratory, non-preregistered analysis approach as part of a larger multimodal brain imaging project (EudraCT No. 2012–004579-37). The primary objective was neuroimaging investigation, with data collected in two consecutive arms where subjects underwent questionnaire-based, phenomenological, neurocognitive, and electroencephalography (EEG) or functional magnetic resonance imaging (fMRI) measurements. In this analysis, we have utilized the fMRI/MRI data. The EEG data collection (EEG arm) preceded a second arm designed for fMRI/ MRI data collection to minimize stress exposure, as EEG procedures were considered less demanding than the fMRI environment (Viktorin et al. 2022; Bravermanova et al. 2018; Dudysova et al. 2020; Nikolic et al. 2023). Within each arm, participants received both psilocybin and placebo treatments in a double-blind, placebo-controlled crossover design. Each participant completed four dosing sessions total: one psilocybin and one placebo session in each arm. Treatment order was balanced across participants to control for order effects, with a minimum 28-day interval between sessions for safety reasons as requested per legal authority and to eliminate residual effects (Studerus et al. 2011; Aday et al. 2020). Each dosing session began with clinical screening including somatic examination, assessment of current living situation, vital signs monitoring, and negative breathalyzer 1 3 Psychopharmacology and urine drug screens. For the fMRI/MRI arm, participants underwent three scanning sessions (baseline, 90 min, and 240 min post-dosing) lasting 45–60 min each. For purposes of this paper we utilized structural MRI data from baseline, 40 min before drug administration. Throughout each session, participants were continuously monitored by two healthcare professionals (one psychiatrist), with additional nursing support available. Follow-up contacts were conducted on days 2 and 4 post-session to assess psychological and physical well-being (Johnson et al. 2008). Treatments consisted of oral psilocybin (0.26 mg/kg, THC Pharm GmbH, > 98% purity) or placebo (wheat starch) in identical capsules prepared by an independent pharmacy. Dosing was weight-adjusted using combinations of 1 mg and 5 mg capsules, with doses ranging from 16 to 24 mg (mean: 19.2 mg). Capsules were administered on an empty stomach with 200 ml water. Participants Forty healthy volunteers were recruited using peer-to-peer snowball sampling and underwent comprehensive screening including Minnesota Multiphasic Personality Inventory-2 (Butcher et al. 2001) and Mini-International Neuropsychiatric Interview (Sheehan et al. 1998) to exclude psychopathology. Complete inclusion and exclusion criteria are provided in Supplementary Material S1. Fifteen participants discontinued after the EEG arm primarily due to fMRI-related anxiety and scheduling concerns. Twenty-five participants completed the fMRI/MRI arm and were included in this analysis (15 males, 10 females; mean age 36.4 ± 7.9 years). Demographic characteristics are presented in Table 1. Psychometric assessment of altered states of consciousness Subjects rated their experience via the Altered States of Consciousness scale (ASCs) using Dittrich’s revised questionnaire (Dittrich 1998). The instrument consists of 72 items assessing the subjective acute effects of the altered state of consciousness, grouped into three primary subscales and one global composite score: Oceanic Boundlessness (OBN) — reflecting experiences of unity, transcendence, and dissolution of personal boundaries; Dread of Ego Dissolution (DED) — capturing feelings of anxiety, loss of self-control, and fear associated with ego dissolution; Visionary Restructuralization (VRS) — describing perceptual alterations, visual distortions, and changes in the meaning of percepts; and the General Altered States of Consciousness score (GASC) — a composite measure derived from the three primary subscales (49 items) plus 23 additional items from Dittrich’s ASCglo secondary scale, summarizing the overall intensity across all experiential domains. The questionnaire was administered 360 min post-administration to capture peak psilocybin effects. ASC scores were calculated as delta scores (ΔASC = ASC psilocybin − ASC placebo) to account for baseline variability in subjective experiences. Lewis et al. (2020) utilized in their analysis the FiveDimensional Altered States of Consciousness Scale (5DASC) and focused exclusively on four emotionally salient subscales: “experience of unity,” “spiritual experience,” “blissful state,” and “insightfulness”. These subscales correspond with higher order subscale Oceanic Boundlessness (Studerus et al. 2010). Because these four subscales used by Lewis et al. (2020) cannot be extracted from the original ASC version, for the purposes of our replication study we worked primarily with OBN alongside other factors (DED, VRS, GASC). Both studies maintained methodological consistency by utilizing visual analog scales and delta scores. MRI data acquisition Structural MRI scans were acquired 40 min before drug administration on the first session within the fMRI arm of the study using a 3 Tesla Siemens Prisma scanner with a 20-channel HeadNeck coil. High-resolution T1-weighted anatomical images were collected using a magnetization-prepared rapid acquisition gradient-echo (MPRAGE) sequence (repetition time = 2300 ms, echo time = 1.33 ms, field of view = 256 mm, inversion time = 1100 ms, flip angle = 7°). The isotropic 1 mm voxel size was identical to Lewis et al. (2020) to ensure methodological consistency and minimize partial volume effects. Participants were positioned comfortably in the scanner, with foam padding to minimize head movement during the scan. Cortical thickness measurement Cortical thickness was measured from placebo session images using the FreeSurfer image analysis suite version 7.4.0 (Fischl 2012). T1-weighted images were processed to extract cortical surfaces and compute cortical thickness for Table 1 Demographic and study parameters of participants (n = 25). Continuous variables are presented as Mean ± SD (range) Characteristic Value Sex 15 males (60%), 10 females (40%) Age (years) 36.4 ± 7.9 (28–53) Education Level University: 22 (88%), Secondary: 2 (8%), Unspecified: 1 (4%) Years of Education 19.8 ± 2.4 (15–23) Weight (kg) 73.8 ± 11.9 (56–99) Drug Dose (mg) 19.2 ± 2.3 (16–24) Administration Time Range: 9:27 − 11:53 1 3 Psychopharmacology 10,000 times while maintaining the predictor matrix (brain regions and covariates) unchanged. For each permutation, we re-fitted the regression model and extracted regression coefficients for the brain regions. The permutation p-value for each coefficient was calculated as the proportion of permuted coefficients with absolute values greater than or equal to the absolute value of the observed coefficient, providing a two-tailed test. FDR correction was applied to permutation p-values using the same procedure as parametric tests (Benjamini-Hochberg within each hemisphere). Then we extended Lewis et al.‘s methodology by: (1) conducting principal component(PC) analysis to identify underlying data structure; (2) performing component-based analysis using partial correlations to control for covariates; and (3) applying FDR correction to our final test set. This multi-step approach allows for direct comparison with the original findings while providing methodological rigor. For brain-experience PC correlations, we computed partial correlations controlling for the same covariates used in the multivariate regression analyses (age, sex, and average postcentral gyrus thickness). This ensures methodological consistency between the regression-based and component-based analyses. Partial correlations were computed via residualization: both PC scores were regressed on the covariates, and Pearson correlations were computed between the residuals. Results Lewis et al. Replication Results Applying Lewis et al.‘s multivariate regression approach failed to replicate their primary findings that rostral anterior each participant using the Desikan-Killiany atlas, consistent with Lewis et al. (2020) methodology. All processed images were visually inspected for quality control. Regions of interest (ROIs) included the rostral anterior cingulate cortex (rACC), caudal anterior cingulate cortex (cACC), and posterior cingulate cortex (PCC) in both hemispheres (Fig. 1). These regions are known to have high density of 5HT2A receptors and are implicated in emotional processing (Beliveau et al. 2016; Lewis et al. 2020). The postcentral gyrus was included as a control region due to its low expression of 5HT2A receptors, allowing us to account for non-specific effects of global cortical thickness. Statistical analysis We employed linear regression models following Lewis et al.‘s methodology. For each hemisphere, we fitted four separate ordinary least squares (OLS) regression models—one for each ASC outcome (DED, OBN, VRS, GASC)—using the three cingulate regions (rACC, cACC, PCC) plus control variables (age, sex, postcentral gyrus thickness) as predictors. Analyses were implemented using Python statsmodels package (v0.14.0). Note that unlike Lewis et al., we did not include dose as a control variable since our experimental design used weight-adjusted dosing, eliminating dose variability. Regarding the multiple comparison correction, we followed their approach to include Benjamini-Hochberg False Discovery Rate correction within each hemisphere (12 tests: 3 cingulate regions × 4 emotional subscales). To verify findings under relaxed distributional assumptions, we performed permutation tests on the multivariate regression models. For each outcome variable, we randomly permuted the dependent variable (delta ASC scores) Fig. 1 Anatomical Locations of Regions of Interest: Left - rostral anterior cingulate cortex (purple), caudal anterior cingulate cortex (orange), posterior cingulate cortex (green). Right - post central gyrus (red) as control region 1 3 Psychopharmacology and detailed in Supplementary Table 1, the majority of associations did not reach statistical significance. The only effect that survived false discovery rate correction was a negative association between left posterior cingulate cortex thickness and Visionary Restructuralization (β = − 0.928, p = 0.003, p_FDR = 0.031). To verify our findings under relaxed distributional assumptions and to address any potential concerns about normality (Supplementary Fig. 1), we performed permutation tests on the full multivariate regression models. This approach maintains the same model structure as the parametric analysis (including covariates for age, sex, and postcentral thickness) while testing significance through data resampling rather than distributional assumptions. For each outcome, we permuted the dependent variable 10,000 times and re-fitted the regression model, computing p-values as the proportion of permuted coefficients exceeding the observed values in absolute value. FDR correction was applied within each hemisphere (12 tests per hemisphere) following the same procedure as the parametric analysis. Permutation tests provided a more conservative assessment of statistical significance than parametric regression (Supplementary Table 2). While the parametric analysis identified one association surviving FDR correction (left posterior cingulate thickness predicting Visionary Restructuralization: β = −0.928, parametric p_FDR = 0.031), this did not survive permutation testing (permutation p_ FDR = 0.216). No brain-experience associations survived FDR correction (p < 0.05) in the permutation analysis. Notably, effect sizes (regression coefficients) were identical cingulate cortex thickness positively predicts emotional psychedelic experiences, even before multiple comparison correction (Table 2). This multivariate regression analysis tested Altered States of Consciousness (ASC) scales (OBN, DED, VRS, GASC) in relation to cortical thickness across cingulate subregions (rACC, cACC, PCC), while controlling for age, sex, and postcentral gyrus thickness. As presented in Fig. 2 Table 2 Comparison of rostral anterior cingulate cortex associations with emotional psychedelic experiences between Lewis et al.‘s original study (n = 55, 5D-ASC emotional subscales) and our replication attempt (n = 25, ASC - Oceanic Boundlessness). RH = right hemisphere; LH = left hemisphere; rACC = rostral anterior cingulate cortex; OBN = Oceanic Boundlessness; FDR = false discovery rate. *p < 0.05, **p < 0.01, *p < 0.001 Study Brain Region Dimension Beta p-value FDR p-value Lewis et al. RH rACC Unity 0.324 0.027* 0.027* Lewis et al. RH rACC Spiritual 0.465 0.001*** 0.002** Lewis et al. RH rACC Bliss 0.386 0.008** 0.011* Lewis et al. RH rACC Insight 0.572 < 0.001*** < 0.001*** Replication RH rACC OBN (emotional subscales equivalent) 0.523 0.297 0.445 Replication LH rACC OBN (emotional subscales equivalent) 0.13 0.687 0.686 Fig. 2 Results of multivariate regression testing associations between cingulate cortex subregions thicknesses and ASC scales (replication of Lewis et al.). Heatmap shows standardized coefficients (left) and FDR-corrected p-values (right) for all tested associations. Analyses controlled for age, sex, and postcentral gyrus thickness. p < 0.05 after FDR correction 1 3 Psychopharmacology showed a clear anterior-posterior gradient: positive loadings for anterior regions (rACC, cACC) and negative loadings for posterior regions (PCC). Component-based analysis results Based on the PCA results, we tested associations between ASC PC1 (“Psychedelic experience”) and both brain structural components (“General Cingulate Thickness” and “Anatomical Gradient”). We focused on these components as they represented clear, interpretable anatomical patterns. Component-based analysis tested associations between brain structural components and psychedelic experience using both parametric and non-parametric methods (Table 3). For the primary parametric analysis, partial correlations controlling for age, sex, and postcentral thickness revealed that the anatomical gradient (greater anterior relative to posterior cingulate thickness) showed a strong positive association (r = 0.676, p_FDR = 0.0004). This finding was corroborated by non-parametric Spearman correlation (ρ = 0.598, p_FDR = 0.002), demonstrating convergent validity across methods. General cingulate thickness showed significance only in the parametric analysis with covariate control (r = −0.505, p_FDR = 0.010) but not in the non-parametric test (ρ = −0.234, p_FDR = 0.261), suggesting method-dependence. We therefore focus our interpretation on the anatomical gradient finding, which demonstrates consistent effects regardless of statistical approach and represents a theoretically meaningful anatomical pattern. between parametric and permutation approaches, with close agreement on uncorrected p-values (e.g., LH PCC → VRS: parametric p = 0.003, permutation p = 0.036). This pattern suggests that parametric findings may be sensitive to distributional assumptions. Data structure analysis Given the limited significant findings from the direct replication approach and the potential for multicollinearity among both brain regions and ASC scales, we proceeded with principal component analysis to identify interpretable anatomical patterns that capture the underlying data structure. Principal component analysis was conducted on the three primary ASC subscales (DED, OBN, VRS); GASC was excluded because it correlates extremely highly with the mean of these three subscales (r = 0.983, p < 0.001), creating statistical redundancy. The three subscales showed substantial intercorrelations (mean r = 0.612), with a single component (PC1) explaining 74.3% of variance (Fig. 3). We labeled this component “Psychedelic Experience” given its uniform positive loadings across all three subscales (DED: 0.547, OBN: 0.621, VRS: 0.562), capturing the general intensity of psychedelic effects. For brain structure, PCA of the six cingulate regions (Fig. 4) identified interpretable anatomical patterns. The first component (PC1, 48.2% variance) showed uniform positive loadings across all regions, representing general cingulate thickness. The second component (PC2, 21.1% variance) Fig. 3 Principal component analysis of the three primary ASC subscales. PC1 (“Psychedelic experience”) explains 74.3% of variance with uniform positive loadings (DED = 0.547, OBN = 0.621, VRS = 0.562), representing overall psychedelic experience intensity. GASC is excluded to avoid redundancy (r = 0.983 with mean of three subscales) 1 3 Psychopharmacology and independent replication efforts in establishing reliable neurobiological markers of psychedelic experience. Anatomical gradient of cingulate cortex in psychedelic experience Our most significant finding was the identification of an anatomical gradient pattern within the cingulate cortex that strongly predicts psychedelic experience intensity (r = 0.676, p_FDR = 0.0004). This gradient represents a neuroanatomical organization where individuals with relatively greater anterior cingulate thickness compared to posterior cingulate thickness (positive gradient scores) experience more intense psychedelic effects across all experiential domains. Conversely, individuals with relatively greater posterior compared to anterior thickness (negative gradient scores) show attenuated psychedelic responses. This pattern suggests that the organizational structure of the cingulate cortex—rather than absolute thickness in any single region—determines psychedelic sensitivity. The mechanisms underlying this gradient effect remain to be determined, but may reflect differences in cortical organization, functional connectivity, or network integration. The Discussion Replication Findings We attempted to replicate Lewis et al.‘s association between right rostral anterior cingulate cortex thickness and emotional psychedelic experiences. Our effect size closely matched their findings (β = 0.523 vs. their range 0.324–0.572), but we did not achieve statistical significance (p = 0.297, FDR p = 0.445). This represents a partial replication where effect direction and magnitude align with the original study, though significance was not reached. Several factors may contribute to this discrepancy. Our smaller sample size (N = 25 vs. N = 55) reduces statistical power, which may explain the non-significant p-value despite matching effect magnitude. Additionally, methodological differences between studies—including scanner specifications, preprocessing pipelines, sample characteristics, and experimental protocols—may influence results. Population-specific factors and the presence of influential outliers in smaller neuroimaging samples can also substantially impact correlation estimates. These considerations underscore the importance of robust analytical approaches Table 3 Brain-experience associations using principal component analysis. Parametric: pearson partial correlations controlling for age, sex, and postcentral thickness (primary analysis). Non-parametric: spearman zero-order correlations without covariates (robustness check for outlier influence). FDR correction applied within each method (2 tests per method, α = 0.05). *p < 0.05 Brain Component ASC Component Parametric r p-value FDR p-value Non-parametric ρ p-value FDR p-value General Thickness Psychedelic Experience −0.505 0.010 0.010* −0.234 0.261 0.261 Anatomical Gradient Psychedelic Experience 0.676 0.0002 0.0004* 0.598 0.002 0.003* Fig. 4 Principal component analysis of cingulate regions reveals two meaningful components: PC1 (General Thickness, 48.2% variance) with uniform positive loadings across all regions, and PC2 (Anatomical Gradient, 21.1% variance) with positive loadings for anterior regions and negative loadings for posterior regions 1 3 Psychopharmacology Finally, the cross-sectional nature of our structural measurements prevents assessment of whether these thickness patterns represent stable individual differences or plastic changes related to psychedelic exposure. Future research should validate this biomarker in clinical populations and investigate mechanistic relationships with 5-HT2A receptor function. Conclusions While Lewis et al.‘s primary finding showed a comparable effect size in our study, we lacked statistical power for confirmation given our smaller sample. Most notably, we discovered an anatomical gradient pattern (greater anterior relative to posterior cingulate thickness) that strongly predicts psychedelic experience intensity (r = 0.676, p_ FDR = 0.0004). This spatial organization pattern represents a potential neuroanatomical marker for understanding individual differences in psychedelic response. Supplementary Information The online version contains supplementary material available at h t t p s : / / d o i . o r g / 1 0 . 1 0 0 7 / s 0 0 2 1 3 - 0 2 5 - 0 6 9 8 3 - 9 . Acknowledgements This work was supported by the Czech Health Research Council (Project Nos. NU21-04-00307 and NW24-04-00413), Long-term conceptual development of research organization (Grant No. RVO 00023752), European Regional Development Fund Project Brain dynamics (Project No. CZ.02.01.01/00/22_008/0004643), project VVI CZECRIN (Project No. LM2023049), Horizon Europe project PsyPal (Grant Agreement No. 101095146) and Charles University Cooperatio Neurosciences research program, and private funds obtained via the Psychedelic Research Foundation (PSYRES) ( h t t p s : / / p s y r e s f o u n d a t i o n . e u ) . We acknowledge the assistance of Claude AI (Anthropic Inc.) in debugging analytical code and providing grammatical corrections during manuscript preparation. The authors carefully reviewed and validated all AI-assisted content to ensure scientific accuracy and reliability. Author contributions T.P. and J. Horáček conceived and designed the project and supervised the experiments. J. Hlinka developed and oversaw the statistical approach. D.G. analyzed the data and wrote the manuscript. F.T., V.V., M.V., A.B., R.A., V.A., J.K., M.N., P.A., and M.B. contributed to data collection. All authors reviewed and approved the final version of the manuscript. Funding This work was supported by the Czech Health Research Council (Project Nos. NU21-04-00307 and NW24-04-00413), Longterm conceptual development of research organization (Grant No. RVO 00023752), European Regional Development Fund Project Brain dynamics (Project No. CZ.02.01.01/00/22_008/0004643), project VVI CZECRIN (Project No. LM2023049), Horizon Europe project PsyPal (Grant Agreement No. 101095146) and Charles University Cooperatio Neurosciences research program, and private funds obtained via the Psychedelic Research Foundation (PSYRES) ( h t t p s : / / p s y r e s f o u n d a t i o n . e u ) . Data availability The datasets generated and analyzed during the current study are not publicly available due to privacy considerations and institutional policies but are available from the corresponding author upon reasonable request and with appropriate ethical approval. relative balance between anterior and posterior regions appears more critical for psychedelic experience intensity than thickness in any single subregion. The robustness of this finding across both parametric and non-parametric methods likely reflects its measurement of relative structural patterns (anterior-to-posterior ratios) rather than absolute thickness levels. Pattern-based measures are inherently less sensitive to individual differences in overall brain size, outliers, and demographic confounds, which may explain why the gradient finding demonstrated convergent validity while the general thickness association was method-dependent. This suggests the anterior-posterior gradient represents a stable neuroanatomical signature of psychedelic responsiveness. Importantly, our gradient finding converges with prior research on chronic exposure to psychedelics. Bouso et al. (2015) reported the same directional pattern in regular ayahuasca users: relative thickening in anterior cingulate regions and thinning in posterior cingulate cortex. Implications Our findings suggest that individual differences in cingulate cortical organization contribute to variability in psychedelic experience intensity. The identification of an anatomical gradient pattern as a predictor of response highlights that spatial organization of cortical structure, rather than absolute regional thickness, may be a more informative neurobiological marker. This has implications for future psychedelic neuroscience research: accounting for baseline structural variance may reduce noise in experimental designs, and spatial patterns across regions may be more replicable markers than single-region measures. Whether these structural patterns in healthy individuals generalize to clinical populations or predict therapeutic outcomes remains to be determined. Future studies should test the replicability of the gradient pattern in independent samples and examine its relationship to treatment response in patient populations. Limitations and future directions Several limitations should be acknowledged. Our sample size (n = 25), while comparable to other psychedelic neuroimaging studies, limits statistical power and generalizability. 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