This double-blind, placebo-controlled study (n=58) investigated the effect of two doses of psilocybin (11mg-15mg/70kg) on cerebral blood flow. The larger dose led to significantly higher ratings on 4 of 11 scales of altered consciousness. It also showed which specific brain regions became more, and less, active.
Psilocybin, the active compound in psychedelic mushrooms, is an agonist of various serotonin receptors. Seminal psilocybin positron emission tomography (PET) research suggested regional increases in glucose metabolism in frontal cortex (hyperfrontality). However, a recent arterial spin labeling (ASL) study suggests psilocybin may lead to hypo-perfusion in various brain regions. In this placebo-controlled, double-blind study we used pseudo-continuous ASL (pCASL) to measure perfusion changes, with and without adjustment for global brain perfusion, after two doses of oral psilocybin (low dose: 0.160 mg/kg; high dose: 0.215 mg/kg) in two groups of healthy controls (n = 29 in both groups, total N = 58) during rest. We controlled for sex and age and used family-wise error corrected p values in all neuroimaging analyses. Both dose groups reported profound subjective drug effects as measured by the Altered States of Consciousness Rating Scale (5D-ASC) with the high dose inducing significantly larger effects in four out of the 11 scales. After adjusting for global brain perfusion, psilocybin increased relative perfusion in distinct right hemispheric frontal and temporal regions and bilaterally in the anterior insula and decreased perfusion in left hemispheric parietal and temporal cortices and left subcortical regions. Whereas, psilocybin significantly reduced absolute perfusion in frontal, temporal, parietal, and occipital lobes, and bilateral amygdalae, anterior cingulate, insula, striatal regions, and hippocampi. Our analyses demonstrate consistency with both the hyperfrontal hypothesis of psilocybin and the more recent study demonstrating decreased perfusion, depending on analysis method. Importantly, our data illustrate that relative changes in perfusion should be understood and interpreted in relation to absolute signal variations.
Papers cited by this study that are also in Blossom
Bernasconi, F. ;., Schmidt, A. ;., Pokorny, T. ;. et al. · Cerebral Cortex (2013)
Carhart-Harris, R. L., Erritzoe, D., Williams, T. et al. · PNAS (2012)
Gasser, P., Holstein, D., Michel, Y. et al. · Journal of Nervous and Mental Disease (2014)
Geyer, M. A., Vollenweider, F. X. · Trends in Pharmacological Sciences (2008)
The serotonin (5-HT) system modulates perception, cognition, mood and consciousness, and classic psychedelics such as psilocybin produce profound alterations in these domains. Neuroimaging studies of psilocybin and related 5-HT agonists have reported apparently conflicting results: early PET studies, after correction for global fluctuations, found regional increases in frontal metabolism (a "hyperfrontal" pattern), whereas a more recent arterial spin labelling (ASL) study reported widespread decreases in cerebral blood flow (CBF). Because serotonergic drugs can also alter vascular tone, disentangling regional neuronal effects from global haemodynamic changes is important for interpreting pharmacological neuroimaging findings. Lewis and colleagues set out to resolve this contradiction by measuring cerebral perfusion after two oral doses of psilocybin using pseudo-continuous ASL (pCASL) in a randomised, double-blind, placebo-controlled design. They compared two analytic strategies: one that normalised for global perfusion to identify relative regional CBF (rCBF) changes, and one non-adjusted analysis to capture absolute or global CBF (gCBF) effects. The authors hypothesised that normalised analyses would reproduce the hyperfrontal pattern seen in PET, that non-adjusted analyses would show global decreases similar to the recent ASL report, and that the higher dose would produce larger effects than the lower dose.
Study design was randomised, double-blind and placebo-controlled. Two separate participant groups received either a lower medium dose (0.16 mg/kg) or a higher medium dose (0.215 mg/kg) of oral psilocybin in single-dose sessions; placebo (maltose) was administered in the alternate session. Sessions were at least 10 days apart. Because regulatory approval was limited to single-dose administration per participant, the low and high dose conditions were implemented in separate groups rather than within-subject. An anatomical MRI was acquired about 60 minutes after drug intake, followed by a resting-state pCASL perfusion scan. Subjective effects were assessed using the 5D-ASC questionnaire at 360 minutes post-dose. Participants abstained from nicotine and caffeine on test days. Participants were recruited via university advertisements and underwent medical screening (medical history, physical examination, blood tests and ECG), urine toxicology and pregnancy testing; prior heavy psychedelic use was an exclusion criterion. The extracted text does not clearly report the sample size in the Participants section, though inferential statistics reported elsewhere in the paper are consistent with a total sample of 58 participants (two groups). All participants provided written informed consent and the study was approved by Swiss regulatory and ethics bodies. Neuroimaging data were acquired on a Philips Achieva 3.0T scanner. The resting pCASL sequence parameters included TR 4400 ms, TE 20 ms, field of view 240 x 240 mm, matrix 80, 23 slices at 7 mm thickness, post-labeling delay 1525 ms, label duration 1650 ms and 50 dynamics (each dynamic a control and a labelled image). High-resolution T1-weighted images were also collected. Preprocessing (performed with the ASLtoolbox and SPM12) comprised realignment, smoothing (6 x 6 x 14 mm kernel), perfusion-weighted image construction, calibration using M0 images and conversion to absolute mean CBF images; data were visually inspected for motion and all scans were reported to have less than one voxel of movement. Statistical analysis of psychometrics used repeated-measures ANOVA on 11 validated 5D-ASC scales with within-subject factors of drug and scale, a between-subject factor of dose (Low vs High) and covariates age and sex; significant effects were followed by Bonferroni-corrected pairwise comparisons. Group-level ASL analyses used a flexible-factorial general linear model in SPM12. Age and sex were included as covariates throughout and family-wise error (FWE) correction was applied at peak-level p < 0.05. To probe dose effects, the High and Low groups were compared and a t-test was applied to subtraction images (psilocybin minus placebo). The investigators used two approaches to assess relative CBF: the normalization option within the flexible-factorial design (global covariate as nuisance) and an analysis that included each participant’s mean whole-brain CBF (from an AAL mask) as an additional covariate; both yielded similar results and the normalized ANOVA is reported. Non-adjusted analyses were used to characterise gCBF changes. Treatment order effects were also tested on extracted quantitative CBF values.
On subjective measures, the repeated-measures ANOVA (drug x scale x dose, controlling for age and sex) produced significant main effects of drug, scale and dose, and significant interactions including scale*dose, drug*dose, scale*drug and the three-way interaction. Pairwise comparisons showed the High dose produced significantly larger subjective effects than the Low dose on four of the 11 5D-ASC scales: Disembodiment (p = 0.017), Complex Imagery (p = 0.026), Elementary Imagery (p = 0.016) and Audiovisual Synesthesia (p = 0.005). Simple main effects indicated all scales increased after psilocybin relative to placebo (all p < 0.001). Analyses of cerebral perfusion found no dose-dependent effects on either rCBF or gCBF even at uncorrected thresholds (p < 0.001), and both dose groups exhibited similar spatial patterns; consequently, subsequent imaging analyses were collapsed across dose groups. Sex had no detectable effect on rCBF or gCBF. Age showed a negative relationship with gCBF; a pCASL SPM map reported two significant age-related clusters (right frontal superior/inferior orbital area at 24 28 -18, k = 73, t = 6.57; middle frontal gyrus at 36 8 34, k = 93, t = 6.26). When the global signal was regressed out (rCBF analysis), psilocybin produced significant local increases and decreases (FWE p < 0.05). Increases were observed in bilateral inferior frontal gyrus and insula, with right-sided increases in hippocampus, operculum and superior temporal gyrus. Decreases were found predominantly in left-hemispheric structures including the amygdala, lentiform nucleus, globus pallidus, putamen, thalamus, insula and portions of frontal, parietal, occipital and temporal cortices. In the non-adjusted gCBF analysis, psilocybin elicited widespread decreases in absolute perfusion across frontal, parietal, temporal, limbic and occipital cortices as well as cingulate, insula, caudate, putamen, pallidum, amygdala, hippocampus and thalamus. No regions showed increased gCBF under psilocybin compared with placebo. Finally, testing for treatment order effects on extracted quantitative CBF values found no significant differences for placebo or psilocybin (placebo t(56) = 0.84, p = 0.84; psilocybin t(56) = -1.17, p = 0.24).
Using both global-normalized and non-adjusted ASL analyses, Lewis and colleagues reconcile prior discrepant findings by showing that analytic choice determines the apparent direction of psilocybin effects on perfusion. Normalized analyses (rCBF) identified regionally specific increases and decreases, including right-lateralised frontal and temporal increases and left-sided decreases in parietal regions and subcortical structures, a pattern consistent with early PET reports of hyperfrontality after global correction. The non-adjusted analyses demonstrated broad reductions in absolute perfusion across cortical and subcortical regions, aligning with a previous ASL study that reported widespread CBF decreases following intravenous psilocybin. The investigators emphasise that ASL remains an indirect measure of neuronal activity and that psilocybin’s vasoactive properties complicate interpretation: observed gCBF decreases could partly reflect global vascular tone changes rather than purely neuronal suppression. The absence of a dose effect on CBF, despite larger subjective effects at the higher dose, suggests that the mechanisms mediating phenomenology may be subtler than what ASL captures in this dose range or that neurovascular factors decouple subjective ratings from perfusion measures. The authors discuss potential receptor-level explanations, noting that 5-HT2A activation on excitatory pyramidal cells may produce regional increases in activity and blood flow, whereas 5-HT1A actions and indirect inhibition via interneurons could produce regionally opposing decreases; they caution that receptor-specific contributions cannot be disentangled in the present design. Methodological implications are highlighted: inclusion or omission of a global covariate meaningfully changes phMRI inferences, and reporting both normalized and non-normalized results can clarify the distinct regional and global consequences of serotonergic challenges. Limitations acknowledged by the authors include the between-subjects dose comparison, the relatively narrow dose range tested, and differences in route of administration across studies they compare. They conclude that understanding both neuronal and neurovascular effects of psilocybin is important for interpreting neuroimaging results and for informing clinical applications, and they recommend careful selection and transparent reporting of analysis approaches in pharmacological ASL research.
The serotonin (5-hydroxytryptamine, 5-HT) system is implicated in neuronal processes regulating perception, cognition, mood, sleep-wake transitions, and consciousness. Abnormalities in 5-HT may be involved in the etiology of many psychiatric conditions and treatments modulating this system have been used for decades. Psilocybin (4-phosphoryloxy-N,N-dimethyltryptamine) and the structurally related DMT (N,N-dimethyltryptamine) and LSD (lysergic acid diethylamide) are indolamine psychedelics, all of which produce an Altered State of Consciousness (ASC) characterized by profound changes in sensory perception, emotion, thought, and sense of self. Furthermore, psilocybin and LSD induce sensory perturbations reminiscent of early stage schizophrenia spectrum disorders in healthy participantsand disrupt sensorimotor gating in healthy subjects, inducing behavior similar to first-episode schizophrenia patients. Hence, further elucidation of classic psychedelics may improve our understanding of the pathophysiology of psychotic states. Alternatively, recent research suggests moderate doses of psilocybin enhance positive mood and attenuate the BOLD response to negative stimuli such as threat-related scenes, facial expressions, and social rejection in healthy participants. These acute effects may be related to the long-term antidepressant or anxiolytic effects reported in earlier and recent studies with psilocybin or LSD. Despite psilocybin's high potential as a neuroscience research tool and possible efficacy as psychiatric treatment, we still have a limited understanding of psilocybin's actions in the human brain. The recent resurgence of interest in the clinical use of classic psychedelics, such as psilocybin, highlights the need to further elucidate their brain effects. Neuroimaging studies investigating the influence of psilocybin, and other 5-HT modulators, on brain activity have found seemingly contradicting results that necessitate clarification. Two PET studies measuring cerebral metabolism of glucose found oral administration of psilocybin produced hypermetabolism, after correction for global fluctuations, in prefrontal brain regions including the ACC, with hypometabolism in subcortical and occipital brain regions. Moreover,showed oral administration of DMT induces similar hyperfrontal effects to psilocybin, predominantly in the right ACC/medial frontal cortex and bilateral insula. Oral administration of the hallucinogenic 5-HT2A agonist mescaline also produces increased cerebral blood flow (CBF) in right hemispheric anterior regions. However, a recent study found contradicting results in that intravenous psilocybin administration induced a widespread decrease of CBF including the frontal cortex during rest. While these studies measured different physiological properties and should not be directly compared, the neuronal processes are coupled; thus, divergent reports necessitate further investigation in order to ascertain the effects of psilocybin on neuronal activity. As interest in the clinical use of psilocybin grows it is imperative to clarify existing contradicting literature on the neural effects. Quantitative neuroimaging techniques, such as PET and ASL, allow us to measure local changes in neural activity in response to a pharmacological challenge. However, a pharmacological challenge with a highly psychoactive drug that also affects vascular tone, such as psilocybin, demands special attention to analysis and interpretation. Namely, since serotonergic drugs generally decrease vascular tone, this could be considered a "nonspecific" cerebral hemodynamic psilocybin effect superimposed on true changes in local neuronal activity. In order to localize regional cerebral effects induced by the pharmacological challenge, the "nonspecific" change would need to be accounted for by a normalization procedure in the analysis. For this reason, we employed two separate analyses: one controlling for global perfusion in order to localize regional, or relative, CBF (rCBF), and one non-adjusted analysis to capture the effects on global, or absolute, CBF (gCBF). Furthermore, since these analyses probe two different effects, we propose unique hypotheses for rCBF and gCBF effects. We investigated two doses of oral psilocybin administration (0.16 mg/kg and 0.215 mg/kg) compared to placebo using pseudo continuous arterial spin labeling (pCASL) fMRI. ASL provides good spatial and temporal resolution while also being non-invasive and quantitative, and is arguably preferable to BOLD in measuring drug actions. We predicted that 1) after controlling for global perfusion, psilocybin would induce a similar hypermetabolic frontal rCBF pattern observed in glucose metabolism PET studies, 2) non-adjusted analysis would reveal a similar pattern of general decreased gCBF seen in the more recent ASL study, and 3) more pronounced effects would result from the higher dose than the lower dose.
Participants were recruited through advertisements in local universities for two separate groups, either low dose (0.16 mg/kg: Low) or a higher dose (0.215 mg/kg: High). All participants were healthy based on medical history, physical examination, blood analysis, and electrocardiography, had normal or corrected-to-normal vision, right hand dominant, and were urine tested for drug use and pregnancy. Prior heavy psychedelic use was an exclusion criterion (Table). Since approval was only obtained for single dose administration, two different groups received the low and high dose. Additionally, to allow for comparability with previous studies and current clinical trialswe chose a lower medium and a higher medium dose for this first neuroimaging dose comparison study.) years, range 20-37 years]. Participants received written and oral descriptions of the study procedures as well as the effects and possible risks of psilocybin administration. All participants provided written informedconsent statements in accordance with the declaration of Helsinki before participation in the study. The Swiss Federal Office of Public Health, Bern, Switzerland authorized the use of psilocybin in humans. Furthermore, the study was approved by the Cantonal Ethics Committee of Zurich.
In a randomized, double blind, and placebo-controlled study, participants received either Placebo (maltose) or oral psilocybin (0.16 mg/kg or 0.215 mg/kg) in two separate sessions at least 10 days apart. These doses reflect the low and high end of the medium dose range commonly used in the literature). An anatomical scan was conducted 60 min after drug administration followed by the resting state pCASL scan. The 5D-ASC, a self-report questionnaire retrospectively assessing the subjective experience after drug intake, was completed 360 min after intake. Participants had to abstain from nicotine and caffeine during the test day.
All MR data were acquired on a Philips Achieva 3.0T whole-body scanner (Best, The Netherlands). Inflatable pillows (Multipad, Pearltec AG, Zurich, Switzerland) were used to increase participant comfort in the scanner and to reduce motion induced artifacts. Each session consisted of a resting state pCASL perfusion-weighted scan and several task-related fMRI BOLD scans (published elsewhere:. Due to superior signal-to-noise ratio, pCASL is considered a more reliable method, compared to other ASL sequences, for measuring phMRI. The imaging parameters for the perfusion-weighted scan (pCASL) were based on the sequence developed byThe plane was positioned parallel to the imaging volume with a 20 mm labeling gap between the imaging volume and the labeling volume. ASL parameters were: repetition time (TR): 4400 ms; minimum echo time (TE): 20 ms; FOV: 240 Â 240 mm 2 ; matrix size: 80, 23 slices with slice thickness of 7 mm and no gap; gradient echo single shot EPI; SENSE 2.5; post-labeling delay of 1525 ms; label duration: 1650 ms; number of dynamics: 50. One dynamic consisted of a control and a labeled image. Additionally, high-resolution anatomical images (voxel size, 1 Â 1 Â 1 mm) were acquired using a standard T1-weighted 3D magnetization prepared rapid gradient echo sequence (MP-RAGE). Image data processing and analysis were carried out using the ASLtoolboxFor each participant, image preprocessing was conducted including realignment, smoothing (6 Â 6 Â 14 mm kernel), perfusion weighted image construction and calculation, and normalization. For ASL data, CBF calculations should be performed prior to spatial normalization. All data was visually inspected for motion artifacts, and all participants were confirmed to have less than one voxel of movement for each ASL scan. Equilibrium brain tissue magnetization (M0) images were recorded in a separate run for each subject using the same parameters as described for the pCASL sequence, apart from the TR (10,000 ms). Next, unique cerebral spinal fluid M0 values were calculated per participant for each session (and corrected for T2* decay;and considered in the calculation of each perfusion weighted image. Perfusion weighted image series were generated by simple subtraction of the label and control images, followed by conversion to absolute mean CBF image series.
Questionnaires: The 5D-ASC comprises 94 items to be answered on visual analogue scales. Recently, scores were calculated for 11 validated scales: experience of unity, spiritual experience, blissful state, insightfulness, disembodiment, impaired control and cognition, anxiety, complex imagery, elementary imagery, audio-visual synesthesia, and changed meaning of percepts. A repeated-measures ANOVA was conducted with two within-subject factors of drug and scale and a between-subject factor of dose (Low vs. High) controlling for age and sex. Significant main effects or interactions were followed by Bonferroni-corrected pairwise comparisons and simple main effects analyses. CBF: Group-level analysis of the ASL images were performed using a general linear model implemented in SPM12. Specifically, the individual mean CBF series (see above) were entered into a second-level group analysis using a flexible-factorial design, and gCBF was analyzed as a comparison between psilocybin and placebo conditions (see Inline Supplementary Fig.). Since both sex and age influence CBF, both were included as covariates. Family-wise error (FWE) corrections were used in all analyses at a peak-level corrected threshold of p < 0.05. To investigate the dose effect, we compared the High and Low dose. In order to thoroughly test this relationship, we also used a t-test to compare subtraction images (psilocybinplacebo) from the Low and High dose groups. To assess rCBF, we ran two separate analyses. First, we used the normalization ANOVA option in our flexible-factorial design which uses a global covariate as a nuisance variable in the general linear model (see Inline Supplementary Fig.). Second, we extracted quantitative CBF values from a whole brain Anatomical Automatic Labeling (AAL) mask to calculate a mean CBF value for each participant and condition. This value was then used as an additional covariate in the analysis described above. Both procedures controlled for global signal and produced similar effects, therefore, we only report the ANOVA with normalization results. Results were visualized using MRIcron and xjView toolbox ().
A repeated-measures ANOVA (drug* scale*dose) controlling for age and sex revealed significant main effects for dose (F(1, 54) ¼ 4.30, p ¼ 0.043), scale (F(10, 560) ¼ 25.55, p < 0.001), and drug (F(1, 56) ¼ 163.86, p < 0.001). Additionally, there was a significant scale*dose (F(10, 560) ¼ 2.79, p ¼ 0.002), drug*dose (F(1, 56) ¼ 6.33, p ¼ 0.015), scale*drug (F(10, 560) ¼ 26.33, p < 0.001), and a scale*drug*dose interaction (F(10, 560) ¼ 2.83, p ¼ 0.002). Bonferroni-corrected pairwise comparisons revealed participants in the high dose group reported significantly larger effects compared to the Low dose group in four out of the 11 scales: Disembodiment (p ¼ 0.017), Complex Imagery (p ¼ 0.026), Elementary Imagery (p ¼ 0.016), and Audiovisual Synesthesia (p ¼ 0.005) (Fig.). Simple main effects analyses showed ratings on all 5D-ASC scales were increased after psilocybin treatment compared to placebo (all p 0.001).
We failed to observe any effect of dose on rCBF or gCBF even at the more lenient p-value of <0.001 (uncorrected). Furthermore, when analyzed separately, both the Low and High dose groups display the same pattern of results; therefore, all other analysis was collapsed across dose groups.
Within our sample, we failed to find an effect of sex in rCBF or gCBF. We did not find an effect of age in rCBF but there was a negative relationship between age and gCBF. The pCASL SPM map revealed two significant clusters (p < 0.05, FWE corrected), in the right frontal superior and inferior orbital area with peak coordinates at 24 28 À18 (cluster size (k) ¼ 73, t ¼ 6.57) and the middle frontal gyrus with peak coordinates at 36 8 34 (k ¼ 93, t ¼ 6.26).
After normalization, the pCASL SPM map revealed significant (p < 0.05, FWE corrected) local drug effects (i.e. across both doses) in the following areas: drug-induced increases in rCBF in bilateral inferior frontal gyrus and insula with right side hippocampus, operculum, and the temporal superior gyrus (Fig.). Additionally, we found decreases in left amygdala, lentiform nucleus, globus pallidus, putamen, thalamus, insula, and portions of the frontal, parietal, occipital, and temporal left cortex (Fig.). A complete list of regions is shown in Tablesand.
Regions with significant drug-induced decreases in gCBF included portions of frontal, parietal, temporal, limbic, and occipital cortex, cingulate, insula, caudate, putamen, pallidum, amygdala, hippocampus, and thalamus. No increases in gCBF were detected (Fig.). A complete list of regions is shown in Table.
Although the participants were blind to treatment order, one can presume that many participants had insight as to the substance administered at their first visit. Therefore, we investigated whether the anticipation of receiving a psychoactive or placebo substance had an effect on CBF by testing the effect of session order on extracted quantitative CBF values. No significant differences were found for placebo (t(56) ¼ 0.84, p ¼ 0.84) or psilocybin (t(56) ¼ À1.17, p ¼ 0.24).
By approaching this data with two analytic procedures we have reconciled a seeming contradiction in the psilocybin neuroimaging literature. First, we controlled for the global signal in order to localize unique relative effects and found psilocybin induces both increases and decreases in rCBF, mirroring results from previous PET studies (. Increases in rCBF were found in right frontal and temporal regions and bilaterally in the anterior insula, while decreases were found in left parietal and occipital regions and the amygdala, globus pallidus, insula, and thalamus. Next, we confirmed a recent ASL investigation by demonstrating psilocybin reduces gCBF when compared to placebo. However, while ASL is more directly associated with neuronal function than BOLD, it is still an indirect measurement. In our results, it is not possible to parse out definitively the effect of psilocybin on neuromodulation via various 5-HT receptor action or the effect on vascular tone. We did not detect a dose effect in either rCBF or gCBF, suggesting the mechanism driving differences in subjective effects might be too subtle to be captured by ASL data in this dose range. Our analysis that controls for global variance (rCBF) aligns well with the seminal psilocybin investigations using PET, which demonstrated general hyperfrontal effects. For example, Vollenweiderfound 15-20 mg dose of oral psilocybin increased metabolic activity in frontal regions, anterior cingulate, medial temporal cortex, and the thalamus in healthy participants. The bilateral regional increase in frontomedial and frontolateral metabolism remained significant after correction for global variance, while decreases were found in the ventral striatum, putamen, and occipital areas. Another group found regional increased glucose metabolism in the right anterior cingulate, frontal operculum, and inferior temporal regions, and hypometabolism in subcortical areas after correction for global variance. Furthermore, our findings of decreases in rCBF as indexed by ASL partially overlap with the activation pattern seen in PET studies corrected for global variance, which report decreased metabolism bilaterally in occipital regions, ventral striatum, and right putamenas well as in the left precentral region and bilateral thalamus.also reports a trend towards decreased rCBF in the thalamus after mescaline. Using resting-state pharmacological ASL designs, we can measure unique regional effects likely driven by drug action on local receptors (Fig.). Other studies using SPECT to assess CBF have found both the mixed 5-HT1A/2A agonist DMT, and the 5-HT2A agonist mescaline to also induce hyperfrontal effects particularly in the right anterior cingulate/frontomedial cortex and in the bilateral insula. The collection of findings that various 5-HT2A agonists induce hyperfrontal activity may reflect actions on the large population of 5-HT2A receptors located on apical dendrites of glutamatergic pyramidal cells, leading to increased neuronal activity and recruitment of blood flow. Especially interesting, our results replicate a more robust right laterality (Fig.) in metabolic hyperfrontalitydespite higher 5-HT2A receptor expression rates in left frontal, cingulate, and orbital cortex. This could suggest that the different ratio of 1A/2A receptor stimulation by psilocybin in the right hemisphere, or complex adaptations due to psychological and emotional processes, might be responsible for driving increased activity. Perhaps the psilocybin induced regional decreases in our results reflect neuromodulation of 5-HT1A receptors. 5-HT1A receptors dosedependently reduce local blood volume in the mouse prefrontal cortex, hippocampus, and amydgala, and inhibit pyramidal cell firing. Furthermore, GABAergic interneurons of the cortex express 5-HT2A receptors, thus, 5-HT can indirectly inhibit pyramidal neurons through this mechanism as well, and rodent studies suggest 1A and 2A receptors have a high co-expression rate (80%) in cortical tissue (Amarg os-). These possible opposing and complex effects of 1A/2A receptor activation by psilocybin may explain regionally opposing neuroimaging findings. It is impossible to delineate these receptor specific responses from our current study, thus, more research using selective receptor blocking pre-drug treatment is warranted to clarify this relationship. Consistent with the literature, psilocybin-induced scores on the 5D-ASC scales (previously reported inall differed from placebo. The High dose produced significantly stronger alterations on four of the 11 scales compared to Low dose psilocybin (Fig.). Interestingly, the High dose group did not report elevated levels of anxiety compared to the Low dose group. Perhaps, the dose effect on subjective experiences in absence of a dose effect in CBF is evidence of a neurovascular factor, in addition to the neuronal effects, in our observed rCBF results. Since we failed to find an effect of sex and a minimal effect of age, these are likely not the reason for no dose effect. Interpreting drug-induced changes in CBF in the context of neurovascular coupling is of important consideration. There are multiple pushpull neurovascular control mechanisms including local vasodilation and constriction via inhibitory interneuron direct actions on smooth muscle, local excitatory neurons, interactions with astrocytes acting to either dilate or constrict vessels, and global pathways involving vasodilator actions of acetylcholine as well as constrictor actions of serotonin. In his review, Bruce Jenkins (2012) highlights the difficulties and limitations in interpreting phMRI data using known vasoactive drugs. Serotonin earned its name for robust vascular modulating properties, thus when perturbing this system, it is of upmost importance to include a discussion of possible direct vascular effects. Possibly, the decreased gCBF we observed, similar to that seen after i.v. administration, is in part capturing the effect of psilocybin on general vascular tone. This suggests that after normalizing the dose to be roughly equivalent, the effects of psilocybin on gCBF may be quite similar across routes of administration. Psilocybin affects neuronal glucose metabolism which is highly correlated with neuronal activity and has been functionally coupled with rCBF. Our rCBF analysis may be more similar to glucose metabolism PET data by capturing an indirect measurement of local neuronal activity by removing the signal noise from blood flow not related to specific recruitment related to neuronal activity. However, psilocybin effects on vascular tone also affect rCBF, possibly due to regional differences in brain microvasculature, therefore, the observed changes in rCBF are likely due to changes in neuronal activation in combination with changes in vascular tone. There has been a wide discussion on the validity of using a global covariate, either as a confound or nuisance variable, in neuroimaging analyses but much of the discussion was based on task-based fMRI data. With BOLD data, the use of a global covariate can reduce sensitivity and introduce spurious deactivations or anticorrelations in the case of task-free designs. However, it is common practice for phMRI ASL resting state investigations to report both analyses with and without a global covariate. Often, including both analyses provides a clearer picture of the true actions of the pharmacological challenge and may improve the analysis by removing inter-individual differences (for examples see:. However, not all reports clearly state if a global covariate was used, leaving the reader to assume no global signal was regressed out. Therefore, we find this topic to be an important point of consideration for the field of phMRI resting state ASL research. Our study also has limitations, such as the between-subjects dose comparison, however the two groups were well matched for age and sex. Furthermore, as this is the first neuroimaging psilocybin dose comparison, we chose to compare a small range of doses and future studies evaluating a larger range of doses is warranted. Lastly, while our gCBF results are similar to another ASL study, it should be noted that the routs of administration differed between studies. In summary, our results demonstrate increased frontal and temporal rCBF, decreased parietal rCBF, and wide range reduction of gCBF in response to two doses of psilocybin. Surprisingly, we show the two doses tested do not differ in their effect on CBF although they produce different subjective effects, suggesting that subjective effects are not captured by changes in CBF. Our results provide a possible explanation for the apparent discrepancy in the literature concerning psilocybin effects on CBF. As research into the clinical applications of psilocybin increases it is important to clarify neuronal and neurovascular effects in brain regions associated with psychiatric disorders. Understanding psilocybin action will aid future clinical work in matching treatment options with disorders involving known brain regions and maximize its full potential as a research tool. Further, we provide an example of the importance of evaluating, choosing, and reporting analysis type employed on pharmacological ASL data. Specifically, our results add to the existing literature the importance of understanding and interpreting CBF in relation to global signal variations. This is a vital contribution as the ASL method is becoming more common in neuroscience research.
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