Dose-related changes in overall brain activity and communication after psilocybin exposure in awake rats

Adult male (n = 24) and female (n = 24) Sprague Dawley rats were purchased from Charles River Laboratories (Wilmington, MA, United States). Animals were housed in Plexiglas cages (two per cage) and maintained in ambient temperature (22–24°C). Animals were maintained on a reverse L-D cycle with lights off at 0900 h and studied during the dark phase when they are normally active. All experiments were conducted between 1000 and 1800 h to avoid the transitions between the L-D dark cycles. Food and water were provided ad libitum. All animals were acquired and cared for in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals. All methods and procedures described below were pre-approved by the Northeastern University Institutional Animal Care and Use Committee under protocol number 23-0407R. Northeastern University’s animal care and use program and housing facilities are fully accredited by AAALAC, International. The protocols used in this study followed the ARRIVE guidelines for reporting in vivo experiments in animal research (Kilkenny et al., 2010). Animals were monitored daily over the duration of the study for general health, food and water consumption. A 15% loss in body weight was set as a humane endpoint.

Psilocybin was acquired through the National Institute on Drug Abuse (NIDA) Drug Supply Program and distributed by the Research Triangle Institute. On the day of imaging PSI was prepared in sterile saline (0.9% NaCl). To deliver drug remotely during the imaging session, a poly-ethylene tube (PE-20), approximately 30 cm in length, was positioned in the peritoneal cavity. The range of doses of PSI were taken from the literature (Higgins et al., 2021; Wojtas et al., 2022; Spain et al., 2015). Rats were randomly assigned to one of four dose groups (vehicle, 0.03, 0.3, 3.0 mg/kg PSI), ensuring each group had a similar distribution of body weights. Each group consisted of 12 rats divided equally between males and females. Due to motion artifact five rats were excluded from the study. Two male rats from the vehicle treatment, one male from the low dose treatment and one male and one female from the high dose group. Following the original study with 48 rats, we ran a pilot study with seven male Sprague Dawley rats to evaluate the effect of the 5-HT_2A receptor antagonist ketanserin on changes in brain activity in response to the 3.0 mg/kg dose of PSI. Rats were housed and acclimated as described above. One hour prior to imaging, rats were given an I.P injection of ketanserin (2.0 mg/kg in saline).

To mitigate the stress associated with head restraint, rats underwent an acclimation protocol to familiarize them with the restraining system, which consisted of a head holder and body tube. This system was designed to exclude surgery and included a cushioned head support, eliminating the need for ear bars and, in turn, reducing discomfort to the animals while minimizing any unintended motion artifacts. These acclimation sessions were conducted daily for five consecutive days. During these sessions, rats were briefly anesthetized with 1–2% isoflurane for placement into the restraining system. Their forepaws were fastened using surgical tape.

Once fully conscious, the rats were positioned within an opaque black box, a “mock scanner,” for 60 min. Inside the mock scanner, a tape recording of the MRI pulse sequence was played to simulate the environment of the magnet bore and the imaging protocol. Under these conditions there is a significant decreases in respiration, heart rate, motor activity, and plasma corticosterone levels when comparing the first and last acclimation sessions, as reported by King et al. (2005). This reduction in autonomic and somatic signs of arousal and stress improved signal resolution and image quality.

Five to six rats were imaged in a day. Each day had a mix of the different experimental groups known by all the investigators. Rats were scanned at 300 MHz using a quadrature transmit/receive volume coil built into the rat head holder and restraining system for awake animal imaging (Ekam Imaging, Boston, MA, United States). A video of the rat preparation for imaging is available at www.youtube.com/watch?v=JQX1wgOV3K4↗. The design of the coil provided complete coverage of the brain from olfactory bulbs to brain stem. Radio frequency signals were sent and received with a quadrature volume coil built into the animal restrainer (Ekam Imaging, Boston MA, United States) (Ferris, 2022). Imaging sessions were conducted using a Bruker Biospec 7.0 T/20-cm USR horizontal magnet (Bruker, Billerica, MA, USA) and a 2 T/m magnetic field gradient insert (ID = 12 cm) capable of a 120-μs rise time. At the beginning of each imaging session, a high-resolution anatomical data set was collected RARE factor 8; 25 slices, 1 mm; FOV 3.0 cm²; data matrix 256 × 256; TR 3 s; TE 12 ms; effective TE 48 ms; NEX 3; 4.48 min acquisition time; in-plane resolution 117.2 μm². Functional images were captured using HASTE pulse sequence: 22 slices, 1.1 mm, FOV of 3.0 cm²; data matrix 96 × 96, TR 6 s, TE 3.75 ms, effective TE 22.5 ms, 25 min acquisition time, in-plane resolution of 312.5 μm². It should also be emphasized that high neuroanatomical fidelity and spatial resolution are critical in identifying distributed neural circuits in any animal imaging study. Many brain areas in the segmented rat atlas have in-plane boundaries of less than 400 μm² and may extend for over 1,000 μm in the rostral/caudal plane. With the development of a segmented, annotated 3D Rat Brain Atlas^© for rats (Ekam Solutions, Boston, MA, United States), it was possible to localize functional imaging data to precise 3D “volumes of interest” in clearly delineated brain areas. This spatial resolution was sufficient to identify the bilateral habenula, with approximately 4–5 voxels on each side, but not to differentiate between the lateral and medial habenula.

The fMRI data analysis consisted of three main steps: pre-processing, processing, and post-processing. All these steps were executed using SPM-12 (available at https://www.fil.ion.ucl.ac.uk/spm/↗) and in house Matlab software. In the pre-processing stage, several operations were performed, including co-registration, motion correction, smoothing, and detrending. Co-registration was carried out with specific parameters: Quality set at 0.97, Smoothing at 0.6 mm Full Width at Half Maximum (FWHM), and Separation at 0.4 mm. Additionally, Gaussian smoothing was applied with a FWHM of 0.8 mm.

The processing step involved aligning the data to a rat atlas, followed by segmentation and statistical analysis. To achieve registration and segmentation, all images were initially aligned and registered to the 3D Rat Brain Atlas^©, which included 169 segmented and annotated brain areas. This alignment was performed using the GUI-based EVA software developed by Ekam Solutions (Boston, MA). The image registration process encompassed translation, rotation, and scaling adjustments, performed independently in all three dimensions (see Supplementary Data Sheet 1). All spatial transformations applied were compiled into a matrix [Tj] for each subject. Each transformed anatomical pixel location was tagged with its corresponding brain area, resulting in fully segmented representations of individual subjects within the atlas.

Each scanning session consisted of 250 data acquisitions with a period of 6 s each for a total lapse time of 25 min. The first 50 scans (5 min) were the control window while the stimulation window was 200–240 (for ca 20 min post-injection) scans. Statistical t-tests were performed on each voxel (~ 36,000 voxels in the whole brain) of each subject within their original coordinate system. Before statistical testing, a baseline signal change threshold of 1% was applied to identify voxels with potentially meaningful signal changes. This excluded voxels that never exceeded ±1% BOLD signal change from baseline during the post-injection period. This 1% cutoff was chosen as a conservative floor to eliminate very small fluctuations that likely reflect noise or minor physiological drift, ensuring that only voxels with at least a minimal response were considered for significance testing. Importantly, this thresholding did not automatically deem 1% as significant; it was simply a preprocessing step. All voxels meeting the threshold were then subjected to statistical analysis using a one sample t-test against zero change. The reference for these t-tests was a null hypothesis of zero change from baseline. The t test statistics used a 95% confidence level (p < 0.05), two-tailed distributions, and heteroscedastic variance assumptions. As a result of the multiple t-test analyses performed, a false-positive detection controlling mechanism was introduced. We set the acceptable False Discovery Rate (FDR) at Q = 0.05 (5%). After performing voxel-wise tests, an FDR correction across all voxels in the brain was applied to control for multiple comparisons using the Benjamini–Hochberg procedure. This subsequent filter guaranteed that the FDR rate was below our cutoff of 0.05. The formulation of the filter satisfied the following expression:

In the equation, Pi represents the p-value derived from the t-test conducted at the i-th pixel within the region of interest, comprising V pixels, with each pixel ranked according to its probability value. For our analysis, we set the false-positive filter value q at 0.2, and we fixed the predetermined constant c(V) at unity, following a conservative approach for assessing significance (Genovese et al., 2002).

To create composite maps displaying the percent changes in the BOLD signal for each experimental group, we mapped each composite pixel location (in terms of rows, columns, and slices) to a voxel within the j-th subject using the inverse transformation matrix [Tj]-1. A trilinear interpolation method was used to determine the contribution of subject-specific voxel values to the composite representation. The use of inverse matrices ensured that the entire composite volume was populated with subject inputs. The average of all contributions was assigned as the percentage change in the BOLD signal at each voxel within the composite representation of the brain for the respective experimental group.

In the post-processing phase, we compared the number of activated voxels in each of the 169 brain areas between the control and PSI doses using a Kruskal–Wallis test statistic. The data were ranked in order of significance, as detailed in Tables 1–4. We generated activation maps, depicted in Figure 1, showing brain areas with significant differences when comparing two groups, e.g., veh vs. 3.0 mg/kg PSI. For Figures 2, 3 we limited statistical comparisons to either global metrics or a priori regions of interest (e.g., somatosensory cortex) rather than every individual brain area.

In an attempt to determine if there were sex differences in the sensitivity to PSI we divided the each of the experimental groups into their male and female cohorts. The final distributions were Vehicle (4 males, 6 females), 0.03 mg/kg (5 males, 6 females), 0.3 mg/kg (6 males, 6 females), and 3.0 mg/kg (5 males, 5 females) PSI. While underpowered a two-way ANOVA comparing the sexes across all doses for whole brain activation was significant (p < 0.0001) as reported in the Results. Dividing each experimental group into male and female cohorts for rsFC analysis was unsuccessful as the analysis requires a minimum of 7–8 subjects as determined from previous studies (Harris-Blum et al., 2024; Ghaw et al., 2024).

The autosampler (Shimadzu SIL-40C) was kept at room temperature (25^o C). Shimadzu pumps (SIL-40D) provided control of mobile phase percentages. The column oven, which housed the mixer and analytical column, was maintained at 40^o C. A Phenomenex Luna C18 (Banushi and Polito, 2023) analytical column was used for chromatographic separation (50 mm length, 2 mm internal diameter, 3 μm particle size, 100 angstrom pore size). The analytical column was preceded by a Zorbax Eclipse XDB-C8 guard column.

Areas comprising the different body-specific parts of the primary somatosensory cortex (e.g., barrel field, hindlimb, truck, forelimb) were activated. Indeed, the primary somatosensory cortices showed both a significant increase in volume of activation as well as percent change in BOLD signal over time, two separate ways to assess functional activity. Liu et al. (2023) using anesthetized male rats also reported that 2.0 mg/kg PSI given IP during the scanning session causes widespread positive BOLD activation and increased functional connectivity. Spain et al. (2015) also imaged male rats under anesthesia given a low (0.03 mg/kg) or high (2.0 mg/kg) dose of psilocin during the scanning session. The low dose of psilocn had little or no effect. However, the high dose of psilocn caused a significant increase in BOLD signal in the olfactory system, limbic and visual cortices but a decrease in the somatosensory cortex, thalamus, and hippocampus. Using a laser scanning technique, these same rats were also studied for changes in localized blood flow to the barrel field of the somatosensory cortex in response to whisker stimulation in the absence and presence of psilocn while also recording local field potentials from implanted electrodes in the same site. The 2.0 mg/kg dose of psilocn enhanced blood flow during whisker stimulation, a response that was augmented by psilocin. Paradoxically, the drug-evoked increase in blood flow occurred during a decrease in neural activity raising questions about neurovascular coupling in the presence of PSI. However, vascular reactivity in response to a hypercapnic challenge was normal when comparing vehicle and psilocin treatments. Golden and Chadderton (2022) reported recordings from the anterior cingulate cortex on awake mice showing an increase in neuronal firing in response to a dose of 2.0 mg/kg of PSI. In addition there was a decrease in low frequency band power but an increase in gamma band power evidence of cortical desynchronization. These studies by Liu, Spain and Golden showing regional increases in BOLD signal and electrical activity and ours showing a global increase to PSI are in sharp contrast to the initial human BOLD imaging study performed by Carhart-Harris et al. (2012a). In healthy volunteers with a history of hallucinogen exposure there was a sharp decrease in cerebral blood flow (CBF) measured with arterial spin labeling in all areas of the brain. The same study was repeated with BOLD imaging again finding a decrease particularly in the prefrontal cortex and anterior cingulate. The magnitude of the decrease in CBF and BOLD was inversely correlated with the subjective effects of PSI. In a subsequent publication on data collected from the same subjects, Carhart-Harris reported PSI enhanced BOLD signal in the visual cortex and somatosensory areas in response to memory recall enacted during the scanning session showing a difference between resting and task driven effects of PSI (Carhart-Harris et al., 2012b). However, the global decrease in hemodynamic measures of brain activity in humans under resting conditions is contrary to PET imaging studies using [F-18] FDG to follow glucose metabolism. Vollenweider et al. (1997) reported a global increase in glucose metabolism following PSI treatment with the greatest changes occurring in the prefrontal and anterior cortical areas. The increase in metabolic hyperfrontality was also reported by Gouzoulis-Mayfrank et al. (1999) in healthy volunteers in response to PSI exposure. The Vollenweider group followed up their PET imaging study with another using arterial spin labeling to follow changes in CBF in response to PSI (Lewis et al., 2017). Correcting for global CBF in each subject, they reported regional differences in blood flow highlighted by an increase in the frontal cortex but a decrease in other brain areas. N,N-dimethyltryptamine (DMT) a hallucinogen with comparable chemistry and activity to PSI, when tested in human volunteers also increases CBF to the frontal, limbic, and anterior cingulate cortices (Riba et al., 2006).

5-HT_2A receptors typically have high affinity in the low nanomolar range, so a plasma psilocin concentration of ~5 nM at the 0.03 mg/kg suggests minimal occupancy/activation of 5-HT_2A and likely below the threshold for measurable effect (Nichols, 2004). In contrast, ~5 μM at 3.0 mg/kg is orders of magnitude higher – enough to occupy nearly all 5-HT_2A receptors and even engage lower-affinity sites like 5-HT_1B and 5-HT_1D receptors. The pharmacological breadth at the high PSI dose could explain why we see widespread activation as the drug is hitting multiple receptor systems, possibly including vasoconstrictive 5-HT₁ receptors, as discussed by Spain et al. (2015). This dose-dependent transition from no effect to hyperactivation might reflect the steep occupancy-response curve of 5-HT_2A and recruitment of additional targets at high dose. In addition to the direct effects on 5-HT_2A receptors, we show here that even the lowest dose of 0.03 mg/kg had a profound effect on plasma levels of the endocannabinoid, 2-AG. There is a growing literature on the relationship of 2-AG and serotonin regulation (Oakes et al., 2019; Parrish and Nichols, 2006; Granberg et al., 2001) that may underly some of the functional changes observed here at the 0.03 mg/kg dose.

Dividing each of the experimental groups into male and female cohorts enabled us to test for sex differences to the sensitivity of different doses of PSI. Neither the vehicle nor the high 3.0 mg/kg dose of PSI presented with a significant sex difference. However, the low 0.03 mg/kg dose of PSI revealed a modest (only six areas activated) but significant male sensitivity to this hallucinogen. More compelling was the sensitivity of females to the medium 0.3 mg/kg dose of PSI with 45/169 brain areas showing a sex difference. The anatomical organization of the female sensitivity was most interesting with clearly defined activation of the ventral tegmental area and its dopaminergic efferent connections to the ventral striatum, accumbens and ventral pallidum (see Figure 6). Almost all of the thalamus was similarly activated together with key areas of the ascending reticular activating system. What was missing from this pattern of global brain activation was almost the entire somatosensory cortex and pons and all the hippocampus, amygdala and olfactory system. Notably, male rats exhibited far less activation in these areas compared to females. Behavioral studies showing a sex differences in response to PSI or psilocin are sparse. Tyls and coworkers treated male and female rats SQ with 0.25 mg/kg, 1.0 mg/kg or 4.0 mg/kg of psilocin and 15 min later measured locomotor activity in an open field (Tyls et al., 2016). There was a dose-dependent decrease in locomotion for both males and diestrus females but not estrus females. However, when tested for acoustic startle for prepulse inhibition there were no sex differences. Adult male mice but not female mice treated IP with PSI in doses ranging from 0.1 mg/kg to 2.0 mg/kg show reduced voluntary ethanol consumption (Alper et al., 2022). In a recent study Effinger et al. (2023) reported the central amygdala of female rats is more sensitive to a 2.0 mg/kg dose of psilocin as compared to males in measures of c-fos immunostaining and photometry in response to an aversive stimulus. There is no clear evidence in the clinical literature of sex difference in sensitivity to PSI (Shadani et al., 2024).

One of the more compelling findings in this study was the dose-dependent increase in global functional connectivity. Reinwald et al. (2023) in a cross-over experimental design, imaged females rats treated with 1.0 mg/kg PSI and vehicle during the MRI scanning session. The studies were done under anesthesia and during the light phase of the L-D cycle. Unfortunately, the cerebellum was not included in the study. There was an overall global decrease in connectivity with PSI treatment, in stark contrast to our findings. The most plausible explanation for this disparity is the confound of anesthesia and impact of circadian biology. The importance of circadian timing and brain function cannot be underestimated. Perivascular clearance of an MRI contrast agent from the brain of awake rats is regulated by the circadian clock being highest during the light phase and lowest during the dark phase of the L-D cycle (Cai et al., 2020). Brain temperature is a circadian rhythm entrained by the L-D cycle (Refinetti and Menaker, 1992; Scheer et al., 2005). These concerns notwithstanding, the data from the Reinwald study was consistent with many human imaging studies that report a general decrease in intra-network connectivity, particularly in association areas like the default mode and salience networks in healthy volunteers treated with PSI (Carhart-Harris et al., 2013; Lebedev et al., 2015; Muthukumaraswamy et al., 2013). Preller et al. (2020) reported a time-dependent change in global connectivity in healthy volunteers following PSI treatment. At 20 min post treatment these was hyperconnectivity in the occipital cortex. At later time points, 40 and 70 min post treatment, the pattern of brain activity evolved into hypoconnectivity in association areas and hyperconnectivity in sensory networks. Preller et al. further demonstrated that the altered brain connectivity was positively correlated with brain areas high in 5-HT_2A gene expression and negatively correlated with the localization of 5-HT_1A gene expression. Preller’s findings align with ours, demonstrating that PSI increases global brain connectivity across sensory networks. In a just published study Siegel and coworkers followed healthy volunteers prior to, during, and three weeks following a high 25 mg/kg dose of PSI (Siegel et al., 2024). PSI had a dramatic effect on disrupting rsFC particularly in the default mode network. The data from global graph theory analysis presented in Table 5 align with the “entropic brain hypothesis” proposed by Carhart-Harris et al. (2014) where psychedelics like PSI increase brain network integration and reduce segregation between normally distinct networks. The PSI induced increased entropy (disorder/uncertainty) in brain activity, particularly in high-level networks may explain the altered states of consciousness associated with classical hallucinogens.

One brain area highlighted in this study and previously overlooked in both preclinical and clinical literature is the cerebellum. Its involvement was anticipated, given the numerous awake animal imaging studies demonstrating alterations in cerebellar activity following the administration of cannabinoids, psychostimulants, ketamine, opioids, and neuropeptides (Demaree et al., 2021; Sadaka et al., 2021; Coleman et al., 2022; Kawazoe et al., 2022; Moore et al., 2016) and most recently the hallucinogen LSD (Ghaw et al., 2024). Traditionally, the cerebellum has been considered primarily responsible for motor coordination. However, it also plays a role in autonomic physiological functions such as heart rate, blood pressure, and respiration (Krohn et al., 2023; Cavdar et al., 2001; Haines et al., 1984; Snider et al., 1976). Additionally, the cerebellum is recognized for its significant involvement in emotional and cognitive functions (Zhang et al., 2023; Schmahmann, 2019; Adamaszek et al., 2017), feeding (Low et al., 2021), and addiction (Miquel et al., 2016). The cerebellum’s connectivity is unique, with all efferent information passing through the fastigial, interposed, and dentate nuclei. These nuclei have extensive bidirectional connections with various brain regions, including the thalamus, hypothalamus, limbic cortex, amygdala, hippocampus, and brainstem (Kebschull et al., 2023). The cerebellum also receives a substantial portion of its nerve connections from the vestibular complex, which relays auditory information from the ear to the cortex. In this study, cerebellar connections extended to the olfactory bulb, frontal association cortex, and hippocampus. This extensive network of connections and its involvement in various behavioral and sensory functions raise the question—could the cerebellum play a role in hallucinogenic effects that mimic psychosis? Notably, given the cerebellum’s involvement in cognitive, emotional, and sensory processes, it may contribute to the wide range of symptoms and cognitive impairments observed in schizophrenia (Andreasen and Pierson, 2008).

This study would have benefited from a suite of behavioral assays measuring cognition, locomotion and emotionality for each dose of PSI. These measures would have been particularly relevant to understanding the region specific pattern of brain activation shown in females to the medium 0.3 mg/kg dose of PSI. The involvement of the ascending reticular activating system and dopaminergic neural circuitry would suggest an increase in motivation.

This study did not include pharmacokinetics. While we analyzed plasma for PSI and its metabolite psilocin these samples were collected at single time point 30 min following IP injection. The drug-induced changes in BOLD signal were recorded 20–24 min after IP injection while the rsFC data were collected ca 35 min post injection. Would the data have differed if we chose later time points, e.g., 45–60 min post injection of PSI? In a recent studying looking at the pharmacokinetics of 1.0 and 3.0 mg/kg PSI in mice injected IP, the levels of plasma psilocin peak at 15 with a half-life of ca 30 min (Passie et al., 2002). Hence, waiting a longer period of time for the acute drug-induced effect of PSI may have been limiting. The rapid rise of psilocin in plasma may account for the early rise in the BOLD signal observed in the time course series shown in Figure 6 for the somatosensory ctx.

This phMRI study testing the effects of PSI on brain activity is similar to many others used to characterize a drug’s effect on the brain (Kawazoe et al., 2022; Sathe et al., 2023; Brems et al., 2023), yet the robust dose-dependent increase in global BOLD signal and connectivity are unprecedented and at odds with much of the preclinical and clinical literature. Spain and coworkers raised concerns around the potential confounding effects of 5-HT agonists and vascular tone with respective to the hemodynamic changes that contribute to the BOLD signal. Psilocin, psilocybin’s active metabolite, primarily acts through the 5-HT_2A receptor. Psilocin also binds with lower, but potentially relevant, affinity for the 5-HT_1B and 5-HT_1D receptors. Serotonin receptors lining the vasculature may modulate vascular tone and causes subsequent changes to BOLD signal independent of metabolism. Spain and colleagues also noted that 5-HT_2A receptors on cortical inhibitory interneurons may induce vasoconstriction and vasodilation, constituting a scenario where increased neuronal activity may lead to either positive or negative BOLD. Conscious, unrestrained rats treated with the hallucinogen DOI (2,5-dimethoxy-4-iodoamphetamine), a 5-HT_2A receptor agonist, reduces blood flow to the cutaneous circulation of the tail through vasoconstriction, an effect blocked by ketanserin pretreatment (Blessing and Seaman, 2003). Whether this vasoconstriction in the peripheral cutaneous circulation generalizes to the cerebral vasculature is unknown. The dose-dependent increase in BOLD signal in this study is not consistent with a decrease in cerebral blood flow; however, it aligns with the decreases in cerebral blood flow and BOLD signal reported in the Carhart-Harris study (Carhart-Harris et al., 2012a).

How do we reconcile these differences? Imaging done in awake animals and during the circadian L-D cycle when they are normally active could account for the discrepancies in the acute BOLD response between our study and that of Spain and Reinwald. When comparing our results to the human literature many of the differences could be explained by the stress of the imaging protocol. Psilocybin and other 5-HT_2A agonists are known to acutely activate the hypothalamic–pituitary–adrenal (HPA) axis. For example, administration of serotonergic psychedelics in rodents triggers robust increases in corticosterone via 5-HT_2A receptor activation (Romanova et al., 2022; Van de Kar et al., 2001). Notably, this effect can be blocked by a 5-HT_2A antagonist (Van de Kar et al., 2001), indicating it is mediated by the same receptor involved in psychedelic effects. In humans, PSI similarly elevates cortisol levels for an hour or more post-administration (Constantino et al., 2025). While rats have been acclimated to the imaging procedure there still maybe some restraint stress and corticosterone that, in combination with PSI, may lead to hypermetabolism or different network engagement than a relatively calm human during the scanning session.

Differences in neurobiology may also account for the discrepancies. There are inherent species differences in serotonin receptor distribution and neural circuitry (Nichols, 2004). Rodents have differences in cortical architecture and potentially in the baseline oscillatory activity compared to humans. For example, the rat default mode network (DMN) is not as pronounced as in humans (Liang et al., 2011), which might affect how PSI modulates “global connectivity.” Differences in 5-HT_2A receptor density (e.g., rats have high densities in certain retrosplenial and frontal areas) could lead to different activation patterns. Additionally, dosing relative to metabolism differs as our rats were scanned during the peak drug effect (within 30 min of injection), whereas human fMRI studies often capture a slightly later timeframe of PSI effects when subjective effects plateau. Comparisons between rodents and humans must be made cautiously and with these confounds in mind. Our results in rats – robust hyperactivation and hyperconnectivity – might reflect an interaction between PSI’s direct pharmacological effects and the stress/arousal state of the animal.

Drawing comparisons between awake rats and awake humans when trying to explain the complex psychedelic effect of psilocybin or any hallucinogen by the pattern of brain activity may be a difficult endeavor. Commercially bred rats are, by most measures, genetically identical. All share a common restricted environment minimizing cognitive, emotional and sensory experience. This neurobiological portrait of their environment is limited to a short window of life—only 3–4 months. The food they eat is identical. They are drug naïve. Hence the variance between experimental doses may be small enough to observe dose-dependent changes across subjects as shown here. All of these controlled conditions are ideal for preclinical discovery but impossible in the world of human neuroscience. The psychedelic experience, by all accounts, is a personalized, untethered, and unfiltered emergence of experience and perception leading to “oceanic boundlessness” and “ego dissolution” (Vollenweider and Preller, 2020). This complex subjective experience cannot be modeled on a rodent. It is unlikely that the head twitch in a rat is a proxy for a hallucinogenic experience (Robbins, 2017; Corne et al., 1963). At face value it is a peripheral measure of a motor response to the CNS effect of 5-HT_2A receptor activation (Gonzalez-Maeso et al., 2007). Nonetheless PSI activation of 5-HT_2A receptor in this study did favor enhanced BOLD signal in the sensorimotor cortex and other brain regions with a high density of 5-HT_2A receptors. Indeed, ketanserin treatment in this study blocked the activation of the somatosensory cortices by PSI. Brain areas hypothesized to be involved in loss of sensory filtering and organization of sensory motor stimuli such as the claustrum (Barrett et al., 2020) and the cortico-basal ganglia-thalamic-cortical loop are all affected by PSI in a dose-dependent manner (Banushi and Polito, 2023). So much of the neuroanatomical circuitry described in the human literature is affected in this study but not in the direction or pattern reported in the clinic.

The original contributions presented in the study are included in the article/, further inquiries can be directed to the corresponding author. Supplementary material