Single-dose psilocybin rapidly and sustainably relieves allodynia and anxiodepressive-like behaviors in mouse models of chronic pain

Hammo, Ahmad; Wisser, Stephen; Cichon, Joseph

doi:10.1038/s41593-025-02068-0

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Published: 02 October 2025

Single-dose psilocybin rapidly and sustainably relieves allodynia and anxiodepressive-like behaviors in mouse models of chronic pain

Nature Neuroscience volume 28, pages 2285–2295 (2025) Cite this article

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Abstract

Chronic pain and mood disorders co-occur, exacerbate one another and share neurobiological mechanisms, but whether a single intervention could promptly alleviate both conditions remains unclear. Here, in two chronic pain models, we show that a single dose of psilocybin induces a rapid and sustained reversal of both mechanical allodynia and anxiodepression-like states in adult male and female mice. Using local psilocin injections, the key active metabolite of psilocybin, we show that the engagement of prefrontal cortical circuits is critical for the concurrent alleviation of both conditions. Two-photon calcium imaging reveals that psilocin rapidly normalizes chronic pain-associated hyperactivity in anterior cingulate cortex layer 2/3 pyramidal neurons. Pharmacologic manipulations with full agonists of 5-HT_2A and 5-HT_1A receptors replicated some, but not all, of psilocin’s cellular and behavioral effects, suggesting that psilocin’s actions arise from partial agonism at these receptors within shared circuits governing pain and mood processing.

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Main

Chronic pain commonly co-occurs with symptoms of depression and anxiety^1,2. This comorbidity may compound pain perception, accelerate the transition from acute to chronic pain, discourage patients from following treatment plans and increase risks of prescribed opioid medications³. The net effect is associated with greater functional limitations and poorer outcomes⁴. At present, there are limited therapeutic options for managing the co-occurring state⁵, but many recognize that the simultaneous assessment and treatment of pain and mood dysfunction is necessary for improved conditions. The convergence of chronic pain and depression involves overlapping dysfunctions in key brain regions, particularly the medial prefrontal cortex (PFC), anterior cingulate cortex (ACC), amygdala and insular cortex^6,7,8. In chronic pain, the ACC often shows hyperactivity⁹ which has been linked to the affective and attentional dimensions of pain¹⁰. Hyperactivity is thought to arise from a disrupted excitatory and inhibitory balance, with increased glutamatergic neurotransmission¹¹, and disinhibition (reduced GABAergic inhibition) amplifying neuronal firing¹². Additionally, maladaptive synaptic plasticity and enhanced connectivity between the ACC and other pain-related regions, such as the thalamus and insular cortex, contribute to persistent signaling¹⁰. Furthermore, attenuating ACC neuronal activity via lesions or inactivation techniques relieves aversive responses to pain^13,14,15. In depressed patients, the ACC exhibits a heterogeneous activity pattern, exhibiting both hyperactivity and hypoactivity^16,17, reflecting the complexity of the mood state itself and/or its comorbidity with other conditions, such as depressed mood in chronic pain or pain in depression¹⁸. These neurobiological alterations in the ACC position it as a critical bridge between chronic pain and depression, suggesting that targeting ACC function could offer an integrated therapeutic approach to alleviating symptoms of both conditions.

Psilocybin, an indole alkaloid, is a naturally occurring psychoactive compound that has been recognized for its analgesic properties since antiquity¹⁹. Psilocybin itself is pharmacologically inactive but is rapidly converted in the body to its active metabolite, psilocin. Psilocin primarily targets serotonergic receptors, including 5-HT_2A, 5-HT_1A and 5-HT_2C, where it acts as a partial agonist, mediating its psychoactive and presumed therapeutic effects²⁰. More recently, psilocybin and other serotonergic partial agonists have shown early therapeutic promise in managing pain symptoms accompanying chronic neuropathic pain^21,22,23, cancer pain, psychiatric distress²⁴, fibromyalgia, migraines and even phantom limb pain²⁵. However, psilocybin’s effectiveness in treating chronic pain has yet to be rigorously and systematically studied. Moreover, emerging trials suggest psilocybin has rapid and durable therapeutic effects in treatment-resistant depression, anxiety and addiction²⁶. Herein, we examine whether a single dose of psilocybin could treat chronic pain and its associated anxiodepressive-like phenotype, using two well-established rodent models of chronic pain.

Results

A single dose of psilocybin relieves chronic neuropathic and inflammatory pain-like states

To produce a chronic pain-like state, we subjected wild-type C57BL/6J mice to either a spared nerve injury (SNI) of the sciatic nerve (sparing the sural nerve) or a single large-volume subcutaneous injection of complete Freund’s adjuvant (CFA; consisting of mycobacterial components, ~80–100 µl) to a hindlimb footpad, which model neuropathic and inflammatory pain-like states, respectively²⁷^,²⁸ (Fig. 1a). We opted for a large-volume CFA injection, rather than smaller volumes (10–20 µl)^27,29, to establish a chronic inflammatory pain-like state (Extended Data Fig. 1). The sham conditions for SNI consisted of an incision at the mid-thigh level through the biceps femoris muscle followed by sciatic nerve exposure without nerve manipulation, while CFA controls received a saline injection of equal volume into the hindlimb footpad.

**Fig. 1: A single dose of psilocybin induces rapid and durable reversal of mechanical hypersensitivity in SNI and CFA mice.**

Immediately following SNI or CFA injection, we observed a substantial increase in mechanical hypersensitivity in the affected hindlimb compared with contralateral hindlimb or sham/saline controls, as assessed via repeated electronic von Frey (vF) measurements (Fig. 1d (SNI) and 1f (CFA)). This mechanical hypersensitivity affected both males and females similarly and persisted for 27 days, suggesting a sex-independent chronic state of mechanical allodynia in this preliminary sex-specific analysis (Fig. 1e (SNI) and 1g (CFA)). Once mechanical hypersensitivity stabilized (~4 weeks), mice received a single intraperitoneal injection (i.p.) of psilocybin. A dose–response curve for psilocybin in head-twitch response (HTR) and open field test demonstrated a divergent effect of psilocybin at 0.5 mg kg⁻¹ dose, showing peak HTR alongside hypoactivity (Fig. 1b,c and Extended Data Fig. 2). We selected the 0.5 mg kg⁻¹ dose for subsequent testing because it produced a maximal effect at a lower dosage as compared with 1 mg kg⁻¹. On the following day (experimental day 28) when animals were reassessed, we detected a complete reversal of mechanical hypersensitivity in both SNI and CFA animals (Fig. 1d,e (SNI) and 1f,g (CFA)). This restorative effect induced by psilocybin persisted in both sexes of SNI and CFA animals for a minimum of 12 days, marking the experiment’s endpoint. These results suggest that a single dose of psilocybin induces a rapid and durable analgesic effect in two distinct conditions associated with chronic pain.

Psilocybin alleviates mood changes associated with chronic neuropathic and inflammatory pain-like states

We next assessed whether SNI- and CFA-induced mechanical allodynia was associated with co-occurring anxiodepressive-like behaviors. Trends in anxiety-related behavior were made by tracking animal’s performance in the elevated plus maze (EPM), open field test (OFT) and light/dark box test, while behavioral despair was assessed in the forced swim test (FST) and tail suspension test (TST) before and after SNI/CFA procedures (Fig. 2a). Consistent with chronic pain-like states driving robust affective changes in preclinical models⁸, both SNI and CFA mice displayed substantial changes in both depression and anxiety associated assays compared with sham/saline controls on day 20 (Fig. 2b–e (depression testing) and 2f–o (anxiety testing)). Within SNI and CFA mice, we did not observe any substantial sex-specific differences across FST, TST, EPM, light/dark box, or OFT (Extended Data Fig. 3). These behavioral shifts were accompanied by deficits in motor skill performance, as evidenced by reduced latency to fall on an accelerating rotarod test, suggesting either fatigue or impaired motor performance related to the behavioral state (allodynia and/or anxiodepressive-like behaviors; Extended Data Fig. 4). Collectively, these results indicate that SNI/CFA-induced chronic mechanical hypersensitivity contributes to the emergence of affective disturbances, including features consistent with behavioral despair, anxiety-like behaviors and impaired motor skill performance.

**Fig. 2: Psilocybin reverses anxiodepressive-like behaviors associated with SNI and CFA pain models.**

To determine if these SNI/CFA-induced anxiodepressive-like behaviors could be cotreated by psilocybin, mice received a single dose of psilocybin on day 27, followed by repeat behavioral testing. Here we found that psilocybin treatment substantially reversed SNI/CFA-induced affective-related behaviors and motor function deficits, while sham/saline controls remained unaffected (Fig. 2 and Extended Data Fig. 4). The normalization of mechanical sensitivity in both SNI and CFA mice paralleled an increase in open-arm activity in EPM and mobility in FST (Extended Data Fig. 5). Examination of the surgical site in SNI mice treated with psilocybin revealed the presence of a neuroma—an anticipated consequence of common peroneal nerve and tibial nerve transection (Extended Data Fig. 6). This finding suggests that psilocybin-induced reversal of behavioral outcomes is unlikely to result from peripheral nerve regeneration within the post-treatment timeframe. Taken together, these experiments suggest that psilocybin acts as a multimodal therapeutic agent, inducing both analgesic and antidepressant/anti-anxiety-like effects following a single treatment.

SNI and CFA animals show preference for psilocybin treatment

To assess whether psilocybin elicited analgesic effects independent of mechanical testing, we subjected SNI/CFA mice to a conditioned place preference (CPP) paradigm (Fig. 3a). On day 27, animals were preconditioned to a two-chamber apparatus, where baseline preferences were established by allowing free exploration of both chambers without any interventions. Following this, mice were conditioned over the next day by receiving saline injections paired with one chamber (‘A’) and psilocybin injections (0.5 mg kg⁻¹ i.p.) paired with the opposite chamber (‘B’). Each conditioning session was conducted with the mice confined to the respective chamber for 15 min immediately following injection. Following the conditioning phase, a post-testing session was conducted, during which mice were allowed to freely explore both chambers without any further drug administration. The time spent in each chamber during post-testing was recorded to assess the development of a conditioned preference for a particular chamber. Consistent with psilocybin driving an analgesic state, both SNI and CFA mice treated with psilocybin exhibited a substantial increase in time spent in the psilocybin-paired chamber (chamber ‘B’; Fig. 3b,d). Sham or saline controls failed to show a preference for psilocybin. This behavior, reflected in the preference index (Fig. 3c for SNI/sham and 3e for CFA/saline), indicates that psilocybin’s alleviation of mechanical allodynia (Fig. 1) fosters a positive behavioral association for the environment associated with analgesia. This effect also aligns with observed improvements in anxiodepressive-like behaviors (Fig. 2), highlighting the interconnected nature of the analgesic state and improved mood changes.

**Fig. 3: Psilocybin promotes preference for the treatment chamber in the CPP test.**

Local application of psilocin to the ACC is sufficient for the rapid restorative effect of SNI-induced mechanical allodynia and anxiodepressive-like behaviors

The simultaneous reduction of mechanical allodynia (Fig. 1) and anxiodepressive-like behaviors (Fig. 2) could arise at multiple locations along the neuraxis^30,31,32. To further pinpoint the critical cohort of presumed serotonergic receptors and circuits mediating psilocybin’s therapeutic effects, we locally administered psilocin, the active metabolite of psilocybin produced following hepatic dephosphorylation, either to the lumbosacral region of the spinal cord or bilaterally to the ACC on day 27 (Fig. 4a,e). Given that the intact sural nerve, primarily deriving its sensory fibers from L4 to S2, becomes hypersensitive following SNI, we specifically targeted psilocin to this region via a single intrathecal injection (Fig. 4a). At both injection locations, psilocin was co-injected with Rhodamine 6G to visualize its presence and local spread within the respective regions (Fig. 4b,f). A dose–response curve of intrathecal psilocin (vehicle control—1, 200 and 500 µM; ~5 µl total) revealed a dose of 200 µM increased the immediate early gene cFos expression, a slow integrator of neuronal activity, in dorsal horn lamina I–II of the lumbosacral spinal cord (Fig. 4c,d). Similarly, local application of psilocin to the ACC region (vehicle control—1, 100, 200, 500 µM; ~2 µl total), assessed using two-photon calcium imaging of pyramidal neurons in awake head-fixed mice expressing GCaMP6f under the CaMK-II promoter, revealed a progressive spontaneous recruitment of layer 2/3 (L2/3) pyramidal neurons with peak activity at a dose of ~200 µM over baseline measurements (Fig. 4g,h and Extended Data Fig. 7). Given the inverted U-shaped response observed in these experiments, we reasoned that the 200 μM dose might effectively engage local postsynaptic serotonergic receptor signaling to modulate neuronal activity involved in pain processing.

**Fig. 4: Local application of psilocin to the ACC reverses SNI-induced mechanical allodynia and anxiodepressive-like behaviors.**

Next, we evaluated whether the local application of psilocin to the ACC or the intrathecal space of the lumbosacral spinal cord in SNI mice would be sufficient to drive the simultaneous restorative effect of mechanical hypersensitivity and associated anxiodepressive-like behaviors. SNI mice receiving bilateral psilocin injections experienced a rapid and persistent reversal of mechanical hypersensitivity when assessed on day 28, lasting until day 40 (Fig. 4i). When the same mice were tested for anxiodepressive-like changes, we observed a substantial improvement in the FST, marked by a decrease in immobile time (Fig. 4j). However, we observed no improvement in time spent on the open arm in EPM compared with prepsilocin treatment measurements (Fig. 4k). In contrast, mice treated with intrathecal psilocin showed persistent allodynia, with no substantial changes in withdrawal thresholds (Fig. 4i). These same mice also exhibited no substantial changes in either EPM or FST behaviors (Fig. 4j,k). Taken together, these results suggest that psilocin’s effects on mechanical hypersensitivity and mood-related dysfunction are likely mediated by supraspinal circuits, with the ACC having a key role.

Local psilocin normalizes spontaneous hyperactivity of pyramidal neurons in both neuropathic and inflammatory pain-like states

To investigate how local application of psilocin to the ACC might reverse these behaviors, we used two-photon calcium imaging in awake, head-restrained SNI/CFA and control mice expressing GCaMP6f in pyramidal neurons after establishing chronic pain-like states. In SNI/CFA mice, L2/3 pyramidal neurons exhibited a substantial elevation in spontaneous activity as compared with sham and saline-injected controls (Fig. 5a,c). Due to the sustained nature of these transients, we quantified calcium activity as the area under the curve of the ΔF/F₀ trace for individual neurons, capturing changes in both frequency and amplitude. On day 20, SNI and CFA mice displayed comparable levels (Mann–Whitney test, P = 0.70) of heightened neuronal activity, suggesting neuropathic and inflammatory pain signals similarly converge and restructure ACC neuronal activity into a hyperactive state (Fig. 5b,d (left)). Remarkably, local psilocin application (200 µM; ~2 µl total) rapidly suppressed (SNI, −38 ± 5.8%; CFA, −44 ± 4.8%) the SNI/CFA-associated hyperactivity to control levels (Fig. 5a–d). SNI/CFA mice receiving vehicle controls showed persistent hyperactivity (Fig. 5b,d). This psilocin-induced reconfiguration of L2/3 neuronal activity was unexpected, as psilocin induced an activating effect in control wild-type mice in Fig. 4h and sham/saline controls in Fig. 5a,c (top). We hypothesize that psilocin’s capacity to suppress neuronal hyperactivity in the ACC may stem from its multifaceted pharmacological actions on postsynaptic serotonergic receptors expressed by cortical neurons.

**Fig. 5: Local psilocin normalizes the spontaneous hyperactivity of pyramidal neurons in both neuropathic and inflammatory pain states.**

Both 5-HT_2A and 5-HT_1A receptor signaling is necessary for psilocybin’s restorative effect on mechanical hypersensitivity and anxiodepressive-like behaviors

To evaluate whether psilocin’s suppression of neuronal activity is related to activation of a specific serotonergic receptor subtype, we subjected SNI mice to either 5-HT_2A or 5-HT_1A receptor blockade before systemic psilocybin administration (Fig. 6a). Here SNI mice on day 27 were given either pimavanserin (1 mg kg⁻¹ i.p. dose), a 5-HT_2A receptor inverse agonist³³ or WAY-100635 (1 mg kg⁻¹ i.p. dose), a selective 5-HT_1A antagonist³⁴, 30 min before receiving psilocybin (0.5 mg kg⁻¹). Pimavanserin pretreatment occluded the spontaneous generation of psilocybin-induced head twitches and its rapid analgesic effects, as withdrawal thresholds remained persistently low on day 28 (Fig. 6b and Extended Data Fig. 8). Similarly, pretreatment with WAY-100635 also prevented psilocybin’s reduction of mechanical hypersensitivity (Fig. 6e). Furthermore, the blockade induced by either pimavanserin or WAY-100635 persisted for up to 40 days, indicating that both antagonists also abolished psilocybin’s long-lasting effects (Fig. 6b,e).

Fig. 6: Activation of both 5-HT1A and 5-HT2A receptors is necessary for psilocybin’s reversal of SNI-induced mechanical hypersensitivity and anxiodepressive-like behaviors. — **Fig. 6: Activation of both 5-HT_1A and 5-HT_2A receptors is necessary for psilocybin’s reversal of SNI-induced mechanical hypersensitivity and anxiodepressive-like behaviors.**

When tested in the EPM and FST, both inhibitors blocked psilocybin’s anti-anxiety and antidepressant-like effects (Fig. 6c,d,f,g). Time spent on the open arms of the EPM decreased in both groups (Fig. 6c,f), while time spent immobile in the FST remained higher compared with baseline levels (Fig. 6d,g). Control mice injected with saline of equal volume 30 min before psilocybin still received the full therapeutic effect in all behavior assays (Fig. 6b–g). Mice receiving only inhibitors (vehicle controls) showed no improvement in any behavioral assays (Fig. 6b–g). Taken together, psilocybin’s rapid and long-lasting analgesic effects are tied to its immediate pharmacological action that depends on initial receptor activation—5-HT_2A targeted by pimavanserin and 5-HT_1A targeted by WAY-100635. Since blocking these receptors at the time of administration prevented prolonged analgesia, this suggests that psilocybin’s therapeutic effects require specific serotonergic receptor engagement during its acute phase to initiate long-term changes.

Therapeutic effect of psilocybin partially replicated by pharmacologically induced full agonism of 5-HT_2A and 5-HT_1A receptors

The blockade of psilocybin-induced improvements in mechanical hypersensitivity and affective behaviors by 5-HT_2A and 5-HT_1A receptor antagonists suggests that activation of both receptor types is necessary to mediate these effects. To this end, we first explored the effect of 5-HT_2A and 5-HT_1A receptor activation on cortical neuronal activity induced by local application of either the phenylalkylamine 2,5-dimethoxy-4-iodoamphetamine (DOI), 5-HT_2A receptor full agonist or 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a 5-HT_1A receptor full agonist. Calcium imaging of L2/3 neurons before and after DOI revealed that DOI induced a strong activation of neuronal activity in both SNI (44% increase) and sham (93% increase) mice (Fig. 7a,b). Conversely, 8-OH-DPAT caused a reduction in neuronal activity in both SNI (64% decrease) and sham (25% decrease) mice (Fig. 7a,b). The combined local application of DOI and 8-OH-DPAT produced an intermediate effect in both SNI (22% increase) and sham (2.8% decrease) mice relative to the perturbations induced by each drug alone (Fig. 7a,b).

Fig. 7: Partial reduction of mechanical hypersensitivity by pharmacologically induced full agonism of 5-HT2A and 5-HT1A receptors. — **Fig. 7: Partial reduction of mechanical hypersensitivity by pharmacologically induced full agonism of 5-HT_2A and 5-HT_1A receptors.**

To determine if these local serotonergic-induced changes in L2/3 neuronal activity might relate to the psilocybin-induced analgesic state and associated anxiodepressive-like behaviors, we pharmacologically agonized the same cohorts serotonergic receptors in four treatment groups in SNI mice—5-HT_1A with 8-OH-DPAT (1 mg kg⁻¹ i.p.), 5-HT_2A with DOI (0.5 mg kg⁻¹ i.p.), both 5-HT_1A + 5-HT_2A with 8-OH-DPAT/DOI (8-OH-DPAT 1 mg kg⁻¹ i.p., DOI 0.5 mg kg⁻¹ i.p.) and vehicle control (Fig. 7c and Extended Data Fig. 9a). Across these treatment groups, we did not observe a psilocybin-like (complete) abolishment effect on mechanical hypersensitivity (Fig. 7d). We observed a modest improvement in mechanical allodynia in the 8-OH-DPAT and 8-OH-DPAT + DOI treatment groups on post-treatment day 1 (Fig. 7d and Extended Data Fig. 9b). When these SNI mice treated with 8-OH-DPAT or 8-OH-DPAT + DOI were tested in the EPM and TST there was no statistically significant improvement (Fig. 7e,f and Extended Data Fig. 9c). SNI mice treated with DOI only displayed increased mobility time in TST (P = 0.028) without any improvement in mechanical hypersensitivity or EPM (Fig. 7c-f (top)). Thus, taken together, our data indicate that the therapeutic effects of psilocybin on mechanical hypersensitivity and anxiodepressive-like behaviors are mediated by the coactivation of 5-HT_2A and 5-HT_1A receptors (Figs. 6 and 7g). However, achieving these effects appears to depend on psilocybin’s partial agonist activity, as full agonism did not produce similar behavioral responses (Fig. 7e–h).

Discussion

Chronic pain and mood dysfunction are interconnected, often forming a vicious cycle that poses substantial challenges for patients in clinical settings¹. The presence of pain can complicate the treatment of depression, as poorly managed or treatment-resistant pain is frequently associated with worse depression outcomes². Conversely, depression in patients experiencing pain is linked to increased pain complaints and greater functional impairment. Reflecting this interplay, we observed a strong correlation between mechanical allodynia and anxiodepressive-like behaviors in all tested SNI and CFA mice (Figs. 1 and 2). While intraplantar CFA injections are commonly used to model sub-acute inflammatory pain, we modified established protocols^27,29 by increasing the CFA injection volume beyond conventional limits to induce a chronic mechanical allodynic state (Extended Data Fig. 1b). The resulting CFA phenotype exhibited comparable levels of mechanical hypersensitivity, affective-related behavior changes and impaired motor skill performance to those observed in SNI mice (Extended Data Fig. 1c). We suspect this transformation of an acute inflammatory pain model to a chronic one is likely driven by an overwhelming inflammatory response, persistent immune signaling and/or alterations in peripheral nerve function.

Consistent with emerging observations of psilocybin’s therapeutic potential in treating chronic pain^22,23, we found behavioral evidence that a single dose of psilocybin induced a rapid and sustained reversal of chronic neuropathic and inflammatory pain-like states and its associated affective state. Building on recent findings mentioned in ref. ²², which demonstrated that administering psilocybin immediately after a pain-inducing procedure (formalin injection) attenuates the development of a chronic pain-like state, and ref. ²³ showing psilocybin can reverse pain-like behaviors in both chemotherapy-induced peripheral neuropathy and CFA rodent models, psilocybin appears to have the potential to both block the onset of chronic pain syndromes through an acute mechanism and reverse established chronic pathology (Fig. 1). In this study, we selected a psilocybin dose of 0.5 mg kg⁻¹, representing the inflection points on the dose–response curve in the HTR assay and open field test (Fig. 1b,c). This dose is half of what was used in the aforementioned rodent studies, allowing us to explore efficacy at a lower threshold. These pain experiments are congruent with other studies; the 0.5 mg kg⁻¹ dose is likely to induce neural plasticity, as behavioral improvements (and forms of learning) and neuronal structural remodeling have been observed in this dose range, 0.3–1.0 mg kg⁻¹ (refs. ^35,36). Future studies in both human and rodent models of chronic pain will be crucial for identifying optimal dosing strategies that elicit behavioral and neurophysiological evidence of neural plasticity.

The concurrent alleviation of both pain models and behaviors associated with affective states indicates the likely convergence of pathological processes within supraspinal regions. Several lines of evidence argue that psilocybin’s reversal of SNI- and CFA-induced chronic pain states stem from neocortical mechanisms. We show that (1) SNI/CFA mice developed a strong association (formation and recall of preference) with the psilocybin-paired chamber as compared with saline (Fig. 3), (2) local application of psilocin to the ACC normalizes chronic pain-associated hyperactivity (Fig. 5) and (3) local application of psilocin to bilateral ACC rapidly reverses mechanical allodynia and depression-like behavior (Fig. 4i,j). Thus, it appears that the neurophysiological and behavioral effects of chronic mechanical allodynia and its reversal induced by psilocin are, to a large degree, cortical phenomena. We suspect that psilocin-induced suppression of heightened spontaneous activity in ACC pyramidal neurons may act to rapidly recalibrate hypersensitivity behavior¹⁵. It has been shown that inhibiting ACC pyramidal neurons³⁷ or activating inhibitory neurons³⁸ can acutely reduce hypersensitivity induced by CFA or formalin. Simultaneously, psilocin may engage additional cortical neurons and circuits, both locally and across broader connected networks tied to descending pain modulation³⁹ through its complex pharmacology and alterations of neuronal excitability⁴⁰. The duration of this acute activity reconfiguration and its potential to facilitate other forms of circuit adaptations, which may underlie the observed sustained improvements—such as dendritic growth and new synapse formation—remain important areas for further investigation.

Psychedelics are widely recognized as 5-HT_2A receptor agonists, yet their mechanism of action is far more complex and multifaceted than initially understood⁴¹. Psilocin is now considered to be relatively nonselective for 5-HT₂ receptors, displaying moderate to high affinity for 5-HT_1A and 5-HT_2A subtypes⁴². Both 5-HT_2A and 5-HT_1A receptors are strongly expressed in the PFC^32,43,44 with comparable distribution across excitatory (~50–70%) and inhibitory (~20–30%) cell types⁴⁵. Single-cell analyses of PFC neurons suggest coexpression of 5-HT_2A and 5-HT_1A receptors⁴⁶ implying that serotonin and psychedelics may exert dynamic excitatory (via 5-HT_2A) or inhibitory (via 5-HT_1A) effects within the same cell depending on their expression ratio and subcellular localization. Selective pharmacological agonism of 5-HT_2A and 5-HT_1A receptors would drive pyramidal neuronal excitability⁴⁷ and hyperpolarization⁴⁸, respectively. In our study, pharmacologically blocking either 5-HT_2A or 5-HT_1A receptors before psilocybin administration completely abolished its rapid and sustained effects on mechanical hypersensitivity and anxiodepressive-like behaviors (Fig. 6). This suggests that altering the balance between 5-HT_2A and 5-HT_1A receptor activity—whether by favoring 5-HT_2A over 5-HT_1A (as with WAY-100635) or 5-HT_1A over 5-HT_2A (as with pimavanserin)—is insufficient to produce therapeutic effects.

Supporting this conclusion, administration of selective full agonists such as DOI (highly selective for 5-HT₂ receptors (A, B, C) with a 1000-fold selectivity over 5-HT1_A receptors⁴⁹) or 8-OH-DPAT (highly selective for 5-HT_1A agonist) alone failed to produce psilocin-like effects on either mechanical allodynia or behaviors assessed in the EPM and TST (Fig. 7). Interestingly, combined full agonism with DOI and 8-OH-DPAT failed to replicate psilocybin’s effects (Fig. 7). This might suggest that partial agonist activity, as seen with psilocin, is critical for achieving the nuanced modulation of receptor activity required for therapeutic efficacy. Additional studies coupling receptor-specific antagonists to psilocybin or new partial agonists⁴¹ in chronic pain patients will clarify whether acute activation of 5-HT_2A or 5-HT_1A receptors is necessary for therapeutic effect.

While selective activation of 5-HT_2A or 5-HT_1A receptors alone failed to replicate psilocybin’s effects on mechanical allodynia and anxiodepressive-like behaviors in SNI mice, two-photon calcium imaging with these specific agonists revealed receptor-specific trends on neuronal activity consistent with prior electrophysical recordings⁵⁰. Local application of DOI-induced activation in L2/3 pyramidal neurons, whereas 8-OH-DPAT produced a net suppressive effect (Fig. 7a,b). Interestingly, the combination of DOI and 8-OH-DPAT elicited a psilocin-like response, characterized by an intermediate effect that inhibited spontaneously hyperactive neurons and activated previously silent neurons. We propose that in SNI/CFA, chronic pain-like states, hyperactive circuits emerge in pain-related affective nodes, like in the ACC. This process of chronification may also modify gene expression and/or sensitivity of serotonergic receptors, characterized by increased 5-HT_2A and decreased 5-HT_1A receptor activity in these regions, thereby contributing to hyperexcitable state (Fig. 7g). Psilocin, acting as a nonselective partial agonist at 5-HT receptors, may (1) rapidly attenuate excessive neuronal firing via 5-HT_1A receptor activation, (2) restore neuronal activity in other cell types that support descending pain modulation and enhanced mood processing facilitated by 5-HT_2A receptor activation and (3) reestablish balanced 5-HT_2A and 5-HT_1A receptor expression and signaling within individual neurons (Fig. 7h). Some of these effects could be due to psilocin’s signaling at other 5-HT receptors including 2B, 2C and even directly at TrkB. Nevertheless, this interplay between receptor subtypes underscores the unique mechanisms by which psilocybin exerts its therapeutic effects in chronic pain-like states with comorbid mood dysfunction.

Methods

Experimental animals

All mice were maintained at the University of Pennsylvania, Perelman School of Medicine, John Morgan animal facility with normal dark/light cycle, controlled room temperature and humidity conditions and had free access to food and water. All animal handling was under guidelines set forth by the School of Medicine’s Institutional Animal Care and Use Committee, approved protocol 807237. C57/BL6 males at an age range of 8–12 weeks were used for behavioral experiments. SNI/CFA and sham controls were housed in cages with five in each cage, with ample food and water. Animals were assigned to groups randomly and no animals were excluded from analysis.

Modeling chronic pain and von Frey testing

SNI of the sciatic nerve or sham operation (sham) was performed on C57BL/6 adult mice (8–12 weeks)⁵¹ In brief, surgeries were performed under strict sterile conditions. Mice were deeply anesthetized, and a small incision in the left thigh was made to expose the sciatic nerve. The tibial and common peroneal nerves were then axotomized/ligated, leaving the sural nerve intact. Care was taken to avoid any contact with or stretching of the intact sural nerve. Muscle and skin were closed in two layers. For sham surgery, the sciatic nerve was exposed but not ligated or cut. For inducing a model of inflammatory pain, undiluted CFA (Sigma-Aldrich, F5881) was subcutaneously injected unilaterally into the intraplantar surface of one hind paw in the mice (~80–100 µl), whereas control mice were injected with 0.9% saline solution (~80 µl). The von Frey test was used to assess the onset and maintenance of mechanical allodynia over time. In all animal groups, mechanical threshold was examined using an Electronic von Frey anesthesiometer (IITC, Life Science Instruments) that measures the precise minimum pressure at which paw withdrawal occurs. Specifically, a von Frey tip of suitable rigidity was attached to an electronic probe and used to apply an increasing pressure to the lateral plantar aspect (the sural nerve skin territory) of the hind paw. The anesthesiometer displays the pressure at which the mouse retracts from the von Frey tip. Three trials for paw withdrawal were recorded for each day tested and an average was reported. The von Frey test was performed during the light cycle by the same researcher.

Behavioral tests

HTR assessment

HTR is described as a rotational/side-to-side head movement that spontaneously occurs following psychedelic administration⁴¹. HTR was assessed in individual animals in an Open Field chamber (25 × 25cm) for 30 min immediately after psilocybin injection. A 30-min session was recorded with a video camera and subsequently scored offline by a trained observer.

EPM

EPM consisted of two open arms without walls and two closed arms with walls. Time spent within open and closed arms was measured using ANY-maze software for a period of 5 min. Mice were placed in the center of an elevated maze, facing an open arm, to start the test. The maze is cleaned with ethanol and dried with paper towels before each test.

FST

A cylindrical glass tank (30 cm height × 20 cm diameter) was filled with water (maintained at 23–25 °C) to a height of 15 cm. Each tank is separated from an adjacent tank using a divider. Animals were lowered gently into water by tail and allowed to swim for 6 min. Only the last 4 min of the test were analyzed for periods of mobility, which were considered to be any movements other than those required to tread water.

OFT

The test was conducted in a 50 × 50 cm arena with a 25 × 25 cm central zone marked in the center. Before each experiment, the chamber was cleaned with 70% ethanol and allowed to dry completely. At the start of the trial, the animal was gently placed in the center of the arena, and behavioral recording using the ANY-maze system was initiated. During the 5-min session, the following parameters were measured: the number of entries into the central zone, the total distance traveled and the time spent in the central zone.

Light/dark box test

The test was conducted in a 45 × 30 × 27 cm box, divided into two compartments—one-third was a covered, dark chamber and the remaining two-thirds was an open, light chamber. The compartments were connected by a 7.5 × 7.5 cm opening, allowing the mouse to move freely between them. White lights were illuminated above the light chamber during the test. The time spent in the light and dark chambers was recorded using ANY-maze software over 10 min. At the start of the trial, the mouse was gently placed in the opening between the compartments, facing the dark chamber. Before each test, the arena was cleaned with 70% ethanol and thoroughly dried with paper towels.

TST

A hollow plastic cylinder is placed around the base of the mouse’s tail to prevent tail climbing. The mouse is then suspended by its tail using a long piece (~15 cm) of strong adhesive tape to the top of the testing system. The test lasts for 6 min but only the final 4 min of the test were analyzed for periods of mobility using ANY-maze software.

Rotarod test

Using the AccurRotor EzRod system, set up rotarod to accelerate from 1 to 45 rpm over 80 s for a total period of 180 s. Gently place the mouse on the rod and start the experiment. The trial begins with the start of the rotation and ends when a mouse falls off the rod. If a mouse clings to the rod and completes a full rotation (while hanging on to the rod), the time for the mouse is stopped and counts as a fall. The test is repeated for three trials and the average of the trials is used. If the mouse falls within the first 10 s of the test, the test is repeated. The latency to fall is then analyzed. Rod is cleaned with 70% ethanol and dried with delicate task wipes before each trial.

CPP

CPP apparatus consists of two chambers with distinct visual cues. One chamber with rectangular-shaped cues and another with circular cues on the walls of the chamber. The chambers are connected by a neutral third chamber with partitions on either side. During the preconditioning phase, the partitions were removed and the mouse was gently placed in the neutral chamber, allowing it to explore the different areas. Immediately after, time spent in each chamber over 15 min was recorded using ANY-maze software. The time spent in each chamber is then analyzed to verify that there is no preconditioning preference. Animals spending more than 80% or less than 20% of total time in one chamber are eliminated from the study. On the conditioning day, mice receive vehicle treatment (saline) and are then immediately placed in the appropriate pairing chamber for 30 min with no access to other chambers (using the partitions to block access). The mouse is then removed from the chamber. Four hours after vehicle treatment injection, the mouse is injected with psilocybin and immediately (within 2 min) is gently placed in the opposite pairing chamber for 30 min. Chamber pairings are counterbalanced (one mouse receives psilocybin in the rectangular chamber while another receives it in the circular chamber) to verify that no bias exists towards cues. On the test day (one day following the conditioning day), the mouse is gently placed in the neutral chamber. The partitions are removed such that the mouse has access to all chambers. Immediately after, time spent in each chamber over 15 min was recorded using ANY-maze software. Increased time spent in the psilocybin-paired chamber indicates a preference for the treatment.

Systemic and local drug delivery

In Fig. 1b,c, mouse head-twitch dose–response curves were recorded following intraperitoneal injection of psilocybin (Usona Institute) at doses of 0 mg kg⁻¹ (saline), 0.25 mg kg⁻¹, 0.50 mg kg⁻¹ and 1.00 mg kg⁻¹. In Fig. 1d–g, following the development of chronic pain, mice were treated with psilocybin on day 27 via a single i.p. (0.5 mg kg⁻¹) or saline (same volume). In Fig. 6, SNI mice received either pimavanserin (Selleck Chemicals, S8183; 1 mg kg⁻¹ i.p. dose) or WAY-100635 (Selleck Chemicals, S2663; 1 mg kg⁻¹ i.p. dose) 30 min before psilocybin (0.5 mg kg⁻¹). In Fig. 7b–m, SNI mice received either 8-OH-DPAT (Selleck Chemicals, S8447; 1 mg kg⁻¹ i.p. dose), DOI (Cayman Chemical, 13885; 1 mg kg⁻¹ i.p. dose) or DOI/DPAT (co-injected with DOI and DPAT; both 1 mg kg⁻¹ i.p. dose).

For behavior experiments in Fig. 4, local psilocin injection (Cayman Chemical, 9003135; 1–500 μM mixed with Rhodamine 6G) to either ACC or spinal cord required a brief period of general anesthesia induced by isoflurane (4–5% for induction, 1–2% for maintenance). To ensure proper anesthetic depth, animal reactivity was assessed periodically by gently pinching the hind paws. For bilateral ACC injection, hair over the scalp was shaved, cleaned thrice with ethanol and a midline scalp incision was made using microsurgical tools. We next removed the soft tissue attached to the skull over ACC regions with fine forceps and a microblade. The mouse skull was immobilized by gluing (cyanoacrylate glue) it to a thin metal plate. Once the glue solidified (2 min), a high-speed microdrill was used to thin a small circular area ( ~ 0.5 mm in diameter) over the ACC bilaterally (+0.5–1.0 mm anterior to the bregma and 0.3–0.5 mm lateral to midline). Intermittent drilling and artificial cerebrospinal fluid (aCSF) immersion minimized heat-induced damage. After the bone thins to ~10–20 μm in thickness, a small bone tear was created with a beveled needle (27G) tip. At this point, a glass electrode filled with psilocin in aCSF (total volume of ~2 μl) was slowly injected into the cortex (depth 200–300 μm) via pressure application with a picospritzer (20 p.s.i., 50 ms per pulse, 1 Hz, 5–10 pulses). Upon completion of injection (~10–15 min), the electrode was rested for ~5–10 min followed by a slow retraction with burr hole closure via a small amount of cyanoacrylate glue. The procedure was repeated for the other hemisphere. The metal plate was then removed, scalp skin reapproximated and sutured.

For the intrathecal psilocin injections, hair overlaying the lower spine was shaved (3 × 3 cm area near base of tail) and cleaned thrice by ethanol. A 15-ml conical tube was placed under the abdomen of the mouse to expose a bigger area of the interspinous ligament, which was punctured by the needle to access the intradural space. Positioning the mouse on a 15-ml conical tube also made it easier to locate and grasp the iliac crest, which is identified by finding the two pits formed at the interface between the muscle and the hip bone. The injection site (L5–L6) was determined by identifying the most protruding spinal process (L6). Of note, injection at this level reduces concern for spinal injury as the spinal cord ends above this level. Next, the animal was grasped just above the hips and a psilocin-loaded Hamilton syringe (1–500 μM, total volume of ~5 μl mixed with Rhodamine 6G) was inserted between the groove of L5 and L6 at a 30° angle and to a depth of 0.5 cm. Once the needle successfully enters the intradural space, the mouse’s tail should move or flick. After observation of tail flick (confirmation of correct space), psilocin was slowly injected, and once completed, the syringe was left in place for 10 s to prevent backflow. The syringe was removed, and the animal was returned to its cage for monitoring.

Spinal cord collection and cFos immunohistochemistry

After perfusion, the entire mouse spinal column, including bone and muscles, was extracted by cutting along the length of the animal from the head to the tail. Small portions of the ribs were retained to help identify the thoracic region. The spinal column was cleaned by removing excess muscle from the spinal column using small scissors. Once cleaned to reveal the bone, the spinal column was placed in a dish with PBS under a magnifying lens. Using blunt tweezers to hold the cord, a blade was used to scrape off the remaining muscle until vertebrae and discs were visible. The lumbosacral region was identified (by counting vertebrae from the ribs - the reference for the thoracic region) and isolated. The spinal cord was extracted by using blunt tweezers to hold one end and pointed tweezers to carefully break the discs on the other end.

Lumbosacral spinal cord samples for subsequent cFos immunostaining were embedded in a 7% agar gel and sectioned in 1× PBS on a vibratome (Leica VT1000S) to obtain 30-μm-thick coronal sections. Intact sections were first blocked for 1 h at room temperature in blocking buffer (5% normal goat serum, 0.1% Triton X-100 in 1× PBS), followed by overnight incubation (~16 h) in primary cFos antibody solution (EnCor Biotechnology, RPCA-c-FOS; 1:1,000 rabbit polyclonal) at room temperature. Sections were washed 3× for 10 min each in 1× PBS before a 2 h room temperature incubation in secondary Alexa-Fluor 568 goat anti-rabbit antibody solution (Invitrogen by ThermoFisher Scientific, A-11011; 1:300). A final wash (3× for 10 min each in 1× PBS) was performed before slide mounting with DAPI Fluoromount-G (Southern Biotech, 0100-20).

Fixed sections were imaged on a fluorescence microscope (Keyence BZ-X810) at ×4 (Plan Apochromat, NA = 0.2) using a red cube filter (ex, 545/25 nm; em, 605/70 nm) or by confocal microscope (Thor Cerna confocal system) at ×10 (Plan Fluor, NA = 0.3) using a fixed wavelength 561 nm laser (Coherent OBIS). Confocal images were stitched using Fiji’s Grid/collection stitching plug-in⁵². Laminae I and II of the dorsal horn of the spinal cord were manually identified in each section using Fiji (version 2.9.0), and cFos-positive cells were identified in Fiji using a particle analyzer pipeline. Briefly, within each lamina I–II dorsal horn region of interest (ROI), the ROI was manually thresholded, converted to a mask, and then processed with a watershed algorithm to aid in the detection of individual cells. Binarized images were then processed with Fiji’s particle detector with size parameters 2–13 pixels² to calculate the number of cFos-positive cells within each ROI. Each plotted sample in Fig. 4d represents the average cell count from 3–6 ROIs in one animal.

Two-photon calcium imaging in awake animals

The genetically encoded calcium indicator GCaMP6f was used for calcium imaging of pyramidal neurons and interneurons in the cortex. GCaMP6f expression was performed using intracranial AAV injections in neonates. Pyramidal neurons were labeled using recombinant AAV9-CaMKII-Cre (Addgene, 105558; 10 nl of AAV per mouse) and AAV9-CAG-FLEX-GCaMP6f (Addgene, 100835; 100 nl of AAV per mouse). A glass micropipette (Drummond, 50,001,001 × 10) was pulled and beveled. A plunger was lightly oiled and inserted into the micropipette to pull the AAV mix. Subsequently, pups at postnatal days 1–2 were anesthetized by hypothermia (typically ~ 2–3 min on ice) and the micropipette was used (freehand) to penetrate the skin and skull and deliver ~200 nl of the virus mix. Medial PFC injection site was determined using landmarks—skull suture lines and head veins—as reference points. Imaging was performed in 1–2-month-old mice, using both sexes, after at least 4 weeks of AAV expression. Mice were group-housed in temperature and humidity-controlled rooms on a 12-h light/12-h dark cycle after injections.

In preparation for imaging, mice underwent a surgical procedure to attach a head holder mount and create an imaging window for two-photon microscopy. In brief, mice were anesthetized with a mixture of 100% oxygen at 2 l min⁻¹ and 1–4% isoflurane. A heating pad was used to maintain the animal’s body temperature at approximately 37 °C. The mouse’s head was shaved, and its skull surface was exposed with a midline scalp incision. The periosteal tissue over the skull surface was removed without damaging the temporal and occipital muscles. A head holder consisting of two parallel metal bars was attached to the animal’s skull. In Cre-positive mice injected with AAV, <1% of mice were negative for GCaMP, suggesting off-target injection was a rare event. In positive mice, a small skull region (~2–4mm in diameter) located over the interfrontal suture was removed, and a round glass coverslip (approximately the same size as the bone being removed) was affixed to the skull with Loctite 495, followed by dental acrylic cement. This window enabled imaging of ACC (+0.5–1.0 mm anterior to the bregma and 0.1–0.3 mm lateral to midline). On recovering from surgical anesthesia, mice with head mounts were habituated daily (two sessions of 30 min with 15-min break) starting on postoperative day 1 in a custom-built body support to minimize potential stress effects of head restraining and imaging. No obvious distress was observed in habituated animals during imaging experiments. Mice tolerated surgery and stress related to the perioperative period as indicated by a 0–10% drop in weight. Imaging experiments were started on postoperative days 2–3 after window implantation.

On the day of imaging, awake mice were positioned in the custom head holder device under the two-photon microscope. In vivo two-photon imaging was performed with an Olympus DIY RS two-photon system (tuned to 910–920 nm) equipped with a Coherent Discovery NX laser. We minimized movement-associated image artifacts by head (secured metal head bars) and body (with a plastic sleeve) restraint on the imaging platform. Mice were head-restrained and imaged for <1 h in total, imaging across several regions in L2/3. To avoid potential interference from psilocin-induced activity-dependent plasticity, animals received a single local dose of psilocin via a small bone flap lateral to the cranial window, which was large enough to accommodate the tip of a glass electrode loaded with Rhodamine 6G and psilocin. Pyramidal neurons in ACC region were randomly chosen and recorded for 2-min sessions under awake conditions and once again after local application of psilocin. All experiments were performed using a ×20 Olympus objective (XLUMPLFLN; NA = 1.00, 2.0 mm working distance) immersed in aCSF, with ×2 digital zoom. Images were acquired at a frame rate of 2–4 Hz (2-μs pixel dwell time). Image acquisition was performed using Olympus Fluoview software and analyzed post hoc using ImageJ software version 2.1.0.

During recordings, motion-related artifacts were typically less than 2 µm. Vertical movements were infrequent and minimized by two metal bars attached to the animal’s skull (described above) and a custom-built body support. All time-lapse images from each field of view were motion-corrected and referenced to a single template frame using cross-correlation image alignment (TurboReg plugin for ImageJ version 2.1.0). ROIs corresponding to visually identifiable somas (pyramidal cells) were selected manually from the field of view. Imaging planes were acquired from L2/3 corresponding to cells positioned ~150–350 μm from the pial surface, respectively. Note that our neonatal injections produced sparse to moderate labeling of GCaMP6. As shown in Fig. 5, 5–20 neurons per L2/3 imaging region were common. A typical experiment would include 3–4 (randomly chosen) imaging regions per animal, yielding ~20–40 cells per animal. Somas that could be identified in all imaging sessions were included in the dataset.

In this study, we used GCaMP6f, an indirect reporter of neuronal spiking activity. All the pixels inside the ROI were averaged to obtain a fluorescence trace for each ROI. Background fluorescence was calculated as the average pixel value per frame from a region without GCaMP expression (blood vessel) and subtracted from the time-series fluorescence traces. The baseline (F₀) of the fluorescence trace was estimated as the average of inactive portions of the traces (~2 s). We did not smooth the raw fluorescence trace (raw traces are presented throughout the paper in each figure). The ΔF/F₀ (%) was calculated as ΔF/F₀ = (F − F₀)/F₀ × 100. GCaMP6f can produce large fluorescence transients (20% ΔF/F) in response to single action potentials, and individual spikes within a burst result in stepwise increases in fluorescence. However, when neuronal firing rates are high, it becomes difficult to resolve the number of action potentials owing to the long decay time constant of GCaMP6 fluorescence. We found that there was a diversity in calcium traces of pyramidal cells, which likely reflects burst and nonburst firing. To compare neuronal activity among different cells with activity patterns, we performed an integrated measurement of a cell’s output activity over 2 min recording, termed area under the curve (%), as well as measuring peak fluorescence signal. In Figs. 4h and 7b, we report calcium activity changes following drug administration as response ratio, calculated as response_drug/response_baseline. To express the relative change from baseline, we computed the response ratio of −1, where positive values indicate an increase in activity relative to baseline and negative values indicate a decrease.

Statistics

Summary data were presented as mean ± s.e.m. Power analysis was used to determine sample sizes for Figs. 1 and 2, while sample sizes among other figures are similar to those reported in our previous publications^53,54,55 and others^9,56,57. Data distribution was assumed to be normal, but this was not formally tested. The interventions were not blinded, as nearly all experiments were carried out and analyzed by A.H. However, J.C., who was blinded to all experimental groups, conducted independent analyses on all datasets. Detailed statistical methods are included in Supplementary Table 1. Exact P values and common levels of significance (not significant, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001) are reported in Figs. 1–7 and Supplementary Table 1. All statistical analyses were performed using GraphPad Prism.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data needed to evaluate the conclusions and replicate the figures presented in this article are provided in the source data and associated data will be available to the scientific community via figshare at https://figshare.com/s/aae99484130e22266bbc (ref. ⁵⁸). Source data are provided with this paper.

Code availability

No new code was used in processing of data presented.

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Acknowledgements

We thank M. Fina for help with animal breeding and management and A. Proekt for helpful discussions and feedback. This work was supported by the National Institutes of Health (R35GM151160-01) and the ASRA Chronic Pain Medicine Research (awarded to J.C.).

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Authors and Affiliations

Department of Anesthesiology and Critical Care, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Ahmad Hammo, Stephen Wisser & Joseph Cichon
Department of Neuroscience, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
Stephen Wisser & Joseph Cichon

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Ahmad Hammo
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Stephen Wisser
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Joseph Cichon
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Contributions

J.C. and A.H. initiated the project and wrote the paper with input from all authors. A.H. acquired, processed and analyzed the behavioral data. J.C. performed the calcium imaging in ACC and spinal cord injections. S.W. collected and analyzed the cFos data.

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Correspondence to Joseph Cichon.

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Nature Neuroscience thanks Rohini Kuner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 High volume CFA injection into the hindlimb footpad induces persistent mechanical allodynia.

a, Representative photograph of a mouse injected with high volume of CFA in the left hind paw (circled in yellow), showing visible fullness and swelling compared to the contralateral hindlimb footpad at day 20 post-injection. The inset highlights the difference between the CFA-injected (left) and the contralateral hindlimb footpad (right). Data are represented as mean ± s.e.m. b, Normalized mechanical withdrawal thresholds for high (n = 22) (red) and low (green) (n = 5) CFA volume animals over the experimental timeline. High CFA animals display a persistent reduction in withdrawal thresholds as compared to the low volume CFA animals. Low CFA animals display a transient mechanical hypersensitivity with thresholds returning to baseline by day 7 post-CFA injection compared to the contralateral limb (two-way ANOVA, P > 0.322 after day 7). c, Comparison of multiple mood-related behavioral responses (top: FST, TST; bottom: EPM, light/dark test, and OFT) between SNI (n = 20) and high-volume CFA (n = 22) mice. CFA mice display similar behavior to SNI mice across all mood-related tests (Kruskal–Wallis followed by Dunn’s multiple comparison: P > 0.10). Data are represented as mean ± s.e.m. **P < 0.01, ns = nonsignificant. Detailed statistics are reported in Supplementary Table 1h.

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Extended Data Fig. 2 Different doses of psilocybin elicit distinct behavioral responses following injection.

a, Intraperitoneal injection of psilocybin induces a transient head twitch response (HTR) over a dosing range of 0.25 mg/kg (n = 6), 0.5 mg/kg (n = 6), and 1 mg/kg (n = 6). Head twitches are summed every 5 min over the first 30 min post-injection. b, Cumulative distance traveled in the open field in animals receiving different psilocybin doses. Higher doses of psilocybin (both 0.5 mg/kg and 1 mg/kg) elicit a hypoactivity state as compared to vehicle controls and psilocybin at 0.25 mg/kg. Data are represented as mean ± s.e.m.

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Extended Data Fig. 3 Sex-specific mood-related behavioral responses in SNI and CFA mice.

a, FST immobility time for SNI (left) and CFA (right) males (SNI: n = 10; CFA: n = 11) and females. (SNI: n = 10; CFA: n = 11). No significant differences were found pre-injury (SNI: P = 0.296; CFA: P = 0.274), post-injury (SNI: P > 0.999; CFA: P = 0.419), and post-treatment (SNI: P > 0.999; CFA: P = 0.446). b, TST immobility time for SNI (left) and CFA (right) males (SNI: n = 6; CFA: n = 5) and females (SNI: n = 4; CFA: n = 5). No significant differences were found on day 0 (SNI: P > 0.999; CFA: P > 0.999), 20 (SNI: P > 0.999; CFA: P > 0.999), 28 (SNI: P > 0.999; CFA: P > 0.999), and 40 (SNI: P > 0.999; CFA: P > 0.999). c, EPM open arm time for SNI (left) and CFA (right) males (SNI: n = 10; CFA: n = 11) and females (SNI: n = 10; CFA: n = 11). No significant differences were found pre-injury (SNI: P = 0.755; CFA: P > 0.999), post-injury (SNI: P > 0.999; CFA: P = 0.344), and post-treatment (SNI: P > 0.999; CFA: P > 0.999). d, Time in light compartment of light/dark box test for SNI (left) and CFA (right) males (SNI: n = 6; CFA: n = 5) and females (SNI: n = 4; CFA: n = 5). No significant differences were found on day 20 (SNI: P > 0.999; CFA: P = 0.235), 28 (SNI: P > 0.999; CFA: P > 0.999), and 40 (SNI: P > 0.999; CFA: P > 0.999). e, Time in center of open field test for SNI (left) and CFA (right) males (SNI: n = 6; CFA: n = 5) and females (SNI: n = 4; CFA: n = 5). No significant differences were found on day 0 (SNI: P > 0.999; CFA: P = 0.441), 20 (SNI: P > 0.999; CFA: P > 0.999), 28 (SNI: P > 0.999; CFA: P > 0.999). CFA females displayed a significant reduction in OPT center time on day 40 compared to males, while no significant differences were found in SNI mice (SNI: P > 0.999; CFA: P = 0.041). Data are represented as mean ± s.e.m. *P < 0.05, ns = not significant. Detailed statistics are reported in Supplementary Table 1,i.

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Extended Data Fig. 4 Motor skill deficits induced by SNI and CFA interventions are reversed by psilocybin.

a,b,d,e, Trajectory for latency to fall (seconds) for individual SNI (a, n = 10, red), sham (b, n = 11, blue), CFA (d, n = 10, gray) and saline (e, n = 10, green) animals over experimental timeline. Thicker line shows the population average. Motor performance declines at day 20, followed by a rapid recovery on day 28 following psilocybin injection. Sham and saline animals show consistent performance across all time points. c,f, Normalized latency to fall on an accelerating rotarod from baseline (day 0). Latency to fall decreases post-injury in (c) SNI (n = 10) and (f) CFA (n = 10) conditions, then increases post-psilocybin injection on day 28 (one-way ANOVA followed by Bonferroni’s multiple comparisons test, SNI: P = 7.9 ×10⁻⁵, CFA: P = 1.6 × 10⁻⁸). Data are represented as mean. ****P < 0.0001. Detailed statistics are reported in Supplementary Table 1,j.

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Extended Data Fig. 5 Simultaneous tracking of mechanical hypersensitivity and mood dysfunction across individual mice in SNI and CFA conditions.

a,b, Measurements of open arm activity in elevated plus maze (EPM) and withdrawal thresholds across multiple time points (day 0 (baseline); day 20 (post-injury); day 40 (after psilocybin treatment)) in individual (a) SNI (n = 20) and (b) CFA (n = 22) mice. c,d, Measurements of immobility time in forced swim test (FST) and withdrawal thresholds across multiple time points in individual (c) SNI (n = 20) and (d) CFA (n = 22) mice.

Source Data

Extended Data Fig. 6 Psilocybin treatment does not regenerate previous severed peripheral sciatic nerves.

Top, photograph of SNI (left) location showing healed surgical site at level of skin. Bottom, representative photographs of sciatic nerve exposure in 3 sham (left) and 3 SNI (right) mice at day 40. SNI procedure results in a neuroma (isolated with blunt dissection) consisting of the common peroneal nerve (CPN) and tibial nerve (TN), while SN remains in normal position intact. Psilocybin treatment at day 28 in SNI mice (3 examples shown) does not induce any gross changes in the appearance of neuroma.

Extended Data Fig. 7 L2/3 neuronal activity changes induced by local serotonergic receptor modulation in ACC.

a, Representative GCaMP6f traces of individual neurons under wakefulness and following local (L-) psilocin (green-shaded area) at different doses (0 µM, 1 µM, 100 µM, 500 µM). Summary shown in Fig. 4h. b, L2/3 peak calcium responses in sham and SNI mice following saline and L-psilocin (200 µM; sham saline, n = 143 from 3 mice; sham L-psilocin, n = 144 from 3 mice; SNI sham, n = 194 from 3 mice; SNI L-psilocin, n = 276 from 4 mice). SNI condition increased the L2/3 peak responses compared to sham, which was reversed by L-psilocin (one-sided Kruskal–Wallis (98), P = 3.4 ×10⁻²¹ followed by Dunn’s multiple comparison: sham versus SNI, P = 7.6 × 10⁻¹¹; sham saline versus sham L-psilocin, P = 5.2 ×10⁻⁴, SNI saline versus SNI L-psilocin, P = 1.3 × 10⁻¹⁹). c, L2/3 peak calcium responses in sham and SNI mice following vehicle control (saline) and local application of DOI (sham n = 189 from 4 mice, SNI n = 222 from 4 mice), 8-OH-DPAT (sham n = 100 from 3 mice, SNI n = 126 from 3 mice), or DOI + 8-OH-DPAT (sham n = 224 from 5 mice, SNI n = 171 from 5 mice). Drug concentration was 200 µM for each condition; one-sided Kruskal–Wallis (430), P = 9.3 × 10⁻⁸⁹ followed by Dunn’s multiple comparisons shown in c. Data are represented as mean ± s.e.m in b,c.

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Extended Data Fig. 8 Pretreatment with pimavanserin before psilocybin blocks the rotational head-twitch response.

a,b, Psilocybin injection (a) induces the prototypical rotational ‘wet-dog’ head-twitch response, which was blocked by pimavanserin administration (b) before psilocybin. The cartoons in a and b (left) are created with BioRender.com.

Extended Data Fig. 9 Pharmacological activation of 5-HT_2A and 5-HT_1A receptors, individually or in combination, affects mechanical withdrawal thresholds and mood behavioral responses.

a, Normalized hindlimb withdrawal thresholds (g) in SNI mice across different conditions. No difference is observed in DOI (n = 6), 8-OH-DPAT (n = 5), DOI + 8-OH-DPAT (n = 6) groups compared to vehicle controls (n = 6) (DOI: P > 0.088, 8-OH-DPAT: P > 0.077, DOI + 8-OH-DPAT: P > 0.064). b, Comparison of hindlimb withdrawal thresholds measured at day 28 normalized to day 26 across different treatment groups. A slight but significant improvement in withdrawal threshold was observed in the DOI + 8-OH-DPAT group compared to the VC group (n = 6, P = 0.0157). However, no significant improvement was observed in the DOI (n = 6, P = 0.9985) or 8-OH-DPAT (n = 5, P > 0.999) groups compared to VC. c, EPM open arm (%) pre-injury, post-injury, and post-treatment. No statistically significant improvement is observed in performance on the EPM post-injection across all drug groups. Data are presented as mean ± s.e.m. **P < 0.01,*P < 0.05, ns, non-significant. Detailed statistics are reported in Supplementary Table 1,k.

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Statistical data for Figs. 1–7 and Extended Data Figs. 1, 3, 4 and 7.

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Hammo, A., Wisser, S. & Cichon, J. Single-dose psilocybin rapidly and sustainably relieves allodynia and anxiodepressive-like behaviors in mouse models of chronic pain. Nat Neurosci 28, 2285–2295 (2025). https://doi.org/10.1038/s41593-025-02068-0

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Received: 28 March 2024
Accepted: 20 August 2025
Published: 02 October 2025
Version of record: 02 October 2025
Issue date: November 2025
DOI: https://doi.org/10.1038/s41593-025-02068-0

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Results

A single dose of psilocybin relieves chronic neuropathic and inflammatory pain-like states

Psilocybin alleviates mood changes associated with chronic neuropathic and inflammatory pain-like states

SNI and CFA animals show preference for psilocybin treatment

Local application of psilocin to the ACC is sufficient for the rapid restorative effect of SNI-induced mechanical allodynia and anxiodepressive-like behaviors

Local psilocin normalizes spontaneous hyperactivity of pyramidal neurons in both neuropathic and inflammatory pain-like states

Both 5-HT2A and 5-HT1A receptor signaling is necessary for psilocybin’s restorative effect on mechanical hypersensitivity and anxiodepressive-like behaviors

Therapeutic effect of psilocybin partially replicated by pharmacologically induced full agonism of 5-HT2A and 5-HT1A receptors

Discussion

Methods

Experimental animals

Modeling chronic pain and von Frey testing

Behavioral tests

HTR assessment

EPM

FST

OFT

Light/dark box test

TST

Rotarod test

CPP

Systemic and local drug delivery

Spinal cord collection and cFos immunohistochemistry

Two-photon calcium imaging in awake animals

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Reporting summary

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Both 5-HT_2A and 5-HT_1A receptor signaling is necessary for psilocybin’s restorative effect on mechanical hypersensitivity and anxiodepressive-like behaviors

Therapeutic effect of psilocybin partially replicated by pharmacologically induced full agonism of 5-HT_2A and 5-HT_1A receptors