Prior trauma shapes the effects of psilocybin on stress sensitization in mice

Abstract

Exposure to traumatic events induces persistent alterations in defensive behavior that extend beyond associative fear memories, including heightened responses to novel stimuli (stress sensitivity) and sustained defensive states in neutral contexts (stress sensitization). While serotonergic psychedelics such as psilocybin have been shown to facilitate fear extinction in associative paradigms, their effects on trauma-induced non-associative stress adaptations remain poorly understood. Here, we developed a behavioral paradigm in mice that dissociates contextual fear recall from stress sensitivity and stress sensitization within the same subjects. Male C57BL/6J mice were exposed to an acute foot-shock stressor, followed by assessment of trauma recall, administration of psilocybin (1 mg/kg) or vehicle, and subsequent evaluation of responses to a novel auditory stimulus and context. Trauma-exposed mice displayed robust associative fear and increased freezing in response to a novel auditory stimulus, consistent with enhanced stress sensitivity. However, psilocybin did not significantly alter this stimulus-evoked response. In contrast, psilocybin produced bidirectional, experience-dependent effects on stress sensitization – it reduced baseline freezing in non-traumatized mice but increased freezing in trauma-exposed animals when re-exposed to a novel context in the absence of a stimulus. Together, these findings demonstrate that psilocybin does not uniformly attenuate trauma-related behaviors, but instead modulates non-associative defensive responses in a manner dependent on prior experience. This work highlights the importance of distinguishing between associative and non-associative components of fear and suggests that psychedelic-induced plasticity may amplify latent trauma-related states under certain conditions.

1. Introduction

Exposure to traumatic or highly stressful events can produce persistent alterations in defensive behavior that extend beyond the original experience [1,15,33]. In addition to associative fear responses – where animals display defensive behaviors upon re-exposure to trauma-associated cues – stress can also induce non-associative changes such as heightened sensitivity to novel stimuli and fear generalization to otherwise neutral environments [14,39,40,50]. These maladaptive responses are core features of stress-related psychiatric disorders, including post-traumatic stress disorder (PTSD), where individuals often exhibit exaggerated responses to previously innocuous stimuli long after the initial trauma [27,31,36,37,48,49].

It is important to note that the term “fear” encompasses not only physiological responses to threat, but also complex subjective emotional experiences. While the canonical “fight-or-flight” response represents an evolutionarily conserved adaptation for survival, the subjective experience of fear in humans involves additional cognitive and affective components that are difficult to model directly in animals [3,55]. Accordingly, in the context of the present study, “fear” memory and expression are operationally defined as trauma-associated defensive behaviors – such as freezing – observed in rodent models.

Traditional preclinical models have largely focused on associative fear conditioning paradigms, in which defensive behaviors such as freezing are measured in the same context in which the stressor occurred. These paradigms typically encompass two related but distinct forms of associative memory – contextual fear memory, in which the entire environment or background context becomes associated with the aversive event, and cued fear memory, in which a discrete stimulus (e.g., a tone) predicts the stressor [6,43]. While both forms of learning have provided critical insights into the neural mechanisms of fear acquisition and recall, they do not fully recapitulate the complexity and unpredictability of fear responses observed in clinical populations. As highlighted in recent work, stress can produce dissociable behavioral adaptations – including associative fear, anxiety-like behavior, and heightened stress sensitivity – that are mediated by partially distinct neural systems [19,45,47,50,51]. These findings underscore the need for experimental paradigms that can disentangle contextual trauma recall from trauma-induced non-associative stress adaptations.

Stress-induced adaptations can manifest at multiple levels, including increased stress sensitivity and stress sensitization. Stress sensitivity refers to an increased behavioral response to an acute, novel stimulus following prior trauma exposure, reflecting a lowered threshold for detecting and reacting to potential threats [49,53]. In contrast, stress sensitization reflects a more persistent adaptation in which defensive behavior is expressed even in the absence of an explicit stressor, indicating lasting, non-associative changes in how organisms interpret and respond to their environment [31,36,39]. Unlike associative fear, which depends on retrieval of a specific memory, these processes are not tied to the original trauma context or cue, but instead reflect broader alterations in threat processing. Importantly, such phenomena may better model aspects of human psychopathology, where symptoms are often triggered by novel or ambiguous environmental cues and can persist even without direct provocation [30,46]. Thus, developing translational models that distinguish between stress sensitivity (enhanced responses to a subsequent acute challenge) and stress sensitization (persistent defensive responding in the absence of an explicit threat) is essential for understanding the mechanisms underlying maladaptive fear and for identifying effective therapeutic strategies.

In parallel, serotonergic psychedelics such as psilocybin, lysergic acid diethylamide (LSD), N,N-dimethyltryptamine (DMT), and mescaline have emerged as promising candidates for the treatment of stress-related disorders [22,24,28]. Psilocybin in particular, a non-selective serotonin 5-HT_2A receptor agonist and active compound found in “magic mushrooms,” has been shown to produce rapid and sustained improvements in mood and anxiety in clinical populations [21], with effects thought to be mediated by enhanced neural plasticity and modulation of discrete circuits within the frontal cortex [13,34,52]. Preclinical studies further suggest that psychedelics can reduce conditioned fear expression and facilitate fear extinction across both contextual and cued fear memories in classical fear-conditioning paradigms [4,13,59]. However, it remains unclear whether these effects extend to non-associative processes such as stress sensitivity and stress sensitization, or whether psychedelic-induced plasticity may instead amplify previously encoded behavioral responses in a state-dependent manner.

To address this gap, we developed and applied a behavioral paradigm designed to dissociate contextual trauma recall from non-associative defensive responses in a novel environment. In this framework, animals are first exposed to an acute stressor, followed by assessment of associative fear expression and, subsequently, responses to a novel stimulus in a distinct context. This approach enables the evaluation of fear acquisition, stress sensitivity, and stress sensitization within the same subjects, providing a more comprehensive model of trauma-induced behavioral adaptations.

In the present study, we asked whether a single administration of psilocybin – given after trauma recall – modulates subsequent trauma-induced maladaptive stress responses in mice. Specifically, we examined whether psilocybin attenuates stimulus-evoked responses (stress sensitivity) as well as sustained defensive behavior following that stimulus (stress sensitization), and whether these effects parallel its reported ability to facilitate fear extinction within the original trauma-associated context. By comparing trauma-exposed and non-traumatized animals and relating post-treatment behavioral outcomes to prior levels of associative fear expression, we sought to determine whether the effects of psychedelics generalize across contexts or instead selectively influence distinct components of stress-induced behavioral plasticity.

2. Materials and methods

2.1. Animals

Adult male C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME), 8–10 weeks of age, were housed in an AAALAC-accredited temperature- and humidity-controlled environment (22 ± 1°C, 60–70% relative humidity) with ad libitum access to food (7012 Teklad LM-485 Mouse/Rat Sterilizable Diet, Harlan Laboratories Inc., Indianapolis, IN) and water throughout the study. Upon arrival and throughout the duration of the stress-sensitization paradigm (days 1–5; Fig. 1), mice were individually housed to prevent potential confounding effects of stress and/or drug exposure among cage mates. Prior to behavioral assays, mice were allowed to acclimate to the vivarium for one week, followed by one week of habituation to handling and transportation to the testing location. All procedures involving animals were approved by the Institutional Animal Care and Use Committee (IACUC) of Virginia Commonwealth University and followed the guidelines of the National Institutes of Health Care and Use of Laboratory Animals. Behavioral testing took place between 9 a.m. and 6 p.m. All efforts were made to minimize animal suffering and the number of animals used.

Fig. 1.

Effect of psilocybin on trauma-induced sensitization to a novel stressor (timeline of the experimental design). Mice were assigned to non-trauma (NT) or trauma (T) groups. Trauma recall (Context A) was assessed 24 h later. Mice then received (i.p.) psilocybin (1 mg/kg) or vehicle, and 24 h later were exposed to a novel context (Context B) with an auditory startle stimulus to assess stress sensitivity. Persistent sensitization was evaluated in Context B 24 h later.

2.2. Stress-sensitization, novel stressor, and sensitivity recall paradigm.

To evaluate stress-induced changes in defensive behavior, mice underwent contextual fear conditioning consisting of a trauma acquisition phase followed by an expression test 24 h later. The following day, mice then received a single administration of psilocybin (1 mg/kg) or vehicle. After an additional 24 h, animals were exposed to a novel startle stimulus in a distinct context, and sensitivity recall was assessed 24 h later (Fig. 1). For trauma induction, a conventional contextual fear acquisition and extinction paradigm was used, as previously described [13], with minor modifications. Briefly, fear conditioning was conducted using two commercially available Near Infrared Video Freeze Systems (MED-VFCNIR-M, Med Associates, Inc., St. Albans, VT), each controlling four test chambers. Each polycarbonate chamber (32 × 25 × 25 cm) was equipped with a metal grid floor and enclosed within a sound-attenuating box (63.5 × 35.5 × 76 cm). Video images were collected at 30 fps (640 × 480 pixels, down-sampled to ~1 pixel per visible mm²). Freezing was automatically defined as the absence of movement for ≥1 s (i.e., negligible variation in pixel composition across 30 consecutive frames). Data are expressed as the percentage of time spent freezing throughout. On Day 1 (Fig. 1), mice were transported from the vivarium to the testing room and allowed to habituate for at least 1 h under continuous white noise, which was maintained throughout all testing procedures. Context A consisted of a standard polycarbonate chamber (dimensions as above), illuminated by bright white light (6000K) and scented with lemon (Watkins Pure Lemon Extract). Mice were randomly assigned to either non-trauma (NT) or trauma (T) groups. Following a 5-min baseline recording period, mice in the T group received 10 unconditioned stimuli consisting of 1-s scrambled foot shocks (1.0 mA) delivered through the grid floor, with 30-s inter-shock intervals. NT mice were placed in the same context for an equivalent duration but did not receive foot shocks. On Day 2 (24 h later; Fig. 1), mice were returned to Context A, and freezing behavior was recorded for 8 min in the absence of any stimulus to assess associative fear expression. Mice were then returned to their home cages. On Day 3, mice received a single administration of psilocybin (1 mg/kg; Usona Institute) or vehicle (0.9% saline). On Day 4 (24 h later), mice were returned to the testing room and placed in a distinct context (Context B), characterized by the absence of white light (940 nm NIR light only) and the presence of a vanilla scent (Private Selections Pure Vanilla Extract). After a 5-min baseline period, mice were exposed to a novel stressor consisting of a single auditory stimulus (125–130 dB, 5000 Hz, 3 s). Freezing behavior was then recorded for an additional 3 min in the same context, after which mice were returned to their home cages. On Day 5 (24 h later), mice were again placed in Context B, and freezing behavior was recorded for 8 min in the absence of any stimulus to assess generalization of trauma-associated defensive behavior and persistent stress sensitization (Fig. 1).

2.4. Head twitch behavior

Detection of head-twitch responses (HTRs) in mice was performed as previously reported [11,12,25]. Briefly, mice were ear-tagged with neodymium magnets (N50; 3 mm diameter × 1 mm height; 50 mg) glued to the top surface of aluminum ear tags for rodents (Las Pias Ear Tag, Stoelting Co.), with the magnetic south pole in contact with the tag. Following ear-tagging, animals were returned to their home cages and allowed to acclimate to the tags for one week. Data acquisition and processing were performed as previously described [11,12,25], using our signal analysis pipeline in combination with a deep learning-based approach based on scalograms. Testing occurred no more than once per week, with at least 7 days between sessions. On test days, mice were placed individually into the monitoring chamber for 15 min to acclimate to the environment and establish baseline HTR. Subsequently, animals received (i.p.) psilocybin (0.3, 1.0 or 2 mg/kg) or vehicle, and HTRs were recorded for 30 min.

2.3. Data Analysis

Animals were randomly allocated into the different experimental groups. No statistical methods were used to predetermine sample sized, but our sample sizes are similar to those in our previous publications [23,25]. For behavior assays, experimenters were blinded to the treatment groups. Potential statistical outliers were identified using the ROUT method (Q = 1%) [42]; however, no individual values were identified as outliers, and therefore no data points were excluded from the analysis. Statistical analysis was assessed using Student’s t-test, and two-way, two-way repeated measures, or three-way ANOVA, depending upon the number of experimental conditions and independent variables. Following a significant ANOVA, specific comparisons were made using Bonferroni’s post-hoc test. All values represent mean ± S.E.M. All statistical analysis were performed with GraphPad Prism software version 11, and comparisons were considered statistically significant if p < 0.05.

3. Results

We first sought to develop a behavioral protocol in which a single acute stressor produces lasting changes in defensive behavior, adapting a previously established model that we and others have used extensively to assess contextual fear expression in mice [13,59] and rats [4]. Male mice were placed in a distinct environment (Context A) where they received 10-foot shocks during a 5-min period (“trauma” [T]) or in the same environment without receiving foot shocks (Fig. 1). As previously reported [13], continuous exposure to foot shocks during this brief period produced a cumulative increase in freezing behavior across successive shocks, an effect not observed in the non-trauma (NT) group (Fig. 2A). A similar effect was observed when comparing the average freezing time across all foot shocks (Fig. 2B). To assess contextual fear expression (trauma recall), mice were returned to their home cage and then re-exposed to the trauma environment in the absence of foot shocks (Fig. 1). As expected [13], when returned to this environment (Context A) the following day, trauma-exposed mice exhibited substantially higher freezing compared to the NT group (Fig. 2C).

Fig. 2.

Trauma-exposed mice display increased freezing time during foot-shock exposure and trauma recall (fear expression). A, Freezing time during foot-shock exposure (Shock number F[9, 414] = 39.62, p < 0.001; NT vs T F[1, 46] = 242.1, p < 0.001; Interaction (F[9, 414] = 35.61, p < 0.001). Two-way repeated measures ANOVA (+++p < 0.001). B, Mean freezing time during shock exposure (t₄₆ = 12.71). C, Mean freezing time during trauma recall (t₄₆ = 8.80). NT, n = 22 mice; T, n = 26 mice. Statistical analysis was performed using two-way repeated measured ANOVA (A) (⁺⁺⁺p < 0.001), or unpaired Student’s t-test (B,C) (***p < 0.001). Data show mean ± S.E.M.

Next, we tested whether a single administration of psilocybin alters behavioral responses to novel environmental stimuli that are not typically stressful in naïve mice but may elicit maladaptive responses following prior trauma exposure. To this end, mice received a single administration of psilocybin (1 mg/kg) in their home cages 24 h after trauma recall testing. This dose was selected based on its inverted dose-response effect on HTR (a rodent behavioral proxy of hallucinogenic potential [9,17,20]), with maximal effects observed at 1 mg/kg (Fig. S1), consistent with our previous reports [25]. Twenty-four hours after psilocybin (or vehicle administration), stress sensitivity was assessed by placing the animals in a novel environment (Fig. 1). At baseline, neither trauma-exposed (T) nor non-trauma (NT) mice exhibited appreciable freezing, and psilocybin had no effect on this measure (Fig. 3A). Immediately thereafter, a loud auditory startle stimulus was delivered (Fig. 1). Trauma-exposed mice displayed a marked increase in freezing compared with NT controls (Fig. 3B), consistent with enhanced stress sensitivity following prior trauma. However, psilocybin did not significantly alter this response in either group (Fig. 3B). A similar effect of prior trauma was observed when comparing freezing behavior before and after the startle stimulus, with increased freezing in T but not NT mice (Fig. 3C and Table 1). Again, psilocybin administered 24 h earlier had no significant effect relative to vehicle-treated controls (Fig. 3C and Table 1).

Fig. 3.

Psilocybin does not affect the impact of auditory startle stimulus on freezing behavior in trauma-exposed mice. A, Mean freezing time before auditory startle stimulus (NT vs T F[1, 44] = 0.52, p > 0.05; Psilocybin F[1, 44] = 0.15, p > 0.05; Interaction F[1, 44] = 0.92, p > 0.05). B, Mean freezing time after auditory startle stimulus (NT vs T F[1, 44] = 7.36, p < 0.01; Psilocybin F[1, 44] = 0.49, p > 0.05; Interaction F[1, 44] = 0.48, p > 0.05). C, Mean freezing time before vs after auditory startle stimulus (see Table 1 for three-way ANOVA analysis). NT-saline, n = 16 mice; NT-psilocybin, n = 6 mice; T-saline, n = 12 mice; T-psilocybin, n = 14 mice. Statistical analysis was performed using two-way (A, B) (⁺⁺p<0.01, n.s., not significant) or three-way (C) (⁺p<0.05) ANOVA, followed by Bonferroni’s post-hoc test (***p<0.01, n.s., not significant). Data show mean ± S.E.M.

Table 1.

Three-way ANOVA analysis of post-acute psilocybin effects on startle-induced freezing in trauma-exposed mice

Source of variation	ANOVA	p value
Before vs After	F[1, 88] = 20.96	p < 0.001 (***)
NT vs T	F[1, 88] = 6.80	p < 0.05 (*)
Psilocybin	F[1, 88] = 0.38	p > 0.05
Before vs After × NT vs T	F[1, 88] = 6.60	p < 0.05 (*)
Before vs After ×	F[1, 88] = 0.52	p > 0.05
NT vs T × Psilocybin	F[1, 88] = 0.83	p > 0.05
Before vs After × NT vs T × Psilocybin	F[1, 88] = 0.16	p > 0.05

Finally, we assessed the expression of trauma-associated defensive behavior (stress sensitization) by re-exposing mice, 24 h later, to a novel context (Context B) in the absence of an auditory startle stimulus (Fig. 1). We observed a significant interaction between trauma exposure and prior psilocybin administration (Fig. 4). Specifically, in vehicle-treated animals, T mice exhibited a non-significant trend (p = 0.09) toward lower freezing compared to NT controls (Fig. 4). In contrast, psilocybin pretreatment reduced freezing in the NT group, whereas it increased freezing in the T group (Fig. 4).

Fig. 4.

Psilocybin affects the generalization of trauma-associated defensive behavior. Data show mean freezing in response to a novel context (Context B) in the absence of an auditory startle stimulus (NT vs T F[1, 44] = 0.16, p > 0.05; Psilocybin F[1, 44] = 0.16, p > 0.05; Interaction F[1, 44] = 5,72, p < 0.05). NT-saline, n = 16 mice; NT-psilocybin, n = 6 mice; T-saline, n = 12 mice; T-psilocybin, n = 14 mice. Statistical analysis was performed using two-way ANOVA (⁺p<0.05), followed by Bonferroni’s post-hoc test (*p<0.05, n.s., not significant). Data show mean ± S.E.M.

4. Discussion

In the present study, we validated a behavioral paradigm in which a single acute stressor produces persistent and dissociable alterations in defensive behavior. Consistent with prior reports [13], trauma-exposed mice exhibited robust fear acquisition during foot-shock exposure and elevated freezing during trauma recall, confirming that this model reliably captures associative fear processes. Importantly, this paradigm also enabled the dissociation of non-associative adaptations, operationally defined here as responses to a novel auditory stimulus in a new context (stress sensitivity) and the subsequent expression of defensive behavior in that same context in the absence of the stimulus (stress sensitization). Extending this framework, stress sensitivity reflects an acute, stimulus-driven increase in defensive responding, whereas stress sensitization reflects a sustained and context-dependent defensive state that persists beyond the immediate threat and does not require ongoing external input [31,36,39,49,53]. Together, these measures provide a translationally relevant approach to modeling distinct dimensions of trauma-induced behavioral adaptations.

Using this paradigm, we found that trauma exposure enhanced freezing responses to an acute auditory stimulus in a novel context, consistent with increased stress sensitivity. This behavioral phenotype has strong translational relevance, as individuals with trauma-related panic disorders often exhibit symptoms primarily in response to salient external stimuli (e.g., loud sirens or alarms). Notably, a single administration of psilocybin following trauma recall did not significantly modify this response. These findings indicate that, under the conditions tested, psilocybin does not attenuate heightened reactivity to externally driven, acute stressors. This is notable given previous reports that psychedelics facilitate fear extinction in associative paradigms [4,13,59], and suggests that their effects may be more selective for processes involving memory updating or extinction rather than stimulus-evoked defensive responses in a novel environment.

In contrast, psilocybin produced bidirectional and experience-dependent effects on stress sensitization. Trauma-exposed mice treated with vehicle did not exhibit any change in freezing compared to non-trauma controls, indicating that prior trauma did not produce elevated persistent defensive behavior under baseline conditions, at least within the experimental parameters used in this study. Nevertheless, in non-traumatized animals, psilocybin reduced freezing behavior during re-exposure to the novel context, suggesting a decrease in baseline defensive tone or anxiety-like behavior. In contrast, in trauma-exposed mice, psilocybin increased freezing under the same conditions, leading to higher levels of defensive behavior relative to vehicle-treated controls. These findings indicate that psilocybin does not uniformly suppress or enhance defensive responses, but rather modulates their expression in a manner that depends critically on prior experience.

One interpretation of these findings is that stress sensitization may not manifest as a stable increase in baseline defensive behavior, but instead as a latent or context-dependent state that can be differentially expressed depending on internal or pharmacological conditions. Under baseline conditions, trauma-exposed animals may not display overt sensitization; however, psilocybin may reveal or amplify underlying trauma-related representations, resulting in increased defensive responding. In contrast, in non-traumatized animals, psilocybin may reduce baseline defensive tone, consistent with anxiolytic-like effects reported in other contexts. Psychedelics are often framed as inherently therapeutic, yet neuroplasticity is not intrinsically beneficial. For example, both psilocybin [25] and drugs with high abuse liability, such as cocaine [38], induce dendritic structural plasticity in subcortical regions including the nucleus accumbens. Together, these results support the idea that psychedelic-induced plasticity does not simply normalize behavior, but may increase the flexibility or expression of pre-existing behavioral states in a history-dependent manner.

The model of trauma-induced stress sensitivity developed in this study may have translational relevance to psychiatric disorders such as PTSD. Specifically, our data demonstrate that exposure to a novel stressor (a single auditory stimulus) does not affect freezing behavior in otherwise naïve mice, whereas the same stimulus elicits a robust increase in freezing in trauma-exposed animals. Because psilocybin did not alter this stimulus-evoked response under the experimental conditions used here, future studies could explore additional paradigms that capture complementary aspects of trauma-related psychopathology, including innate defensive behaviors (e.g., predator odor exposure) and associative fear overgeneralization paradigms, thereby distinguishing non-associative stress adaptations from generalized fear processes.

Several limitations of the present study should be considered. First, all experiments were conducted in male mice, despite increasing evidence that psychedelics can produce sex-dependent effects on behavior, synaptic plasticity, and receptor signaling [25,26,54,57]. Future studies will be necessary to determine whether the bidirectional effects observed here generalize to females, particularly given known sex differences in stress responsivity and serotonergic systems [7]. Second, although the dose of psilocybin was selected based on our dose-response studies in the head-twitch response assay, it remains unknown whether higher or lower doses, or repeated administration rather than a single injection, would produce similar effects in this or other models of trauma-induced sensitization to novel stressors. Third, our findings are limited to psilocybin, and it remains unclear whether similar effects would be observed with other serotonergic psychedelics such as LSD, DMT, or mescaline, which differ in pharmacokinetics and receptor binding profiles [44]. Lastly, although psilocin (the active metabolite of psilocybin) is commonly described as a 5-HT_2A receptor agonist, it interacts with multiple GPCRs, including 5-HT_1A [8,58], 5-HT_1B [16,35] and 5-HT_2C [5] receptors, as well as non-GPCR pathways, including TrkB signaling [41]. Therefore, the receptor mechanisms underlying these effects remain to be determined. Future studies using receptor-selective antagonists or genetic models will be essential to establish whether these behavioral effects are mediated by 5-HT_2A receptor signaling or involve contributions from other serotonergic or non-serotonergic pathways.

An additional consideration is that mice were individually housed from arrival and throughout the 5-day behavioral protocol to prevent potential confounding effects of social interactions or drug exposure among cage mates. Although this approach enhances experimental control [17,18], single housing itself can represent a mild but chronic stressor, particularly in social species such as mice. Social isolation has been reported to influence baseline anxiety-like behavior, stress reactivity, and neural plasticity [2,32], which could in turn affect measures of freezing behavior and sensitivity to subsequent stressors. Therefore, it is possible that individual housing contributed to the behavioral phenotype observed here, potentially interacting with both trauma exposure and psilocybin treatment. Future studies incorporating group-housed conditions or directly comparing housing paradigms will be important to determine the extent to which social isolation influences stress sensitivity and sensitization in this model.

At the circuit level, the dissociation between stress sensitivity and stress sensitization suggests the involvement of partially distinct neural substrates. Stress sensitivity likely engages circuits involved in rapid threat detection and sensory processing, including the amygdala and brainstem pathways, whereas stress sensitization may reflect sustained alterations in cortico-limbic networks such as the prefrontal cortex, hippocampus, and extended amygdala [10,29,56]. The experience-dependent effects of psilocybin observed here further suggest that these compounds may differentially modulate top-down versus bottom-up components of defensive behavior, potentially through changes in cortical excitability, synaptic plasticity, or network-level integration. Future work combining this behavioral paradigm with circuit-specific manipulations, in vivo recordings, or activity-dependent tagging approaches will be important to determine how psychedelic compounds interact with trauma-encoded neural ensembles.

In conclusion, our findings demonstrate that psilocybin does not attenuate stress sensitivity to acute stimuli under the conditions tested, but exerts complex and experience-dependent effects on stress sensitization. These results highlight the importance of distinguishing between associative and non-associative components of defensive behavior and suggest that the effects of psychedelics may depend strongly on prior experience and behavioral state. More broadly, these findings underscore the need for experimental paradigms that capture the multidimensional nature of trauma-related behavioral adaptations and for therapeutic approaches that can selectively target both stimulus-driven and persistent components of maladaptive fear.

Supplementary Material

1

Fig. S1. Dose-response effect of psilocybin on HTR behavior. HTR counts correspond to the first 30 min after injection with psilocybin at the indicated dose (i.p.), or vehicle (F[3, 16] = 16.58, p < 0.001; n = 6 mice per group). Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post-hoc test (*p<0.05, ***p<0.001). Data show mean ± S.E.M.

Significance statement Psilocybin differentially modulates trauma-related behaviors in mice – leaving stimulus evoked stress sensitivity unchanged while bidirectionally altering non-associative stress sensitization in an experience-dependent manner, highlighting that psychedelic-induced plasticity can amplify or reduce defensive states depending on prior trauma exposure.