Towards Novel Antidepressant Strategies: A Comparative Study of Ketamine, Psilocybin, and Fluoxetine in a Chronic Stress Model

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The chronic social defeat (CSD) stress model in mice is a well-established preclinical paradigm for inducing depression-like behaviors and evaluating antidepressants effectiveness. This study compared the efficacy of both acute and chronic fluoxetine with acute ketamine and psilocybin treatment in male C57BL/6J mice subjected to CSD. Fluoxetine showed no significant effects 24 hours after a single dose or following 7 days of repeated administration; antidepressant-like effects only appeared after 14 days of continuous treatment. In contrast, a single dose of either ketamine or psilocybin significantly reversed social avoidance behavior at 24 hours, with sustained effects observed at 7- and 14-days post-treatment. These findings suggest that ketamine and psilocybin elicit rapid and durable, antidepressant-like responses in this preclinical model, in contrast to traditional SSRIs, like fluoxetine, which requires extended treatment duration, mirroring clinical efficacy patterns. The results support the utility of the CSD model in evaluating antidepressant efficacy and highlight the therapeutic potential of fast-acting agents such as ketamine and psilocybin as alternatives to conventional treatments for major depressive disorder. Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Biological sciences/Neuroscience Biological sciences/Psychology Social science/Psychology Chronic social defeat treatment-resistant depression antidepressant efficacy ketamine psilocybin fluoxetine Figures Figure 1 Figure 2 Figure 3 Introduction Major depressive disorder (MDD) is a prevalent health condition that is associated with significant health and socioeconomic burdens. Globally, it is estimated that 5% of the adult population − 280 million individuals - experiences depression [ 1 ]. In light of the escalating prevalence of MDD, including treatment-resistant depression (TRD) cases, there is a critical need to develop novel therapeutic interventions and more effective pharmacological agents to address this condition. A major challenge in developing novel antidepressants lies in identifying rapid-acting compounds that offer immediate relief, particularly for individuals with severe or treatment-resistant depression. The complex and multifaceted nature of anxiety disorders and depression, in combination with the paucity of effective treatment options, highlights the importance of animal models in elucidating the underlying aetiology and pathophysiology of depression. Animal models provide a valuable platform for asses sing the efficacy of emerging therapeutic strategies. Among them, the chronic social defeat (CSD) mouse model represents a robust, stress-induced paradigm that recapitulates the core features of human depression. Notably, its selective responsiveness to clinically effective antidepressants enhances its translational relevance for preclinical research [ 2 ]. Social avoidance is a hallmark endpoint of the CSD model and can be reversed following acute or chronic antidepressant treatments. Classic selective serotonin reuptake inhibitors (SSRIs) have been evaluated in the CSD model [ 3 ] however, their delayed onset of action has led to increased interest in identifying compounds capable of producing a more rapid antidepressant effect. Unlike many other depression models utilized to assess the effectiveness of rapid-acting antidepressants, social defeat stress serves as a compelling framework for concurrently evaluating the impact of novel fast-acting compounds alongside traditional, long-acting SSRI antidepressants. While fluoxetine is one of the most widely prescribed antidepressants and has proven efficacy in treating MDD, its use is associated with several significant limitations. A major drawback is its delayed onset of action; therapeutic effects often require 2 to 6 weeks to manifest, which can be problematic for patients in acute distress [ 4 ]. Furthermore, approximately one-third of patients fail to achieve remission, even after adequate trials, highlighting issues with treatment resistance [ 5 ]. The mechanism of action, which involves the selective inhibition of the serotonin transporter (SERT), does not fully account for the complex neurobiology of depression, and chronic use may result in compensatory receptor downregulation and diminished neuroplasticity over time [ 6 ]. Emerging evidence supports the efficacy of rapid-acting antidepressants, particularly subanesthetic doses of ketamine and serotonergic psychedelics like psilocybin, in treating MDD and related conditions. Ketamine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, exerts its antidepressant effects by modulating glutamatergic transmission. Specifically, ketamine blocks NMDA receptors on GABAergic interneurons, leading to disinhibition of glutamate release, which in turn activates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [ 7 , 8 ]. This cascade results in the activation of downstream signalling pathways, including brain-derived neurotrophic factor (BDNF) release and mammalian target of rapamycin (mTOR) signalling, which promote synaptic plasticity and neurogenesis [ 7 , 8 ]. Clinically, ketamine has demonstrated a rapid onset of antidepressant effects, often within hours, and sustained efficacy for up to a week following a single intravenous infusion, particularly in patients with TRD [ 9 , 10 , 11 ]. Similarly, psilocybin—a serotonergic psychedelic and prodrug of psilocin—acts primarily as a partial agonist at the 5-HT2A receptor, which is highly expressed in cortical regions involved in mood, cognition, and self-referential thought. This receptor activation leads to a temporary disruption of the default mode network (DMN), increased neural connectivity, and alterations in emotional processing and self-perception [ 12 , 13 ]. In recent randomized controlled trials, psilocybin-assisted psychotherapy has produced rapid and enduring reductions in depressive symptoms, with effects sustained for weeks to months after just one or two sessions [ 14 , 15 ]. These findings highlight psilocybin’s potential as a paradigm-shifting intervention for MDD, particularly for individuals who do not respond to conventional pharmacotherapy. Together, these agents challenge traditional models of antidepressant action by demonstrating that robust symptom relief can occur rapidly through mechanisms beyond monoaminergic modulation. Their efficacy in both clinical and preclinical settings underscores the need for continued exploration into neuroplasticity-based treatments for mood disorders. The present study provides a comparative analysis of a single and chronic administration of the traditional SSRI fluoxetine versus a single administration of fast-acting antidepressants (i.e. ketamine and psilocybin) in a rodent model of psychosocial stress. The present study seeks to facilitate the development of more effective and rapid-acting treatments for MDD, particularly targeting patients who do not respond to conventional therapies. Materials and methods Animals and housing The study was conducted using adult C57BL/6J male (7–10 weeks, n = 82) and adult CD-1 male mice (11–13 weeks, n = 144) from Charles River (DE). All C57BL/6J mice were housed in groups of 5–8 per cage, and all CD-1 mice were singly housed upon arrival. Mice were maintained under controlled conditions (21 ± 1°C, 37 ± 1% of humidity, 12/12 hours light/dark cycles, lights on at 7 a.m.) with food and water available ad libitum. All testing was conducted at the beginning of the light cycle. The experimental procedures were conducted in accordance with the European Communities Council Directive (Directive 2010/63 EU into the Polish Directives (Journal of Laws of 2021, item 1331, as amended)). The experimental procedures adhered to principles described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and abided by the ARRIVE guidelines. The experimental protocol was reviewed and approved by the Local Ethics Committee in Olsztyn, Poland (approval numbers 33/2021 and 104/2023). The experiment was conducted according to the design outlined in Fig. 1 . Aggression screening and chronic social defeat procedure The aggressive CD1 mice were selected using an aggression screening that had been previously described by Golden et al. (2011) [ 2 ]. In this test, resident CD-1 mice were exposed to intruders for three consecutive days in order to confirm consistent aggressive behavior. Twenty-four hours prior to the first defeat session, each CD-1 aggressor mouse selected for the study was placed into one side of a divided cage separated by a custom-made perforated partition. Each C57BL/6J male mouse was exposed to a social defeat session lasting a maximum of 10 min once a day for 10 consecutive days. During the 10 minutes of physical interaction, CD-1 mice initiated multiple direct confrontations. Continued aggressive attacks were interrupted when necessary to avoid physical injuries. At the end of each defeat session, the aggressor and intruder were separated by the perforated divider into the two halves of the cage. The mice were housed in their respective areas of the cage for the next 24 hours. For each subsequent daily defeat, the intruder animals (C57BL/6J) were exposed to a new resident CD-1 mouse to prevent habituation effect. The control animals (n = 10) were pair-housed in identical cages, with one animal on each side of the partition. They were rotated to a new cage daily, but never experienced direct physical contact with their cage mates. Animal welfare was assessed daily after each defeat session. Social Preference (SP) test The day after the last defeat session, all C57BL/6J mice were tested for social aversion during the social preference (SP) test, as previously described by Golden et al. (2011). The test was performed in dim lighting conditions (30 lux) in rectangular white plexiglass chambers (42 cm × 42 cm × 42 cm). Each SP test consisted of two separate sessions, each lasting 2.5 minutes: “no target session” with an empty cylinder, and “target session” with an unfamiliar CD1 mouse in the cylinder. In each session, the time spent in the “interaction zone”, defined as the area (4 cm in diameter) surrounding the perforated cylinder, was analyzed. The social preference score was calculated by dividing the time spent in the “interaction zone” when the target was present by the time spent in the “interaction zone” when the target was absent. Data were captured in video file formats using ANY-maze software (Stoelting Co). Based on the results from the first SP test (pre-treatment), mice were divided into either stress-resilient or stress-susceptible groups. Following the administration of the assigned treatment, the mice underwent subsequent SP tests at 24 hours, 7- and 14-days post-administration to assess both the acute and long-term effects of the treatment. Pharmacological Treatment Stress-susceptible C57BL/6J mice were randomly assigned to treatment groups, ensuring comparable baseline social preference scores across groups. The mice received either a single subcutaneous (s.c.) injection of ketamine hydrochloride (10 mg/kg, LGC, MM0144.00-0250) or an intraperitoneal injection (i.p.) of psilocybin (10 mg/kg, LGC, CAY-14041). Fluoxetine hydrochloride (20 mg/kg/day, Sigma-Aldrich, F132) was administered via i.p. injection once daily for 2 consecutive weeks. All drugs were dissolved in 0.9% NaCl prior to administration. Statistical analysis All data were analyzed using the GraphPad Prism 9 statistical software (GraphPad Software, Inc.). An alpha level of 0.05 was used as a criterion for statistical significance. All data were presented as mean ± SEM (standard error of the mean). Outliers of each data set were identified using the ROUT method, which is based on the False Discovery Rate (FDR), where the “Q” value determines the maximum desired FDR. A Q value of 5% was used in the analysis. Normality was assessed using the Shapiro-Wilk normality test. Data from the first SP test, determining the stress-resilient vs. stress-susceptible phenotype of animals, was analyzed by a One-way ANOVA test with Tukey’s post hoc analysis. Social preference was analyzed by Mixed Effect Analysis with Uncorrected Fisher’s LSD test when comparing scores between the first, second, third, and fourth SP tests. Results Bimodal phenotype distribution after the Chronic Social Defeat procedure The CSD procedure led to a clear phenotypic division between stress-susceptible and stress-resilient animals. Approximately 51% of C57BL/6J mice developed a significant (p < 0.0001) social avoidance phenotype, as indicated by reduced social preference scores, whereas the remaining 49% demonstrated resilience, exhibiting higher social preference scores compared to their stress-susceptible counterparts. This phenotypic division mirrors human variability in stress responsivity, emphasizing that exposure to stress does not uniformly result in the development of depressive pathology. These findings further support the translational validity of the chronic social defeat paradigm. Comparative antidepressant efficacy of ketamine, psilocybin, and fluoxetine over time The study investigated the effects of ketamine (10 mg/kg, s.c.), psilocybin (10 mg/kg, i.p.), and fluoxetine (20 mg/kg/day, i.p.) on social avoidance behavior in C57BL/6J mice subjected to chronic social defeat (CSD) stress. Ketamine and psilocybin each produced rapid and sustained antidepressant-like effects, significantly (p < 0.05) reducing social avoidance behavior at 24 hours, 7 days, and 14 days following a single administration. In contrast, fluoxetine required chronic administration over a period of 14 days to elicit a comparable behavioral effect. Neither acute (24 hours post-single dose) nor sub-chronic (7-day) fluoxetine treatment significantly improved social behaviour in stress-susceptible mice, underscoring the delayed onset of action associated with conventional selective serotonin reuptake inhibitors (SSRIs). The CSD procedure robustly reduced the social preference score in vehicle-treated stress-susceptible mice in comparison to non-stressed controls, and this stress-induced phenotype remained stable across the 14-day observation period. These findings highlight the rapid and sustained antidepressant-like actions of ketamine and psilocybin, contrasting with the delayed onset of conventional SSRIs like fluoxetine. The results support growing evidence that psychedelic and NMDA receptor-based therapies may offer faster-acting and longer-lasting treatment options for depression, particularly in patients exhibiting treatment-resistant symptoms or social withdrawal. These compounds may overcome key limitations of current antidepressants, including the delayed therapeutic onset and can be identified with the CSD model. Discussion The social defeat paradigm (CSD) models both chronic physical and psychosocial stress, providing enhanced translational relevance and pharmacological validity compared to other commonly used models that rely on artificial stressors [ 16 ], pharmacological manipulations [ 17 – 19 ], or acute stress paradigms [ 20 , 21 ]. Previous studies have demonstrated that chronic social defeat stress induces a range of emotion-related behavioural alterations, including social withdrawal, increased risk assessment behaviour, and reduced sucrose preference—an indicator of anhedonia [ 22 , 23 ]. Susceptible mice exhibit heightened activation in several emotion-regulating brain regions, such as the prefrontal cortex, bed nucleus of the stria terminalis, ventral hippocampus, and periaqueductal grey matter [ 24 ]. In addition, CSD leads to region-specific impairments in oligodendrogenesis [ 25 ] and hyperactivation of dopamine (DA) neurons in the ventral tegmental area (VTA) [ 25 ]. These chronic neurobiological alterations are thought to underlie the observed behavioural consequences following prolonged stress exposure. In this study, we confirm the persistence of the stress-susceptible phenotype in C57BL/6J mice induced by a 10-day social defeat procedure. This phenotype was characterised by sustained behavioural alterations—specifically, social interaction avoidance—that remained stable for up to two weeks post-defeat. In addition, we report that treatment with ketamine or psilocybin following CSD stress rapidly reversed the persistent social avoidance phenotype. This effect was evident within 24 hours post-treatment and remained detectable for up to 14 days. In contrast, fluoxetine required 14 days of continuous administration to elicit a comparable effect, with no significant behavioural changes observed at earlier timepoints. Fluoxetine is a prototypical SSRI with therapeutic applications extending beyond major depressive disorder [ 26 ]. Its primary mechanism of action involves inhibition of the serotonin transporter (SERT), thereby increasing extracellular serotonin (5-HT) levels in response to serotonergic neuronal activity [ 27 ]. Consistent with the typical profile of SSRIs, fluoxetine requires 2 to 4 weeks of continuous administration before its antidepressant effects become apparent—a delayed onset that was also observed in our study. Previous reports have shown that fluoxetine reverses behavioural despair, social avoidance, and anxiety-like behaviours induced by chronic social defeat stress in mice [ 28 ]. Moreover, chronic fluoxetine treatment has been associated with enhanced cell proliferation and increased levels of BDNF in the hippocampus and frontal cortex [ 29 ]. Both fluoxetine and ketamine have been shown to increase the firing activity of glutamatergic pyramidal neurons in the medial prefrontal cortex (mPFC), a mechanism associated with their antidepressant effects [ 29 , 30 ]. However, while ketamine produces this effect rapidly, fluoxetine requires approximately 21 days of chronic administration to achieve a comparable outcome [ 30 ]. The dynamic interaction between excitatory pyramidal neurons (which release glutamate (Glu)) and inhibitory interneurons (which release γ-aminobutyric acid, GABA) is believed to be fundamental to the rapid onset of antidepressant-like responses. Notably, antidepressants that modulate serotonin levels may also influence the balance between glutamatergic and GABAergic neurotransmission. Indeed, chronic fluoxetine treatment has been shown to significantly alter Glu and GABA concentrations in key brain regions [ 31 ]. The delayed therapeutic onset of fluoxetine may therefore reflect the time required for downstream or indirect effects—such as modulation of excitatory/inhibitory signalling balance—to manifest following initial serotonergic modulation. While fluoxetine offers proven long-term benefits in the treatment of depression, its slow onset remains a substantial clinical limitation. This delay can carry serious implications, including a heightened risk of suicidal behaviour during the initial weeks of treatment before therapeutic effects emerge [ 32 ]. As such, there is increasing interest in exploring combination therapies that integrate conventional SSRIs with rapid-acting agents such as ketamine or psilocybin to address this critical gap in early symptom relief. Ketamine, a (NMDAR antagonist, has been shown to reverse the social avoidance phenotype in the chronic social defeat model following a single administration, producing both rapid and sustained antidepressant-like effects [ 25 ]. This finding is consistent with our results, which demonstrated that a single dose of ketamine elicited a marked behavioural shift from a stress-susceptible to a resilient phenotype as early as 24 hours post-administration, with effects persisting for up to 14 days. Ketamine has demonstrated rapid and robust antidepressant effects in patients with TRD[ES1.1][IC1.2]. A single subanaesthetic dose of intravenous ketamine (0.5 mg/kg) has been shown to alleviate depressive symptoms within hours, with effects lasting up to one week in many patients [ 10 , 33 ]. Ketamine's clinical efficacy has led to the FDA’s approval of esketamine, the S-enantiomer of ketamine, for TRD in 2019 [ 34 ]. Although the precise mechanisms underlying ketamine's antidepressant action remain incompletely understood, recent evidence by Cai et al. (2023) suggests that its effects may involve modulation of hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels [ 25 ]. An increase in HCN channel-mediated current has been associated with pathogenic hyperactivation of ventral tegmental area (VTA) DA neurons, a process linked to the emergence of depressive-like behaviours. Notably, ketamine has been shown to reverse this hyperactivation, suggesting that modulation of HCN channel function may contribute to its antidepressant effects [ 25 ]. Interestingly, genetic deletion of N-methyl-D-aspartate receptors (NMDARs)—non-competitive glutamatergic receptors—results in a reduction of HCN channel activity in thalamic neurons in mice [ 35 ]. This observation aligns with prevailing hypotheses that implicate NMDAR inhibition as a central mechanism underlying ketamine’s rapid antidepressant action [ 36 ]. Additionally, ketamine has been reported to significantly increase extracellular serotonin (5-HT) levels, suggesting a functional link between serotonergic and glutamatergic neurotransmission [ 37 ]. Another proposed mechanism involves the activation of pro-synaptogenic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, enhancing AMPA receptor activity relative to N-methyl-D-aspartate (NMDA) receptors [ 38 , 39 ]. These findings collectively support the notion that ketamine's antidepressant effects arise from a convergence of multiple, interconnected pathways. Despite ketamine's short elimination half-life of approximately 13 minutes in mice [ 40 ], its antidepressant-like effects persist for up to 14 days in our model. This sustained efficacy may be partially explained by its prolonged action at specific neural targets. For instance, a single dose of ketamine has been shown to suppress burst firing in the lateral habenula (LHb) for up to 24 hours, during which the drug remains entrapped within NMDA receptors [ 40 ]. By blocking these receptors, ketamine prevents the inhibition of downstream dopaminergic and serotonergic neurons, facilitating a rapid onset of mood improvement. Ketamine has also been reported to directly bind to TrkB (neurotrophic receptor tyrosine kinase B, Ntrk2)—the high-affinity receptor for BDNF—a property it shares with pharmacologically diverse antidepressants such as fluoxetine [ 29 ] and psilocybin [ 41 ]. This interaction is thought to promote neuroplasticity and may contribute to the therapeutic effects of these compounds. Psilocybin, a serotonergic psychedelic and 5-HT2A receptor partial agonist, has also garnered attention for its potential to treat TRD. Recent randomised controlled trials have shown that a single or two supervised doses of psilocybin can produce rapid, significant, and sustained reductions in depressive symptoms in patients with TRD [ 14 , 15 ]. Unlike ketamine, the antidepressant effects of psilocybin are often accompanied by profound psychological experiences, which may contribute to therapeutic outcomes themselves [ 42 ]. Preclinical studies suggest that psilocybin may stimulate the synthesis of BDNF and induce neuroplastic adaptations through a TrkB receptor-dependent mechanism, thereby contributing to its long-lasting therapeutic effects [ 41 ]. Notably, these sustained effects have also been associated with psilocybin’s ability to initiate a positive feedback loop that enhances empathy and promotes prosocial behaviour [ 41 ]. While psilocybin has demonstrated efficacy in various rodent models of stress and social behaviour, to our knowledge, its effects have not previously been evaluated in the chronic social defeat model in mice. In the present study, we report both acute (24 hours post-administration) and sustained (up to 14 days) antidepressant-like effects of psilocybin in a validated psychosocial model of depression, highlighting its potential for treating stress-induced social withdrawal. Both compounds challenge the traditional paradigm of chronic, daily dosing regimen required for antidepressant efficacy. While ketamine is often administered repeatedly to maintain a therapeutic response, psilocybin may confer longer-lasting effects from fewer sessions. Importantly, both agents represent promising interventions for patients unresponsive to standard therapies and are now the focus of ongoing clinical trials and mechanistic studies. In conclusion, the CSD paradigm represents a valuable translational model for evaluating the efficacy of both conventional and emerging antidepressant therapies. Its relevance lies in its ability to mimic the key features of human depression, including persistent social withdrawal and stress susceptibility. Importantly, it also offers a platform for the development of personalised treatment strategies. To the best of our knowledge, this is the first study to comprehensively and comparatively examine the effects of a single administration of ketamine and psilocybin, alongside repeated fluoxetine treatment, in the mouse model of chronic social defeat. These findings contribute to a growing body of evidence supporting the therapeutic potential of rapid-acting agents in the treatment of stress-induced behavioural phenotypes. Declarations Author Contributions M.D. and E.S. conceived the experiment, M.D. and J.K. conducted the experiment, M.D. analyzed the results, M.D., J.K., I.C., and E.S. wrote the manuscript. All authors reviewed the manuscript. Corresponding author: E.S. 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Additional Declarations Competing interest reported. The experiments described in this study were funded by Transpharmation Ltd. All authors were employees of Transpharmation Ltd. at the time of the study and received their salaries from the company. Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 06 Oct, 2025 Reviews received at journal 01 Oct, 2025 Reviewers agreed at journal 27 Sep, 2025 Reviews received at journal 26 Sep, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviewers invited by journal 25 Sep, 2025 Editor assigned by journal 25 Sep, 2025 Editor invited by journal 09 Sep, 2025 Submission checks completed at journal 06 Aug, 2025 First submitted to journal 06 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7269356","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":525103585,"identity":"9fc5b12f-f470-4be2-87b0-4688219aa85e","order_by":0,"name":"Malgorzata Domzalska","email":"","orcid":"","institution":"Transpharmation Poland Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Malgorzata","middleName":"","lastName":"Domzalska","suffix":""},{"id":525103586,"identity":"4f572f8f-3cb5-4a7a-8482-a4d7a0fdb3ce","order_by":1,"name":"Joanna Kwiatkowska","email":"","orcid":"","institution":"Transpharmation Poland Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Kwiatkowska","suffix":""},{"id":525103587,"identity":"81f78e66-806d-441b-8227-a2380ebd697b","order_by":2,"name":"Iwona Cichon","email":"","orcid":"","institution":"Transpharmation Poland Ltd.","correspondingAuthor":false,"prefix":"","firstName":"Iwona","middleName":"","lastName":"Cichon","suffix":""},{"id":525103588,"identity":"131821c0-7404-43ae-836b-fe7a58a33bf6","order_by":3,"name":"Ewa Sokolowska","email":"data:image/png;base64,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","orcid":"","institution":"Transpharmation Poland Ltd.","correspondingAuthor":true,"prefix":"","firstName":"Ewa","middleName":"","lastName":"Sokolowska","suffix":""}],"badges":[],"createdAt":"2025-08-01 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13:16:07","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":94072,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7269356/v1/2c4ad313635a56c6ad362541.html"},{"id":92951806,"identity":"38bb8915-faf7-4534-84ec-52f8b74f10e7","added_by":"auto","created_at":"2025-10-07 13:16:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":55977,"visible":true,"origin":"","legend":"\u003cp\u003eStudy design.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7269356/v1/bb3998fde0624ddf09452c8b.png"},{"id":92951121,"identity":"8146c769-f57b-4c00-95e5-ddc90f6fe39e","added_by":"auto","created_at":"2025-10-07 13:08:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":116052,"visible":true,"origin":"","legend":"\u003cp\u003eSocial preference distribution after chronic social defeat stress.\u003c/p\u003e","description":"","filename":"floatimage210.png","url":"https://assets-eu.researchsquare.com/files/rs-7269356/v1/5d45099f518ef63ea32a23c7.png"},{"id":92951127,"identity":"17dba6f4-32b0-4fcf-aa4c-e3142a8491d7","added_by":"auto","created_at":"2025-10-07 13:08:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126911,"visible":true,"origin":"","legend":"\u003cp\u003eSocial avoidance phenotype in stress-susceptible (SUS) mice following pharmacological interventions.\u003c/p\u003e","description":"","filename":"floatimage312.png","url":"https://assets-eu.researchsquare.com/files/rs-7269356/v1/1135d10566b6df2d4491e29d.png"},{"id":97179408,"identity":"f746d458-9b6f-44d8-ab1c-92d623882601","added_by":"auto","created_at":"2025-12-01 16:15:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":716458,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7269356/v1/4c556cf0-ae3d-4eb8-b01c-32c4525ab7a0.pdf"}],"financialInterests":"Competing interest reported. The experiments described in this study were funded by Transpharmation Ltd. All authors were employees of Transpharmation Ltd. at the time of the study and received their salaries from the company.","formattedTitle":"Towards Novel Antidepressant Strategies: A Comparative Study of Ketamine, Psilocybin, and Fluoxetine in a Chronic Stress Model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMajor depressive disorder (MDD) is a prevalent health condition that is associated with significant health and socioeconomic burdens. Globally, it is estimated that 5% of the adult population \u0026minus;\u0026thinsp;280\u0026nbsp;million individuals - experiences depression [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In light of the escalating prevalence of MDD, including treatment-resistant depression (TRD) cases, there is a critical need to develop novel therapeutic interventions and more effective pharmacological agents to address this condition. A major challenge in developing novel antidepressants lies in identifying rapid-acting compounds that offer immediate relief, particularly for individuals with severe or treatment-resistant depression.\u003c/p\u003e\u003cp\u003eThe complex and multifaceted nature of anxiety disorders and depression, in combination with the paucity of effective treatment options, highlights the importance of animal models in elucidating the underlying aetiology and pathophysiology of depression. Animal models provide a valuable platform for asses\u003c/p\u003e\u003cp\u003esing the efficacy of emerging therapeutic strategies. Among them, the chronic social defeat (CSD) mouse model represents a robust, stress-induced paradigm that recapitulates the core features of human depression. Notably, its selective responsiveness to clinically effective antidepressants enhances its translational relevance for preclinical research [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSocial avoidance is a hallmark endpoint of the CSD model and can be reversed following acute or chronic antidepressant treatments. Classic selective serotonin reuptake inhibitors (SSRIs) have been evaluated in the CSD model [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e] however, their delayed onset of action has led to increased interest in identifying compounds capable of producing a more rapid antidepressant effect. Unlike many other depression models utilized to assess the effectiveness of rapid-acting antidepressants, social defeat stress serves as a compelling framework for concurrently evaluating the impact of novel fast-acting compounds alongside traditional, long-acting SSRI antidepressants.\u003c/p\u003e\u003cp\u003eWhile fluoxetine is one of the most widely prescribed antidepressants and has proven efficacy in treating MDD, its use is associated with several significant limitations. A major drawback is its delayed onset of action; therapeutic effects often require 2 to 6 weeks to manifest, which can be problematic for patients in acute distress [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Furthermore, approximately one-third of patients fail to achieve remission, even after adequate trials, highlighting issues with treatment resistance [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The mechanism of action, which involves the selective inhibition of the serotonin transporter (SERT), does not fully account for the complex neurobiology of depression, and chronic use may result in compensatory receptor downregulation and diminished neuroplasticity over time [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eEmerging evidence supports the efficacy of rapid-acting antidepressants, particularly subanesthetic doses of ketamine and serotonergic psychedelics like psilocybin, in treating MDD and related conditions. Ketamine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, exerts its antidepressant effects by modulating glutamatergic transmission. Specifically, ketamine blocks NMDA receptors on GABAergic interneurons, leading to disinhibition of glutamate release, which in turn activates α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This cascade results in the activation of downstream signalling pathways, including brain-derived neurotrophic factor (BDNF) release and mammalian target of rapamycin (mTOR) signalling, which promote synaptic plasticity and neurogenesis [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Clinically, ketamine has demonstrated a rapid onset of antidepressant effects, often within hours, and sustained efficacy for up to a week following a single intravenous infusion, particularly in patients with TRD [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eSimilarly, psilocybin\u0026mdash;a serotonergic psychedelic and prodrug of psilocin\u0026mdash;acts primarily as a partial agonist at the 5-HT2A receptor, which is highly expressed in cortical regions involved in mood, cognition, and self-referential thought. This receptor activation leads to a temporary disruption of the default mode network (DMN), increased neural connectivity, and alterations in emotional processing and self-perception [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In recent randomized controlled trials, psilocybin-assisted psychotherapy has produced rapid and enduring reductions in depressive symptoms, with effects sustained for weeks to months after just one or two sessions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. These findings highlight psilocybin\u0026rsquo;s potential as a paradigm-shifting intervention for MDD, particularly for individuals who do not respond to conventional pharmacotherapy.\u003c/p\u003e\u003cp\u003eTogether, these agents challenge traditional models of antidepressant action by demonstrating that robust symptom relief can occur rapidly through mechanisms beyond monoaminergic modulation. Their efficacy in both clinical and preclinical settings underscores the need for continued exploration into neuroplasticity-based treatments for mood disorders.\u003c/p\u003e\u003cp\u003eThe present study provides a comparative analysis of a single and chronic administration of the traditional SSRI fluoxetine versus a single administration of fast-acting antidepressants (i.e. ketamine and psilocybin) in a rodent model of psychosocial stress. The present study seeks to facilitate the development of more effective and rapid-acting treatments for MDD, particularly targeting patients who do not respond to conventional therapies.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eAnimals and housing\u003c/p\u003e\u003cp\u003eThe study was conducted using adult C57BL/6J male (7\u0026ndash;10 weeks, n\u0026thinsp;=\u0026thinsp;82) and adult CD-1 male mice (11\u0026ndash;13 weeks, n\u0026thinsp;=\u0026thinsp;144) from Charles River (DE). All C57BL/6J mice were housed in groups of 5\u0026ndash;8 per cage, and all CD-1 mice were singly housed upon arrival. Mice were maintained under controlled conditions (21\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, 37\u0026thinsp;\u0026plusmn;\u0026thinsp;1% of humidity, 12/12 hours light/dark cycles, lights on at 7 a.m.) with food and water available ad libitum. All testing was conducted at the beginning of the light cycle. The experimental procedures were conducted in accordance with the European Communities Council Directive (Directive 2010/63 EU into the Polish Directives (Journal of Laws of 2021, item 1331, as amended)). The experimental procedures adhered to principles described in the National Institutes of Health Guide for the Care and Use of Laboratory Animals and abided by the ARRIVE guidelines. The experimental protocol was reviewed and approved by the Local Ethics Committee in Olsztyn, Poland (approval numbers 33/2021 and 104/2023).\u003c/p\u003e\u003cp\u003eThe experiment was conducted according to the design outlined in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eAggression screening and chronic social defeat procedure\u003c/p\u003e\u003cp\u003eThe aggressive CD1 mice were selected using an aggression screening that had been previously described by Golden et al. (2011) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In this test, resident CD-1 mice were exposed to intruders for three consecutive days in order to confirm consistent aggressive behavior. Twenty-four hours prior to the first defeat session, each CD-1 aggressor mouse selected for the study was placed into one side of a divided cage separated by a custom-made perforated partition. Each C57BL/6J male mouse was exposed to a social defeat session lasting a maximum of 10 min once a day for 10 consecutive days. During the 10 minutes of physical interaction, CD-1 mice initiated multiple direct confrontations. Continued aggressive attacks were interrupted when necessary to avoid physical injuries.\u003c/p\u003e\u003cp\u003eAt the end of each defeat session, the aggressor and intruder were separated by the perforated divider into the two halves of the cage. The mice were housed in their respective areas of the cage for the next 24 hours. For each subsequent daily defeat, the intruder animals (C57BL/6J) were exposed to a new resident CD-1 mouse to prevent habituation effect. The control animals (n\u0026thinsp;=\u0026thinsp;10) were pair-housed in identical cages, with one animal on each side of the partition. They were rotated to a new cage daily, but never experienced direct physical contact with their cage mates. Animal welfare was assessed daily after each defeat session.\u003c/p\u003e\u003cp\u003eSocial Preference (SP) test\u003c/p\u003e\u003cp\u003eThe day after the last defeat session, all C57BL/6J mice were tested for social aversion during the social preference (SP) test, as previously described by Golden et al. (2011). The test was performed in dim lighting conditions (30 lux) in rectangular white plexiglass chambers (42 cm \u0026times; 42 cm \u0026times; 42 cm). Each SP test consisted of two separate sessions, each lasting 2.5 minutes: \u0026ldquo;no target session\u0026rdquo; with an empty cylinder, and \u0026ldquo;target session\u0026rdquo; with an unfamiliar CD1 mouse in the cylinder. In each session, the time spent in the \u0026ldquo;interaction zone\u0026rdquo;, defined as the area (4 cm in diameter) surrounding the perforated cylinder, was analyzed. The social preference score was calculated by dividing the time spent in the \u0026ldquo;interaction zone\u0026rdquo; when the target was present by the time spent in the \u0026ldquo;interaction zone\u0026rdquo; when the target was absent. Data were captured in video file formats using ANY-maze software (Stoelting Co). Based on the results from the first SP test (pre-treatment), mice were divided into either stress-resilient or stress-susceptible groups.\u003c/p\u003e\u003cp\u003eFollowing the administration of the assigned treatment, the mice underwent subsequent SP tests at 24 hours, 7- and 14-days post-administration to assess both the acute and long-term effects of the treatment.\u003c/p\u003e\u003cp\u003ePharmacological Treatment\u003c/p\u003e\u003cp\u003eStress-susceptible C57BL/6J mice were randomly assigned to treatment groups, ensuring comparable baseline social preference scores across groups. The mice received either a single subcutaneous (s.c.) injection of ketamine hydrochloride (10 mg/kg, LGC, MM0144.00-0250) or an intraperitoneal injection (i.p.) of psilocybin (10 mg/kg, LGC, CAY-14041). Fluoxetine hydrochloride (20 mg/kg/day, Sigma-Aldrich, F132) was administered via i.p. injection once daily for 2 consecutive weeks. All drugs were dissolved in 0.9% NaCl prior to administration.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eAll data were analyzed using the GraphPad Prism 9 statistical software (GraphPad Software, Inc.). An alpha level of 0.05 was used as a criterion for statistical significance. All data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of the mean). Outliers of each data set were identified using the ROUT method, which is based on the False Discovery Rate (FDR), where the \u0026ldquo;Q\u0026rdquo; value determines the maximum desired FDR. A Q value of 5% was used in the analysis. Normality was assessed using the Shapiro-Wilk normality test. Data from the first SP test, determining the stress-resilient vs. stress-susceptible phenotype of animals, was analyzed by a One-way ANOVA test with Tukey\u0026rsquo;s post hoc analysis. Social preference was analyzed by Mixed Effect Analysis with Uncorrected Fisher\u0026rsquo;s LSD test when comparing scores between the first, second, third, and fourth SP tests.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eBimodal phenotype distribution after the Chronic Social Defeat procedure\u003c/p\u003e\u003cp\u003eThe CSD procedure led to a clear phenotypic division between stress-susceptible and stress-resilient animals. Approximately 51% of C57BL/6J mice developed a significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) social avoidance phenotype, as indicated by reduced social preference scores, whereas the remaining 49% demonstrated resilience, exhibiting higher social preference scores compared to their stress-susceptible counterparts.\u003c/p\u003e\u003cp\u003eThis phenotypic division mirrors human variability in stress responsivity, emphasizing that exposure to stress does not uniformly result in the development of depressive pathology. These findings further support the translational validity of the chronic social defeat paradigm.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eComparative antidepressant efficacy of ketamine, psilocybin, and fluoxetine over time\u003c/p\u003e\u003cp\u003eThe study investigated the effects of ketamine (10 mg/kg, s.c.), psilocybin (10 mg/kg, i.p.), and fluoxetine (20 mg/kg/day, i.p.) on social avoidance behavior in C57BL/6J mice subjected to chronic social defeat (CSD) stress. Ketamine and psilocybin each produced rapid and sustained antidepressant-like effects, significantly (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) reducing social avoidance behavior at 24 hours, 7 days, and 14 days following a single administration. In contrast, fluoxetine required chronic administration over a period of 14 days to elicit a comparable behavioral effect. Neither acute (24 hours post-single dose) nor sub-chronic (7-day) fluoxetine treatment significantly improved social behaviour in stress-susceptible mice, underscoring the delayed onset of action associated with conventional selective serotonin reuptake inhibitors (SSRIs). The CSD procedure robustly reduced the social preference score in vehicle-treated stress-susceptible mice in comparison to non-stressed controls, and this stress-induced phenotype remained stable across the 14-day observation period.\u003c/p\u003e\u003cp\u003eThese findings highlight the rapid and sustained antidepressant-like actions of ketamine and psilocybin, contrasting with the delayed onset of conventional SSRIs like fluoxetine. The results support growing evidence that psychedelic and NMDA receptor-based therapies may offer faster-acting and longer-lasting treatment options for depression, particularly in patients exhibiting treatment-resistant symptoms or social withdrawal. These compounds may overcome key limitations of current antidepressants, including the delayed therapeutic onset and can be identified with the CSD model.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe social defeat paradigm (CSD) models both chronic physical and psychosocial stress, providing enhanced translational relevance and pharmacological validity compared to other commonly used models that rely on artificial stressors [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], pharmacological manipulations [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], or acute stress paradigms [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Previous studies have demonstrated that chronic social defeat stress induces a range of emotion-related behavioural alterations, including social withdrawal, increased risk assessment behaviour, and reduced sucrose preference\u0026mdash;an indicator of anhedonia [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Susceptible mice exhibit heightened activation in several emotion-regulating brain regions, such as the prefrontal cortex, bed nucleus of the stria terminalis, ventral hippocampus, and periaqueductal grey matter [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. In addition, CSD leads to region-specific impairments in oligodendrogenesis [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and hyperactivation of dopamine (DA) neurons in the ventral tegmental area (VTA) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. These chronic neurobiological alterations are thought to underlie the observed behavioural consequences following prolonged stress exposure.\u003c/p\u003e\u003cp\u003eIn this study, we confirm the persistence of the stress-susceptible phenotype in C57BL/6J mice induced by a 10-day social defeat procedure. This phenotype was characterised by sustained behavioural alterations\u0026mdash;specifically, social interaction avoidance\u0026mdash;that remained stable for up to two weeks post-defeat. In addition, we report that treatment with ketamine or psilocybin following CSD stress rapidly reversed the persistent social avoidance phenotype. This effect was evident within 24 hours post-treatment and remained detectable for up to 14 days. In contrast, fluoxetine required 14 days of continuous administration to elicit a comparable effect, with no significant behavioural changes observed at earlier timepoints.\u003c/p\u003e\u003cp\u003eFluoxetine is a prototypical SSRI with therapeutic applications extending beyond major depressive disorder [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Its primary mechanism of action involves inhibition of the serotonin transporter (SERT), thereby increasing extracellular serotonin (5-HT) levels in response to serotonergic neuronal activity [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Consistent with the typical profile of SSRIs, fluoxetine requires 2 to 4 weeks of continuous administration before its antidepressant effects become apparent\u0026mdash;a delayed onset that was also observed in our study. Previous reports have shown that fluoxetine reverses behavioural despair, social avoidance, and anxiety-like behaviours induced by chronic social defeat stress in mice [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Moreover, chronic fluoxetine treatment has been associated with enhanced cell proliferation and increased levels of BDNF in the hippocampus and frontal cortex [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBoth fluoxetine and ketamine have been shown to increase the firing activity of glutamatergic pyramidal neurons in the medial prefrontal cortex (mPFC), a mechanism associated with their antidepressant effects [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. However, while ketamine produces this effect rapidly, fluoxetine requires approximately 21 days of chronic administration to achieve a comparable outcome [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The dynamic interaction between excitatory pyramidal neurons (which release glutamate (Glu)) and inhibitory interneurons (which release γ-aminobutyric acid, GABA) is believed to be fundamental to the rapid onset of antidepressant-like responses. Notably, antidepressants that modulate serotonin levels may also influence the balance between glutamatergic and GABAergic neurotransmission. Indeed, chronic fluoxetine treatment has been shown to significantly alter Glu and GABA concentrations in key brain regions [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe delayed therapeutic onset of fluoxetine may therefore reflect the time required for downstream or indirect effects\u0026mdash;such as modulation of excitatory/inhibitory signalling balance\u0026mdash;to manifest following initial serotonergic modulation. While fluoxetine offers proven long-term benefits in the treatment of depression, its slow onset remains a substantial clinical limitation. This delay can carry serious implications, including a heightened risk of suicidal behaviour during the initial weeks of treatment before therapeutic effects emerge [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. As such, there is increasing interest in exploring combination therapies that integrate conventional SSRIs with rapid-acting agents such as ketamine or psilocybin to address this critical gap in early symptom relief.\u003c/p\u003e\u003cp\u003eKetamine, a (NMDAR antagonist, has been shown to reverse the social avoidance phenotype in the chronic social defeat model following a single administration, producing both rapid and sustained antidepressant-like effects [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This finding is consistent with our results, which demonstrated that a single dose of ketamine elicited a marked behavioural shift from a stress-susceptible to a resilient phenotype as early as 24 hours post-administration, with effects persisting for up to 14 days. Ketamine has demonstrated rapid and robust antidepressant effects in patients with TRD[ES1.1][IC1.2]. A single subanaesthetic dose of intravenous ketamine (0.5 mg/kg) has been shown to alleviate depressive symptoms within hours, with effects lasting up to one week in many patients [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Ketamine's clinical efficacy has led to the FDA\u0026rsquo;s approval of esketamine, the S-enantiomer of ketamine, for TRD in 2019 [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough the precise mechanisms underlying ketamine's antidepressant action remain incompletely understood, recent evidence by Cai et al. (2023) suggests that its effects may involve modulation of hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. An increase in HCN channel-mediated current has been associated with pathogenic hyperactivation of ventral tegmental area (VTA) DA neurons, a process linked to the emergence of depressive-like behaviours. Notably, ketamine has been shown to reverse this hyperactivation, suggesting that modulation of HCN channel function may contribute to its antidepressant effects [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Interestingly, genetic deletion of N-methyl-D-aspartate receptors (NMDARs)\u0026mdash;non-competitive glutamatergic receptors\u0026mdash;results in a reduction of HCN channel activity in thalamic neurons in mice [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. This observation aligns with prevailing hypotheses that implicate NMDAR inhibition as a central mechanism underlying ketamine\u0026rsquo;s rapid antidepressant action [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAdditionally, ketamine has been reported to significantly increase extracellular serotonin (5-HT) levels, suggesting a functional link between serotonergic and glutamatergic neurotransmission [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Another proposed mechanism involves the activation of pro-synaptogenic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, enhancing AMPA receptor activity relative to N-methyl-D-aspartate (NMDA) receptors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. These findings collectively support the notion that ketamine's antidepressant effects arise from a convergence of multiple, interconnected pathways. Despite ketamine's short elimination half-life of approximately 13 minutes in mice [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], its antidepressant-like effects persist for up to 14 days in our model. This sustained efficacy may be partially explained by its prolonged action at specific neural targets. For instance, a single dose of ketamine has been shown to suppress burst firing in the lateral habenula (LHb) for up to 24 hours, during which the drug remains entrapped within NMDA receptors [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. By blocking these receptors, ketamine prevents the inhibition of downstream dopaminergic and serotonergic neurons, facilitating a rapid onset of mood improvement.\u003c/p\u003e\u003cp\u003eKetamine has also been reported to directly bind to TrkB (neurotrophic receptor tyrosine kinase B, Ntrk2)\u0026mdash;the high-affinity receptor for BDNF\u0026mdash;a property it shares with pharmacologically diverse antidepressants such as fluoxetine [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] and psilocybin [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This interaction is thought to promote neuroplasticity and may contribute to the therapeutic effects of these compounds.\u003c/p\u003e\u003cp\u003ePsilocybin, a serotonergic psychedelic and 5-HT2A receptor partial agonist, has also garnered attention for its potential to treat TRD. Recent randomised controlled trials have shown that a single or two supervised doses of psilocybin can produce rapid, significant, and sustained reductions in depressive symptoms in patients with TRD [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Unlike ketamine, the antidepressant effects of psilocybin are often accompanied by profound psychological experiences, which may contribute to therapeutic outcomes themselves [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePreclinical studies suggest that psilocybin may stimulate the synthesis of BDNF and induce neuroplastic adaptations through a TrkB receptor-dependent mechanism, thereby contributing to its long-lasting therapeutic effects [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Notably, these sustained effects have also been associated with psilocybin\u0026rsquo;s ability to initiate a positive feedback loop that enhances empathy and promotes prosocial behaviour [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. While psilocybin has demonstrated efficacy in various rodent models of stress and social behaviour, to our knowledge, its effects have not previously been evaluated in the chronic social defeat model in mice. In the present study, we report both acute (24 hours post-administration) and sustained (up to 14 days) antidepressant-like effects of psilocybin in a validated psychosocial model of depression, highlighting its potential for treating stress-induced social withdrawal.\u003c/p\u003e\u003cp\u003eBoth compounds challenge the traditional paradigm of chronic, daily dosing regimen required for antidepressant efficacy. While ketamine is often administered repeatedly to maintain a therapeutic response, psilocybin may confer longer-lasting effects from fewer sessions. Importantly, both agents represent promising interventions for patients unresponsive to standard therapies and are now the focus of ongoing clinical trials and mechanistic studies.\u003c/p\u003e\u003cp\u003eIn conclusion, the CSD paradigm represents a valuable translational model for evaluating the efficacy of both conventional and emerging antidepressant therapies. Its relevance lies in its ability to mimic the key features of human depression, including persistent social withdrawal and stress susceptibility. Importantly, it also offers a platform for the development of personalised treatment strategies. To the best of our knowledge, this is the first study to comprehensively and comparatively examine the effects of a single administration of ketamine and psilocybin, alongside repeated fluoxetine treatment, in the mouse model of chronic social defeat. These findings contribute to a growing body of evidence supporting the therapeutic potential of rapid-acting agents in the treatment of stress-induced behavioural phenotypes.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.D. and E.S. conceived the experiment, M.D. and J.K. conducted the experiment, M.D. analyzed the results, M.D., J.K., I.C., and E.S. wrote the manuscript. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eCorresponding author: E.S.\u003c/p\u003e\n\u003cp\u003eCorrespondence to:\u0026nbsp;[email protected]\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that supports the findings of this study are available from the corresponding author upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments described in this study were funded by Transpharmation Ltd. All authors were employees of Transpharmation Ltd. at the time of the study and received their salaries from the company.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiments described in this study were funded by Transpharmation Ltd. 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The use of the psychological flexibility model to support psychedelic-assisted therapy. \u003cem\u003eJ. Contextual Behav. Sci.\u003c/em\u003e \u003cb\u003e15\u003c/b\u003e, 92\u0026ndash;102 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Chronic social defeat, treatment-resistant depression, antidepressant efficacy, ketamine, psilocybin, fluoxetine","lastPublishedDoi":"10.21203/rs.3.rs-7269356/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7269356/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDepression is a debilitating mental disorder affecting millions worldwide, yet current pharmacological treatments, such as selective serotonin reuptake inhibitors (SSRIs), often exhibit delayed onset and limited efficacy. The chronic social defeat (CSD) stress model in mice is a well-established preclinical paradigm for inducing depression-like behaviors and evaluating antidepressants effectiveness. This study compared the efficacy of both acute and chronic fluoxetine with acute ketamine and psilocybin treatment in male C57BL/6J mice subjected to CSD. Fluoxetine showed no significant effects 24 hours after a single dose or following 7 days of repeated administration; antidepressant-like effects only appeared after 14 days of continuous treatment. In contrast, a single dose of either ketamine or psilocybin significantly reversed social avoidance behavior at 24 hours, with sustained effects observed at 7- and 14-days post-treatment. These findings suggest that ketamine and psilocybin elicit rapid and durable, antidepressant-like responses in this preclinical model, in contrast to traditional SSRIs, like fluoxetine, which requires extended treatment duration, mirroring clinical efficacy patterns. The results support the utility of the CSD model in evaluating antidepressant efficacy and highlight the therapeutic potential of fast-acting agents such as ketamine and psilocybin as alternatives to conventional treatments for major depressive disorder.\u003c/p\u003e","manuscriptTitle":"Towards Novel Antidepressant Strategies: A Comparative Study of Ketamine, Psilocybin, and Fluoxetine in a Chronic Stress Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-07 13:08:02","doi":"10.21203/rs.3.rs-7269356/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-06T04:35:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-01T04:21:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"269751722824699388784630453083167373568","date":"2025-09-27T10:34:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-26T07:59:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"27170737375128005204892000360622232266","date":"2025-09-25T12:38:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T10:14:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-25T10:06:32+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-09-09T10:41:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-06T11:31:07+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-08-06T11:28:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8ab2ea82-8ae2-43e5-a455-a44369a9513d","owner":[],"postedDate":"October 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":55802009,"name":"Health sciences/Diseases"},{"id":55802010,"name":"Biological sciences/Drug discovery"},{"id":55802011,"name":"Health sciences/Medical research"},{"id":55802012,"name":"Biological sciences/Neuroscience"},{"id":55802013,"name":"Biological sciences/Psychology"},{"id":55802014,"name":"Social science/Psychology"}],"tags":[],"updatedAt":"2025-12-01T16:11:14+00:00","versionOfRecord":{"articleIdentity":"rs-7269356","link":"https://doi.org/10.1038/s41598-025-29642-7","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-11-26 15:58:26","publishedOnDateReadable":"November 26th, 2025"},"versionCreatedAt":"2025-10-07 13:08:02","video":"","vorDoi":"10.1038/s41598-025-29642-7","vorDoiUrl":"https://doi.org/10.1038/s41598-025-29642-7","workflowStages":[]},"version":"v1","identity":"rs-7269356","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7269356","identity":"rs-7269356","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}