Effects of dietary supplementation with in vitro-cultivated arboreal medicinal mushrooms on stress coping strategies, depressive and anxiety-like behaviour of rats.

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Emil Trofimiuk, Agata Fijałkowska, Hubert Oniszczuk, Halina Car, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6118750/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Preclinical studies show that an edible arboreal medicinal mushroom – Hericium erinaceus (HE), is neuroprotective against high corticosterone levels and modulates stress-coping strategies of rodents. Moreover, other arboreal mushrooms ( Fomitopsis officinalis , FO; and Pleurotus djamor , PDJ) had a similar effect. Here, we explored potential dose-dependency of the effects of dietary supplementation with HE, FO or PDJ on anxiety-like behaviours and stress-coping strategies in rats; and, compared them to a drug commonly prescribed for stress-related psychiatric disorders (fluoxetine, FLX; 20 mg/kg) to evaluate those effects quantitatively and qualitatively. For 3 weeks, male Wistar rats were given 0, 100, 250 or 500 mg/kg ( per os ) of HE, FO or PDJ lyophilizate obtained from in vitro cultures. Subsequently, animals were tested in the battery of behavioral tests: open field (OF), elevated plus maze (EPM) and forced swimming (FST) tests. Middle dose of FO increased rearing in OFT indicates reduced anxiety-like behavior in rodents, and swimming (while decreasing climbing) in FST indicate antidepressant-like and also anti-anxiety-like effects no worse or even superior to those of fluoxetine. At the same time, we concluded that the effects of medicinal mushroom supplementation on stress-related behaviours vary depending on the mushroom species and dosage regimen. Biological sciences/Neuroscience Biological sciences/Neuroscience/Cognitive neuroscience Hericium erinaceus Fomitopsis officinalis Pleurotus djamor rats anxiety depression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Stress is a well-established environmental risk factor implicated in the development of all affective disorders. While the biological mechanisms underlying stress and its impact on the nervous system have been extensively studied 1 , the interplay between stress and other environmental factors in shaping mental health outcomes remains an area of active investigation. Among these factors, diet has recently gained significant attention as a modulatory influence on mental health, with some experts proposing the emergence of a new field: nutritional psychiatry 2 – 3 . For instance, longitudinal studies have demonstrated that diets such as the Mediterranean diet and anti-inflammatory diets are associated with a reduced risk of depressive symptoms 4 – 5 . Furthermore, clinical trials investigating dietary interventions have reported modest but promising therapeutic effects on depressive symptoms, although no significant benefits have been observed for anxiety 6 . Preclinical experimental studies, which allow for direct exploration of the interaction between stress and diet in the pathogenesis of affective disorders, have primarily focused on isolated dietary components. Among these, Hericium erinaceus (HE), an edible medicinal mushroom commonly known as lion’s mane or yamabushitake, has shown notable neuroprotective and behavioral effects. Research has demonstrated that HE mitigates the detrimental effects of elevated corticosterone levels 7 (Lew et al., 2020), promotes hippocampal neurogenesis 8 , and increases brain-derived neurotrophic factor (BDNF) levels 9 . Additionally, HE has been shown to modulate stress-related behaviors in rodents, as evidenced by improvements in tail suspension and forced swimming tests 8 – 9 . Our recent work further revealed that dietary supplementation with HE alters exploratory behavior in healthy mice exposed to anxiety-inducing environments, such as the elevated plus-maze (EPM). Intriguingly, two other medicinal mushrooms, Fomitopsis officinalis (FO, commonly known as agarikon) and Pleurotus djamor (PDJ, commonly known as pink oyster mushroom), exhibited similar behavioral effects 10 . Building on these findings, the present study aimed to explore the dose-dependency of the effects of HE, FO, and PDJ, and to compare their efficacy with that of a selective serotonin reuptake inhibitor (SSRI, fluoxetine), a well-established pharmacological treatment for stress-related behaviors. This comparison provides a qualitative benchmark for evaluating the therapeutic potential of these mushrooms. Additionally, we sought to systematically compare the effects of the three mushrooms to assess the influence of different raw materials and dosages. The rationale for investigating dose-dependency arose from our observation that the outcomes of mushroom supplementation in the EPM varied depending on individual differences in food intake among the animals 10 . Dose-response studies are a standard methodological approach for evaluating the effects of novel substances or raw materials, and administering precise doses via gastric gavage eliminates variability associated with random consumption. Specifically, we noted a potential anxiolytic-like effect with lower doses of HE and FO, and with higher doses of PDJ. Furthermore, we aimed to determine whether these effects could be replicated across rodent species—from mice to rats—as this would enhance the translational relevance of our findings and provide a more robust foundation for future research. By addressing these questions, this study seeks to advance our understanding of the therapeutic potential of medicinal mushrooms in modulating stress-related behaviors and to provide insights into their mechanisms of action. Such findings could pave the way for the development of novel, diet-based interventions for affective disorders, complementing existing pharmacological approaches. Results OFT No effect of experimental manipulations was observed on: 1) the level of locomotor activity measured by the number of crossings through the grid lines (Fig. 1 , F (10, 98) = 1.35; p = 0.214); and 2) anxiety-like behaviour measured by time spent in the central part of the open field (Fig. 2 , F (10, 99) = 1.24, p = 0.278). However, compared to the control group Fomitopsis officinalis at a dose of 250 mg/kg, increased threefold the occurrence of exploratory/orienting behaviour measured by the number of rearings (Fig. 3 , K-W = 35.8; p < 0.0001; Dunn's post hoc test: p = 0.0027). Analogous differences at the same dose (i.e., increased exploratory behaviour) were observed compared to fluoxetine (Fig. 3 , p = 0.0212), Pleurotus djamor at a dose of 500 mg/kg (Fig. 3 , p = 0.0495), and Hericium erinaceus at all 3 doses (Fig. 3 , 100 mg/kg, p = 0.0085; 250 mg/kg, p < 0.0001; 500 mg/kg body weight, p < 0.0001). EPM Rats receiving the lowest dose of Fomitopsis officinalis mycelium (100 mg/kg) and the lowest dose of Pleurotus djamor (100 mg/kg) stayed in the "dangerous" open arms the longest of all groups; and, on average twice as long (Fig. 4 , 70.7 ± 6.8 s and 67.5 ± 11.65 s respectively) as rats in the control group (35 ± 6.8 s, p = 0.0309 and p = 0.0383). In post-hoc tests with Tukey's correction for multiple comparisons for one-way ANOVA model (F (10, 98) = 2.366, p = 0.0149), this effect was statistically significant. There was a significant an anxiolytic-like effect of the lowest dose of F. officinalis compared to: fluoxetine (p = 0.028), vs. highest dose (500 mg/kg) P. djamor (p = 0.0183), vs. the lowest dose of Hericium erinaceus (p = 0.0152) and the lowest dose of P. djamor vs. the highest dose of P. djamor (p = 0.0362). After administration of lyophilized mycelium of Fomitopsis officinalis at a dose of 100 mg/kg rats also visited open arms most often, and did so significantly more often than control animals (Fig. 5 , ANOVA: F (10, 99) = 3.22, p = 0.001; post hoc vs control p = 0.0175) under chronic administration of 20 mg/kg of fluoxetine (p = 0.004); and, the highest dose of Pleurotus djamor (Fig. 5 , p = 0.004). The same tendency was found for comparisons with doses 100 mg/kg (p = 0.059) and 500 mg/kg (p = 0.085) of Hericium erinaceus . No other effects were observed. The above pattern of intergroup differences confirmed the potential anxiolytic effect of low doses of Fomitopsis officinalis and Pleurotus djamor mycelium observed also in studies carried out on mice 10 . FST Chronic administration of fluoxetine 20 mg/kg of body weight of (p = 0.010), as well as lowest (p = 0.0172), medium (p = 0.0135) or high dose (p = 0.0172) of Fomitopsis officinalis mycelium and Pleurotus djamor (250 mg/kg: p = 0.0004; 500 mg/kg: p = 0.0002) lowest (p = 0.0058), medium (p = 0.0006) or high dose (p = 0.0003) of Hericium erinaceus reduced the immobility time of male Wistar rats compared to the control group (Fig. 6 , ANOVA: F (10, 97) = 2.745, p = 0.0051) and this time was similar to that observed after administration of FLX as a positive control. In the case of active stress-coping, ANOVA analysis showed (Fig. 7 ; F (10, 97) = 2.038, p = 0.0373) that control group had a shorter swimming time vs. FLX group (p = 0.0291), vs. Fomitopsis officinalis mycelium (250 mg/kg, p = 0.0014), vs. Fomitopsis officinalis mycelium (500 mg/kg, p = 0.0468), vs. Pleurotus djamor (250 mg/kg; p = 0.0203); vs. Pleurotus djamor (500 mg/kg; p = 0.0389); vs. Hericium erinaceus (250 mg/kg; p = 0.0097) and vs. Hericium erinaceus (500 mg/kg; p = 0.014). Compared to the control group (Fig. 8, ANOVA: F (10, 97) = 3.71, p = 0.0003), the lowest dose of Fomitopsis officinalis mycelium (100 mg/kg; p = 0.012), the lowest dose of Hericium erinaceus (100 mg/kg; p = 0.0002), as well highest dose of Pleurotus djamor (500 mg/kg; p = 0.0059) and Hericium erinaceus (500 mg/kg; p = 0.0126) mycelium increased climbing time of male Wistar rats. All that results means that used treatments have a positive effect on the expression of a stress coping strategy that depends on noradrenergic neurotransmission. The inverse relationship occurred for the middle dose of Fomitopsis officinalis mycelium (250 mg/kg) increased climbing time compared to the positive control, fluoxetine (p = 0.0115), as well as lowest dose of Fomitopsis officinalis mycelium (100 mg/kg, p = 0.0008) and the highest doses of Pleurotus djamor (500 mg/kg; p = 0.0003) and Hericium erinaceus (500 mg/kg; p = 0.0008). Statistical significance was also observed for the positive control, FLX: vs. FO250 (p = 0.0115)) and HE100 (p = 0.0287), in the first case in favour of fluoxetine, and in the second case HE100 in the context of stress coping strategy. Discussion The results of this study suggest that long-term administration of medicinal mushrooms reveals their potential anti-anxiety and antidepressant effects and may influence motivation. We observed that these effects depend on the test conditions and the dosage of the mushrooms. Variations in outcomes also stem from the specifics of the test, the type of motivating stimulus (aversive or appetitive), and the behavior being assessed. A pivotal finding that led to the widespread adoption of the Forced Swimming Test (FST) was that potent human antidepressants increase the use of active coping strategies in animals subjected to the test. Active strategies, such as swimming and climbing, predominate during initial swim training, but over time, passive strategies like floating tend to replace them. Consequently, the primary value of the FST lies in its predictive validity, which enables it to assess a drug’s potential antidepressant efficacy based on its ability to promote active coping behaviors. The FST also demonstrates strong construct validity for evaluating coping strategies in response to acute stress. Results from the FST (Figs. 7 and 8) indicate that chronic administration of 250 mg/kg of Fomitopsis officinalis mycelium induces an active stress-coping profile in rats, characteristic of enhanced serotonergic neurotransmission 11 . This is evidenced by increased swimming time (Fig. 7 ) and a concurrent decrease in climbing time (Fig. 8). HPLC analysis 12 revealed that a 250 mg dose of Fomitopsis officinalis mycelium contains approximately 1.3 mg of 5-hydroxy-L-tryptophan (5-HTP), the immediate precursor to serotonin (5-HT). This raises the question of whether this component contributes to the observed effects, given that boosting serotonergic neurotransmission is known to produce antidepressant outcomes. Supplying this precursor could potentially elevate 5-HT levels in the central nervous system (CNS). Mannucci et al. (2006) 13 demonstrated in mice that acute doses of 12.5 to 50 mg/kg of 5-HTP were effective in the FST. However, the lowest of these doses (~ 12.5 mg/kg) is approximately ten times higher than the 1.3 mg/kg of 5-HTP provided by Fomitopsis officinalis mycelium in our study, with the added distinction that our administration was chronic rather than acute. Furthermore, Mannucci et al. (2006) 13 observed effects on immobility—a parameter unaffected by Fomitopsis officinalis mycelium in our study—complicating direct comparisons. Mannucci et al. (2006) 13 did not assess active stress-coping strategies, as this requires a two-day modified FST protocol in rats 11 . Nonetheless, both studies report antidepressant-like effects. Additionally, Fomitopsis species have been noted for their anti-inflammatory properties 14 in different experimental models, suggesting potential CNS effects beyond peripheral inflammation. Reducing inflammatory cytokines may also contribute to antidepressant outcomes. In this study, administration of 250 mg/kg and 500 mg/kg of Hericium erinaceus and Pleurotus djamor mycelium reduced the time spent on passive stress-coping behaviors (Fig. 6 ). The corresponding 5-HTP levels were 0.38 and 0.76 mg/kg for H. erinaceus (HE) and 1.75 and 3.5 mg/kg for P. djamor (PDJ) 15 . Unlike Fomitopsis officinalis , neither mushroom influenced serotonergic-dependent swimming (Fig. 7 ). Reducing immobility in rats typically undermines passive strategies, which conserve energy in threatening situations, while favoring active strategies. In these groups, active coping predominated, indicating significant antidepressant potential. Notably, this effect did not directly correlate with the amount of 5-HTP supplied, suggesting other mechanisms at play. Phenylalanine, a precursor to norepinephrine, modulates climbing—an alternative active stress-coping strategy. However, phenylalanine was not detected in Pleurotus djamor mycelium from in vitro cultures 16 , consistent with its lack of effect on climbing in this study. In contrast, rats receiving the lowest dose (100 mg/kg) of Hericium erinaceus —equivalent to 0.15 mg/kg of phenylalanine—preferred climbing, yet this low level suggests minimal involvement of this precursor. Additionally, reduced γ-aminobutyric acid (GABA) release in the basolateral amygdala (BLA) has been shown to decrease immobility in the FST, indicating an antidepressant effect via presynaptic GABA B autoreceptor stimulation 17 . Significant GABA content has been reported in Pleurotus and Hericium mycelium 18 – 19 , and our results confirm a notable reduction in immobility with these mushrooms (Fig. 6 ). Furthermore, Pleurotus djamor mycelium polysaccharides have been shown to inhibit proinflammatory cytokines (IL-6 and TNF-α), suggesting an anti-inflammatory role that may limit CNS inflammation 20 . Here, we report for the first time the anxiolytic and anti-stress effects of P. djamor mycelium at the highest dose, comparable to those of H. erinaceus and F. officinalis , highlighting their adaptogenic properties. Bioactive compounds from H. erinaceus fruiting bodies and mycelium enhance neurotrophic factor production, such as nerve growth factor (NGF), linked to cell proliferation 8 . Approximately 150 small molecules have been identified in H. erinaceus , with erinacines and hericenones being the most studied for their neurotrophic and neuroprotective properties. Long-term, high-dose administration of H. erinaceus may improve hippocampal neurogenesis and the survival of new neurons in the dentate gyrus, potentially influencing depression-related mechanisms 20 . Its mycelium activates NGF and BDNF pathways while inhibiting NF-κB signalling in mice 21 . BDNF plays a key role in mood regulation, closely tied to the neurogenic effects of antidepressants. Moreover, H. erinaceus reversed restraint-induced hippocampal changes in mice, including low levels of norepinephrine, dopamine, and serotonin, and high levels of IL-6 and TNF-α 9 . Additionally, prolonged H. erinaceus administration reduced the expression of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and the NLRP3 inflammasome 22 . ASC is a critical adaptor in the inflammasome complex, which releases inflammatory cytokines like IL-1β and IL-18. H. erinaceus also lowered IL-1β and IL-18 levels, suggesting an interaction with neuroinflammation—an additional CNS disease mechanism. This may partly explain the behavioral effects observed in our study, though further assays are needed to confirm this hypothesis. The doses of mushrooms tested here demonstrate a beneficial anti-anxiety and antidepressant profile in the FST, warranting further investigation. The effects of chronic mycelium administration appear to stem from a complex interplay of active compounds rather than reliance on specific neurotransmitter precursors. In the Open Field Test (OFT), "rearing" refers to an animal standing on its hind legs, extending its forelimbs forward or upward, often touching the floor or walls with its paws. This behavior can be interpreted in several ways: 1) Exploration: rearing is often a sign of curiosity and a desire to investigate the environment. 2) Anxiety: frequent rearing may indicate nervousness, possibly as the animal scans for threats or seeks escape from an open space. 3) Stress: excessive rearing can reflect a stress response to a novel environment. 4) Sensory investigation: animals may use their whiskers during rearing to gather sensory information. Increased rearing after administration of 250 mg/kg of Fomitopsis officinalis mycelium suggests a lack of 5-HTP involvement, as studies show that 50 mg/kg of 5-HTP either does not affect rearing 23 or reduces it alongside locomotor activity 24 . Other OFT and Elevated Plus-Maze (EPM) results support the exploratory interpretation, showing no evidence of heightened anxiety or stress in these animals. Rats receiving 100 and 250 mg/kg of Fomitopsis officinalis mycelium more frequently explored the open arms of the EPM (Figs. 4 and 5 ), suggesting a potential anxiolytic effect, consistent with prior mouse studies 10 . In contrast, Hericium erinaceus (lowest dose) and Pleurotus djamor (highest dose) showed opposite interspecies effects (mouse vs. rat) 10 , indicating anti-anxiety effects in rats. These figures highlight differences in anxiolytic effects based on open-arm entries and time spent, though not relative to controls or fluoxetine (FLX). Despite these findings, the lack of a fluoxetine effect in the EPM merits discussion, as SSRIs are prescribed for anxiety disorders. First, SSRIs vary in clinical efficacy, and fluoxetine’s effect on generalized anxiety disorder requires confirmation in larger trials 25 . Second, chronic fluoxetine administration in Wistar rats did not produce an effect in the EPM 26 , consistent with our protocol. Third, meta-analyses question the EPM’s predictive validity for SSRIs, showing anxiogenic-like effects with chronic use in rats 27 . Conclusions This study demonstrates the adaptogenic properties of the tested mushrooms, including anxiolytic and antidepressant effects—reported here for the first time for Pleurotus djamor . These effects do not directly depend on biogenic amine precursors in the mycelium but rather on the complex action of their active compounds. The observed effects were dose-dependent, following a U-shaped or linear pattern, introducing a variable for future research. These results support the potential use of medicinal mushrooms to alleviate anxiety, depression, or as adaptogens in psychosocial stress scenarios. Given their complex biochemical composition, attributing effects to specific mechanisms requires extensive further study. Basic neurotransmitter precursors explain these effects only partially, necessitating research to isolate and test individual active compounds. However, the multi-component nature of these mushrooms likely drives their observed behavioral effects. Materials and methods Mushroom Mycelial Cultures Mycelia of HE (BHe 2020), PDJ (BPdj 1119) and FO (JFo 1397) from in vitro mushroom library of the Department of Pharmaceutical Botany, Jagiellonian University Medical College (Kraków, Poland), were passage from Petri dishes with solid agar growth medium (according to Oddoux) to 250 mL of liquid medium. The liquid medium with mycelium was shaken in Erlenmeyer flasks at 140 rpm for 4 weeks using a rotary shaker (Altel, Poland), under laboratory conditions simulating natural environment, i.e., 16:8-h light–dark cycle, at 23 ± 2°C. Subsequently, the mycelium was transferred to an in-house designed 10-L bioreactor with air–lift system, where it was grown for 10 days in aerated conditions (sterile air and discharged CO 2 ). The resultant biomass was isolated from the substrate, rinsed 4 times with distilled water, frozen and – finally – lyophilized (Labconco, USA). The chemical composition of the in vitro -obtained mycelium of all 3 mushroom species had been studied and the results are available in the following publications of Fijałkowska et al. (2020) 12 , Włodarczyk et al. (2020) 28 , and Krakowska et al. (2020) 16 . Animals The study was conducted on a group of 110 male rats ( Rattus norvegicus ) of the Wistar strain (Cmdb:Wi, CMD, Białystok, Poland) aged between 8 to10 weeks, weighing 200–250 g at the beginning of the study, divided into 11 groups of 10 individuals each. The experiments were carried out at the Centre for Experimental Medicine and the Department of Clinical Pharmacology of the Medical University of Bialystok. The experimental procedures were carried out according to the European Regulation (EU) 2019/1010 and after obtaining a license from a Local Ethical Committee for Animal Experiments in Olsztyn, Poland (consent LEC 60/2021). All methods are reported in accordance with ARRIVE guidelines. Housing conditions Animals were housed in a room with stable conditions of 45–60% humidity, 20–23°C temperature, 15 air exchanges per hour and a 12 h/12 h light-dark cycle (lights on at 7:00 a.m.). Rats (2–3 individuals per Techniplast 1354G cage) were provided with an environmental enrichment, ad libitum access to chow (ssniff Spezialdiäten GmbH or Labofeed B) and filtered drinking water. All animals had been acclimatized, handled and gentled before experimental procedures begun. Supplementation Supplementation lasted 21 days. Doses were determined on the basis of previously-reported data and were set at 100, 250 and 500 mg mycelium/kg of body weight for all of the mushroom species. Mycelium was suspended in a volume of 2 mL of 2% CMC (carboxymethyl cellulose) every day at 8.00 a.m., and administered using disposable soft intragastric probes with polypropylene caps and a ball-tipped syringe port to prevent tissue damage. Experimental groups received one of 3 doses of mycelium of one of 3 mushrooms species. Positive control animals received fluoxetine 20 mg/kg of body weight daily at 2% CMC (2 mL/kg); rats in the negative control group received the 2% CMC vehicle alone in the same volume (2 mL/kg). Animals were weighed daily throughout the study. Open Field Test (OFT) On the 21st day of supplementation and one hour after the administration of supplementation material, rats were placed in an open-field apparatus (square box: 100x100x47 cm with white floor divided by eight lines into 25 equal squares) to assess locomotor activity. After a 1 min of habituation to the apparatus, rats were assessed for 5 min. The parameters measured in the test were: number of rearing episodes, fields crossed, and time spent in the central and side zones - counted automatically using the EthoVision XT system (Noldus, NL). Elevated Plus Maze test (EPM) After the OFT, anxiety-like behaviour was tested in the elevated plus maze (EPM) test. Rats were placed individually in the centre of a cross-shaped platform with shoulder dimensions of 50 x 11 cm (length x width), located 75 cm above the floor. The two opposite arms were covered on their sides with walls 50 x 41 cm (length x height), while the remaining 2 were uncovered. Time spent by the rats in the closed and open arms of the platform was measured - counted automatically using the EthoVision XT system (Noldus, NL). Forced Swimming Test (FST) The Forced Swimming Test (FST) was performed to determine the effective active dose of the supplemented mushroom material. It was carried out after 21 days of supplementation, on the 22nd day of the procedure, after performing the OFT and the EPM test. The test lasted two days and consisted of two trials. During the test, rats were placed in a glass, transparent cylinder with a diameter of 25 cm and a height of 50 cm, filled 2/3 with water (so that the animal did not touch the bottom) at a temperature of 23 ± 1ºC, without the possibility of escape. The water in the cylinder was changed after each animal. On the first day of the test, the animals were placed in water for 15 minutes. Next day, 24 h after the first trial, the procedure was repeated for 5 minutes 29 (Porsolt et al., 1977). During the test, following measurements were obtained: immobility time, time spent on climbing activities and time spent on swimming activities. Statistics All results are presented as means ± standard error of the mean (SEM). The normality of data distributions was checked with the Shapiro-Wilk test, and the homogeneity of variances with the Brown-Forsythe test. Statistical analysis was performed using one-way ANOVA and post hoc test with Tukey's correction for multiple comparisons. If the assumptions for the one-way ANOVA parametric test were not met, the Kruskal-Wallis test with Dunn's post hoc test were used. A p -value < 0.05 was considered statistically significant. Abbreviations ASC - apoptosis-associated speck-like protein containing a caspase recruitment domain, BDNF - brain-derived neurotropic factor, BLA - basolateral amygdala, CMC - carboxymethyl cellulose, CNS – central nervous system, EPM - elevated plus maze test, EU - European Regulation, FLX - fluoxetine, FO - Fomitopsis officinalis , FST - forced swimming test, GABA - γ-aminobutyric acid HE - Hericium erinaceus, HPLC – high pressure liquid chromatography 5-HT - 5-hydroxy-tryptamine, serotonin IL-1β – interleukin 1β, IL-6 – interleukin 6, IL-18 – interleukin 18, NGF - nerve growth factor, NLRP3 - NOD-, LRR- and pyrin domain-containing protein 3 OF - open field test, PDJ - Pleurotus djamor , SEM - standard error of the mean, SSRI - selective serotonin reuptake inhibitor, TNF-α – tumour necrosis factor α Declarations Funding: Medical University of Bialystok (Grant number: SUB/1/DN/22/001/1166). Data availability: Data supporting these findings are available within this article. Conflict of interest : All authors have no conflicts of interests to declare. The authors acknowledge study funding and the supply of investigation the design of this study. No actual or perceived conflicts of interest are reported. Ethical approval: The experimental procedures were carried out in accordance with the European Regulation (EU) 2019/1010 and approved by Local Ethical Committee for Animal Experiments in Olsztyn, Poland (consent LEC 60/2021). All methods are reported in accordance with ARRIVE guidelines. Data availability: The data are available from the corresponding author on reasonable request. Author contributions: ET, AF, BM wrote the research protocol and HO helped conduct the study on draft and design and performed the experimental on rats, and HC wrote the manuscript, BM also served as supervisor for this manuscript. All authors have read and approved the final manuscript. References McEwen, B. S. & Akil, H. 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The beneficial effects of Fomitopsis pinicola chloroform extract on a dextran sulfate sodium-induced ulcerative colitis mice model. Ann. Transl Med. 11 , 35. 10.21037/atm-22-5143 (2023). Fijałkowska, A. et al. Edible mushrooms as a potential component of dietary interventions for major depressive disorder. Foods 11 , 1489. 10.3390/foods11101489 (2022). Krakowska, A. et al. Muszyńska, B. Selected edible medicinal mushrooms from Pleurotus genus as an answer for human civilization diseases. Food Chem. 327 , 127084. 10.1016/j.foodchem.2020.127084 (2020). Andolina, D., Maran, D., Valzania, A., Conversi, D. & Puglisi-Allegra, S. Prefrontal/amygdalar system determines stress coping behavior through 5-HT/GABA connection. Neuropsychopharmacology 38 , 2057–2067. 10.1038/npp.2013.107 (2013). Cohen, N. et al. Chemical composition and nutritional and medicinal value of fruit bodies and submerged cultured mycelia of culinary-medicinal higher Basidiomycetes mushrooms. Int. J. Med. Mushrooms . 16 , 273–291. 10.1615/intjmedmushr.v16.i3.80 (2014). Chaiyasut, C. et al. Lactobacillus fermentum HP3-Mediated Fermented Hericium erinaceus Juice as a Health Promoting Food Supplement to Manage Diabetes Mellitus. J. Evid. Based Integr. Med. 23 , 1–9. 10.1177/2515690X18765699 (2018). Cordaro, M. et al. Mechanisms and Potential Implications of Hericium erinaceus in NLRP3 Inflammasome Activation by Reactive Oxygen Species during Alzheimer's Disease. Antioxid. (Basel) . (10), 1664. 10.3390/antiox10111664 (2021). Szućko-Kociuba, I., Trzeciak-Ryczek, A., Kupnicka, P. & Chlubek, D. Neurotrophic and Neuroprotective Effects of Hericium erinaceus. Int. J. Mol. Sci. 24 , 15960. 10.3390/ijms242115960 (2023). Protti, M. P. & De Monte, L. Dual Role of Inflammasome Adaptor ASC in Cancer. Front. Cell. Dev. Biol. 8 , 40. 10.3389/fcell.2020.00040 (2020). Pranzatelli, M. R. The comparative of the behavioral syndromes induced by TRH and by 5–HT in the rat. Gen. Pharmacol. 19 , 205–211. 10.1016/0306-3623(88)90062-6 (1988). Oliveira, G. H. & Palermo-Neto, J. Effects of 2, 4-Dichlorophenoxyacetic Acid (2, 4-D) on open-field behaviour and neurochemical parameters of rats. Pharmacol. Toxicol. 73 , 79–85. 10.1111/j.1600-0773.1993.tb01540.x (1993). Slee, A., Nazareth, I., Bondaronek, P., Liu, Y. & Cheng, Z. Freemantle, N. Pharmacological treatments for generalised anxiety disorder: A systematic review and network meta–analysis. Lancet 2019 , 393 , 768–777. 10.1016/S0140-6736(18)31793-8 Griebel, G., Cohen, C., Perrault, D. & Sanger, D. J. Behavioral effects of acute and chronic fluoxetine in Wistar-Kyoto rats. Physiol. Behav. 67 , 315–320. 10.1016/s0031-9384(98)00298-4 (1999). Kryst, J., Majcher-Maślanka, I. & Chocyk, A. Effects of chronic fluoxetine treatment on anxiety- and depressive-like behaviors in adolescent rodents – systematic review and meta-analysis. Pharmacol. Rep. 74 , 920–946. 10.1007/s43440-022-00420-w (2022). Włodarczyk, A. et al. Edible and medicinal mushroom Hericium erinaceus as a potential natural material with influence on brain functions. Med. Int. Rev. 29 , 4–10 (2020). https://www.researchgate.net/publication/344323885 Porsolt, R. D., Le Pichon, M. & Jalfre, M. Depression: a new animal model sensitive to antidepressant treatments. Nature 266 , 730–732. 10.1038/266730a0 (1977). Additional Declarations No competing interests reported. Supplementary Files Graphicalabstract.pdf Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. 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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-6118750","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":427051661,"identity":"d5032d44-1981-43b8-943c-18c9d7b936b7","order_by":0,"name":"Emil Trofimiuk","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/UlEQVRIiWNgGAWjYFACxsYDIIq9mYHhAMMBGwYGCSCPB4j5cGtpOMCQAFR0GKwlDaGFDY89EC1guw4cJqyFX+xwwwHGH/fkeNi5Ew/znDmfuHZ2A+ODt20Mebi0SM5OBDms2JiHmXfDYZ4btxO33TnAbDi3jaEYlxaD22AtCYn7wVo+ALXcSGCT5m1jSGzDocUepqUHouUcSAv7b3xaDKRRtNw4ALaFGZ8WCZAtCWkJYL8cnHMm2XjbnYPNknPOSeD0C//s9IcPPtgkyPHwn9384c0xO9ltt5sPfnhTZpPHj0MLGCSgchkbQNYnYKojBMjQMgpGwSgYBcMUAABo7GLooEH2EAAAAABJRU5ErkJggg==","orcid":"","institution":"Medical University of Białystok","correspondingAuthor":true,"prefix":"","firstName":"Emil","middleName":"","lastName":"Trofimiuk","suffix":""},{"id":427051662,"identity":"6a09d2ca-7dfd-495a-899e-6e31c9648a29","order_by":1,"name":"Agata Fijałkowska","email":"","orcid":"","institution":"Jagiellonian University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Agata","middleName":"","lastName":"Fijałkowska","suffix":""},{"id":427051663,"identity":"9720102d-c75b-4303-8ed3-afb6a49e9310","order_by":2,"name":"Hubert Oniszczuk","email":"","orcid":"","institution":"Medical University of Bialystok","correspondingAuthor":false,"prefix":"","firstName":"Hubert","middleName":"","lastName":"Oniszczuk","suffix":""},{"id":427051664,"identity":"6b9154eb-b42c-4ffe-a4af-7bc83b12e960","order_by":3,"name":"Halina Car","email":"","orcid":"","institution":"Medical University of Białystok","correspondingAuthor":false,"prefix":"","firstName":"Halina","middleName":"","lastName":"Car","suffix":""},{"id":427051665,"identity":"d2285dd7-d297-47a3-9b77-a0fa8dbde957","order_by":4,"name":"Bożena Muszyńska","email":"","orcid":"","institution":"Jagiellonian University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Bożena","middleName":"","lastName":"Muszyńska","suffix":""}],"badges":[],"createdAt":"2025-02-27 08:08:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6118750/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6118750/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78430112,"identity":"15b04d59-f9f9-47be-940b-89957d7912e9","added_by":"auto","created_at":"2025-03-13 07:09:39","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":146397,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of supplementation with 3 doses (100, 250 and 500 mg/kg) of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO), \u003cem\u003ePleurotus djamor\u003c/em\u003e (PDJ) or \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE) mycelium, as well as fluoxetine (FLX, 20 mg/kg) on number of crosses through the grid lines (locomotor activity) after 21 days of administration \u003cem\u003eper os\u003c/em\u003e assessed in the open field test (OFT).\u003c/p\u003e","description":"","filename":"image1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/05ba3b948bfe8a0d4c9805ba.jpg"},{"id":78430562,"identity":"8bab04c4-0d4b-40e7-bc9d-1e89dc612b6a","added_by":"auto","created_at":"2025-03-13 07:17:39","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":119838,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of supplementation with 3 doses (100, 250 and 500 mg/kg) of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO), \u003cem\u003ePleurotus djamor\u003c/em\u003e (PDJ) or \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE) mycelium, as well as fluoxetine (FLX, 20 mg/kg) on the time spent in the central part of the apparatus (antianxiety-like behaviour) after 21 days of administration \u003cem\u003eper os \u003c/em\u003eassessed in the open field test (OFT).\u003c/p\u003e","description":"","filename":"image2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/44455f37e37d8ba504223090.jpg"},{"id":78428985,"identity":"51cdb82b-eedf-4aa8-a562-0b29d0ebffab","added_by":"auto","created_at":"2025-03-13 07:01:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":136300,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the effect of 21-day supplementation with 3 doses (100, 250 and 500 mg/kg, \u003cem\u003eper os\u003c/em\u003e) of mycelium of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO), \u003cem\u003ePleurotus djamor\u003c/em\u003e (PDJ) or \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE), and fluoxetine (FLX, 20 mg/kg) on exploratory/orienting behaviour (number of rearings) assessed in OFT. FO250 vs. control group (**p\u0026lt;0.01), vs fluoxetine (*p\u0026lt;0.05) and in comparison, with PDJ500 (*p\u0026lt;0.05) and all doses of HE (**p\u0026lt;0.01 and ****p\u0026lt;0.0001 respectively).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/87f9203eaa0402816e2abb41.png"},{"id":78430561,"identity":"0749d277-518d-4513-b4dc-690fd8356911","added_by":"auto","created_at":"2025-03-13 07:17:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":124774,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the anxiolytic-like effect, time spent in the open arms of the EPM, after 21 days of supplementation with 3 doses (100, 250 and 500 mg/kg, \u003cem\u003eper os\u003c/em\u003e) of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO), \u003cem\u003ePleurotus djamor\u003c/em\u003e (PDJ) or \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE) with an anxiolytic drug (fluoxetine, FLX, 20 mg/kg). Statistically significant differences were found when comparing: 100 mg/kg of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e vs control (*p\u0026lt;0.05); vs. FLX p\u0026lt;0.05), vs. the highest dose of \u003cem\u003ePleurotus djamor\u003c/em\u003e (*p\u0026lt;0.05); vs. low dose of \u003cem\u003eHericium erinaceus \u003c/em\u003e(*p\u0026lt;0.05) and comparing low dose of \u003cem\u003ePleurotus djamor\u003c/em\u003e vs highest dose of \u003cem\u003ePleurotus djamor \u003c/em\u003e(*p\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/3454678e7e5dce2c722eca62.png"},{"id":78428982,"identity":"7b36222f-2b1d-4623-ac87-519d0662080f","added_by":"auto","created_at":"2025-03-13 07:01:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":108933,"visible":true,"origin":"","legend":"\u003cp\u003eComparison of the anxiolytic-like effect (number of visits to open EPM arms) after 21 days of supplementation with 3 doses (100, 250 and 500 mg/kg, \u003cem\u003eper os\u003c/em\u003e) of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO), \u003cem\u003ePleurotus djamor\u003c/em\u003e (PDJ) or \u003cem\u003eHericium erinaceus\u003c/em\u003e(HE) with an antidepressant drug (fluoxetine, FLX, 20 mg/kg). Compared to the control group, the dose of 100 mg/kg \u003cem\u003eFomitopsis officinalis\u003c/em\u003e visited open arms significantly more often (*p\u0026lt;0.05). In comparison, the dose of 100 mg/kg of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium was significantly significant to fluoxetine (*** p \u0026lt; 0.001) and the highest dose of \u003cem\u003ePleurotus djamor\u003c/em\u003e(*** p\u0026lt;0.001).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/a4f4d84c1a00dbad29615f6c.png"},{"id":78428991,"identity":"2defacc2-1d5f-4675-83c4-b217670a7602","added_by":"auto","created_at":"2025-03-13 07:01:39","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":181004,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of 23-day supplementation with 3 doses (100, 250 and 500 mg/kg, per os) of mycelium of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO), \u003cem\u003ePleurotus djamor\u003c/em\u003e(PDJ) or \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE), and fluoxetine (FLX, 20 mg/kg) on the passive stress-coping strategy (time of immobility) in the forced swimming test (FST). Compared to the control group, FLX (*p\u0026lt;0.01), all doses of FO (*p\u0026lt;0.05), both higher doses of PDJ (*** p\u0026lt;0.001) and all doses of HE (respectively: **p,0.01 and ***p\u0026lt;0.001) effectively reduced the immobility time of male Wistar rats i.e., negatively influenced the expression of a passive strategy, the main benefit of which is the conservation of energy in a threat situation.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/b88c2848b813cebd5197ee92.png"},{"id":78428997,"identity":"ca40a58c-72e4-471d-8446-6f3b55010c52","added_by":"auto","created_at":"2025-03-13 07:01:39","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":176849,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of 23-day supplementation with 3 doses (100, 250 and 500 mg/kg, \u003cem\u003eper os\u003c/em\u003e) of the mycelium of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO), \u003cem\u003ePleurotus djamor\u003c/em\u003e(PDJ) or \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE), and fluoxetine (FLX, 20 mg/kg) on active stress-coping strategy (swimming) in the forced swimming test (FST). Compared to the control group FLX (* p\u0026lt;0.05) and all the middle and highest doses of FO (** p\u0026lt;0.01; *p\u0026lt;0.05) PDJ (*p\u0026lt; 0.05, *p\u0026lt;0.05) as well as HE (**p\u0026lt;0.01; *p\u0026lt;0,05) increased the swimming time of male Wistar rats, which means that it has a positive effect on the expression of a stress coping strategy that depends on serotonergic neurotransmission.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/c1f10ebf0969c3989551cd56.png"},{"id":78430563,"identity":"84da2f01-1ac6-47bc-b8b7-cf275d04f477","added_by":"auto","created_at":"2025-03-13 07:17:39","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":156944,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of 23-day supplementation with 3 doses (100, 250 and 500 mg/kg, \u003cem\u003eper os\u003c/em\u003e) of mycelium of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO), \u003cem\u003ePleurotus djamor\u003c/em\u003e (PDJ) or \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE), and fluoxetine (FLX, 20 mg/kg) on an active stress-coping strategy (climbing) in the forced swimming test (FST). Compared to the control group, the lowest dose of FO (* p\u0026lt;0.05) and HE (*** p\u0026lt;0.001), as well highest dose of PDJ (** p\u0026lt;0.01) and HE (* p\u0026lt;0.05) mycelium increased climbing time of male Wistar rats, which means that it has a positive effect on the expression of a stress coping strategy that depends on noradrenergic neurotransmission. The inverse relationship occurred for the middle dose of FO (\u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e p\u0026lt;0.01) compared to the lowest dose of FO and the highest doses of PDJ (\u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e p\u0026lt;0.001) and HE100 (\u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003ep\u0026lt;0.01) and HE500 (\u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e p\u0026lt;0.001). Significance was also noted for the positive control, FLX: vs. FO250 and HE100 (\u003csup\u003e#\u003c/sup\u003ep\u0026lt;0.05).\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/cb663fcbaada41c0b2964cd4.png"},{"id":80608623,"identity":"81a800d7-c8b7-41d0-9a1e-469f80f6299c","added_by":"auto","created_at":"2025-04-15 07:23:19","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1865751,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/92bc2887-bebc-4f79-b0d1-65489d625be3.pdf"},{"id":78428992,"identity":"e374369f-75fc-4012-beb3-116b301a646b","added_by":"auto","created_at":"2025-03-13 07:01:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":218513,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6118750/v1/fa6cc36b5323b3e10d015c5c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of dietary supplementation with in vitro-cultivated arboreal medicinal mushrooms on stress coping strategies, depressive and anxiety-like behaviour of rats.","fulltext":[{"header":"Introduction","content":"\u003cp\u003eStress is a well-established environmental risk factor implicated in the development of all affective disorders. While the biological mechanisms underlying stress and its impact on the nervous system have been extensively studied\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, the interplay between stress and other environmental factors in shaping mental health outcomes remains an area of active investigation. Among these factors, diet has recently gained significant attention as a modulatory influence on mental health, with some experts proposing the emergence of a new field: nutritional psychiatry \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. For instance, longitudinal studies have demonstrated that diets such as the Mediterranean diet and anti-inflammatory diets are associated with a reduced risk of depressive symptoms \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Furthermore, clinical trials investigating dietary interventions have reported modest but promising therapeutic effects on depressive symptoms, although no significant benefits have been observed for anxiety \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePreclinical experimental studies, which allow for direct exploration of the interaction between stress and diet in the pathogenesis of affective disorders, have primarily focused on isolated dietary components. Among these, \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE), an edible medicinal mushroom commonly known as lion\u0026rsquo;s mane or yamabushitake, has shown notable neuroprotective and behavioral effects. Research has demonstrated that HE mitigates the detrimental effects of elevated corticosterone levels \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e(Lew et al., 2020), promotes hippocampal neurogenesis \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and increases brain-derived neurotrophic factor (BDNF) levels \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Additionally, HE has been shown to modulate stress-related behaviors in rodents, as evidenced by improvements in tail suspension and forced swimming tests \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Our recent work further revealed that dietary supplementation with HE alters exploratory behavior in healthy mice exposed to anxiety-inducing environments, such as the elevated plus-maze (EPM). Intriguingly, two other medicinal mushrooms, \u003cem\u003eFomitopsis officinalis\u003c/em\u003e (FO, commonly known as agarikon) and \u003cem\u003ePleurotus djamor\u003c/em\u003e (PDJ, commonly known as pink oyster mushroom), exhibited similar behavioral effects \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBuilding on these findings, the present study aimed to explore the dose-dependency of the effects of HE, FO, and PDJ, and to compare their efficacy with that of a selective serotonin reuptake inhibitor (SSRI, fluoxetine), a well-established pharmacological treatment for stress-related behaviors. This comparison provides a qualitative benchmark for evaluating the therapeutic potential of these mushrooms. Additionally, we sought to systematically compare the effects of the three mushrooms to assess the influence of different raw materials and dosages.\u003c/p\u003e \u003cp\u003eThe rationale for investigating dose-dependency arose from our observation that the outcomes of mushroom supplementation in the EPM varied depending on individual differences in food intake among the animals \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Dose-response studies are a standard methodological approach for evaluating the effects of novel substances or raw materials, and administering precise doses via gastric gavage eliminates variability associated with random consumption. Specifically, we noted a potential anxiolytic-like effect with lower doses of HE and FO, and with higher doses of PDJ. Furthermore, we aimed to determine whether these effects could be replicated across rodent species\u0026mdash;from mice to rats\u0026mdash;as this would enhance the translational relevance of our findings and provide a more robust foundation for future research.\u003c/p\u003e \u003cp\u003eBy addressing these questions, this study seeks to advance our understanding of the therapeutic potential of medicinal mushrooms in modulating stress-related behaviors and to provide insights into their mechanisms of action. Such findings could pave the way for the development of novel, diet-based interventions for affective disorders, complementing existing pharmacological approaches.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOFT\u003c/h2\u003e \u003cp\u003eNo effect of experimental manipulations was observed on: \u003cb\u003e1)\u003c/b\u003e the level of locomotor activity measured by the number of crossings through the grid lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, F \u003csub\u003e(10, 98)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.35; p\u0026thinsp;=\u0026thinsp;0.214); and \u003cb\u003e2)\u003c/b\u003e anxiety-like behaviour measured by time spent in the central part of the open field (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e,\u003c/p\u003e \u003cp\u003eF\u003csub\u003e(10, 99)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.24, p\u0026thinsp;=\u0026thinsp;0.278). However, compared to the control group \u003cem\u003eFomitopsis officinalis\u003c/em\u003e at a dose of 250 mg/kg, increased threefold the occurrence of exploratory/orienting behaviour measured by the number of rearings (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, K-W\u0026thinsp;=\u0026thinsp;35.8; p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Dunn's \u003cem\u003epost hoc\u003c/em\u003e test: p\u0026thinsp;=\u0026thinsp;0.0027). Analogous differences at the same dose (i.e., increased exploratory behaviour) were observed compared to fluoxetine (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, p\u0026thinsp;=\u0026thinsp;0.0212), \u003cem\u003ePleurotus djamor\u003c/em\u003e at a dose of 500 mg/kg (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, p\u0026thinsp;=\u0026thinsp;0.0495), and \u003cem\u003eHericium erinaceus\u003c/em\u003e at all 3 doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, 100 mg/kg, p\u0026thinsp;=\u0026thinsp;0.0085; 250 mg/kg, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; 500 mg/kg body weight, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEPM\u003c/h3\u003e\n\u003cp\u003eRats receiving the lowest dose of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium (100 mg/kg) and the lowest dose of \u003cem\u003ePleurotus djamor\u003c/em\u003e (100 mg/kg) stayed in the \"dangerous\" open arms the longest of all groups; and, on average twice as long (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, 70.7\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8 s and 67.5\u0026thinsp;\u0026plusmn;\u0026thinsp;11.65 s respectively) as rats in the control group (35\u0026thinsp;\u0026plusmn;\u0026thinsp;6.8 s, p\u0026thinsp;=\u0026thinsp;0.0309 and p\u0026thinsp;=\u0026thinsp;0.0383). In \u003cem\u003epost-hoc\u003c/em\u003e tests with Tukey's correction for multiple comparisons for one-way ANOVA model (F\u003csub\u003e(10, 98)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.366, p\u0026thinsp;=\u0026thinsp;0.0149), this effect was statistically significant. There was a significant an anxiolytic-like effect of the lowest dose of \u003cem\u003eF. officinalis\u003c/em\u003e compared to: fluoxetine (p\u0026thinsp;=\u0026thinsp;0.028), vs. highest dose (500 mg/kg) \u003cem\u003eP. djamor\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0183), vs. the lowest dose of \u003cem\u003eHericium erinaceus\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0152) and the lowest dose of \u003cem\u003eP. djamor vs.\u003c/em\u003e the highest dose of \u003cem\u003eP. djamor\u003c/em\u003e (p\u0026thinsp;=\u0026thinsp;0.0362).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter administration of lyophilized mycelium of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e at a dose of 100 mg/kg rats also visited open arms most often, and did so significantly more often than control animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, ANOVA: F\u003csub\u003e(10, 99)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.22, p\u0026thinsp;=\u0026thinsp;0.001; \u003cem\u003epost hoc\u003c/em\u003e vs control p\u0026thinsp;=\u0026thinsp;0.0175) under chronic administration of 20 mg/kg of fluoxetine (p\u0026thinsp;=\u0026thinsp;0.004); and, the highest dose of \u003cem\u003ePleurotus djamor\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, p\u0026thinsp;=\u0026thinsp;0.004). The same tendency was found for comparisons with doses 100 mg/kg (p\u0026thinsp;=\u0026thinsp;0.059) and 500 mg/kg (p\u0026thinsp;=\u0026thinsp;0.085) of \u003cem\u003eHericium erinaceus\u003c/em\u003e. No other effects were observed. The above pattern of intergroup differences confirmed the potential anxiolytic effect of low doses of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e and \u003cem\u003ePleurotus djamor\u003c/em\u003e mycelium observed also in studies carried out on mice \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eFST\u003c/h3\u003e\n\u003cp\u003eChronic administration of fluoxetine 20 mg/kg of body weight of (p\u0026thinsp;=\u0026thinsp;0.010), as well as lowest (p\u0026thinsp;=\u0026thinsp;0.0172), medium (p\u0026thinsp;=\u0026thinsp;0.0135) or high dose (p\u0026thinsp;=\u0026thinsp;0.0172) of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium and \u003cem\u003ePleurotus djamor\u003c/em\u003e (250 mg/kg: p\u0026thinsp;=\u0026thinsp;0.0004; 500 mg/kg: p\u0026thinsp;=\u0026thinsp;0.0002) lowest (p\u0026thinsp;=\u0026thinsp;0.0058), medium (p\u0026thinsp;=\u0026thinsp;0.0006) or high dose (p\u0026thinsp;=\u0026thinsp;0.0003) of \u003cem\u003eHericium erinaceus\u003c/em\u003e reduced the immobility time of male Wistar rats compared to the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, ANOVA: F\u003csub\u003e(10, 97)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.745, p\u0026thinsp;=\u0026thinsp;0.0051) and this time was similar to that observed after administration of FLX as a positive control.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the case of active stress-coping, ANOVA analysis showed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e; F\u003csub\u003e(10, 97)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.038, p\u0026thinsp;=\u0026thinsp;0.0373) that control group had a shorter swimming time vs. FLX group (p\u0026thinsp;=\u0026thinsp;0.0291), vs. \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium (250 mg/kg, p\u0026thinsp;=\u0026thinsp;0.0014), vs. \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium (500 mg/kg, p\u0026thinsp;=\u0026thinsp;0.0468), vs. \u003cem\u003ePleurotus djamor\u003c/em\u003e (250 mg/kg; p\u0026thinsp;=\u0026thinsp;0.0203); vs. \u003cem\u003ePleurotus djamor\u003c/em\u003e (500 mg/kg; p\u0026thinsp;=\u0026thinsp;0.0389); vs. \u003cem\u003eHericium erinaceus\u003c/em\u003e (250 mg/kg; p\u0026thinsp;=\u0026thinsp;0.0097) and vs. \u003cem\u003eHericium erinaceus\u003c/em\u003e (500 mg/kg; p\u0026thinsp;=\u0026thinsp;0.014).\u003c/p\u003e \u003cp\u003eCompared to the control group (Fig.\u0026nbsp;8, ANOVA: F\u003csub\u003e(10, 97)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.71, p\u0026thinsp;=\u0026thinsp;0.0003), the lowest dose of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium (100 mg/kg; p\u0026thinsp;=\u0026thinsp;0.012), the lowest dose of \u003cem\u003eHericium erinaceus\u003c/em\u003e (100 mg/kg; p\u0026thinsp;=\u0026thinsp;0.0002), as well highest dose of \u003cem\u003ePleurotus djamor\u003c/em\u003e (500 mg/kg; p\u0026thinsp;=\u0026thinsp;0.0059) and \u003cem\u003eHericium erinaceus\u003c/em\u003e (500 mg/kg; p\u0026thinsp;=\u0026thinsp;0.0126) mycelium increased climbing time of male Wistar rats. All that results means that used treatments have a positive effect on the expression of a stress coping strategy that depends on noradrenergic neurotransmission. The inverse relationship occurred for the middle dose of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium (250 mg/kg) increased climbing time compared to the positive control, fluoxetine (p\u0026thinsp;=\u0026thinsp;0.0115), as well as lowest dose of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium (100 mg/kg, p\u0026thinsp;=\u0026thinsp;0.0008) and the highest doses of \u003cem\u003ePleurotus djamor\u003c/em\u003e (500 mg/kg; p\u0026thinsp;=\u0026thinsp;0.0003) and \u003cem\u003eHericium erinaceus\u003c/em\u003e (500 mg/kg; p\u0026thinsp;=\u0026thinsp;0.0008). Statistical significance was also observed for the positive control, FLX: vs. FO250 (p\u0026thinsp;=\u0026thinsp;0.0115)) and HE100 (p\u0026thinsp;=\u0026thinsp;0.0287), in the first case in favour of fluoxetine, and in the second case HE100 in the context of stress coping strategy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of this study suggest that long-term administration of medicinal mushrooms reveals their potential anti-anxiety and antidepressant effects and may influence motivation. We observed that these effects depend on the test conditions and the dosage of the mushrooms. Variations in outcomes also stem from the specifics of the test, the type of motivating stimulus (aversive or appetitive), and the behavior being assessed.\u003c/p\u003e \u003cp\u003eA pivotal finding that led to the widespread adoption of the Forced Swimming Test (FST) was that potent human antidepressants increase the use of active coping strategies in animals subjected to the test. Active strategies, such as swimming and climbing, predominate during initial swim training, but over time, passive strategies like floating tend to replace them. Consequently, the primary value of the FST lies in its predictive validity, which enables it to assess a drug\u0026rsquo;s potential antidepressant efficacy based on its ability to promote active coping behaviors. The FST also demonstrates strong construct validity for evaluating coping strategies in response to acute stress.\u003c/p\u003e \u003cp\u003eResults from the FST (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e and 8) indicate that chronic administration of 250 mg/kg of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium induces an active stress-coping profile in rats, characteristic of enhanced serotonergic neurotransmission \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. This is evidenced by increased swimming time (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) and a concurrent decrease in climbing time (Fig.\u0026nbsp;8). HPLC analysis \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e revealed that a 250 mg dose of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium contains approximately 1.3 mg of 5-hydroxy-L-tryptophan (5-HTP), the immediate precursor to serotonin (5-HT). This raises the question of whether this component contributes to the observed effects, given that boosting serotonergic neurotransmission is known to produce antidepressant outcomes. Supplying this precursor could potentially elevate 5-HT levels in the central nervous system (CNS).\u003c/p\u003e \u003cp\u003eMannucci et al. (2006) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e demonstrated in mice that acute doses of 12.5 to 50 mg/kg of 5-HTP were effective in the FST. However, the lowest of these doses (~\u0026thinsp;12.5 mg/kg) is approximately ten times higher than the 1.3 mg/kg of 5-HTP provided by \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium in our study, with the added distinction that our administration was chronic rather than acute. Furthermore, Mannucci et al. (2006) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e observed effects on immobility\u0026mdash;a parameter unaffected by \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium in our study\u0026mdash;complicating direct comparisons. Mannucci et al. (2006) \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e did not assess active stress-coping strategies, as this requires a two-day modified FST protocol in rats \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Nonetheless, both studies report antidepressant-like effects. Additionally, \u003cem\u003eFomitopsis\u003c/em\u003e species have been noted for their anti-inflammatory properties \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e in different experimental models, suggesting potential CNS effects beyond peripheral inflammation. Reducing inflammatory cytokines may also contribute to antidepressant outcomes.\u003c/p\u003e \u003cp\u003eIn this study, administration of 250 mg/kg and 500 mg/kg of \u003cem\u003eHericium erinaceus\u003c/em\u003e and \u003cem\u003ePleurotus djamor\u003c/em\u003e mycelium reduced the time spent on passive stress-coping behaviors (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The corresponding 5-HTP levels were 0.38 and 0.76 mg/kg for \u003cem\u003eH. erinaceus\u003c/em\u003e (HE) and 1.75 and 3.5 mg/kg for \u003cem\u003eP. djamor\u003c/em\u003e (PDJ) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Unlike \u003cem\u003eFomitopsis officinalis\u003c/em\u003e, neither mushroom influenced serotonergic-dependent swimming (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Reducing immobility in rats typically undermines passive strategies, which conserve energy in threatening situations, while favoring active strategies. In these groups, active coping predominated, indicating significant antidepressant potential. Notably, this effect did not directly correlate with the amount of 5-HTP supplied, suggesting other mechanisms at play.\u003c/p\u003e \u003cp\u003ePhenylalanine, a precursor to norepinephrine, modulates climbing\u0026mdash;an alternative active stress-coping strategy. However, phenylalanine was not detected in \u003cem\u003ePleurotus djamor\u003c/em\u003e mycelium from in vitro cultures \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, consistent with its lack of effect on climbing in this study. In contrast, rats receiving the lowest dose (100 mg/kg) of \u003cem\u003eHericium erinaceus\u003c/em\u003e\u0026mdash;equivalent to 0.15 mg/kg of phenylalanine\u0026mdash;preferred climbing, yet this low level suggests minimal involvement of this precursor. Additionally, reduced γ-aminobutyric acid (GABA) release in the basolateral amygdala (BLA) has been shown to decrease immobility in the FST, indicating an antidepressant effect via presynaptic GABA B autoreceptor stimulation \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Significant GABA content has been reported in \u003cem\u003ePleurotus\u003c/em\u003e and \u003cem\u003eHericium mycelium\u003c/em\u003e \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, and our results confirm a notable reduction in immobility with these mushrooms (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Furthermore, \u003cem\u003ePleurotus djamor\u003c/em\u003e mycelium polysaccharides have been shown to inhibit proinflammatory cytokines (IL-6 and TNF-α), suggesting an anti-inflammatory role that may limit CNS inflammation \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Here, we report for the first time the anxiolytic and anti-stress effects of \u003cem\u003eP. djamor\u003c/em\u003e mycelium at the highest dose, comparable to those of \u003cem\u003eH. erinaceus\u003c/em\u003e and \u003cem\u003eF. officinalis\u003c/em\u003e, highlighting their adaptogenic properties.\u003c/p\u003e \u003cp\u003eBioactive compounds from \u003cem\u003eH. erinaceus\u003c/em\u003e fruiting bodies and mycelium enhance neurotrophic factor production, such as nerve growth factor (NGF), linked to cell proliferation \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Approximately 150 small molecules have been identified in \u003cem\u003eH. erinaceus\u003c/em\u003e, with erinacines and hericenones being the most studied for their neurotrophic and neuroprotective properties. Long-term, high-dose administration of \u003cem\u003eH. erinaceus\u003c/em\u003e may improve hippocampal neurogenesis and the survival of new neurons in the dentate gyrus, potentially influencing depression-related mechanisms \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Its mycelium activates NGF and BDNF pathways while inhibiting NF-κB signalling in mice \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. BDNF plays a key role in mood regulation, closely tied to the neurogenic effects of antidepressants. Moreover, \u003cem\u003eH. erinaceus\u003c/em\u003e reversed restraint-induced hippocampal changes in mice, including low levels of norepinephrine, dopamine, and serotonin, and high levels of IL-6 and TNF-α \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, prolonged \u003cem\u003eH. erinaceus\u003c/em\u003e administration reduced the expression of caspase-1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and the NLRP3 inflammasome \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. ASC is a critical adaptor in the inflammasome complex, which releases inflammatory cytokines like IL-1β and IL-18. \u003cem\u003eH. erinaceus\u003c/em\u003e also lowered IL-1β and IL-18 levels, suggesting an interaction with neuroinflammation\u0026mdash;an additional CNS disease mechanism. This may partly explain the behavioral effects observed in our study, though further assays are needed to confirm this hypothesis.\u003c/p\u003e \u003cp\u003eThe doses of mushrooms tested here demonstrate a beneficial anti-anxiety and antidepressant profile in the FST, warranting further investigation. The effects of chronic mycelium administration appear to stem from a complex interplay of active compounds rather than reliance on specific neurotransmitter precursors.\u003c/p\u003e \u003cp\u003eIn the Open Field Test (OFT), \"rearing\" refers to an animal standing on its hind legs, extending its forelimbs forward or upward, often touching the floor or walls with its paws. This behavior can be interpreted in several ways: 1) Exploration: rearing is often a sign of curiosity and a desire to investigate the environment. 2) Anxiety: frequent rearing may indicate nervousness, possibly as the animal scans for threats or seeks escape from an open space. 3) Stress: excessive rearing can reflect a stress response to a novel environment. 4) Sensory investigation: animals may use their whiskers during rearing to gather sensory information. Increased rearing after administration of 250 mg/kg of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium suggests a lack of 5-HTP involvement, as studies show that 50 mg/kg of 5-HTP either does not affect rearing \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e or reduces it alongside locomotor activity \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. Other OFT and Elevated Plus-Maze (EPM) results support the exploratory interpretation, showing no evidence of heightened anxiety or stress in these animals.\u003c/p\u003e \u003cp\u003eRats receiving 100 and 250 mg/kg of \u003cem\u003eFomitopsis officinalis\u003c/em\u003e mycelium more frequently explored the open arms of the EPM (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting a potential anxiolytic effect, consistent with prior mouse studies \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In contrast, \u003cem\u003eHericium erinaceus\u003c/em\u003e (lowest dose) and \u003cem\u003ePleurotus djamor\u003c/em\u003e (highest dose) showed opposite interspecies effects (mouse vs. rat) \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, indicating anti-anxiety effects in rats. These figures highlight differences in anxiolytic effects based on open-arm entries and time spent, though not relative to controls or fluoxetine (FLX).\u003c/p\u003e \u003cp\u003eDespite these findings, the lack of a fluoxetine effect in the EPM merits discussion, as SSRIs are prescribed for anxiety disorders. First, SSRIs vary in clinical efficacy, and fluoxetine\u0026rsquo;s effect on generalized anxiety disorder requires confirmation in larger trials \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Second, chronic fluoxetine administration in Wistar rats did not produce an effect in the EPM \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, consistent with our protocol. Third, meta-analyses question the EPM\u0026rsquo;s predictive validity for SSRIs, showing anxiogenic-like effects with chronic use in rats \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study demonstrates the adaptogenic properties of the tested mushrooms, including anxiolytic and antidepressant effects\u0026mdash;reported here for the first time for \u003cem\u003ePleurotus djamor\u003c/em\u003e. These effects do not directly depend on biogenic amine precursors in the mycelium but rather on the complex action of their active compounds. The observed effects were dose-dependent, following a U-shaped or linear pattern, introducing a variable for future research. These results support the potential use of medicinal mushrooms to alleviate anxiety, depression, or as adaptogens in psychosocial stress scenarios. Given their complex biochemical composition, attributing effects to specific mechanisms requires extensive further study. Basic neurotransmitter precursors explain these effects only partially, necessitating research to isolate and test individual active compounds. However, the multi-component nature of these mushrooms likely drives their observed behavioral effects.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eMushroom Mycelial Cultures\u003c/h2\u003e \u003cp\u003eMycelia of HE (BHe 2020), PDJ (BPdj 1119) and FO (JFo 1397) from \u003cem\u003ein vitro\u003c/em\u003e mushroom library of the Department of Pharmaceutical Botany, Jagiellonian University Medical College (Krak\u0026oacute;w, Poland), were passage from Petri dishes with solid agar growth medium (according to Oddoux) to 250 mL of liquid medium. The liquid medium with mycelium was shaken in Erlenmeyer flasks at 140 rpm for 4 weeks using a rotary shaker (Altel, Poland), under laboratory conditions simulating natural environment, i.e., 16:8-h light\u0026ndash;dark cycle, at 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C. Subsequently, the mycelium was transferred to an in-house designed 10-L bioreactor with air\u0026ndash;lift system, where it was grown for 10 days in aerated conditions (sterile air and discharged CO\u003csub\u003e2\u003c/sub\u003e). The resultant biomass was isolated from the substrate, rinsed 4 times with distilled water, frozen and \u0026ndash; finally \u0026ndash; lyophilized (Labconco, USA). The chemical composition of the \u003cem\u003ein vitro\u003c/em\u003e-obtained mycelium of all 3 mushroom species had been studied and the results are available in the following publications of Fijałkowska et al. (2020) \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e, Włodarczyk et al. (2020) \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e, and Krakowska et al. (2020) \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAnimals\u003c/h3\u003e\n\u003cp\u003eThe study was conducted on a group of 110 male rats (\u003cem\u003eRattus norvegicus\u003c/em\u003e) of the Wistar strain (Cmdb:Wi, CMD, Białystok, Poland) aged between 8 to10 weeks, weighing 200\u0026ndash;250 g at the beginning of the study, divided into 11 groups of 10 individuals each. The experiments were carried out at the Centre for Experimental Medicine and the Department of Clinical Pharmacology of the Medical University of Bialystok. The experimental procedures were carried out according to the European Regulation (EU) 2019/1010 and after obtaining a license from a Local Ethical Committee for Animal Experiments in Olsztyn, Poland (consent LEC 60/2021). All methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHousing conditions\u003c/h2\u003e \u003cp\u003eAnimals were housed in a room with stable conditions of 45\u0026ndash;60% humidity, 20\u0026ndash;23\u0026deg;C temperature, 15 air exchanges per hour and a 12 h/12 h light-dark cycle (lights on at 7:00 a.m.). Rats (2\u0026ndash;3 individuals per Techniplast 1354G cage) were provided with an environmental enrichment, \u003cem\u003ead libitum\u003c/em\u003e access to chow (ssniff Spezialdi\u0026auml;ten GmbH or Labofeed B) and filtered drinking water. All animals had been acclimatized, handled and gentled before experimental procedures begun.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eSupplementation\u003c/h2\u003e \u003cp\u003eSupplementation lasted 21 days. Doses were determined on the basis of previously-reported data and were set at 100, 250 and 500 mg mycelium/kg of body weight for all of the mushroom species. Mycelium was suspended in a volume of 2 mL of 2% CMC (carboxymethyl cellulose) every day at 8.00 a.m., and administered using disposable soft intragastric probes with polypropylene caps and a ball-tipped syringe port to prevent tissue damage.\u003c/p\u003e \u003cp\u003eExperimental groups received one of 3 doses of mycelium of one of 3 mushrooms species. Positive control animals received fluoxetine 20 mg/kg of body weight daily at 2% CMC (2 mL/kg); rats in the negative control group received the 2% CMC vehicle alone in the same volume (2 mL/kg). Animals were weighed daily throughout the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOpen Field Test (OFT)\u003c/h2\u003e \u003cp\u003eOn the 21st day of supplementation and one hour after the administration of supplementation material, rats were placed in an open-field apparatus (square box: 100x100x47 cm with white floor divided by eight lines into 25 equal squares) to assess locomotor activity. After a 1 min of habituation to the apparatus, rats were assessed for 5 min. The parameters measured in the test were: number of rearing episodes, fields crossed, and time spent in the central and side zones - counted automatically using the EthoVision XT system (Noldus, NL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eElevated Plus Maze test (EPM)\u003c/h2\u003e \u003cp\u003eAfter the OFT, anxiety-like behaviour was tested in the elevated plus maze (EPM) test. Rats were placed individually in the centre of a cross-shaped platform with shoulder dimensions of 50 x 11 cm (length x width), located 75 cm above the floor. The two opposite arms were covered on their sides with walls 50 x 41 cm (length x height), while the remaining 2 were uncovered. Time spent by the rats in the closed and open arms of the platform was measured - counted automatically using the EthoVision XT system (Noldus, NL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eForced Swimming Test (FST)\u003c/h2\u003e \u003cp\u003eThe Forced Swimming Test (FST) was performed to determine the effective active dose of the supplemented mushroom material. It was carried out after 21 days of supplementation, on the 22nd day of the procedure, after performing the OFT and the EPM test.\u003c/p\u003e \u003cp\u003eThe test lasted two days and consisted of two trials. During the test, rats were placed in a glass, transparent cylinder with a diameter of 25 cm and a height of 50 cm, filled 2/3 with water (so that the animal did not touch the bottom) at a temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026ordm;C, without the possibility of escape. The water in the cylinder was changed after each animal.\u003c/p\u003e \u003cp\u003eOn the first day of the test, the animals were placed in water for 15 minutes. Next day, 24 h after the first trial, the procedure was repeated for 5 minutes \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e (Porsolt et al., 1977).\u003c/p\u003e \u003cp\u003eDuring the test, following measurements were obtained: immobility time, time spent on climbing activities and time spent on swimming activities.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eAll results are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). The normality of data distributions was checked with the Shapiro-Wilk test, and the homogeneity of variances with the Brown-Forsythe test. Statistical analysis was performed using one-way ANOVA and \u003cem\u003epost hoc\u003c/em\u003e test with Tukey's correction for multiple comparisons. If the assumptions for the one-way ANOVA parametric test were not met, the Kruskal-Wallis test with Dunn's \u003cem\u003epost hoc\u003c/em\u003e test were used. A \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eASC - apoptosis-associated speck-like protein containing a caspase recruitment domain,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBDNF - brain-derived neurotropic factor,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBLA - basolateral amygdala,\u003c/p\u003e\n\u003cp\u003eCMC - carboxymethyl cellulose,\u003c/p\u003e\n\u003cp\u003eCNS – central nervous system,\u003c/p\u003e\n\u003cp\u003eEPM - elevated plus maze test,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eEU - European Regulation,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFLX - fluoxetine,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFO\u003cem\u003e\u0026nbsp;- Fomitopsis officinalis\u003c/em\u003e,\u003c/p\u003e\n\u003cp\u003eFST - forced swimming test,\u003c/p\u003e\n\u003cp\u003eGABA - γ-aminobutyric acid\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHE\u003cem\u003e\u0026nbsp;- Hericium erinaceus,\u003c/em\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHPLC – high pressure liquid chromatography\u003c/p\u003e\n\u003cp\u003e5-HT - 5-hydroxy-tryptamine, serotonin\u003c/p\u003e\n\u003cp\u003eIL-1β – interleukin 1β,\u003c/p\u003e\n\u003cp\u003eIL-6 – interleukin 6,\u003c/p\u003e\n\u003cp\u003eIL-18 – interleukin 18,\u003c/p\u003e\n\u003cp\u003eNGF - nerve growth factor,\u003c/p\u003e\n\u003cp\u003eNLRP3 -\u0026nbsp;NOD-, LRR- and pyrin domain-containing protein 3\u003c/p\u003e\n\u003cp\u003eOF - open field test,\u003c/p\u003e\n\u003cp\u003ePDJ\u003cem\u003e\u0026nbsp;- Pleurotus djamor\u003c/em\u003e,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSEM - standard error of the mean,\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSSRI - selective serotonin reuptake inhibitor,\u003c/p\u003e\n\u003cp\u003eTNF-α – tumour necrosis factor α\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e Medical University of Bialystok (Grant number: SUB/1/DN/22/001/1166).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e Data supporting these findings are available within this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e: All authors have no conflicts of interests to declare. The authors acknowledge study funding and the supply of investigation the design of this study. No actual or perceived conflicts of interest are reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval: \u003c/strong\u003eThe experimental procedures were carried out in accordance with the European Regulation (EU) 2019/1010 and approved by Local Ethical Committee for Animal Experiments in Olsztyn, Poland (consent LEC 60/2021). All methods are reported in accordance with ARRIVE guidelines.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability:\u003c/strong\u003e The data are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions: \u003c/strong\u003eET, AF, BM wrote the research protocol and HO helped conduct the study on draft and design and performed the experimental on rats, and HC wrote the manuscript, BM also served as supervisor for this manuscript. 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Depression: a new animal model sensitive to antidepressant treatments. \u003cem\u003eNature\u003c/em\u003e \u003cb\u003e266\u003c/b\u003e, 730\u0026ndash;732. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/266730a0\u003c/span\u003e\u003cspan address=\"10.1038/266730a0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (1977).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Hericium erinaceus, Fomitopsis officinalis, Pleurotus djamor, rats, anxiety, depression","lastPublishedDoi":"10.21203/rs.3.rs-6118750/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6118750/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePreclinical studies show that an edible arboreal medicinal mushroom \u0026ndash; \u003cem\u003eHericium erinaceus\u003c/em\u003e (HE), is neuroprotective against high corticosterone levels and modulates stress-coping strategies of rodents. Moreover, other arboreal mushrooms (\u003cem\u003eFomitopsis officinalis\u003c/em\u003e, FO; and \u003cem\u003ePleurotus djamor\u003c/em\u003e, PDJ) had a similar effect.\u003c/p\u003e \u003cp\u003eHere, we explored potential dose-dependency of the effects of dietary supplementation with HE, FO or PDJ on anxiety-like behaviours and stress-coping strategies in rats; and, compared them to a drug commonly prescribed for stress-related psychiatric disorders (fluoxetine, FLX; 20 mg/kg) to evaluate those effects quantitatively and qualitatively.\u003c/p\u003e \u003cp\u003eFor 3 weeks, male Wistar rats were given 0, 100, 250 or 500 mg/kg (\u003cem\u003eper os\u003c/em\u003e) of HE, FO or PDJ lyophilizate obtained from \u003cem\u003ein vitro\u003c/em\u003e cultures. Subsequently, animals were tested in the battery of behavioral tests: open field (OF), elevated plus maze (EPM) and forced swimming (FST) tests.\u003c/p\u003e \u003cp\u003eMiddle dose of FO increased rearing in OFT indicates reduced anxiety-like behavior in rodents, and swimming (while decreasing climbing) in FST indicate antidepressant-like and also anti-anxiety-like effects no worse or even superior to those of fluoxetine.\u003c/p\u003e \u003cp\u003eAt the same time, we concluded that the effects of medicinal mushroom supplementation on stress-related behaviours vary depending on the mushroom species and dosage regimen.\u003c/p\u003e","manuscriptTitle":"Effects of dietary supplementation with in vitro-cultivated arboreal medicinal mushrooms on stress coping strategies, depressive and anxiety-like behaviour of rats.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-13 07:01:34","doi":"10.21203/rs.3.rs-6118750/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"12ae6059-846a-4aa6-8bfe-f982b8d88e86","owner":[],"postedDate":"March 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":45502749,"name":"Biological sciences/Neuroscience"},{"id":45502750,"name":"Biological sciences/Neuroscience/Cognitive neuroscience"}],"tags":[],"updatedAt":"2025-04-15T07:23:09+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-13 07:01:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6118750","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6118750","identity":"rs-6118750","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}