Introduction

The development of stress resilience requires some degree of stress exposure1,2. For instance, young adults who experience work-related stress during adolescence tend to show fewer deleterious health effects during the transition from school to the workforce2. Exploratory analyses suggest that the relationship between childhood adversity and stress reactivity follows a curvilinear pattern: children raised in moderately stressful environments show lower sympathetic reactivity than children in either highly or minimally stressful environments1. While severe stress is undoubtedly harmful, resilience cannot develop without some exposure to manageable stressors.

The concept of stress inoculation proposes that prior exposure to mild, controllable stressors—referred to as “inocula”—enhances an individual’s ability to control future stress responses. This effect has also been shown in animal models3,4,5,6. In rodents, stress inoculation paradigms such as brief maternal deprivation during the neonatal period, or physical restraint and social defeat during adulthood have been shown to reduce depression- or anxiety-like behaviors and promote neuronal growth in the hippocampus4,5,7,8. Although stress inoculation is a potential preventive intervention for psychiatric disorders, it remains less studied than other nonpharmacological approaches, such as physical exercise, probiotic diets, and light therapy9,10,11,12. Consequently, little information is available regarding the specific types and degrees of stress exposure that effectively promote stress resilience.

In this study, we developed a novel stress inoculation model for rodents, termed environmental chronic mild stress (ECMS), by modifying the conventional chronic mild stress (CMS) paradigm. CMS is a well-established approach for inducing behavioral phenotypes associated with clinical depression13 and anxiety9,14, involving rodent exposure to different stressors each day, such as water deprivation, food deprivation, wet bedding, radio noise, cold swimming, restraint and others14,15,16. A critical distinction of ECMS is the deliberate exclusion of stressors that provoke hunger, thirst, or direct physical distress. Instead, rodents in the ECMS protocol were group-housed and exposed daily to mild environmental disturbances intended as inoculative stressors. These stressors include crowding, tilted cages, partial disruption of the light–dark cycles, and wet bedding. Although such stressors reduce environmental comfort, their overall stress intensity is expected to be lower than that of the CMS protocol. Based on the proposed inverted U-shaped relationship between stress intensity and stress responsiveness in organisms1,6, ECMS is expected to promote stress resilience, contrasting with the effects of CMS. We examined the effects of ECMS stress inoculation on body weight, estrous cycle, depression- and anxiety-like behaviors, social interaction (SI), and corticosterone levels in adult female rats, and examined which stress elements may function beneficially as inoculation. Given that females are at a significantly higher risk than males for developing psychiatric disorders17, we conducted this study using female rats. Anxiety- and depression-like behaviors induced by CMS have been reported in both male and female rodents13,18,19, but knowledge of stress inoculation in females remains limited. The findings may offer insights into differentiating between harmful and beneficial stressors and identifying optimal conditions for fostering stress resilience.

Furthermore, we examined the interaction between stress inoculation and supplementation with Lactococcus cremoris H61, a beneficial bacterium. Strain H61 is expected to improve the gut microbiota profile and exert antidepressive effects under stress20 through the microbiota–gut–brain axis21,22,23. Although multiple nonpharmacological approaches can be implemented simultaneously in everyday human settings, their combined effects have rarely been verified. Combining such therapies with different mechanisms may help address a broader range of maladaptive behaviors and could exert synergistic effects. In this study, we examined conditions for the combined use of stress inoculation and H61 intake in anticipation of stronger synergistic effects.

Results

Effects on body weight, food intake, and estrous cycle

ECMS treatment and the H61 diet were initiated at 7 weeks of age (Fig. 1). Body weights increased similarly across all groups from 7 to 15 weeks of age (Fig. 2a), during which the ECMS and H61 treatments were administered. No significant differences in body weight changes were observed among the groups. Neither ECMS nor H61 treatment significantly affected food intake or estrous cycle (Fig. 2b,c). Overall, the treatments did not produce measurable positive or negative effects on body weight, food intake, or estrous cycle.

Fig. 1
figure 1

Experimental timeline. The test diet intervention was administered beginning at 7 weeks of age and continued until the end of the experiment. The ECMS-Control and ECMS-H61 groups were exposed to one of five randomly selected stressors (indicated within a dashed line) for 5 days each week over an 8 week period. Immediately after the 8-week treatment, the estrous cycle, behavioral tests, and serum corticosterone levels were examined in the sequence shown in the figure. To minimize the influence of the estrous cycle on behavioral tests and serum corticosterone levels, only rats identified as being in the metestrus or diestrus phase were included in each test. OFT open field test, EPM elevated plus maze, SI social interaction, SPT sucrose preference test, FST forced swim test.

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Fig. 2
figure 2

Changes in body weight, food intake, and estrous cycle length. (a) Rate of body weight change. (b) Weekly food intake per rat from the beginning to the end of the 8 week ECMS. (c) Estrous cycle length at 14 weeks of age after ECMS treatment. Non-ECMS-Cont.: ECMS was not provided with the control diet, non-ECMS-H61: ECMS was not provided with the H61-containing diet, ECMS-Cont.: ECMS was provided with the control diet, ECMS-H61: ECMS was provided with the H61-containing diet. All data are presented as means ± SEM. n = 12 per group.

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Effects on anxiety-like behaviors in the open field test (OFT) and elevated plus maze (EPM) test

Two-way ANOVAs revealed significant main effects of both the ECMS and H61 diet on time spent in the center and periphery in the OFT. ECMS-treated rats spent significantly more time in the central area (Fig. 3a, F (1, 44) = 6.38, P < 0.05) and less time in the peripheral area (Fig. 3b, F (1, 44) = 4.15, P < 0.05) than non-ECMS-treated rats. Similarly, rats fed the H61 diet spent more time in the central area (Fig. 3a, F (1, 44) = 7.57, P < 0.01) and less time in the peripheral area (Fig. 3b, F(1, 44) = 9.59, P < 0.01) than those fed the control diet. Among ECMS-treated rats, the number of grooming behaviors was significantly reduced (Fig. 3c, F (1, 44) = 6.80, P < 0.05), and the total distance traveled was significantly increased (Fig. 3d, F (1, 44) = 8.78, P < 0.01) compared to non-ECMS-treated rats. Anxiolytic-like changes in grooming and locomotor activity were observed only in the ECMS group, suggesting broader anti-anxiety effects of ECMS than of the H61 diet. Although no significant interaction effect was observed between treatments, ECMS and H61 diet independently reduced anxiety-like behaviors in the OFT. In the EPM test, there were no significant differences in the time spent in the open and closed arms or in the number of entries into the open-end areas (Fig. 4).

Fig. 3
figure 3

Effects of treatments in the OFT. (a) Time spent in the center area. (b) Time spent in the peripheral area. (c) Number of self-grooming behaviors. (d) Total distance traveled. All data are presented as means ± SEM. *P < 0.05, **P ≤ 0.01. n = 12 per group.

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Fig. 4
figure 4

Effects of treatments in the EPM test. (a) Time spent in the open arms. (b) Time spent in the closed arms. (c) Number of reaches toward the open ends. All data are presented as means ± SEM. n = 12 per group.

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Effects on social behavior in the social interaction (SI) test

Data from two animals were excluded because behavioral tests could not be conducted during the metestrus or diestrus periods. Across all groups, rats spent significantly more time near the stimulus cage and within the stimulus zone than the empty cage and empty zone, respectively (Fig. 5a,b, F (1, 42) = 160.90, P < 0.01). No significant differences were observed among groups in the social approaching ratio (Fig. 5c) or the social staying ratio (Fig. 5d). And, both ratios were significantly higher than the expected value of 0.5 (one-sample t-test; Fig. 5c,d). For the social approaching ratio: Non-ECMS-Cont., t (20) = 9.90, P < 0.01; Non-ECMS-H61, t (20) = 4.63, P < 0.01; ECMS-Cont., t (22) = 8.42, P < 0.01; and ECMS-H61, t (22) = 7.60, P < 0.01. For the social staying ratio: Non-ECMS-Cont., t (20) = 7.98, P < 0.01; Non-ECMS-H61, t (20) = 4.15, P < 0.01; ECMS-Cont., t (22) = 6.41, P < 0.01; and ECMS-H61, t (22) = 6.91, P < 0.01. The average total distance traveled was also similar across groups (Fig. 5e). These findings indicate that all rats showed adequate interest in other individuals, and that neither ECMS nor H61 treatment affected social motivation or general locomotor activity.

Fig. 5
figure 5

Effects of treatments in the SI test. (a) Time spent approaching the stimulus and empty cages. (b) Staying time. (c) Social approaching ratio. (d) Social staying ratio. (e) Total distance traveled. All data are presented as means ± SEM. **P ≤ 0.01. n = 11–12 per group.

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Effects on depression-like behaviors in the sucrose preference test (SPT) and forced swim test (FST)

Six rats were excluded from the SPT data analysis, and two from the FST due to misalignment of the estrous cycle on the test day. In the SPT, no significant differences were observed among the groups for any index: sucrose intake (Fig. 6a) and water intake (Fig. 6b). All groups showed sucrose preference ratios significantly above the expected value of 0.5 (Fig. 6c; one-sample t-test). Non-ECMS-Cont., t (20) = 11.46, P < 0.01; Non-ECMS-H61, t (18) = 5.53, P < 0.01; ECMS-Cont., t (18) = 3.63, P < 0.01; and ECMS-H61, t (20) = 5.02, P < 0.01. Similarly, in the FST, there were no significant differences in average immobility time across groups (Fig. 7). These findings indicate that neither ECMS nor H61 treatment had a measurable effect on depression-like behaviors, including anhedonia.

Fig. 6
figure 6

Effects of treatments in the SPT. (a) Amount of sucrose intake. (b) Amount of water intake. (c) Sucrose preference ratio. All data are presented as means ± SEM. *P < 0.05. **P ≤ 0.01. n = 10–11 per group.

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Fig. 7
figure 7

Effects of treatments on immobility time in the FST. All data are presented as means ± SEM. n = 10–12 per group.

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Effects on corticosterone levels

A two-way ANOVA revealed a tendency for an interaction between ECMS and H61 diet treatments (F (1, 35) = 3.65, P < 0.10). Post hoc tests showed that among rats fed the control diet, corticosterone levels were significantly higher in the ECMS-treated group (ECMS-Cont.) than in the non-ECMS-treated group (non-ECMS-Cont.) (Fig. 8, t (35) = 2.34, P < 0.05). Furthermore, after ECMS treatment, rats fed the H61 diet (ECMS-H61) exhibited significantly lower corticosterone levels than those fed the control diet (ECMS-Cont.) (Fig. 8, t (35) = 2.59, P < 0.05). These findings suggest that ECMS treatment increased corticosterone responsiveness to stress, and that this increase could be suppressed by administration of the H61 probiotic diet.

Fig. 8
figure 8

Effects of treatments on corticosterone levels. All data are presented as means ± SEM. *P ≤ 0.05. n = 7–12 per group.

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Discussion

As expected, ECMS contributed to the reduction of anxiety-like behaviors without affecting body weight, food intake, estrous cycle, or sociability in female rats. Continuous exposure to mild environmental stress produced effects opposite to those typically induced by CMS, which evokes anxiety-like behavior, depression-like behavior, or alters the estrous cycle in female rats18,19,24. Our findings, together with previous reports on CMS, support a curvilinear relationship between stress intensity and stress reactivity in females3,25,26, suggesting that mild stress exposure can promote the development of stress resilience. Although ECMS inoculation led to increased corticosterone levels following acute stress exposure, this heightened hormonal response was inhibited by H61 treatment. The combination of ECMS and probiotic supplementation appears to enhance stress resilience without overstimulating the hypothalamic–pituitary–adrenal (HPA) axis.

Successful stress inoculation is typically associated with a moderate elevation in corticosterone or cortisol levels in both males and females, which appears necessary to activate negative feedback circuits in the hippocampus and subsequently reduce the responsiveness of the HPA axis to future stress6,26. In this study, it was suggested that corticosterone levels were elevated during ECMS treatment, thereby contributing to the development of stress resilience. For example, the light–dark cycle disruptions implemented as part of the ECMS protocol may have induced elevated corticosterone secretion. Abnormal light–dark conditions, such as acute constant light exposure for 38 h, have been reported to increase corticosterone levels by affecting hippocampal function in male rodents27. These findings are consistent with previous studies showing that repeated administration of low-dose corticosterone enhances stress resilience28,29. While a modest increase in corticosterone may be necessary for effective stress inoculation, sustained high levels should be avoided due to potential adverse effects. To further optimize the therapeutic efficacy of ECMS, reducing the duration of treatment may be beneficial. In previous rodent studies, stress inoculation protocols lasted 3–4 weeks in males4,5 and 2 weeks in females25. Therefore, ECMS interventions of 4 weeks or less may be sufficient to reduce anxiety-like behaviors without inducing excessive corticosteroid stress responses.

The type of stress resilience appears to depend on the nature of stress exposure used for inoculation. ECMS-treated rats subjected to varying housing sizes and brightness as environmental stressors showed reduced anxiety-like behavior in the OFT. However, because ECMS did not include exposure to heightened conditions, these rats did not show reduced anxiety in the EPM test. A previous study on stress inoculation reported that male mice exposed to nonphysical contact with other individuals showed increased contact time in SI tests but did not increase time spent in the central area in the OFT4. These findings suggest that rodents can develop resilience primarily to stressors similar to those experienced during inoculation, which is consistent with the inoculation mechanism described in humans30. Thus, ECMS may be a useful animal model for examining the neural and biological mechanisms underlying stress inoculation. Additionally, stress inoculation may be more effective than probiotic treatment with beneficial bacteria when the subsequent stress is evident, as ECMS-treated rats showed stronger anti-anxiety behaviors than those treated with the H61 probiotic in the OFT. Similarly, in humans, stress inoculation techniques appear to facilitate coping with predictable environmental stressors, such as starting a new job or transitioning to a new school31,32. To summarize, three key elements are important for effective stress inoculation and resilience acquisition: the mildness of the stress, the nature of the stress, and the duration of treatment. In rodents, stress paradigms such as ECMS that exclude physical distress may be suitable for promoting anti-anxiety effects in OFT. However, prolonged treatment may lead to the overactivation of the HPA axis. Therefore, the duration of therapy should be minimized.

The current ECMS treatment did not reduce immobility time in the FST. In contrast, Parihar et al.8 reported decreased immobility following 5 min of daily restraint stress for 28 days. It has also been reported that despite reduced activity levels in the open field, female rats exhibited active swimming in the FST after conventional CMS that included physical stressors such as food and water deprivation18. Physical forms of CMS, such as restraint or deprivation, may sometimes promote broader resilience, potentially including antidepressant- and anxiolytic-like effects8,18,29. However, repeated restraint for only 20 min over seven consecutive days has been shown to alter neural activation in the basolateral amygdala33. The adverse effects of restraint stress are reportedly more pronounced during adolescence than adulthood34. Therefore, stress inoculation protocols involving physical stressors require careful consideration of both duration and developmental stage, particularly when applied during adolescence.

Understanding the boundary between beneficial and harmful stressors is important. This boundary likely varies across strains and sexes because sensitivity to stress differs among them. In CMS, for example, female Wistar rats are more vulnerable than males18, whereas male Long Evans rats are more vulnerable than females35. The present results with female Wistar–Imamichi rats alone do not clearly define this boundary. To effectively apply stress inoculation for building resilience, further research involving multiple strains and both sexes is needed. Another limitation of this study is that the order of behavioral tests was not randomized. Because behavioral tests themselves are stressful, they were conducted in a fixed order from relatively less to more stressful to minimize carryover effects. However, this fixed order may have influenced the results. Because the OFT was conducted first, ECMS showed an anxiolytic effect, but this effect may have diminished in subsequent tests. Randomizing the order of behavioral tests should be considered in future studies.

We found a complementary effect between ECMS inoculation and diet supplementation with the strain H61, wherein H61 intake attenuated the increased corticosterone reactivity induced by ECMS. These results suggest that consuming effective supplements may promote stress resilience without elevating the physiological burden following stress inoculation. The anxiolytic effects of ECMS and H61 treatments appear to arise from distinct neural mechanisms. Stress inoculation may modulate negative feedback circuits of the HPA axis6, whereas probiotic diets have been associated with suppression of excessive neuroinflammation9,21. These mechanisms may act simultaneously, producing complementary effects. Notably, no synergistic effect was observed between the ECMS and H61 treatments. This may be adaptive for rats, as anxiety-like behavior represents an evolutionarily conserved defensive response that aids danger avoidance and survival in new environments. Excessive suppression of anxiety may not always be biologically advantageous. Thus, combining multiple nonpharmacological therapies may promote safer and more versatile stress tolerance rather than merely maximizing resilience.

Methods

Animals and treatments

Forty-eight 6 week-old female Wistar–Imamichi rats were purchased from the Institute for Animal Reproduction (Ibaraki, Japan). The rats were housed in groups of three per plastic cage (23 × 38 × 20 cm) under standard laboratory conditions (23 °C ± 1 °C; a 12/12 h light–dark cycle) with water and food provided ad libitum. All housing and experimental protocols were approved by the University of Tsukuba Committee on Animal Research (Approval No. 22-408) and were conducted in compliance with the Guidelines for the Care and Use of Laboratory Animals of the Japanese Ministry of the Environment (Notification No. 88, 2006) and related regulations. This study is reported in accordance with the ARRIVE guidelines. The rats were randomly assigned to four experimental groups (n = 12 per group). The first group received a control diet without ECMS treatment (non-ECMS-Cont.), and the second group, which did not receive ECMS but was fed a diet containing strain H61 (non-ECMS-H61). The third and fourth groups were both treated with ECMS and received either the control diet (ECMS-Cont.) or the H61 diet (ECMS-H61). To maintain consistency, rats housed in the same cage were assigned to the same experimental group. The control diet consisted of 100% MF standard rodent feed (MF; Oriental Yeast, Tokyo, Japan). The H61 diet was prepared by supplementing the control diet with 0.05% (w/w) heat-killed strain H61 (Toa-shinyaku).

Experimental schedule and environmental chronic mild stress

Following a 1 week acclimatization period, the non-ECMS-H61 and ECMS-H61 groups began receiving the H61 diet starting at 7 weeks of age. Throughout the ECMS treatment and behavioral testing period, rats were continuously fed either the control diet or the H61 diet. The ECMS treatment also commenced at 7 weeks of age and was administered over an 8-week period. During the ECMS period, the ECMS-Cont. and ECMS-H61 groups were exposed once daily to one of five randomly selected stressors, 5 days per week (Fig. 1). The stressors were designed to mildly disrupt environmental comfort and included (1) cage tilting, in which the cage floor was tilted at a 45° angle for 24 h (10:00 a.m. to 10:00 a.m.); (2) overcrowding, in which three rats were housed in a small transparent plastic cage (26 × 15 × 13 cm) for 24 h (10:00 a.m. to 10:00 a.m.); (3) cage flooding, in which bedding was dampened with 300 ml of tap water for 24 h (10:00 a.m. to 10:00 a.m.); (4) illumination during the dark phase, in which rats were exposed to light during their usual dark period for 12 h (8:00 p.m. to 8:00 a.m.); and (5) darkening during the light phase, in which lights were turned off for 3 h (10:00 a.m. to 1:00 p.m.). After the ECMS period, a series of behavioral tests were conducted, followed by measurement of serum corticosterone levels. All tests were conducted during the light phase (10:00 a.m. to 6:00 p.m.). To minimize the influence of the estrous cycle on both behavioral tests and serum corticosterone levels, only rats in the metestrus or diestrus phases—characterized by low estrogen levels36—were included in each test. Additionally, vaginal smears were collected from all rats at 14 weeks of age (immediately after the 8 week ECMS period) over at least four consecutive days to assess the potential impact of each treatment on the estrous cycle periodicity. Previous studies have reported that stressors, such as CMS, can readily disrupt the estrous cycle24.

Estrous cycle observation

The estrous cycle stage was determined each morning by vaginal smears observed under a microscope, following the method described by McLean et al.37. This process involved inserting a cotton swab moistened with distilled water into the vaginal canal of each rat to collect samples from the vaginal wall, which were then smeared onto slides. For staining, the slides were submerged in 0.1% cresyl violet for 5 min at room temperature, rinsed with tap water, and air-dried. This procedure allowed the identification and categorization of the estrous cycle into four stages: proestrus, estrus, metestrus, and diestrus. The proestrus and estrus stages, both associated with high estrogen levels, were characterized by the presence of nucleated and cornified epithelial cells, respectively. Conversely, metestrus, indicative of low estrogen levels, was characterized by a combination of cornified epithelial cells and leukocytes, while diestrus was marked predominantly by the presence of migrating leukocytes.

OFT

The OFT was performed using a gray polyvinyl chloride apparatus measuring 90 × 90 × 45 cm, with a central area defined as a 54 × 54 cm square. The apparatus was enclosed by a black curtain and illuminated at 47 lx. Each rat was placed in the apparatus facing either the front left or right corner, and its free movement was recorded using a digital camera for 10 min. The total movement distance (Total distance) and time spent in the central area (Center time) and peripheral area (Peripheral time) were analyzed using ANY-maze software (Version 6.16, Stoelting Co., IL, USA). Additionally, the frequency of grooming behavior—defined as the use of paws or tongues to clean or scratch the body—was recorded.

EPM test

The EPM test was performed using an apparatus made of gray acrylic resin, elevated 40 cm above the floor, and illuminated at 680 lx. The open and closed arms of the maze each measured 50 cm in length and 10 cm in width. The closed arms were enclosed by walls 40 cm in height. A central platform was defined as a 10 × 10 cm square at the intersection of the open and closed arms. Each rat was placed on the central platform and allowed to freely explore the maze for 10 min. Behavior was recorded using a digital camera and analyzed using the ANY-maze software. The following parameters were measured: the time spent within the open arms (Time in Open Arms), the time spent in the closed arms (Time in Closed Arms), and the number of times the rat reached the end of the open arms (Number of Reaches Open-end). A reach to the end of an open arm was defined as the extension of a body part, such as the nose or front limbs, beyond the end of the open arm.

SI test

The SI test was performed in a gray acrylic box measuring 40 × 80 × 50 cm. The apparatus was divided into the left, right, and middle zones and featured two cylindrical wire cages positioned at each end to house the stimulus animals. Each wire cage (15 cm in diameter and 18 cm in height) was made of stainless steel, with a 0.5 × 0.5 cm mesh, which allowed for physical interaction between the experimental and the stimulus individual. The apparatus was enclosed by a black curtain and illuminated at 80 lx. Rat behavior was recorded using a camera positioned above the SI setup.

Eight 8 week-old female Wistar–Imamichi rats, purchased from the Institute for Animal Reproduction (Ibaraki, Japan), were used as stimulus individuals. Before the SI test, both the experimental rats and stimulus individuals underwent area habituation. During the area habituation, the experimental animals were allowed 5 min of free exploration in the apparatus, which contained two empty wire cages. Stimuli individuals were placed within the wire cages for 10 min. The following day, each experimental rat was introduced into the middle area of the apparatus and allowed to explore freely for 10 min. One wire cage contained a stimulus individual (Stimulus cage), and the other was left empty (Empty cage). The following behavioral parameters were analyzed using ANY-maze software: duration of approach to each wire cage (approaching time), time spent within each zone (staying time), and total distance traveled. The approaching time was defined as the rat facing a wire cage with its nose within 2 cm of the mesh. The staying time was defined as the duration the rat remained in either the left or right zones. The social approaching ratio and social staying ratio were calculated as follows: social approaching ratio = approaching time to stimuli cage / (approaching time to stimuli cage + empty cage), social staying ratio = staying time in zone with stimuli cage/(staying time in zone with stimuli cage + empty cage).

SPT

The SPT spanned four consecutive days, with sucrose preference assessed on the fourth day. Rats were habituated to the sucrose solutions by placing two bottles of 1% sucrose solutions for 24 h, followed by replacing one of the bottles with water for the next 24 h. On the third day, the rats underwent 24 h of food and water deprivation. On the fourth day, each cage was provided with two bottles of sucrose and water; the volume consumed after 24 h was measured, and the sucrose preference was calculated as a percentage.

FST

The FST was performed using a cylindrical apparatus made of acrylic resin, measuring a diameter of 20 cm and a height of 45 cm. The apparatus was filled to a height of 35 cm with water to prevent the rat’s tail from touching the bottom, and the water temperature was maintained at 23 °C ± 2 °C. The FST consisted of two swim sessions: a 15 min pretest followed by a 5-min test, conducted 24 h apart. In each session, each test rat was placed into the apparatus, and their movements were recorded using a digital camera to detect immobility as an indicator of depression-like behavior. Immobility time was defined as the duration during which the entire body, including all four limbs, ceased movement and sank20. Floating responses were excluded from immobility scoring, as they have been indicated not to be a depression-like state but rather an adaptive coping mechanism to escape from the water38,39. Floating was defined as a state in which the body remained buoyant with immobile limbs while the rat’s nose stayed above the water surface. Behavioral data were analyzed using ANY-maze video-tracking software.

Corticosterone assay

To assess serum corticosterone levels following acute stress exposure, tail blood samples were collected 20 min after the initiation of the FST. Each rat was placed in a cylindrical polyethylene restraint apparatus, and its tail was immediately incised using a razor blade to obtain the blood sample. To minimize the confounding effects of restraint-induced stress on serum corticosterone levels, all procedures were completed within 150 s. If blood sampling was not completed within this timeframe, the process was discontinued. Following the blood sampling, the rats were euthanized by an overdose of pentobarbital sodium. A total of 39 blood samples were successfully collected and subjected to hormonal analysis from the following groups: non-ECMS-Cont. (n = 7), non-ECMS-H61 (n = 9), ECMS-Cont. (n = 12), and non-ECMS-H61 (n = 11). Samples from rats with insufficient blood volume were excluded. The collected blood was centrifuged at 10,000 rpm for 15 min, after which the supernatant serum was harvested and stored at − 60 °C. The concentration of corticosterone in the blood serum was measured using a commercial ELISA kit (YK240 Corticosterone EIA Kit; Yanaihara Institute Inc., Shizuoka, Japan), following the manufacturer’s instructions.

Statistical analysis

Statistical analyses were conducted using SPSS. Data were analyzed using two-way or three-way ANOVA, followed by Holm–Sidak post hoc tests or one-sample t-tests, as appropriate. Differences were considered statistically significant at P < 0.05, and a trend of significance was noted when P < 0.10. All data are presented as means ± SEM.