Modulation of gut microbiota composition due to early weaning stress induces depressive behavior during the juvenile period in mice

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Early weaning (EW), a form of social stress in mice, leads to increased anxiety and an enhanced stress response in the hypothalamic-pituitary-adrenal axis during adulthood. Early life stress also modulates the immune system and increases vulnerability to infection. However, studies investigating the causal relationships among juvenile stress, microbiota changes, and immune and behavioral deficits are limited. Therefore, we hypothesized that EW alters gut microbiota composition and impairs the development of the nervous and immune systems. Results: EW mice moved longer distances in the marble-burying test and had longer immobility times in the tail suspension test than normal weaning (NW) mice. In parallel, the gut microbiome composition differed between NW and EW mice, and the abundance of Erysipelotrichacea in EW mice at 8 weeks of age was lower than that in NW mice. In an empirical study, germ-free mice colonized with the gut microbiota of EW mice (GF-EW mice) demonstrated higher depressive behavior than GF mice colonized with normal weaning microbiota (GF-NW mice). Immune cell profiles were also affected by the EW microbiota colonization; the number of CD4+ T cells in the spleen was reduced in GF-EW mice. Conclusion: Our results suggest that EW-induced alterations in the gut microbiota cause depressive behaviors and modulate the immune system. Microbiome Stress Behavior Depression Germ-Free Mouse Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background It has been shown that stress exposure in early life or inadequate maternal care causes several physiological and behavioral alterations in adulthood. Separation from the dam before weaning is a rodent model of developmental stress, and early weaning (EW) enhances stress responses of the hypothalamic-pituitary-adrenal (HPA) axis and increases anxiety and lowers cognitive function in adulthood, similar to human-abused children [ 1 – 3 ]. Early-weaned pups might be under physiological and psychological stress, and the behavioral and neuroendocrine changes caused by EW have been suggested to be related to changes in gene expression in the brain, probably programmed via epigenetic changes in neurons [ 4 , 5 ]. Furthermore, stress in early life affects immune function [ 6 , 7 ]. Accumulating evidence has implicated crosstalk between physiological stress and gut microbiota. For example, it has been reported that germ-free (GF) mice display excessive stress responsiveness compared with specific-pathogen-free (SPF) mice [ 8 ], and maternal separation alters the gut microbiota, thereby shaping increased stress and elevated anxiety after growth [ 9 ]. In addition, gut microbiota disruption in pregnant mice by antibiotic treatment induces high anxiety behaviors in their offspring [ 10 ]. Dynamic changes in the microbiota during the juvenile period occur concurrently with central nervous system development, suggesting that the gut microbiota in neonates and juveniles can shape stress and anxiety behaviors [ 11 ]. The timing of early life exposure to bacteria and the bacterial community composition have permanent effects on immune system development and symbiotic bacteria composition [ 12 , 13 ]. The immune system communicates with the central nervous system and is involved in mediating anxiety and depression [ 14 ]. Individual differences in the peripheral immune system can predict and promote susceptibility [ 15 ]. The microbiome is a causal factor in autism [ 16 ], and children with autism have a dysregulated immune system [ 17 ]. Maternal immune activation promotes behavioral abnormalities, and these phenotypes in the offspring require maternal intestinal bacteria that promote T helper 17 cell differentiation [ 18 ], suggesting that the gut microbiota regulates behavioral phenotypes via immunological pathways in the host. However, studies investigating the causal relationships among juvenile stress, microbiota changes, and immune and behavioral deficits are limited. In this study, we conducted an empirical analysis of how EW stress affects behavior and gut microbiota, and which of these changes in behavior and the immune system are caused by changes in the gut microbiota in gnotobiotic mice. Results EW mice show more depressive behavior in the juvenile period First, we assessed anxiety-like and depressive behaviors in EW and normal weaning (NW) mice on PD29 and 56 (Fig. 1 A). In the tail suspension test, the immobility time of EW mice was longer than that of NW mice at 4 weeks of age and shorter than that of NW mice at 8 weeks of age (Fig. 1 B: week; F1,55 = 0.150, p = 0.700, group; F1,55 = 0.341, p = 0.562, week × group interaction; F1,55 = 21.886, p < 0.001). The immobility time of EW mice was decreased from 4 to 8 weeks of age, while the immobility time of NW mice was increased from 4 to 8 weeks of age. In the marble burying test, the distance moved by the EW mice was greater than that moved by the NW mice at both 4 and 8 weeks of age (Fig. 1 C: week; F1,59 = 50.742, p < 0.001, group; F1,59 = 0.049, p = 0.826, week × group interaction; F1,59 = 0.049, p = 0.826). Although marble-burying behavior increased from 4 to 8 weeks of age, no differences were observed between the NW and EW mice (Fig. 1 D: week; F1,119 = 22.672, p < 0.001, group; F1,119 = 1.069, p = 0.303, week × group interaction; F1,119 = 0.030, p = 0.864). In the open field test, the distance traveled and center duration of the EW mice were not different from those of the NW mice. (Fig. S1 A and B). EW alters the gut microbiota composition in adulthood 16S ribosomal RNA (rRNA) gene sequencing was performed to assess the fecal microbiome. At 4 weeks of age, there was almost no difference in the gut microbiota composition between NW and EW mice; however, the abundance of Erysipelotrichaceae in EW mice at 8 weeks of age was lower than that in NW mice (Fig. 2 A). Bacterial richness and evenness, estimated using the Shannon index, but not the Chao1 index, were higher in NW mice than in EW mice at 8 weeks of age (Fig. 2 B). Principal coordinate analysis (PCoA) based on weighted UniFrac distances depicted age-related changes in the microbiota composition along the first two principal coordinate axes (PCo1 and PCo2; Fig. 2 C). Changes along the PCo2 axis were diminished in EW mice (Fig. 2 D), implying an immature microbiota in the adult EW mice. Furthermore, linear discriminant analysis effect size (LEfSe) was performed to identify the bacterial taxa enriched in EW and NW mice, and there were significant differences between the NW and EW groups (Fig. 3 A and B). Transplantation of gut microbiota from the EW mice induces more depressive behaviors in the juvenile period To evaluate the effect of gut microbiota modulation by EW on host behavior, we observed the behavior of GF mice colonized with gut microbiota from EW or NW mice (Fig. 4 A). In the tail suspension test, the immobility time of GF-EW mice was longer than that of GF-NW and GF mice at 4 weeks of age, and this difference disappeared at 8 weeks of age (Fig. 4 B: week, F1,92 = 10.649, p < 0.01; group, F2,92 = 5.532, p < 0.01; week × group interaction, F2,92 = 5.905, p < 0.01). At 4 weeks of age, GF-EW mice moved considerably further than GF and GF-NW mice, and this difference disappeared at 8 weeks of age (Fig. 4 C: week, F1,92 = 10.649, p < 0.01; group, F2,92 = 5.532, p < 0.01; week × group interaction, F2,92 = 5.905, p < 0.01). The marble-burying behavior of GF-EW mice was not different from that of GF-NW and GF mice at both 4 and 8 weeks of age, and the marble-burying behavior in all mouse groups increased from 4 to 8 weeks of age (Fig. 4 D: week; F1,78 = 26.955, p < 0.001; group: F2,78 = 0.161, p = 0.851, week × group interaction; F2,78 = 0.484, p = 0.618). In the open field test, there was no significant difference in the distance traveled and center duration between the GF-EW group and the other groups. (Fig. S1 C and D). EW microbiota does not affect fecal corticosterone levels. To observe whether the change in behavioral phenotype caused by the EW microbiota was accompanied by HPA axis enhancement, fecal corticosterone levels were measured over time. Fecal corticosterone levels in the GF-EW mice were comparable to those in the GF-NW mice (Fig. S2: week; F7,63 = 7.591 p < 0.001, group; F1,9 = 0.013, p = 0.911, week × group interaction; F1,63 = 1.953, p = 0.076). GF-EW mice have a different gut microbiota composition from GF-NW mice There were no consistent differences in the microbial composition (Fig. 5 A). The Shannon index of the GF-EW group was lower than that of the GF-NW group at 4 weeks of age (Fig. 5 B). Furthermore, LEfSe identified differentially abundant taxa in the NW and EW groups at both ages (Fig. 5 C and D). The gut microbiota of EW mice reduces the cell number of T cells To elucidate the effects of EW-induced gut microbiota changes on the immune system, we analyzed the spleen and gut immune cells of GF, GF-NW, and GF-EW mice (Fig. 6 ). The GF-EW group had fewer CD4 + T cells in the spleen than the other groups did. Similarly, the GF-EW group had fewer CD4 + T cells, B cells, and dendritic cells in the small intestinal lamina propria (SILP), and fewer CD4 + T cells in the mesenteric lymph nodes (mLN) than the GF-NW group. CD8 + T cell numbers in the SILP of GF mice were lower than those in GF-NW mice, and GF-EW mice were intermediate between those in GF and GF-NW mice. The numbers of CD4 + and CD8 + T cells in the mLN of GF-EW mice were lower than those in GF-NW mice, and the number of CD4 + T cells in GF mice was intermediate between those in GF-EW and GF-NW mice. In addition, the number of B cells was lower in GF-EW mice than in GF mice, with GF-NW mice in the middle. No difference in the number of mLN dendritic cells was observed between the groups. Discussion Studies investigating the causal relationships among juvenile stress, microbiota changes, and immune and behavioral deficits are limited. Here, we conducted an empirical analysis of how EW stress affects behavior and gut microbiota, and which of these changes in behavior and the immune system are caused by changes in the gut microbiota in gnotobiotic mice. In this study, we demonstrated that mice exposed to EW stress exhibit more depression-like behaviors and hyperactivity at 4 weeks of age. The fecal microbiome composition of these mice differed from that of NW mice, especially at 8 weeks of age. In an empirical study using GF mice, GF-EW mice orally administered microbiomes from EW mice demonstrated more depression-like behaviors at 4 weeks of age. GF-EW mice demonstrated different fecal microbiome compositions and immune cell profiles compared with GF-NW mice. These results suggest that changes in the gut microbiota composition due to EW stress may regulate developmental behavioral phenotypes and the immune system. We investigated the effects of EW stress on the behavioral phenotypes of mice. EW mice demonstrated more locomotor activity in the marble-burying test and a higher immobility time in the tail suspension test at 4 weeks of age. The longer travel distance in the marble-burying test in EW mice was maintained up to 8 weeks of age, but the immobility time in the tail suspension test of EW mice was reduced at 8 weeks of age. Our findings are partially supported by a report by George et al. showing that mice exposed to early life stress exhibit hyperactivity and increased depressive behavior [ 19 , 20 ]. However, the reduced depressive behavior of EW mice at 8 weeks of age is inconsistent with the increased depressive behavior of 10-week-old maternally separated mice with early weaning at PD17 [ 19 ]. This discrepancy may be explained by the presence of maternal separation in the neonatal period. There were differences in the gut microbiota composition between EW and NW mice, especially at 8 weeks of age. Moussaoui et al. reported that early life stress-exposed rat pups had decreased fecal microbial diversity, and the composition was characterized by an increased abundance of gram-positive cocci and a reduction in fiber-degrading butyrate-producing bacteria [ 21 ]. In humans, changes in microbiota and supplementation with specific bacteria early in development may mitigate the behavioral and mental health consequences of early bacterial disruption [ 22 ]. The gut microbiota of patients with depression demonstrated an increase in Thermoanaerobacteraceae and a decrease in Prevotellaceae compared to healthy individuals, and administration of the gut microbiota from patients with depression to antibiotic-treated rats resulted in depressive-like behavior and increased inflammatory cytokine levels in the blood [ 23 ]. We also found that depressive behavior at 4 wk of age in GF mice colonized with EW mouse gut microbiota was as high as that in EW mice at 4 weeks of age. These findings support our hypothesis that EW stress alters the gut microbiota composition and that this change is a causal factor of depression-like behavior in mice. EW mice demonstrated a shorter immobilization time in the tail suspension test at 8 weeks of age, but GF-EW mice did not show any difference compared with GF-NW mice at the same age. This discrepancy could be due to the timing of exposure of the bacterial community. EW mice were naturally exposed to the microbiome at birth, whereas GF-EW mice were germ-free until the first exposure to the microbiota on postnatal day (PD) 10. The first colony formation in the gut microbiome has an important effect on host immune development [ 24 ]. This difference in timing may have caused the differences in depression-like behaviors observed in this study. It was found that the EW mice and the NW mice at 4 weeks of age did not show a clearly different gut microbiota composition, but at 8 weeks of age, they demonstrated a different gut microbiota composition. Therefore, it is hypothesized that EW stress exposure induces moderate but critical changes in the gut microbiota composition during the juvenile period; however, these changes become obvious in the gut microbiota composition after growth. The reasons for this time lag between stress exposure and changes in the gut microbiota are not clear; however, it has been reported that early life stress alters the pro-inflammatory cytokine expression and may serve as a primer for a secondary challenge that can induce lifelong immune changes [ 25 ]. Further studies are required to clarify the mechanisms and effects underlying long-lasting changes in the microbiome. Fecal corticosterone levels in GF-EW mice were comparable to those in GF-NW mice, suggesting that the changing gut microbiota associated with EW does not affect the basal activity of the HPA axis. In a previous study, our group reported higher basal and stress-response corticosterone concentrations in EW mice than in NW mice [ 3 ]. Therefore, we hypothesized that transplantation of the EW-gut microbiome would increase HPA activity; however, we did not observe any such effects. In our previous study, the fecal corticosterone concentration of GF mice was lower than that of SPF mice, and treatment of GF mice with fecal-derived bacterial solution from SPF mice increased the fecal corticosterone concentration compared to SPF mice [ 26 ], suggesting that there are bacteria in the feces of SPF mice that increase corticosterone concentration in GF mice. Sudo et al. reported that the higher HPA axis responses, but not basal responses, of sterile mice were suppressed to the same level as those of SPF mice by transplanting the feces of SPF mice into sterile mice by 6 weeks of age [ 8 ]. Therefore, it is feasible that the gut microbiota influences HPA axis development. The composition of the microbiota and the establishment of characteristic bacterial species at any given developmental period need to be clarified in further studies. Our study revealed that GF mice colonized with the gut microbiota of EW mice demonstrated an immune cell profile different from that of GF mice treated with the gut microbiota of NW mice. The gut microbiota alters the immune development and function of the host [ 27 ]. For example, it has been reported that a strain of Clostridium from the human gut microbiota promotes Treg cell differentiation [ 28 ], and short-chain fatty acids from the gut microbiota regulate IL-22 production by CD4 + T cells [ 29 ]. In this study, GF-EW mice exhibited a reduced number of CD4 + T cells, especially in the spleen. The depletion of CD4 + CD25 + T Cells has been reported to increase immobility time in forced swimming tests in non-stress-exposed mice. In humans, low levels of serum CD4 + CD25 + T cells have been observed in patients with major depression [ 30 ]. Depressive behavior is reduced by Lactobacillus rhamnosus JB-1 [ 31 , 32 ]. These effects may alter the behavioral phenotypes of GF-EW mice. Inflammatory cytokines are increased in depressed patients and mouse models of depression [ 33 ], and TNF-α and IL-6 expression is elevated in the prefrontal cortex and orbitofrontal cortex of depressed patients who die by suicide [ 34 ]. Goshen et al. reported that mice exposed to chronic mild stress (CMS) (a model of depression) have increased levels of IL-1, a pro-inflammatory cytokine, in the hippocampus, and that IL-1 receptor knockout (KO) mice no longer exhibit CMS-induced depressive behavior [ 35 ]. Because EW has been reported to impair the mucosal immune response to infectious pathogens in animals, EW manipulation may impair immune function development [ 7 ]. In conclusion, our findings demonstrate that EW-induced alterations in the gut microbiota cause depressive behaviors and modulate the immune system. These results support the possibility that the changes in the gut microbiota of EW mice observed in this study may increase depression-like behavior in mice via the immune system. The present results do not demonstrate that this microbiome-immune system is a causal pathway to modulate depression-like behavior, and future studies are warranted. Materials and Methods Animals We used C57BL/6J mice (CLEA Japan Inc., Tokyo, Japan) and their germ-free (GF) counterparts (CLEA Japan Inc.). All mice were maintained under a standard 12 h: 12 h light-dark cycle and provided with a pelleted diet and water ad libitum. The environment was maintained at a constant temperature (24 ± 1°C), and humidity (50 ± 5%). Two to three mice were housed per cage. All mice were tested in behavioral experiments at 4 and 8 weeks of age and euthanized at 10 weeks of age. All experimental procedures were approved by the Animal Ethics Committee of the Azabu University (# 150316-3). Experimental procedures Experiment 1. Effects of early weaning on behavior and gut microbiota To determine whether EW stress alters the gut microbiota composition and behavioral phenotypes, specific-pathogen-free (SPF) mice were assigned to two groups. Conventional pregnant mice were purchased from CLEA Japan, checked every morning until delivery, and fed a standard pelleted diet (MM3, Funabashi Farm Co., Funabashi, Japan) and tap water. The experimental schedule for Experiment 1 is shown in Fig. 1 A. On postnatal day (PD) 16, half the litter of pups was separated from the dam and divided into females and males, and they were assigned to the EW (n = 21) group. The EW mice were fed a powdered diet until day PD28. On PD28, the remaining pups were weaned (normal weaning; n = 44). At PD29 and PD56, behavioral tests were conducted on all mice in a soundproof room to determine the effects on developing offspring and whether the effects of the bacterial flora were maintained in adults. Experiment 2. Behavioral and immune changes due to EW microbiota We examined the causal relationship between gut microbiota composition modulation by EW and the development of behavior and/or the immune system. Pregnant GF mice were purchased from CLEA Japan and housed in groups in a vinyl isolator (Sanki Kagaku Kougei Co., Kanagawa, Japan). All GF mice were fed a sterile pellet diet (CMF 50kGy, ORIENTAL YEAST, Tokyo, Japan) and sterile water. Immediately after transferring pregnant GF mice to a vinyl isolator, swab tests were conducted as previously described (Kamimura et al., 2019) to ensure a germ-free state. The vinyl isolator was swabbed with an ICR-swab (Merck Millipore, Darmstadt, Alemanha) and then incubated at 25°C for 48 h, and turbidity was measured with an absorptiometer (Shimazu CO., Kyoto, Japan). The schedule for Experiment 2 is shown in Fig. 4 A. When the pups were on PD10, each GF dam was orally administered the gut microbiota from the EW or NW mice on PD56 (GF-EW, n = 22 or GF-NW mice, n = 23). The fecal sample was filtered using a mesh 100µm cell strainer followed by centrifugation. The supernatant was removed and the remaining pellets were dissolved in autoclaved sterilized saline. The solution was transferred to a sterilized vinyl isolator for subsequent oral FMT administration. We administered 100 µL to each mouse. Autoclaved saline was orally administered to dams in the control group of GF mice (n = 6). Donor EW and NW mice from each cohort were the littermates. All mice were weaned on PD28, and the dam was removed from the vinyl isolator. Behavioral tests were performed using a vinyl isolator. After the tests, the mice were moved to a conventional environment and euthanized on PD70. Given the sex differences in response to EW stress reported in our previous studies [ 2 ], feces were collected from male mouse donors. In this experiment, three pairs of litters were examined at different time points using different donor feces. Behavioral tests The behavioral tests in Experiment 1 were conducted in a soundproof room, whereas those in Experiment 2 were conducted in vinyl isolators. In Experiment 2, behavioral tests other than the open field test were performed in a vinyl isolator in which each mouse group was bred, and the animals were subjected to open field testing immediately after being aseptically transferred to a new vinyl isolator per group in an open arena. Sterilized tools (open arena, stainless-steel marble, test cages, and clips) were used in Experiment 2. All behavioral tests were performed under fluorescent light during the light period. Tail suspension test The mouse tail tip was fixed with a clip, the suspended mouse was video-recorded for 6 min, and the duration of immobility was measured. Mice were considered immobile only when they hung passively and were completely motionless. The recorded behavior was coded into numerical numbers to be blinded to the condition, and two experimenters who were blind to the conditions performed the analyses. Open field test The mice were released into an open arena (30 cm × 30 cm) with a defined central area (20 cm × 20 cm), and their behavior was videotaped for 5 min. To analyze motor activity and anxiety, we used the Ethovision XT version 10 (Noldus, Wageningen, Netherlands) to measure the distance traveled and the duration spent in the central area (Kulbeth et al., 2021). Marble burying test Before the marble burying test commenced, the animals were habituated to the test cages (17.5 × 24.5 × 12.5 cm) containing 3 cm deep bedding (Japan SLC, Inc., Sioka, Japan) for 5 min. After habituation, we spaced 12 stainless steel-made marble (φ 9.58 mm) on the bedding at 3 cm intervals. For the experiments, mice were released into the test cage, their behavior was videotaped for 25 min, and the number of buried marbles was recorded. We defined the condition that two-thirds of the marbles were buried in the bedding as "buried.” Ethovision XT version 10 (Noldus, Wageningen, Netherlands) was used to measure the distance traveled. In Experiment 2, because the recorded videos could not be analyzed automatically by Ethovision XT owing to the vinyl isolator cover, we edited them using AviUtl version 1.00 ( http://spring-fragrance.mints.ne.jp/aviutl/ ) to increase detection sensitivity. Fecal sample collection Due to the high-stress responsiveness of GF mice (Sudo et al., 2004), we performed minimally stressed fecal sampling rather than stress-bearing blood sampling. Fresh fecal samples from all mice were collected directly into sterilized collection tubes between 10:00 and 13:00 at PD28 and PD56, suspended in phosphate-buffered saline containing 20% glycerol, immediately frozen using liquid nitrogen, and stored at − 80°C until use. Fecal samples for measuring corticosterone were collected from the three groups of GF-NW (n = 9) and GF-EW (n = 7) mice immediately before the cage changes, and then they were stored at − 20°C until processing and were collected every other week between 10:00 and 11:00 during the period when the mice were 4–8 weeks of age. Microbiome analysis Fecal samples frozen at − 80°C were thawed on ice before use. Bacterial DNA was extracted as described previously (Miyauchi et al., 2020). The 16S ribosomal RNA (rRNA) gene V3-V4 region (341F–806R) was PCR-amplified from the bacterial gene using the 16S metagenomic sequencing library protocol (Kozich et al., 2013). The amplicons were purified and quantified using the AMPure XP (Beckman Coulter, California, USA) and Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Samples were pooled in equal concentrations and sequenced on a MiSeq system (Illumina, California, USA; 2 × 300 bp, paired-end reads). The sequenced reads were demultiplexed using bcl2fastq (v1.8.4), and the resulting fastq files were processed with DADA2 (v.1.18.0) (Callahan et al., 2016) using the parameters described in the tutorial pipeline ( https://benjjneb.github.io/dada2/tutorial_1_8.html ). After trimming the low-quality reads, duplicate reads were used for amplicon sequence variant (ASVs) inference. Chimera-free ASVs were assigned to the SILVA database v138 using a naïve Bayesian classifier implemented in DADA2. The phyloseq package (v.1.34.0) in R (v.4.0.3) was used for the downstream analysis. After the removal of mitochondria and chloroplast reads, we obtained 5,156,243 total reads (37,364 ± 12,252 [mean ± SD]). For alpha and beta diversities, data were rarefied to a minimum number of reads per sample (11,658 reads). Linear discriminant analysis (LDA) effect size (LEfSe) was performed to detect bacterial taxa with significantly different abundances between groups using the LEfSe software (Segata et al., 2011). Fecal corticosterone analysis Fecal corticosterone concentrations were measured according to published protocols (Kamimura et al., 2019). ELISA plates were coated with anti-rabbit IgG antibody solution (Jackson ImmunoResearch Laboratories, PA, USA, Cat # 111-005-003, RRID: AB_2337913) and washed twice with a plate washer (Immunowash Model 1250; Bio-Rad Laboratories, Inc., CA, USA). Aliquots of an anti-corticosterone antibody raised in rabbits (Cosmo Bio Co., Tokyo, Japan, Cat# FKA-420-E, RRID: AB_10708379) and aliquots of HRP-labeled corticosterone (FKA419; Cosmo Bio Co., Tokyo, Japan) were added to each well, and the reaction was stopped by adding N-H2SO4. The absorbance of the samples was measured using an automatic microplate reader (Bio-Rad Model 550; Bio-Rad Laboratories Inc., Hercules, CA, USA). Immunological profiling The spleens, mesenteric lymph nodes (mLN), and Peyer’s patches were processed by homogenization on a 70 µm cell strainer, and the splenocytes were treated with red blood cell lysis buffer (BioLegend, California, USA). Lamina propria lymphocytes from the small intestine were isolated as previously described (Miyauchi et al., 2020). In brief, the epithelial layer was removed by agitating the small intestine in Hank’s balanced salt solution supplemented with 2% fetal bovine serum (FBS), 1 mM dithiothreitol, and 20 mM EDTA for 30 min at 37°C. The tissue was minced and digested in RPMI 1640 medium containing 2% FBS, 400 U/mL collagenase D (Roche, Basel, Switzerland), 0.25 U/mL dispase (BD Biosciences, New Jersey, USA), and 0.1 mg/mL DNase I (Wako, Hiroshima, Japan) for 30 min at 37°C with agitation. Lymphocytes were harvested at 40%/80% Percoll interphase. To stain dead cells, single-cell suspensions were incubated with Zombie Aqua dye (BioLegend, San Diego, CA, USA) for 15 min at room temperature. After Fc receptors were blocked with anti-CD16/32 antibody (BD Biosciences, New Jersey, USA), the cells were stained with anti-CD3ε (APC-Cy7, BioLegend, California, USA), anti-CD4 (APC, BioLegend, California, USA ), anti-CD8α (PerCP–Cy5.5, BioLegend, California, USA), anti-CD19 (FITC, BD Biosciences, New Jersey, USA), anti-CD11c (BV450, BD Biosciences, New Jersey, USA), and anti-MHC class II (biotin, Thermo Fisher, Massachusetts, USA) antibodies, followed by labeling with APC-Cy7-streptavidin conjugate (BioLegend, California, USA). The cells were fixed with the Foxp3 staining buffer set (Thermo Fisher Scientific) and stored at 4 ºC. All data were collected using a FACSCanto II cytometer (BD Biosciences, California, USA) and analyzed using FlowJo (version 10.5, Tree Star). Statistical analysis The data on the behavioral tests were analyzed using two-way repeated-measures ANOVA to evaluate the differences between groups, and we added a post-hoc test using the Bonferroni correction. A generalized linear mixed model was constructed with the week of the experiment and the group as dependent variables when nonparametric tests were applicable. The data on the 16S rRNA analysis were analyzed using a one-way ANOVA to evaluate the differences between groups, and we added a post hoc test using Tukey's multiple comparison test. Dunn's multiple comparisons test was added after the Kruskal-Wallis test when parametric preconditions were not met. Declarations Competing interests The authors declare no conflict of interest. Data availability statement Raw sequencing data for the 16S rRNA gene are available from the DDBJ Sequence Read Archive (DRA013030). The data supporting the findings of this study are available from the corresponding author, TK, upon reasonable request. The raw data were generated at Azabu University. Conflict of Interest The authors declare no conflicts of interest associated with this manuscript. Author Contributions IK and EM, TT carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study, and drafted the manuscript; AU, NT, KT, and IK carried out the in vivo experiments and IK conducted the behavioral statistics. EM, TT, HN, TT, TK, and MK prepared the cell suspensions for FACS analysis. EM and MK contributed to the 16S rRNA gene sequencing. HO, MN and KM, TK conceived of the study, designed the study, coordinated the study, and helped draft the manuscript. All authors provided their final approval for publication and agreed to be held accountable for the work performed. Funding This work was funded by grants from the JPSP KAKENHI (19H00972 to TK and 18H02356 to KM), and this study was supported by the Center for Human and Animal Symbiosis Science, Azabu University. Acknowledgments References Kikusui T, Takeuchi Y, Mori Y. Early weaning induces anxiety and aggression in adult mice. Physiol Behav. 2004;81:37–42. Kikusui T, Mori Y. Behavioral and neurochemical consequences of early weaning in rodents. J Neuroendocrinol. 2009;21:427–31. Kikusui T, Nakamura K, Kakuma Y, Yuji M. Early weaning augments neuroendocrine stress responses in mice. Behav Brain Res. 2006;175:96–103. Nakamura K, Kikusui T, Takeuchi Y, Mori Y.: Changes in social institution-and food restriction-induced aggressive behaviors and hippocampal 5HT1B mRNA receptor expression in male mice from early weaning. Behav Brain Res. 2008;187:442–8. Kikusui T, Kanbara N, Ozaki M, Hirayama N, Ida K, Tokita M, et al. 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Tochitani S, Ikeno T, Ito T, Sakurai A, Yamauchi T, Matsuzaki H.: Administration of non-absorbable antibiotics to pregnant mice to perturb the maternal gut microbiota is associated with alterations in offspring behavior. PLoS One. 2016;11:e0138293. Microbiota N. Microbiota and neurodevelopmental trajectories: role of maternal and early life nutrition. Hansen CHF, Nielsen DS, Kverka M, Zakostelska Z, Klimesova K, Hudcovic T, et al. Patterns of early gut colonization shape the host immune response. PLoS One. 2012;7:e34043. Gomez de Agüero M, Ganal-Vonarburg SC, Fuhrer T, Rupp S, Uchimura Y, Li H, et al. Maternal microbiota drive early postnatal innate immune development. Science. 2016;351:1296–302. Reiche EMV, Nunes SOV and Morimoto HK. Stress, depression, the immune system, and cancer. Lancet Oncol. 2004;5:617–25. Hodes GE, Pfau ML, Leboeuf M, Golden SA, Christoffel DJ, Bregman D, et al. Individual differences in the peripheral immune system promote the resilience and susceptibility to social stress. Proc Natl Acad Sci U S A. 2014;111:16136–41. Sharon G, Cruz NJ, Kang D-W, Gandal MJ, Wang B, Kim Y-M, et al. Human gut microbiota from autism spectrum disorder patients promotes behavioral symptoms in mice. Cell. 2019;177:1600-1618.e17. Meltzer A, Van de Water J. Role of the immune system in autism spectrum disorder. Neuropsychopharmacol. 2017;42:284–98. Kim S, Kim H, Yim YS, Ha S, Atarashi K, Tan TG, et al. Maternal gut bacteria promote neurodevelopmental abnormalities in mouse offspring. Nature. 2017;549:528–32. George ED, Bordner KA, Elwafi HM, Simen AA. Maternal separation with early weaning: A novel mouse model of early life neglect. BMC Neurosci. 2010;11:123. Bangsgaard Bendtsen KM, Krych L, Sørensen DB, Pang W, Nielsen DS, Josefsen K, et al. Gut microbiota composition correlated with grid-floor-induced stress and behavior in BALB/c mice. PLoS One. 2012;7:e46231. Moussaoui N, Jacobs JP, Larauche M, Biraud M, Million M, Mayer E, et al. Chronic early life stress in rat pups alters basal corticosterone levels, intestinal permeability, and fecal microbiota at weaning, which is influenced by sex. J Neurogastroenterol Mtil. 2017;23:135–43. Vogel SC, Brito NH, and Callaghan BL. Early life stress and the development of infant gut Microbiota: Implications for mental health and neurocognitive development. Curr Psychiatry Rep 2020;22:61. Kelly JR, Borre Y, O’ Brien C, Patterson E, El Aidy S, Deane J, et al. Transferring the blue: Depression-associated gut microbiota induce neurobehavioral changes in rats. J Psychiatr Res. 2016;82:109–18. Gensollen T, Iyer SS, Kasper DL, Blumberg RS. How microbiota colonization in early life shapes the immune system. Science. 2016;352:539–44. Duque-Quintero M, Hooijmans CR, Hurowitz A, Ahmed A, Barris B, Homberg JR, et al. Endurance effects of early life adversity on reward processes: A systematic review and meta-analysis of animal studies. Neurosci Biobev Rev. 2022;142:104849. Kamimura I, Watarai A, Takamura T, Takeo A, Miura K, Morita H, et al. Gonadal steroid hormone secretion during the juvenile period depends on the host-specific microbiota and contributes to the development of odor preference. Dev Psychobiol. 2019;61:670–8. Zheng D, Liwinski T, Elinav E. Interactions between microbiota and immunity in health and disease. Cell Res. 2020;30:492–506. Atarashi K, Tanoue T, Oshima K, Suda W, Nagano Y, Nishikawa H, et al. Treg induction by a rationally selected mixture of Clostridia strains from the human microbiota. Nature. 2013;500:232–6. Yang W, Yu T, Huang X, Bilotta AJ, Xu L, Lu Y, et al. Intestinal microbiota-derived short-chain fatty acids regulation by immune cell IL-22 production and gut immunity. Nat Commun. 2020;11:4457. Li Y, Xiao B, Qiu W, Yang L, Hu B, Tian X, et al. Altered expression of CD4(+)CD25(+) regulatory T cells and its 5-HT(1a) receptor in patients with major depression disorder. J Affect Disord. 2010;124:68–75. Liu Y, Mian MF, Neufeld K, et al.. CD4+ CD25+ T cells are essential for the behavioral effects of Lactobacillus rhamnosus JB-1 in Male BALB/c mice. Brain Behav Immun. 2020. Kim S-J, Lee H, Lee G, Oh S-J, Shin M-K, Shim I, et al. CD4+CD25+ regulatory T cell depletion modulates anxiety- and depression-like behaviors in mice. PLoS One. 2012;7:e42054. Dantzer R, O’Connor JC, Freund GG, Johnson RW, Kelley KW. When the immune system is subjugated to the brain, inflammation, illness, and depression occur. Nat Rev Neurosci. 2008;9:46–56. Pandey GN, Rizavi HS, Zhang H, et al.. Abnormal protein and mRNA expression of inflammatory cytokines in the prefrontal cortex of depressed individuals who died by suicide. J Psychiatry Neurosci. 2018;43:376–85. Goshen I, Kreisel T, Ben-Menachem-Zidon O, Licht T, Weidenfeld J, Ben-Hur T, et al. Brain interleukin-1 mediates chronic stress-induced depression in mice via adrenocortical activation and suppression of hippocampal neurogenesis suppression. Mol Psychiatry. 2008;13:717–28. Additional Declarations No competing interests reported. Supplementary Files AddFigAM.pptx Supplementary Fig. S1. Comparison between EW and NW, GF-EW and GF-NW mice in open field tests The graph shows the duration spent in the central area (A, C) and the distance traveled (B, D) during the open-field test. In Experiment 1, the time spent in the central area and the distance traveled by the EW mice did not differ from those of the NW mice. Similarly, there was no difference in the open-field test between the GF-EW and GF-NW groups in Experiment 2. Supplementary Fig. S2. Chronological changes in fecal corticosterone levels in each group (GF-NW, n = 9, GF-EW, n = 7). Supplementary Fig. S4 shows the change in fecal corticosterone concentration in each group over time (two-way repeated measures ANOVA, week: F7,63 = 7.591, p < 0.001; group: F1,9 = 0.013, p = 0.911; week × group interaction: F1,63 = 1.953, p = 0.076). Cite Share Download PDF Status: Published Journal Publication published 20 Jun, 2024 Read the published version in Animal Microbiome → Version 1 posted Editorial decision: Revision requested 21 Apr, 2024 Reviews received at journal 20 Apr, 2024 Reviewers agreed at journal 18 Apr, 2024 Reviews received at journal 18 Apr, 2024 Reviewers agreed at journal 08 Apr, 2024 Reviewers invited by journal 20 Mar, 2024 Editor assigned by journal 13 Mar, 2024 Submission checks completed at journal 11 Mar, 2024 First submitted to journal 09 Mar, 2024 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. 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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-4060437","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":278296419,"identity":"7e6f0743-ea18-4885-a878-36869b6f0f2c","order_by":0,"name":"Itsuka Kamimura","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Itsuka","middleName":"","lastName":"Kamimura","suffix":""},{"id":278296420,"identity":"a7a1aa60-3dd2-4ee6-9f6a-6caf14d1b461","order_by":1,"name":"Eiji Miyauchi","email":"","orcid":"","institution":"RIKEN","correspondingAuthor":false,"prefix":"","firstName":"Eiji","middleName":"","lastName":"Miyauchi","suffix":""},{"id":278296421,"identity":"cb46faff-b34b-4d5b-bcfb-31d6883d5731","order_by":2,"name":"Tadashi Takeuchi","email":"","orcid":"","institution":"RIKEN","correspondingAuthor":false,"prefix":"","firstName":"Tadashi","middleName":"","lastName":"Takeuchi","suffix":""},{"id":278296422,"identity":"74e20307-225b-4da3-a738-b9962a9f2678","order_by":3,"name":"Noriaki Tsuchiya","email":"","orcid":"","institution":"Azabu University","correspondingAuthor":false,"prefix":"","firstName":"Noriaki","middleName":"","lastName":"Tsuchiya","suffix":""},{"id":278296423,"identity":"e8a02f97-0a60-426e-ba90-e9cb25d65ffe","order_by":4,"name":"Kanami Tamura","email":"","orcid":"","institution":"Azabu 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University","correspondingAuthor":true,"prefix":"","firstName":"Takefumi","middleName":"","lastName":"Kikusui","suffix":""}],"badges":[],"createdAt":"2024-03-09 23:14:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4060437/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4060437/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s42523-024-00322-7","type":"published","date":"2024-06-20T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":52624212,"identity":"c9ae71e8-9293-4c10-b8be-a4613512c2a6","added_by":"auto","created_at":"2024-03-13 17:27:43","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":60495,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEW increases depressive-like behaviors and hyperactivity in marble-burying test during the juvenile period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schedule for Experiment 1. The mice were divided into NW and EW groups by weaning. EW mice (n = 21) were weaned on PD16 and NW mice (n = 44) were co-housed with dams until PD28. (B) The total duration of immobility during the tail suspension test in EW and NW mice. Distance moved (C) and number of marbles buried (D) in the marble burying test. The NW is black, and the EW is red. Data were represented as mean ± s.e.m. Comparison of performance in behavioral experiments at 4 and 8 wk of age; ##p \u0026lt; 0.01, ###p \u0026lt; 0.001. Comparison between EW and NW mice; * P \u0026lt; 0.05, *** P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-4060437/v1/581638737e524b783d708b22.png"},{"id":52624213,"identity":"49902dc7-17a0-4169-9df7-7f8b71a43817","added_by":"auto","created_at":"2024-03-13 17:27:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":148449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe effects of early weaning on the gut microbiota\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Bar plot representing the relative abundance of the top 10 families in fecal samples from NW and EW mice. The remainder were labeled as “others.” (\u003cstrong\u003eB\u003c/strong\u003e) Chao1 (left) and Shannon (right) indices of gut microbiota. (\u003cstrong\u003eC\u003c/strong\u003e) Principal coordinate analysis (PCoA) based on weighted UniFrac distances. The density plots show the sample distributions along the PCo1 and PCo2 axes. (\u003cstrong\u003eD\u003c/strong\u003e) PCo1 (left) and PCo2 (right) values for each sample in PCoA plot (C). *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-4060437/v1/975dc58b6f0e02aafe44b5f6.png"},{"id":52625200,"identity":"e120335e-c480-42dc-9000-f9851d4ed5b9","added_by":"auto","created_at":"2024-03-13 17:35:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":185274,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially abundant taxa enriched in NW and EW mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDifferentially abundant taxa between the groups at 4 weeks (A) and 8 weeks (B) of age were identified using LEfSe (p \u0026lt; 0.05, absolute LDA score \u0026gt; 2.5) and visualized using a phylogenetic cladogram (left) and bar plots (right).\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-4060437/v1/955a0a0fb7e03f71d4de1a29.png"},{"id":52625198,"identity":"e035f7ed-e218-4d96-ba1a-4a6001979734","added_by":"auto","created_at":"2024-03-13 17:35:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":77551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGF mice colonized with EW microbiota demonstrate depression-like behavior and hyperactivity during the juvenile period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Schedule for experiment 2. GF dam mice were orally administered EW or NW mice with fecal-derived microbiota or sterilized saline when the pups were PD10 (GF-NW, n = 23; GF-EW, n = 22; GF, n = 6). (B) Immobility time in the tail suspension test. Distance moved (C) and number of marbles buried (D) in the marble-burying test. The same letters in the line graph indicate a lack of significant differences between the groups. Data were represented as mean ± s.e.m. *p \u0026lt; 0.05, **p \u0026lt;0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-4060437/v1/2b285f253aa36e3d32a795d1.png"},{"id":52624219,"identity":"2cab85c4-10f9-43b2-b197-c468a25e2f07","added_by":"auto","created_at":"2024-03-13 17:27:44","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":242692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe gut microbiota of GF-NW and GF-EW mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Bar plot representing the relative abundances of the top 10 families in the fecal samples of GF-NW and GF-EW mice. The remainder were labeled as “others.” (B) Chao1 (left) and Shannon (right) indices of gut microbiota. (\u003cstrong\u003eC\u003c/strong\u003e-\u003cstrong\u003eD\u003c/strong\u003e) Differentially abundant taxa between the groups at 4 weeks (C) and 8 weeks (D) of age were identified by LEfSe (p \u0026lt; 0.05, absolute LDA score \u0026gt; 3.0) and visualized using bar plots.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-4060437/v1/09b05d7951034da80f96f11e.png"},{"id":52624217,"identity":"d54917fa-de70-4561-b1be-11bea8f1b654","added_by":"auto","created_at":"2024-03-13 17:27:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":133939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEW mice microbiota reduces the cell number of T cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell numbers in the spleen, small intestinal lamina propria (SILP), Peyer's patches (PP), and mesenteric lymph nodes (mLN). Data are represented as mean ± s.e.m. *p \u0026lt; 0.05, **p \u0026lt;0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-4060437/v1/37109cb2b58329344e6bea76.png"},{"id":58823768,"identity":"988f2cea-2e69-4a47-b782-4f204f639766","added_by":"auto","created_at":"2024-06-21 17:06:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1568419,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4060437/v1/69d7a969-c4fd-4a42-affa-ae328c219bd9.pdf"},{"id":52624215,"identity":"7c886d8f-b1c3-4fff-9df5-ea2bf7027ae9","added_by":"auto","created_at":"2024-03-13 17:27:43","extension":"pptx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":102636,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S1. Comparison between EW and NW, GF-EW and GF-NW mice in open field tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe graph shows the duration spent in the central area (A, C) and the distance traveled (B, D) during the open-field test. In Experiment 1, the time spent in the central area and the distance traveled by the EW mice did not differ from those of the NW mice. Similarly, there was no difference in the open-field test between the GF-EW and GF-NW groups in Experiment 2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSupplementary Fig. S2. Chronological changes in fecal corticosterone levels in each group (GF-NW, n = 9, GF-EW, n = 7).\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSupplementary Fig. S4 shows the change in fecal corticosterone concentration in each group over time (two-way repeated measures ANOVA, week: F7,63 = 7.591, p \u0026lt; 0.001; group: F1,9 = 0.013, p = 0.911; week × group interaction: F1,63 = 1.953, p = 0.076).\u003c/p\u003e","description":"","filename":"AddFigAM.pptx","url":"https://assets-eu.researchsquare.com/files/rs-4060437/v1/21d22b8397fd84b86d115bb2.pptx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Modulation of gut microbiota composition due to early weaning stress induces depressive behavior during the juvenile period in mice","fulltext":[{"header":"Background","content":"\u003cp\u003eIt has been shown that stress exposure in early life or inadequate maternal care causes several physiological and behavioral alterations in adulthood. Separation from the dam before weaning is a rodent model of developmental stress, and early weaning (EW) enhances stress responses of the hypothalamic-pituitary-adrenal (HPA) axis and increases anxiety and lowers cognitive function in adulthood, similar to human-abused children [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Early-weaned pups might be under physiological and psychological stress, and the behavioral and neuroendocrine changes caused by EW have been suggested to be related to changes in gene expression in the brain, probably programmed via epigenetic changes in neurons [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, stress in early life affects immune function [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccumulating evidence has implicated crosstalk between physiological stress and gut microbiota. For example, it has been reported that germ-free (GF) mice display excessive stress responsiveness compared with specific-pathogen-free (SPF) mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and maternal separation alters the gut microbiota, thereby shaping increased stress and elevated anxiety after growth [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In addition, gut microbiota disruption in pregnant mice by antibiotic treatment induces high anxiety behaviors in their offspring [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Dynamic changes in the microbiota during the juvenile period occur concurrently with central nervous system development, suggesting that the gut microbiota in neonates and juveniles can shape stress and anxiety behaviors [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe timing of early life exposure to bacteria and the bacterial community composition have permanent effects on immune system development and symbiotic bacteria composition [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The immune system communicates with the central nervous system and is involved in mediating anxiety and depression [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Individual differences in the peripheral immune system can predict and promote susceptibility [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The microbiome is a causal factor in autism [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and children with autism have a dysregulated immune system [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Maternal immune activation promotes behavioral abnormalities, and these phenotypes in the offspring require maternal intestinal bacteria that promote T helper 17 cell differentiation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], suggesting that the gut microbiota regulates behavioral phenotypes via immunological pathways in the host. However, studies investigating the causal relationships among juvenile stress, microbiota changes, and immune and behavioral deficits are limited. In this study, we conducted an empirical analysis of how EW stress affects behavior and gut microbiota, and which of these changes in behavior and the immune system are caused by changes in the gut microbiota in gnotobiotic mice.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEW mice show more depressive behavior in the juvenile period\u003c/h2\u003e \u003cp\u003eFirst, we assessed anxiety-like and depressive behaviors in EW and normal weaning (NW) mice on PD29 and 56 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In the tail suspension test, the immobility time of EW mice was longer than that of NW mice at 4 weeks of age and shorter than that of NW mice at 8 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB: week; F1,55\u0026thinsp;=\u0026thinsp;0.150, p\u0026thinsp;=\u0026thinsp;0.700, group; F1,55\u0026thinsp;=\u0026thinsp;0.341, p\u0026thinsp;=\u0026thinsp;0.562, week \u0026times; group interaction; F1,55\u0026thinsp;=\u0026thinsp;21.886, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The immobility time of EW mice was decreased from 4 to 8 weeks of age, while the immobility time of NW mice was increased from 4 to 8 weeks of age. In the marble burying test, the distance moved by the EW mice was greater than that moved by the NW mice at both 4 and 8 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC: week; F1,59\u0026thinsp;=\u0026thinsp;50.742, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, group; F1,59\u0026thinsp;=\u0026thinsp;0.049, p\u0026thinsp;=\u0026thinsp;0.826, week \u0026times; group interaction; F1,59\u0026thinsp;=\u0026thinsp;0.049, p\u0026thinsp;=\u0026thinsp;0.826). Although marble-burying behavior increased from 4 to 8 weeks of age, no differences were observed between the NW and EW mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD: week; F1,119\u0026thinsp;=\u0026thinsp;22.672, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, group; F1,119\u0026thinsp;=\u0026thinsp;1.069, p\u0026thinsp;=\u0026thinsp;0.303, week \u0026times; group interaction; F1,119\u0026thinsp;=\u0026thinsp;0.030, p\u0026thinsp;=\u0026thinsp;0.864). In the open field test, the distance traveled and center duration of the EW mice were not different from those of the NW mice. (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEW alters the gut microbiota composition in adulthood\u003c/h2\u003e \u003cp\u003e16S ribosomal RNA (rRNA) gene sequencing was performed to assess the fecal microbiome. At 4 weeks of age, there was almost no difference in the gut microbiota composition between NW and EW mice; however, the abundance of \u003cem\u003eErysipelotrichaceae\u003c/em\u003e in EW mice at 8 weeks of age was lower than that in NW mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Bacterial richness and evenness, estimated using the Shannon index, but not the Chao1 index, were higher in NW mice than in EW mice at 8 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Principal coordinate analysis (PCoA) based on weighted UniFrac distances depicted age-related changes in the microbiota composition along the first two principal coordinate axes (PCo1 and PCo2; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Changes along the PCo2 axis were diminished in EW mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), implying an immature microbiota in the adult EW mice. Furthermore, linear discriminant analysis effect size (LEfSe) was performed to identify the bacterial taxa enriched in EW and NW mice, and there were significant differences between the NW and EW groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTransplantation of gut microbiota from the EW mice induces more depressive behaviors in the juvenile period\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the effect of gut microbiota modulation by EW on host behavior, we observed the behavior of GF mice colonized with gut microbiota from EW or NW mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). In the tail suspension test, the immobility time of GF-EW mice was longer than that of GF-NW and GF mice at 4 weeks of age, and this difference disappeared at 8 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB: week, F1,92\u0026thinsp;=\u0026thinsp;10.649, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; group, F2,92\u0026thinsp;=\u0026thinsp;5.532, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; week \u0026times; group interaction, F2,92\u0026thinsp;=\u0026thinsp;5.905, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). At 4 weeks of age, GF-EW mice moved considerably further than GF and GF-NW mice, and this difference disappeared at 8 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC: week, F1,92\u0026thinsp;=\u0026thinsp;10.649, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; group, F2,92\u0026thinsp;=\u0026thinsp;5.532, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; week \u0026times; group interaction, F2,92\u0026thinsp;=\u0026thinsp;5.905, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The marble-burying behavior of GF-EW mice was not different from that of GF-NW and GF mice at both 4 and 8 weeks of age, and the marble-burying behavior in all mouse groups increased from 4 to 8 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD: week; F1,78\u0026thinsp;=\u0026thinsp;26.955, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; group: F2,78\u0026thinsp;=\u0026thinsp;0.161, p\u0026thinsp;=\u0026thinsp;0.851, week \u0026times; group interaction; F2,78\u0026thinsp;=\u0026thinsp;0.484, p\u0026thinsp;=\u0026thinsp;0.618). In the open field test, there was no significant difference in the distance traveled and center duration between the GF-EW group and the other groups. (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEW microbiota does not affect fecal corticosterone levels.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo observe whether the change in behavioral phenotype caused by the EW microbiota was accompanied by HPA axis enhancement, fecal corticosterone levels were measured over time. Fecal corticosterone levels in the GF-EW mice were comparable to those in the GF-NW mice (Fig. S2: week; F7,63\u0026thinsp;=\u0026thinsp;7.591 p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, group; F1,9\u0026thinsp;=\u0026thinsp;0.013, p\u0026thinsp;=\u0026thinsp;0.911, week \u0026times; group interaction; F1,63\u0026thinsp;=\u0026thinsp;1.953, p\u0026thinsp;=\u0026thinsp;0.076).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eGF-EW mice have a different gut microbiota composition from GF-NW mice\u003c/h2\u003e \u003cp\u003eThere were no consistent differences in the microbial composition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The Shannon index of the GF-EW group was lower than that of the GF-NW group at 4 weeks of age (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, LEfSe identified differentially abundant taxa in the NW and EW groups at both ages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and D).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eThe gut microbiota of EW mice reduces the cell number of T cells\u003c/h2\u003e \u003cp\u003eTo elucidate the effects of EW-induced gut microbiota changes on the immune system, we analyzed the spleen and gut immune cells of GF, GF-NW, and GF-EW mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The GF-EW group had fewer CD4\u0026thinsp;+\u0026thinsp;T cells in the spleen than the other groups did. Similarly, the GF-EW group had fewer CD4\u0026thinsp;+\u0026thinsp;T cells, B cells, and dendritic cells in the small intestinal lamina propria (SILP), and fewer CD4\u0026thinsp;+\u0026thinsp;T cells in the mesenteric lymph nodes (mLN) than the GF-NW group. CD8\u0026thinsp;+\u0026thinsp;T cell numbers in the SILP of GF mice were lower than those in GF-NW mice, and GF-EW mice were intermediate between those in GF and GF-NW mice. The numbers of CD4\u0026thinsp;+\u0026thinsp;and CD8\u0026thinsp;+\u0026thinsp;T cells in the mLN of GF-EW mice were lower than those in GF-NW mice, and the number of CD4\u0026thinsp;+\u0026thinsp;T cells in GF mice was intermediate between those in GF-EW and GF-NW mice. In addition, the number of B cells was lower in GF-EW mice than in GF mice, with GF-NW mice in the middle. No difference in the number of mLN dendritic cells was observed between the groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eStudies investigating the causal relationships among juvenile stress, microbiota changes, and immune and behavioral deficits are limited. Here, we conducted an empirical analysis of how EW stress affects behavior and gut microbiota, and which of these changes in behavior and the immune system are caused by changes in the gut microbiota in gnotobiotic mice. In this study, we demonstrated that mice exposed to EW stress exhibit more depression-like behaviors and hyperactivity at 4 weeks of age. The fecal microbiome composition of these mice differed from that of NW mice, especially at 8 weeks of age. In an empirical study using GF mice, GF-EW mice orally administered microbiomes from EW mice demonstrated more depression-like behaviors at 4 weeks of age. GF-EW mice demonstrated different fecal microbiome compositions and immune cell profiles compared with GF-NW mice. These results suggest that changes in the gut microbiota composition due to EW stress may regulate developmental behavioral phenotypes and the immune system.\u003c/p\u003e \u003cp\u003eWe investigated the effects of EW stress on the behavioral phenotypes of mice. EW mice demonstrated more locomotor activity in the marble-burying test and a higher immobility time in the tail suspension test at 4 weeks of age. The longer travel distance in the marble-burying test in EW mice was maintained up to 8 weeks of age, but the immobility time in the tail suspension test of EW mice was reduced at 8 weeks of age. Our findings are partially supported by a report by George et al. showing that mice exposed to early life stress exhibit hyperactivity and increased depressive behavior [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, the reduced depressive behavior of EW mice at 8 weeks of age is inconsistent with the increased depressive behavior of 10-week-old maternally separated mice with early weaning at PD17 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. This discrepancy may be explained by the presence of maternal separation in the neonatal period.\u003c/p\u003e \u003cp\u003eThere were differences in the gut microbiota composition between EW and NW mice, especially at 8 weeks of age. Moussaoui et al. reported that early life stress-exposed rat pups had decreased fecal microbial diversity, and the composition was characterized by an increased abundance of gram-positive cocci and a reduction in fiber-degrading butyrate-producing bacteria [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In humans, changes in microbiota and supplementation with specific bacteria early in development may mitigate the behavioral and mental health consequences of early bacterial disruption [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The gut microbiota of patients with depression demonstrated an increase in Thermoanaerobacteraceae and a decrease in Prevotellaceae compared to healthy individuals, and administration of the gut microbiota from patients with depression to antibiotic-treated rats resulted in depressive-like behavior and increased inflammatory cytokine levels in the blood [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. We also found that depressive behavior at 4 wk of age in GF mice colonized with EW mouse gut microbiota was as high as that in EW mice at 4 weeks of age. These findings support our hypothesis that EW stress alters the gut microbiota composition and that this change is a causal factor of depression-like behavior in mice.\u003c/p\u003e \u003cp\u003eEW mice demonstrated a shorter immobilization time in the tail suspension test at 8 weeks of age, but GF-EW mice did not show any difference compared with GF-NW mice at the same age. This discrepancy could be due to the timing of exposure of the bacterial community. EW mice were naturally exposed to the microbiome at birth, whereas GF-EW mice were germ-free until the first exposure to the microbiota on postnatal day (PD) 10. The first colony formation in the gut microbiome has an important effect on host immune development [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This difference in timing may have caused the differences in depression-like behaviors observed in this study.\u003c/p\u003e \u003cp\u003eIt was found that the EW mice and the NW mice at 4 weeks of age did not show a clearly different gut microbiota composition, but at 8 weeks of age, they demonstrated a different gut microbiota composition. Therefore, it is hypothesized that EW stress exposure induces moderate but critical changes in the gut microbiota composition during the juvenile period; however, these changes become obvious in the gut microbiota composition after growth. The reasons for this time lag between stress exposure and changes in the gut microbiota are not clear; however, it has been reported that early life stress alters the pro-inflammatory cytokine expression and may serve as a primer for a secondary challenge that can induce lifelong immune changes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Further studies are required to clarify the mechanisms and effects underlying long-lasting changes in the microbiome.\u003c/p\u003e \u003cp\u003eFecal corticosterone levels in GF-EW mice were comparable to those in GF-NW mice, suggesting that the changing gut microbiota associated with EW does not affect the basal activity of the HPA axis. In a previous study, our group reported higher basal and stress-response corticosterone concentrations in EW mice than in NW mice [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Therefore, we hypothesized that transplantation of the EW-gut microbiome would increase HPA activity; however, we did not observe any such effects. In our previous study, the fecal corticosterone concentration of GF mice was lower than that of SPF mice, and treatment of GF mice with fecal-derived bacterial solution from SPF mice increased the fecal corticosterone concentration compared to SPF mice [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], suggesting that there are bacteria in the feces of SPF mice that increase corticosterone concentration in GF mice. Sudo et al. reported that the higher HPA axis responses, but not basal responses, of sterile mice were suppressed to the same level as those of SPF mice by transplanting the feces of SPF mice into sterile mice by 6 weeks of age [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, it is feasible that the gut microbiota influences HPA axis development. The composition of the microbiota and the establishment of characteristic bacterial species at any given developmental period need to be clarified in further studies.\u003c/p\u003e \u003cp\u003eOur study revealed that GF mice colonized with the gut microbiota of EW mice demonstrated an immune cell profile different from that of GF mice treated with the gut microbiota of NW mice. The gut microbiota alters the immune development and function of the host [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. For example, it has been reported that a strain of Clostridium from the human gut microbiota promotes Treg cell differentiation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and short-chain fatty acids from the gut microbiota regulate IL-22 production by CD4\u0026thinsp;+\u0026thinsp;T cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, GF-EW mice exhibited a reduced number of CD4\u0026thinsp;+\u0026thinsp;T cells, especially in the spleen. The depletion of CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;T Cells has been reported to increase immobility time in forced swimming tests in non-stress-exposed mice. In humans, low levels of serum CD4\u0026thinsp;+\u0026thinsp;CD25\u0026thinsp;+\u0026thinsp;T cells have been observed in patients with major depression [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Depressive behavior is reduced by \u003cem\u003eLactobacillus rhamnosus\u003c/em\u003e JB-1 [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. These effects may alter the behavioral phenotypes of GF-EW mice. Inflammatory cytokines are increased in depressed patients and mouse models of depression [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], and TNF-α and IL-6 expression is elevated in the prefrontal cortex and orbitofrontal cortex of depressed patients who die by suicide [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Goshen et al. reported that mice exposed to chronic mild stress (CMS) (a model of depression) have increased levels of IL-1, a pro-inflammatory cytokine, in the hippocampus, and that IL-1 receptor knockout (KO) mice no longer exhibit CMS-induced depressive behavior [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Because EW has been reported to impair the mucosal immune response to infectious pathogens in animals, EW manipulation may impair immune function development [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn conclusion, our findings demonstrate that EW-induced alterations in the gut microbiota cause depressive behaviors and modulate the immune system. These results support the possibility that the changes in the gut microbiota of EW mice observed in this study may increase depression-like behavior in mice via the immune system. The present results do not demonstrate that this microbiome-immune system is a causal pathway to modulate depression-like behavior, and future studies are warranted.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eWe used C57BL/6J mice (CLEA Japan Inc., Tokyo, Japan) and their germ-free (GF) counterparts (CLEA Japan Inc.). All mice were maintained under a standard 12 h: 12 h light-dark cycle and provided with a pelleted diet and water ad libitum. The environment was maintained at a constant temperature (24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C), and humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;5%). Two to three mice were housed per cage. All mice were tested in behavioral experiments at 4 and 8 weeks of age and euthanized at 10 weeks of age. All experimental procedures were approved by the Animal Ethics Committee of the Azabu University (# 150316-3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eExperimental procedures\u003c/h2\u003e \u003cp\u003eExperiment 1. Effects of early weaning on behavior and gut microbiota\u003c/p\u003e \u003cp\u003eTo determine whether EW stress alters the gut microbiota composition and behavioral phenotypes, specific-pathogen-free (SPF) mice were assigned to two groups. Conventional pregnant mice were purchased from CLEA Japan, checked every morning until delivery, and fed a standard pelleted diet (MM3, Funabashi Farm Co., Funabashi, Japan) and tap water. The experimental schedule for Experiment 1 is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. On postnatal day (PD) 16, half the litter of pups was separated from the dam and divided into females and males, and they were assigned to the EW (n\u0026thinsp;=\u0026thinsp;21) group. The EW mice were fed a powdered diet until day PD28. On PD28, the remaining pups were weaned (normal weaning; n\u0026thinsp;=\u0026thinsp;44). At PD29 and PD56, behavioral tests were conducted on all mice in a soundproof room to determine the effects on developing offspring and whether the effects of the bacterial flora were maintained in adults.\u003c/p\u003e \u003cp\u003eExperiment 2. Behavioral and immune changes due to EW microbiota\u003c/p\u003e \u003cp\u003eWe examined the causal relationship between gut microbiota composition modulation by EW and the development of behavior and/or the immune system. Pregnant GF mice were purchased from CLEA Japan and housed in groups in a vinyl isolator (Sanki Kagaku Kougei Co., Kanagawa, Japan). All GF mice were fed a sterile pellet diet (CMF 50kGy, ORIENTAL YEAST, Tokyo, Japan) and sterile water. Immediately after transferring pregnant GF mice to a vinyl isolator, swab tests were conducted as previously described (Kamimura et al., 2019) to ensure a germ-free state. The vinyl isolator was swabbed with an ICR-swab (Merck Millipore, Darmstadt, Alemanha) and then incubated at 25\u0026deg;C for 48 h, and turbidity was measured with an absorptiometer (Shimazu CO., Kyoto, Japan).\u003c/p\u003e \u003cp\u003eThe schedule for Experiment 2 is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA. When the pups were on PD10, each GF dam was orally administered the gut microbiota from the EW or NW mice on PD56 (GF-EW, n\u0026thinsp;=\u0026thinsp;22 or GF-NW mice, n\u0026thinsp;=\u0026thinsp;23). The fecal sample was filtered using a mesh 100\u0026micro;m cell strainer followed by centrifugation. The supernatant was removed and the remaining pellets were dissolved in autoclaved sterilized saline. The solution was transferred to a sterilized vinyl isolator for subsequent oral FMT administration. We administered 100 \u0026micro;L to each mouse. Autoclaved saline was orally administered to dams in the control group of GF mice (n\u0026thinsp;=\u0026thinsp;6).\u003c/p\u003e \u003cp\u003eDonor EW and NW mice from each cohort were the littermates. All mice were weaned on PD28, and the dam was removed from the vinyl isolator. Behavioral tests were performed using a vinyl isolator. After the tests, the mice were moved to a conventional environment and euthanized on PD70. Given the sex differences in response to EW stress reported in our previous studies [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], feces were collected from male mouse donors. In this experiment, three pairs of litters were examined at different time points using different donor feces.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBehavioral tests\u003c/h2\u003e \u003cp\u003eThe behavioral tests in Experiment 1 were conducted in a soundproof room, whereas those in Experiment 2 were conducted in vinyl isolators. In Experiment 2, behavioral tests other than the open field test were performed in a vinyl isolator in which each mouse group was bred, and the animals were subjected to open field testing immediately after being aseptically transferred to a new vinyl isolator per group in an open arena. Sterilized tools (open arena, stainless-steel marble, test cages, and clips) were used in Experiment 2. All behavioral tests were performed under fluorescent light during the light period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTail suspension test\u003c/h2\u003e \u003cp\u003eThe mouse tail tip was fixed with a clip, the suspended mouse was video-recorded for 6 min, and the duration of immobility was measured. Mice were considered immobile only when they hung passively and were completely motionless. The recorded behavior was coded into numerical numbers to be blinded to the condition, and two experimenters who were blind to the conditions performed the analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eOpen field test\u003c/h2\u003e \u003cp\u003eThe mice were released into an open arena (30 cm \u0026times; 30 cm) with a defined central area (20 cm \u0026times; 20 cm), and their behavior was videotaped for 5 min. To analyze motor activity and anxiety, we used the Ethovision XT version 10 (Noldus, Wageningen, Netherlands) to measure the distance traveled and the duration spent in the central area (Kulbeth et al., 2021).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eMarble burying test\u003c/h2\u003e \u003cp\u003eBefore the marble burying test commenced, the animals were habituated to the test cages (17.5 \u0026times; 24.5 \u0026times; 12.5 cm) containing 3 cm deep bedding (Japan SLC, Inc., Sioka, Japan) for 5 min. After habituation, we spaced 12 stainless steel-made marble (φ 9.58 mm) on the bedding at 3 cm intervals. For the experiments, mice were released into the test cage, their behavior was videotaped for 25 min, and the number of buried marbles was recorded. We defined the condition that two-thirds of the marbles were buried in the bedding as \"buried.\u0026rdquo;\u003c/p\u003e \u003cp\u003eEthovision XT version 10 (Noldus, Wageningen, Netherlands) was used to measure the distance traveled. In Experiment 2, because the recorded videos could not be analyzed automatically by Ethovision XT owing to the vinyl isolator cover, we edited them using AviUtl version 1.00 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://spring-fragrance.mints.ne.jp/aviutl/\u003c/span\u003e\u003cspan address=\"http://spring-fragrance.mints.ne.jp/aviutl/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) to increase detection sensitivity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFecal sample collection\u003c/h2\u003e \u003cp\u003eDue to the high-stress responsiveness of GF mice (Sudo et al., 2004), we performed minimally stressed fecal sampling rather than stress-bearing blood sampling. Fresh fecal samples from all mice were collected directly into sterilized collection tubes between 10:00 and 13:00 at PD28 and PD56, suspended in phosphate-buffered saline containing 20% glycerol, immediately frozen using liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C until use.\u003c/p\u003e \u003cp\u003eFecal samples for measuring corticosterone were collected from the three groups of GF-NW (n\u0026thinsp;=\u0026thinsp;9) and GF-EW (n\u0026thinsp;=\u0026thinsp;7) mice immediately before the cage changes, and then they were stored at \u0026minus;\u0026thinsp;20\u0026deg;C until processing and were collected every other week between 10:00 and 11:00 during the period when the mice were 4\u0026ndash;8 weeks of age.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eMicrobiome analysis\u003c/h2\u003e \u003cp\u003eFecal samples frozen at \u0026minus;\u0026thinsp;80\u0026deg;C were thawed on ice before use. Bacterial DNA was extracted as described previously (Miyauchi et al., 2020). The 16S ribosomal RNA (rRNA) gene V3-V4 region (341F\u0026ndash;806R) was PCR-amplified from the bacterial gene using the 16S metagenomic sequencing library protocol (Kozich et al., 2013). The amplicons were purified and quantified using the AMPure XP (Beckman Coulter, California, USA) and Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Samples were pooled in equal concentrations and sequenced on a MiSeq system (Illumina, California, USA; 2 \u0026times; 300 bp, paired-end reads). The sequenced reads were demultiplexed using bcl2fastq (v1.8.4), and the resulting fastq files were processed with DADA2 (v.1.18.0) (Callahan et al., 2016) using the parameters described in the tutorial pipeline (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://benjjneb.github.io/dada2/tutorial_1_8.html\u003c/span\u003e\u003cspan address=\"https://benjjneb.github.io/dada2/tutorial_1_8.html\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). After trimming the low-quality reads, duplicate reads were used for amplicon sequence variant (ASVs) inference. Chimera-free ASVs were assigned to the SILVA database v138 using a na\u0026iuml;ve Bayesian classifier implemented in DADA2. The phyloseq package (v.1.34.0) in R (v.4.0.3) was used for the downstream analysis. After the removal of mitochondria and chloroplast reads, we obtained 5,156,243 total reads (37,364\u0026thinsp;\u0026plusmn;\u0026thinsp;12,252 [mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD]). For alpha and beta diversities, data were rarefied to a minimum number of reads per sample (11,658 reads). Linear discriminant analysis (LDA) effect size (LEfSe) was performed to detect bacterial taxa with significantly different abundances between groups using the LEfSe software (Segata et al., 2011).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eFecal corticosterone analysis\u003c/h2\u003e \u003cp\u003eFecal corticosterone concentrations were measured according to published protocols (Kamimura et al., 2019). ELISA plates were coated with anti-rabbit IgG antibody solution (Jackson ImmunoResearch Laboratories, PA, USA, Cat # 111-005-003, RRID: AB_2337913) and washed twice with a plate washer (Immunowash Model 1250; Bio-Rad Laboratories, Inc., CA, USA). Aliquots of an anti-corticosterone antibody raised in rabbits (Cosmo Bio Co., Tokyo, Japan, Cat# FKA-420-E, RRID: AB_10708379) and aliquots of HRP-labeled corticosterone (FKA419; Cosmo Bio Co., Tokyo, Japan) were added to each well, and the reaction was stopped by adding N-H2SO4. The absorbance of the samples was measured using an automatic microplate reader (Bio-Rad Model 550; Bio-Rad Laboratories Inc., Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eImmunological profiling\u003c/h2\u003e \u003cp\u003eThe spleens, mesenteric lymph nodes (mLN), and Peyer\u0026rsquo;s patches were processed by homogenization on a 70 \u0026micro;m cell strainer, and the splenocytes were treated with red blood cell lysis buffer (BioLegend, California, USA). Lamina propria lymphocytes from the small intestine were isolated as previously described (Miyauchi et al., 2020). In brief, the epithelial layer was removed by agitating the small intestine in Hank\u0026rsquo;s balanced salt solution supplemented with 2% fetal bovine serum (FBS), 1 mM dithiothreitol, and 20 mM EDTA for 30 min at 37\u0026deg;C. The tissue was minced and digested in RPMI 1640 medium containing 2% FBS, 400 U/mL collagenase D (Roche, Basel, Switzerland), 0.25 U/mL dispase (BD Biosciences, New Jersey, USA), and 0.1 mg/mL DNase I (Wako, Hiroshima, Japan) for 30 min at 37\u0026deg;C with agitation. Lymphocytes were harvested at 40%/80% Percoll interphase.\u003c/p\u003e \u003cp\u003eTo stain dead cells, single-cell suspensions were incubated with Zombie Aqua dye (BioLegend, San Diego, CA, USA) for 15 min at room temperature. After Fc receptors were blocked with anti-CD16/32 antibody (BD Biosciences, New Jersey, USA), the cells were stained with anti-CD3ε (APC-Cy7, BioLegend, California, USA), anti-CD4 (APC, BioLegend, California, USA ), anti-CD8α (PerCP\u0026ndash;Cy5.5, BioLegend, California, USA), anti-CD19 (FITC, BD Biosciences, New Jersey, USA), anti-CD11c (BV450, BD Biosciences, New Jersey, USA), and anti-MHC class II (biotin, Thermo Fisher, Massachusetts, USA) antibodies, followed by labeling with APC-Cy7-streptavidin conjugate (BioLegend, California, USA). The cells were fixed with the Foxp3 staining buffer set (Thermo Fisher Scientific) and stored at 4 \u0026ordm;C. All data were collected using a FACSCanto II cytometer (BD Biosciences, California, USA) and analyzed using FlowJo (version 10.5, Tree Star).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data on the behavioral tests were analyzed using two-way repeated-measures ANOVA to evaluate the differences between groups, and we added a post-hoc test using the Bonferroni correction. A generalized linear mixed model was constructed with the week of the experiment and the group as dependent variables when nonparametric tests were applicable. The data on the 16S rRNA analysis were analyzed using a one-way ANOVA to evaluate the differences between groups, and we added a post hoc test using Tukey's multiple comparison test. Dunn's multiple comparisons test was added after the Kruskal-Wallis test when parametric preconditions were not met.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw sequencing data for the 16S rRNA gene are available from the DDBJ Sequence Read Archive (DRA013030). \u003cstrong\u003eThe data supporting the findings of this study are available from the corresponding author, TK, upon reasonable request. The raw data were generated at Azabu University.\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest associated with this manuscript. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIK and EM, TT carried out the molecular lab work, participated in data analysis, carried out sequence alignments, participated in the design of the study, and drafted the manuscript; AU, NT, KT, and IK carried out the \u003cem\u003ein vivo\u003c/em\u003e experiments and IK conducted the behavioral statistics. EM, TT, HN, TT, TK, and MK prepared the cell suspensions for FACS analysis. EM and MK contributed to the 16S rRNA gene sequencing. HO, MN and KM, TK conceived of the study, designed the study, coordinated the study, and helped draft the manuscript. All authors provided their final approval for publication and agreed to be held accountable for the work performed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was funded by grants from the JPSP KAKENHI (19H00972 to TK and 18H02356 to KM), and this study was supported by the Center for Human and Animal Symbiosis Science, Azabu University.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eKikusui T, Takeuchi Y, Mori Y. Early weaning induces anxiety and aggression in adult mice. Physiol Behav. 2004;81:37\u0026ndash;42.\u003c/li\u003e\n\u003cli\u003eKikusui T, Mori Y. Behavioral and neurochemical consequences of early weaning in rodents. 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CD4+ CD25+ T cells are essential for the behavioral effects of Lactobacillus rhamnosus JB-1 in Male BALB/c mice. Brain Behav Immun. 2020.\u003c/li\u003e\n\u003cli\u003eKim S-J, Lee H, Lee G, Oh S-J, Shin M-K, Shim I, et al. CD4+CD25+ regulatory T cell depletion modulates anxiety- and depression-like behaviors in mice. PLoS One. 2012;7:e42054.\u003c/li\u003e\n\u003cli\u003eDantzer R, O\u0026rsquo;Connor JC, Freund GG, Johnson RW, Kelley KW. When the immune system is subjugated to the brain, inflammation, illness, and depression occur. Nat Rev Neurosci. 2008;9:46\u0026ndash;56.\u003c/li\u003e\n\u003cli\u003ePandey GN, Rizavi HS, Zhang H, et al.. Abnormal protein and mRNA expression of inflammatory cytokines in the prefrontal cortex of depressed individuals who died by suicide. J Psychiatry Neurosci. 2018;43:376\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eGoshen I, Kreisel T, Ben-Menachem-Zidon O, Licht T, Weidenfeld J, Ben-Hur T, et al. Brain interleukin-1 mediates chronic stress-induced depression in mice via adrenocortical activation and suppression of hippocampal neurogenesis suppression. Mol Psychiatry. 2008;13:717\u0026ndash;28.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"animal-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amic","sideBox":"Learn more about [Animal Microbiome](http://animalmicrobiome.biomedcentral.com)","snPcode":"42523","submissionUrl":"https://submission.nature.com/new-submission/42523/3","title":"Animal Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Microbiome, Stress, Behavior, Depression, Germ-Free Mouse","lastPublishedDoi":"10.21203/rs.3.rs-4060437/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4060437/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground:\u003c/strong\u003e The gut microbiota plays an important role in the development of behavior and immunity in infants and juveniles. Early weaning (EW), a form of social stress in mice, leads to increased anxiety and an enhanced stress response in the hypothalamic-pituitary-adrenal axis during adulthood. Early life stress also modulates the immune system and increases vulnerability to infection. However, studies investigating the causal relationships among juvenile stress, microbiota changes, and immune and behavioral deficits are limited. Therefore, we hypothesized that EW alters gut microbiota composition and impairs the development of the nervous and immune systems.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e EW mice moved longer distances in the marble-burying test and had longer immobility times in the tail suspension test than normal weaning (NW) mice. In parallel, the gut microbiome composition differed between NW and EW mice, and the abundance of \u003cem\u003eErysipelotrichacea\u003c/em\u003e in EW mice at 8 weeks of age was lower than that in NW mice. In an empirical study, germ-free mice colonized with the gut microbiota of EW mice (GF-EW mice) demonstrated higher depressive behavior than GF mice colonized with normal weaning microbiota (GF-NW mice). Immune cell profiles were also affected by the EW microbiota colonization; the number of CD4+ T cells in the spleen was reduced in GF-EW mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e Our results suggest that EW-induced alterations in the gut microbiota cause depressive behaviors and modulate the immune system.\u003c/p\u003e","manuscriptTitle":"Modulation of gut microbiota composition due to early weaning stress induces depressive behavior during the juvenile period in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-13 17:27:38","doi":"10.21203/rs.3.rs-4060437/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-04-21T13:23:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-20T04:37:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"8da7ceda-dee8-47ac-9635-f91e13f5103a","date":"2024-04-19T02:48:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-04-18T07:34:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"055bae0d-5d31-43d9-99bb-ea96f33c07b7","date":"2024-04-08T07:33:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-21T03:46:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-13T17:18:40+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-11T13:03:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Animal Microbiome","date":"2024-03-09T23:04:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"animal-microbiome","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"amic","sideBox":"Learn more about [Animal Microbiome](http://animalmicrobiome.biomedcentral.com)","snPcode":"42523","submissionUrl":"https://submission.nature.com/new-submission/42523/3","title":"Animal Microbiome","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23586980-4c6e-4330-9601-8080ac9eb996","owner":[],"postedDate":"March 13th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-06-21T15:56:55+00:00","versionOfRecord":{"articleIdentity":"rs-4060437","link":"https://doi.org/10.1186/s42523-024-00322-7","journal":{"identity":"animal-microbiome","isVorOnly":false,"title":"Animal Microbiome"},"publishedOn":"2024-06-20 15:56:55","publishedOnDateReadable":"June 20th, 2024"},"versionCreatedAt":"2024-03-13 17:27:38","video":"","vorDoi":"10.1186/s42523-024-00322-7","vorDoiUrl":"https://doi.org/10.1186/s42523-024-00322-7","workflowStages":[]},"version":"v1","identity":"rs-4060437","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4060437","identity":"rs-4060437","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}