Possible role of the gut microbiota–brain axis in the antidepressant effects of (R)-ketamine in a social defeat stress model

Accumulating evidence suggests that the gut microbiota–brain axis plays a role in the pathogenesis of depression, thereby contributing to the antidepressant actions of certain compounds. (R)-ketamine has a greater potency and longer-lasting antidepressant effects than (S)-ketamine. Here, we investigated whether the gut microbiota plays a role in the antidepressant effects of these two ketamine enantiomers. The role of the gut microbiota in the antidepressant effects of ketamine enantiomers in a chronic social defeat stress (CSDS) model of depression was examined using 16S ribosomal RNA gene sequencing of fecal samples. At the phylum level, CSDS-susceptible mice showed alterations in the levels of Tenericutes and Actinobacteria; however, neither ketamine enantiomers influenced these alterations. At the class level, both ketamine enantiomers significantly attenuated the increase in the levels of Deltaproteobacteria in the susceptible mice after CSDS. Furthermore, (R)-ketamine, but not (S)-ketamine, significantly attenuated the reduction in the levels of Mollicutes in the susceptible mice. At the genus level, both ketamine enantiomers significantly attenuated the decrease in the levels of Butyricimonas in the susceptible mice. Notably, (R)-ketamine was more potent than (S)-ketamine at reducing the levels of Butyricimonas in the susceptible mice. In conclusion, this study suggests that the antidepressant effects of two enantiomers of ketamine in CSDS model may be partly mediated by the restoration of the gut microbiota. Furthermore, the specific effect of (R)-ketamine on the levels of Mollicutes and Butyricimonas may explain its robust antidepressant action.

The gut microbiota-brain axis is a complex multiorgan bidirectional signaling system between the gut microbiota and brain that plays a crucial role in host physiology, homeostasis, development, and metabolism [33][34][35] . Several studies suggest that the gut microbiota contributes to the pathogenesis of depression and the antidepressant actions of certain compounds [36][37][38][39][40][41][42][43][44][45] . Therefore, the present study examined whether the gut microbiota plays a role in the mechanisms underlying the antidepressant actions of (R)ketamine and (S)-ketamine in a chronic social defeat stress (CSDS) model of depression.

Animals
Male adult C57BL/6 mice, aged 8 weeks (body weight 20-25 g, Japan SLC Inc., Hamamatsu, Japan) and male adult CD1 (ICR) mice, aged 13-15 weeks (body weight >40 g, Japan SLC Inc.) were used. Animals were housed under controlled temperatures and 12 h light/dark cycles (lights on between 0700-1900 hours), with ad libitum food (CE-2; CLEA Japan Inc., Tokyo, Japan) and water. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, USA. The protocol was approved by the Chiba University Institutional Animal Care and Use Committee.

CSDS model
The procedure of CSDS was performed as reported previously 26,29,30,[46][47][48][49][50] . The C57BL/6 mice were exposed to a different CD1 aggressor mouse for 10 min/day, total for 10 days. When the social defeat session ended, the resident CD1 mouse and the intruder mouse were housed in one half of the cage separated by a perforated Plexiglas divider to allow visual, olfactory, and auditory contact for the remainder of the 24-h period. Subsequently, all mice were housed individually 24 h after the last session. On day 11, social interaction test (SIT) was performed to select subgroups of mice that were susceptible and unsusceptible to social defeat stress. This was accomplished by placing mice in an interaction test box (42 × 42 cm) with an empty wire-mesh cage (10 × 4.5 cm) located at one end. The movement of the mice was tracked for 2.5 min, followed by 2.5 min in the presence of an unfamiliar aggressor confined in the wire-mesh cage. The duration of the subject's presence in the "interaction zone" (defined as the 8-cm-wide area surrounding the wire-mesh cage) was recorded by a stopwatch. The interaction ratio was calculated as time spent in an interaction zone with an aggressor/time spent in an interaction zone without an aggressor. An interaction ratio of 1 was set as the cutoff: mice with scores <1 were defined as "susceptible" to social defeat stress and those with scores ≥1 were defined as "unsusceptible". In the experiments,~70-80 % of mice were susceptible after CSDS. Susceptible mice were randomly divided into the subsequent experiments. Control mice without social defeat stress were housed in the same cage before the behavioral tests.

Locomotion
The locomotor activity of mice was measured by an animal movement analysis system SCANET MV-40 (MELQUEST Co., Ltd., Toyama, Japan). The mice were placed in experimental cages (length × width × height: 560 × 560 × 330 mm). The cumulative exercise was recorded for 60 min. Cages were cleaned between testing session.

TST
A small piece of adhesive tape was placed~2 cm from the tip of the tail for mouse. A single hole was punched in the tape and mice were hung individually on a hook. The immobility time was recorded for 10 min. Mice were considered immobile only when they hung passively and completely motionless.

FST
The FST was tested by an automated forced-swim apparatus SCANET MV-40 (MELQUEST Co., Ltd., Toyama, Japan). The mice were placed individually in a cylinder (diameter: 23 cm; height: 31 cm) containing 15 cm of water, maintained at 23 ± 1℃. Immobility time from activity time as (total) − (active) time was calculated by the apparatus analysis software. The immobility time for mouse was recorded for 6 min.
SPT Mice were exposed to water and 1% sucrose solution for 48 h, followed by 4 h of water and food deprivation and a 1 h exposure to two identical bottles, one is water, and another is 1% sucrose solution. The bottles containing water and sucrose were weighed before and at the end of this period. The sucrose preference was calculated as a percentage of sucrose solution consumption to the total liquid consumption.

Statistical analysis
The data show as the mean ± SEM. Analysis was performed using PASW Statistics 20 (formerly SPSS Statistics; SPSS, Tokyo, Japan). The data were analyzed using the one-way analysis of variance (ANOVA) or two-way ANOVA, followed by post hoc Tukey test. Furthermore, Principal Coordinate Analysis (PCoA) was performed to visualize similarities or dissimilarities of the data of four groups. The P values of less than 0.05 were considered statistically significant.

Results
(R)-ketamine showed more potent antidepressant effects than (S)-ketamine in the susceptible mice after CSDS The antidepressant effects of (R)-ketamine and (S)ketamine in the susceptible mice after CSDS were examined (Fig. 1a). There were no changes in the body weight among the four groups (Fig. 1b). There were no significant differences in the locomotion among the four groups (Fig. 1c). In the TST and FST, both (R)-ketamine and (S)-ketamine significantly decreased the increased immobility time in the susceptible mice after CSDS (Figs. Fig. 2 The PCoA analysis of the gut bacterium data. a The PCoA analysis plots of Bray-Curtis dissimilarity among the four groups. b The PCoA analysis plots of Euclidean dissimilarity among the four groups 1d, 1e). Furthermore, (R)-ketamine showed greater antidepressant effects than (S)-ketamine. In the SPT, (R)ketamine exerted more potent anti-anhedonia effects than (S)-ketamine (Fig. 1f). These data indicate that (R)-ketamine exerts more potent antidepressant and antianhedonia effects than (S)-ketamine, consistent with our previous reports 26,29,30 .
The PCoA analysis of the gut bacterium data The PCoA analysis plots of Bray-Curtis dissimilarity among the four groups showed that the dots of control group (a1-a6) were close to the dots of (R)-ketaminetreated group (c1-c6) compared with (S)-ketamine-treated group (d1-d6; Fig. 2a). Furthermore, the PCoA analysis plots of Euclidean dissimilarity showed that the dots of control group (a1-a6) were close to the dots of (R)-ketamine-treated group (c1-c6) compared with (S)-ketamine-treated group (d1-d6; Fig. 2b). Thus, it is likely that (R)-ketamine has more potency to improve the altered composition of gut microbiota after CSDS than (S)ketamine.

Altered composition in the gut bacterium at the phylum level
The phylum levels of gut bacterium 4 days (day 16) after a single dose of saline, (R)-ketamine, or (S)-ketamine are shown (Fig. 3a). Tenericutes in the gut were significantly decreased in the susceptible mice after CSDS, although neither ketamine enantiomers affected the decreased levels of Tenericutes in the susceptible mice (Fig. 3b). Furthermore, the susceptible mice had the increased levels of Actinobacteria, although neither ketamine The phylum levels of Tenericutes (one-way ANOVA: F 3,20 = 23.58, P < 0.001) in the gut. c The phylum levels of Actinobacteria (one-way ANOVA: F 3,20 = 11.39, P < 0.001) in the gut. Data are shown as mean ± SEM (n = 6). **P < 0.01, ***P < 0.001. NS not significant enantiomers affected the increased levels of Actinobacteria in the susceptible mice (Fig. 3c).

Altered composition in the gut bacteria at the class level
The class levels of gut bacterium 4 days after a single dose of saline, (R)-ketamine, or (S)-ketamine are shown (Fig. 4a). Deltaproteobacteria were significantly increased in the susceptible mice after CSDS. Both (R)-ketamine and (S)-ketamine significantly decreased the reduced levels of Deltaproteobacteria in the susceptible mice (Fig.  4b). Furthermore, Mollicutes were significantly decreased in the susceptible mice after CSDS. Interestingly, (R)ketamine, but not (S)-ketamine, significantly attenuated the reduced levels of Mollicutes in the susceptible mice (Fig. 4c).

Altered composition in the gut bacteria at the family level
The family levels of gut bacterium 4 days after a single dose of saline, (R)-ketamine, or (S)-ketamine are shown (Fig. 5a). Desulfovibrionaceae were significantly increased in the susceptible mice after CSDS. Furthermore, (S)ketamine significantly enhanced the increased levels of Desulfovibrionaceae in the susceptible mice, although (R)ketamine did not alter the increased levels of Desulfovibrionaceae (Fig. 5b).

Altered composition in the gut bacteria at the genus level
The genus levels of gut bacterium 4 days after a single dose of saline, (R)-ketamine, or (S)-ketamine are shown (Fig. 6a). Butyricimonas were significantly decreased in the susceptible mice after CSDS. Furthermore, both (R)ketamine and (S)-ketamine significantly increased the In addition, (R)-ketamine was more potent than (S)ketamine (Fig. 6b). Moreover, both (R)-ketamine and (S)ketamine significantly increased the reduced levels of others in the susceptible mice (Fig. 6c).

Discussion
The levels of Tenericutes, a phylum of bacteria, were significantly decreased and the levels of Actinobacteria, a phylum of Gram-positive bacteria, were significantly increased in the susceptible mice after CSDS. Neither of the ketamine enantiomers had an effect on these alterations. In contrast, both ketamine enantiomers significantly attenuated the increased levels of Deltaproteobacteria, a class of Proteobacteria, in the susceptible mice. Interestingly, (R)-ketamine, but not (S)-ketamine, significantly increased the reduced levels of Mollicutes in the susceptible mice. We also observed that (S)-ketamine, but not (R)-ketamine, significantly enhanced the increased levels of Desulfovibrionaceae, a family of Proteobacteria, in the susceptible mice. Lastly, while both ketamine enantiomers significantly attenuated the reduced levels of the genus Butyricimonas in the susceptible mice, (R)-ketamine was more potent than (S)ketamine. These findings suggest that an altered gut microbiota composition plays a role in the depression-like phenotype of CSDS-susceptible mice and that the restoration of the gut microbiota induced by (R)-ketamine partly explains its robust antidepressant effects.
At the phylum level, levels of Actinobacteria were significantly higher in active major depressive disorder (MDD) patients than in healthy control subjects 36,38 . Consistent with these results, we found increased levels of Actinobacteria in CSDS-susceptible mice. Although the exact physiological implications of Actinobacteria in depression are not fully understood, it is likely that increased levels of Actinobacteria in the gut contribute to the pathogenesis of depression. However, neither of the ketamine enantiomers reduced the levels of Actinobacteria in CSDS-susceptible mice.
At the class level, the levels of Deltaproteobacteria and Mollicutes were altered in CSDS-susceptible mice. There are no available reports on altered levels of Deltaproteobacteria and Mollicutes in MDD patients. However, increased levels of sulfite-reducing Deltaproteobacteria were found in ulcerative colitis (UC) patients with higher levels of inflammation 54 . Given the role of inflammation in CSDS-susceptible mice 42 , it is possible that increased levels of Deltaproteobacteria contribute to the depression-like phenotype of these mice through inflammatory responses. In addition, the reduction of levels of Deltaproteobacteria in CSDS-susceptible mice treated with either of the ketamine enantiomers may partly explain their antidepressant actions. Interestingly, (R)ketamine, but not (S)-ketamine, significantly attenuated the reduced levels of Mollicutes in CSDS-susceptible mice. Although the precise physiological implications of Deltaproteobacteria and Mollicutes in depression are unknown, it is likely that changes in the level of these bacteria induced by (R)-ketamine partially mediate its antidepressant action. Nonetheless, further studies on the relationship between (R)-ketamine's antidepressant effects and Deltaproteobacteria and Mollicutes are needed.
At the family level, we observed an increase in the levels of Desulfovibrionaceae in CSDS-susceptible mice. Desulfovibrionaceae are sulfate-reducing, nonfermenting, anaerobic, Gram-negative bacteria characterized by the presence of desulfoviridine 55 . Desulfovibrionaceae in the gut are the main biological source of hydrogen sulfate (H 2 S), which regulates a wide range of physiological functions, including the cardiovascular, neuronal, immune, respiratory, gastrointestinal, liver, and endocrine systems, by influencing cellular signaling pathways and sulfhydration of target proteins 56,57 . Furthermore, the levels of Desulfovibrionaceae were higher in animal models of metabolic syndrome 58 . H 2 S derived from gut microbiota such as Desulfovibrionaceae is associated with gastrointestinal disorders, such as UC, Crohn's disease, and irritable bowel syndrome 56 . Increased levels of Desulfovibrionaceae possibly play a role in the pathogenesis of depression via H 2 S-induced inflammation. In addition, levels of fecal H 2 S were higher in UC patients than in healthy control subjects 59,60 . Therefore, it is of great interest to measure the levels of fecal H 2 S in MDD patients. Indeed, further studies on the role of H 2 Sproducing bacteria, such as Desulfovibrionaceae, in depression are needed.
The genus Butyricimonas are butyrate producers with anti-inflammatory properties. We observed decreased levels of Butyricimonas in CSDS-susceptible mice, and altered levels of Butyricimonas were reported in MDD patients and untreated multiple sclerosis patients 36,61 . Butyricimonas produce butyrate, which reduces inflammation and helps maintain a healthy gut. Therefore, decreased levels of Butyricimonas in the gut may play an inflammation-based role in the pathogenesis of depression. Interestingly, increase in the levels of Butyricimonas induced by (R)-ketamine was more potent than that by (S)-ketamine. Taken together, the improvement in the levels of Butyricimonas may explain the better antidepressant effects of (R)-ketamine than (S)-ketamine. Considering the possible role of the gut microbiota in the antidepressant actions of certain compounds 42,45 , it is likely that the gut microbiota-brain axis plays at least a partial role in robust antidepressant actions of (R)ketamine.
The precise mechanisms underlying antidepressant actions of ketamine and (R)-ketamine are not fully understood. It has been suggested that anti-inflammatory actions of ketamine might play a role in its antidepressant effects [62][63][64][65] . Given the role of gut microbiota in the immune system [33][34][35]42,66 , it is likely that antiinflammatory action of (R)-ketamine might play a role in its antidepressant effects through the immunomodulation by the gut microbiota. Nonetheless, further studies on the anti-inflammatory and antidepressant actions of (R)-ketamine in the gut microbiota-brain axis will be needed.
The present data do not provide direct evidence of the effect of gut microbiota on the antidepressant actions of (R)-ketamine because behavioral experiments using germfree mice were not performed. However, it is well known that the antimicrobial effects of currently available antidepressants are important for the correction of the intestinal dysbiosis observed in MDD patients 45 . Therefore, the gut microbiota-brain axis possibly plays a role in the antidepressant actions of (R)-ketamine. Nonetheless, additional studies elucidating the relationship between the gut microbiota-brain axis and the antidepressant actions of (R)-ketamine are needed. In addition, further studies using germ-free mice will be needed to confirm the role of gut microbiota in the antidepressant effects of (R)ketamine.
In conclusion, the present study suggests that the gut microbiota-brain axis at least partially mediates the antidepressant actions of (R)-ketamine. Furthermore, it is likely that the specific effect of (R)-ketamine on the decreased levels of Mollicutes and Butyricimonas after CSDS may explain its robust antidepressant action.