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.

Materials

(R)-ketamine hydrochloride and (S)-ketamine hydrochloride were prepared by recrystallization of (R,S)-ketamine (Ketalar®, (R,S)-ketamine hydrochloride, Daiichi Sankyo Pharmaceutical Ltd., Tokyo, Japan) and D-(-)-tartaric acid (or L- ( + )-tartaric acid), as reported previously25. The purity of two ketamine enantiomers was determined by a high-performance liquid chromatography (CHIRALPAK® IA, Column size: 250 × 4.6 mm, Mobile phase: n-hexane/dichloromethane/diethylamine (75/25/0.1), Daicel Corporation, Tokyo, Japan). The contamination of another enantiomer was not detected for two ketamine enantiomers. The dose (10 mg/kg as ketamine hydrochloride) of (R)-ketamine and (S)-ketamine was used as previously reported25,26,27,28,29,30,31.

CSDS model

The procedure of CSDS was performed as reported previously26,29,30,46,47,48,49,50. 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.

Treatment and behavioral tests

Saline (10 ml/kg), (R)-ketamine (10 mg/kg), or (S)-ketamine (10 mg/kg) was administered intraperitoneally (i.p.) into the susceptible mice after CSDS. Saline (10 ml/kg) was also administered i.p. into control mice (Fig. 1a). Behavioral tests, including locomotion test, tail suspension test (TST), forced swimming test (FST), and 1% sucrose preference test (SPT), were performed as reported previously26,29,30,46,47,48,49,50. Behavioral tests were also performed by two observers who were blinded to the group assignment of mice. Each treatment group was equally represented in each experimental cohort.

Fig. 1: Antidepressant effects of ketamine enantiomers in susceptible mice after CSDS a The schedule of CSDS model, treatment, behavioral tests, and feces collection. CSDS was performed from day 1 to day 10, and social interaction test (SIT) was performed on day 11. Saline, (R)-ketamine, or (S)-ketamine were administered i.p. into CSDS-susceptible mice. Behavioral tests and SPT were performed from day 12 to day 15. On day 16, mouse feces was collected. b Body weight (time: F 3,15 = 9.3, P < 0.001, treatment: F 3,15 = 0.268, P = 0.848, interaction: F 9,15 = 0.576, P = 0.813). c–e Behavioral tests including locomotion test (LMT; one-way ANOVA, F 3,20 = 0 159, P = 0.923), TST (F 3,20 = 18.362, P < 0.001) and FST (F 3,20 = 15.107, P < 0.001) were performed after treatment. f SPT was performed 3 days after treatment (F 3,20 = 20.287, P < 0.001). Data are shown as mean ± SEM (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001. NS not significant Full size image

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.

16S rRNA analysis of fecal samples

The fecal samples were collected 4 days (day 16) after a single dose of saline (10 ml/kg), (R)-ketamine (10 mg/kg), or (S)-ketamine (10 mg/kg). They placed in 1.5 ml tubes, snap-frozen on dry ice, and stored at −80°C. The 16S rRNA analysis of fecal samples was performed at Takara Bio. Inc. (Shiga, Japan). The DNA extraction was performed using the MoBio Powerlyzer Powersoil DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA). The V4 hypervariable region of the bacterial 16S rRNA gene was amplified from the fecal DNA extracts using modified universal bacterial primer pairs 515 F (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA-3′) and 806 R (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT-3′) with Illumina adaptor overhang sequences. Amplicons were generated, cleaned, indexed, and sequenced according to the Illumina MiSeq 16S Metagenomic Sequencing Library Preparation protocol (http://support.illumina.com/ downloads/16s_metagenomic_sequencing_library_preparation.html) with slight modifications. Sequencing data were combined and sample identification assigned to multiplexed reads using the MOTHUR software environment42,51. The data were denoised; low-quality sequences, pyrosequencing errors, and chimeras were removed, and then sequences were clustered into operational taxonomic units (OTUs) at 97% identity using the CD-HITOTU pipeline (available from http://eeizhong-lab.ucsd.edu/cd-hit-otu)42,52. OTUs containing fewer than four reads per individual diet/animal combination were excluded due to the likelihood of there being a sequencing artifact. The samples were normalized by randomly resampling sequences used to the lowest number of sequences per sample (each diet/animal combination) using Daisychopper (http://www.festinalente.me/bioinf/). Taxonomic classification of OTUs was conducted using the Ribosomal Database Project Classifier42,53.

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.