Lack of metabolism in (R)-ketamine’s antidepressant actions in a chronic social defeat stress model

Since the metabolism of (R,S)-ketamine to (2R,6R)-hydroxynorketamine (HNK) is reported to be essential for ketamine’s antidepressant effects, there is an increasing debate about antidepressant effects of (2R,6R)-HNK. Using pharmacokinetic and behavioral techniques, we investigated whether intracerebroventricular (i.c.v.) infusion of (R)-ketamine or (2R,6R)-HNK show antidepressant effects in a chronic social defeat stress (CSDS) model of depression. Low levels of (2R,6R)-HNK in the brain after i.c.v. infusion of (R)-ketamine were detected, although brain levels of (2R,6R)-HNK were markedly lower than those after i.c.v. infusion of (2R,6R)-HNK. Furthermore, high levels of (2R,6R)-HNK in the blood and liver after i.c.v. infusion of (R)-ketamine or (2R,6R)-HNK were detected. A single i.c.v. infusion of (R)-ketamine showed rapid and long-lasting (7 days) antidepressant effects in a CSDS model. In contrast, i.c.v. infusion of (2R,6R)-HNK did not show any antidepressant effect in the same model, although brain concentration of (2R,6R)-HNK was higher than after i.c.v. infusion of (R)-ketamine. This study suggest that (R)-ketamine in the periphery after washout from the brain is metabolized to (2R,6R)-HNK in the liver, and subsequently, (2R,6R)-HNK enters into brain tissues. Furthermore, it is unlikely that (2R,6R)-HNK is essential for the antidepressant actions of (R)-ketamine in a CSDS model.

To exclude the metabolism of ketamine to HNK in the liver, this study was undertaken to examine whether intracerebroventricular (i.c.v.) infusion of (R)-ketamine or its metabolite (2R,6R)-HNK shows antidepressant effects in a CSDS model of depression. First, using a liquid chromatography tandem mass spectrometry (LC-MS/MS), we determined the concentration of (2R,6R)-HNK in the brain, blood and liver after i.c.v. injection of (R)-ketamine and (2R,6R)-HNK. Second, we examined whether i.c.v. infusion of (R)-ketamine and (2R,6R)-HNK shows antidepressant effects in a CSDS model.

Discussion
In the present study, we found evidence of (2R,6R)-HNK in the brain after i.c.v. infusion of (R)-ketamine, although brain concentration of (2R,6R)-HNK was lower than those after i.c.v. infusion of (2R,6R)-HNK. Furthermore, we detected high concentration of (2R,6R)-HNK in the blood and liver after i.c.v. infusion of (R)-ketamine. These data suggest that (R)-ketamine in the periphery after washout from the brain is metabolized to (2R,6R)-HNK in the liver, and subsequently, (2R,6R)-HNK enters into brain tissues. In addition, we also found high concentration of (2R,6R)-HNK in the blood and liver after i.c.v. infusion of (2R,6R)-HNK, indicating the rapid washout into periphery from the brain. Nonetheless, the concentration of (2R,6R)-HNK in the brain after i.c.v. infusion of (R)-ketamine was lower than that after i.c.v. infusion of (2R,6R)-HNK.
In this study, we found that i.c.v. infusion of (R)-ketamine showed rapid and long-lasting antidepressant effects in a CSDS model, consistent with previous reports using intraperitoneal administration 25,[27][28][29]36,37 . In contrast, i.c.v. infusion of (2R,6R)-HNK did not show any antidepressant effects in a CSDS model, although the concentrations of (2R,6R)-HNK in the brain were higher than those after i.c.v. infusion of (R)-ketamine. Furthermore, we reported that intraperitoneal administration of (R)-ketamine, but not (2R,6R)-HNK, shows rapid and long-lasting antidepressant effects in a CSDS model and an inflammation-induced model 27 . In a rat LH model, we recently reported that (2R,6R)-HNK (20 or 40 mg/kg, 24 h and 5 days) did not elicit any antidepressant effects in LH rats, although (R)-ketamine (20 mg/kg) showed sustained (24 h) and long-lasting (5 days) antidepressant effects in the same model 30 . In addition, we reported that a single bilateral infusion of (R)-ketamine into the infralimbic (IL) region of the medial prefrontal cortex (mPFC), CA3, and dentate gyrus of the hippocampus shows long-lasting (5 days) antidepressant effects in a rat LH model 38 . A previous study also demonstrated that microinfusion of (R,S)-ketamine into IL of mPFC produces antidepressant-like effects in control unstressed rats 39 . Collectively, it is likely that (R)-ketamine itself in the brain can exert antidepressant effects in rodents with depression-like phenotype and that (2R,6R)-HNK in the brain does not have antidepressant effects in rodents with depression-like phenotype.
In  15 . In contrast, many previous reports showed that (+)-MK-801 had antidepressant-like effects in control naïve rodents [41][42][43][44][45][46] . In addition, we reported that (+)-MK-801 induces rapid antidepressant effects in a CSDS model, although this response is not long-lasting 47 . Taking these findings together, it is possible that NMDAR inhibition and other unknown mechanisms may play a role in the long-lasting (7 days) antidepressant actions of ketamine, although NMDAR inhibition may play a role in a rapid antidepressant effect 47 .
Recently, Yao et al. 48 reported that a single intraperitoneal injection of (R,S)-ketamine (10 mg/kg, 1 day) impaired long-term potentiation (LTP) in the nucleus accumbens (NAc) of control mice but had no effects on the basic properties of glutamatergic transmission in this region. This loss of LTP in the NAc was maintained for 7 days, consistent with the long-lasting antidepressant actions of (R,S)-ketamine. Furthermore, a single injection of (2R,6R)-HNK (10 mg/kg, 1 day) also impaired LTP in the NAc of control mice. Interestingly, (R,S)-ketamine (10 mg/kg) and its enantiomers (R)-and (S)-ketamine (10 mg/kg) significantly attenuated reduced dendritic spine density, brain-derived neurotrophic factor (BDNF) and its receptor TrkB signaling, and GluA1/PSD-95 expression in the medial prefrontal cortex (mPFC) and hippocampus (CA3 and DG) of mice with a depression-like phenotype, but did not alter the corresponding elevations in NAc 25,28,49 . In the rat LH model, we also reported that a single bilateral infusion of (R)-ketamine into the infralimbic region of mPFC, CA3, and DG improved depression-like symptoms, whereas a single bilateral infusion of (R)-ketamine into the NAc did not induce antidepressant effects 38 . These findings suggest that (R,S)-ketamine and its enantiomers exert antidepressant effects by normalizing BDNF−TrkB signaling and synaptogenesis in the mPFC and hippocampus, but not NAc. Taken together, it is unlikely that NAc plays a direct role in the antidepressant actions of (R,S)-ketamine and its two enantiomers.
In conclusion, the present study demonstrates that i.c.v. infusion of (R)-ketamine, but not its final metabolite (2R,6R)-HNK, could elicit a rapid and long-lasting antidepressant effect in a CSDS model, although low tissue concentrations of (2R,6R)-HNK were detected in the brain after i.c.v. infusion of (R)-ketamine. The present data argue against the claim made by a paper that stated that (2R,6R)-HNK is essential for the antidepressant actions of (R,S)-ketamine [or (R)-ketamine] 15 . Finally, we propose that (R)-ketamine, through NMDAR inhibition and subsequent unidentified mechanisms (for instance, synaptogenesis via BDNF-TrkB signaling) 25

Methods and Materials
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., Hamamatsu, Japan) were used. Animals were housed under controlled temperatures and 12 hour light/dark cycles (lights on between 07:00-19:00 h), with ad libitum food (CE-2; CLEA Japan, Inc., Tokyo, Japan) and water. The study was approved by the Chiba University Institutional Animal Care and Use Committee (Permission number: ). 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. All efforts were made to minimize suffering.
Materials. (R)-ketamine hydrochloride was prepared as previously reported 24 . The purity of (R)-ketamine was determined by a high-performance liquid chromatography with a chiral column as previously reported 24 . (2R,6R)-HNK hydrochloride was purchased from Sigma-Aldrich Co, Ltd (St Louis, MO, USA).
Determination of (2R,6R)-HNK in the mouse samples was performed at Sumika Chemical Analysis Service, Ltd (Osaka, Japan). A 50-µl aliquot of the plasma or brain (or liver) homogenate specimen was mixed with 25 µl of 1 mM ammonium hydrogen carbonate/acetonitrile (7:3, v/v), 20 µl of acetonitrile/methanol (9:1, v/v) containing 2 H 4 -norketamine (Sigma-Aldrich Co, Ltd, St. Louis, MO, USA) as an internal standard (I.S.) and 100 µl of 1 mM ammonium hydrogen carbonate. And t-butyl methyl ether, 2 mL, was added and vortex-mixed for 1 minute. After centrifugation at 3,000 rpm for 5 minutes, the organic layer was transferred to another empty glass tube. The solvent was evaporated to dryness under a stream of nitrogen gas at 25 °C. The residue was dissolved in 100 μl of 1 mM ammonium hydrogen carbonate/acetonitrile (7:3, v/v) by vortex-mixing for 30 seconds and sonicating for 1 minute. The solution was centrifuged at 3,000 rpm for 5 minutes. A 5-µl aliquot of the supernatant resulting from the pretreatment was subjected to an enantioselective liquid chromatography tandem mass spectrometry (LC-MS/ MS) assay. The LC-MS/MS system was constructed using a Shimadzu LC-20A high-performance liquid chromatography system (Shimadzu, Tokyo, Japan) and API5000 tandem mass spectrometer (AB SCIEX, Foster City, CA, USA). The MS/MS data were acquired and processed using Analyst version 1.6.1 software (AB SCIEX, Foster City, CA). Chromatographic separation was performed at 25 °C on a CHIRALPAK AS-3R analytical column (4.6 mm i.d. × 100 mm, 3 µm particles, Daicel Corporation, Tokyo, Japan) using 1 mM ammonium hydrogencarbonate/acetonitrile (54:46, v/v) as a mobile phase at a flow rate of 1.0 ml/min. The selected reaction monitoring transition of (2R,6R)-HNK was m/z 240.5 → m/z 125.0, and the I.S. was m/z 228.1 → m/z 129.1. The lower limit of quantification (LLOQ) in the brain and liver was 0.5 ng/g tissue. The LLOQ in plasma was 0.1 ng/ml. Chronic social defeat stress (CSDS) model. CSDS was performed as previously reported 25,[27][28][29]36,37,48,54,55 . The C57BL/6 mice were exposed to a different CD1 aggressor mouse for 10 min/day for 10 days. After the social defeat session, the resident CD1 mouse and the intruder C57BL/6 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-hour period. All mice were housed individually 24 hour after the last social defeat stress session. On day 11, a social interaction test (SIT) was performed to divide susceptible group and resilient group to CSDS. The test 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 CD1 mouse 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 wiremesh 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. The cutoff for an interaction ratio was set as 1. Mice with scores < 1 were defined as "susceptible" to social defeat stress, and mice with scores ≥ 1 were defined as "resilient". Only susceptible mice were used in the behavioral experiments. Control C57BL/6 mice not exposed CSDS were housed in the home cage before the behavioral tests.
Tail suspension test (TST). TST was performed 4 hour after i.c.v. infusion. A small piece of adhesive tape placed approximately 2 cm from the tip of the mouse tail. A single hole was punched in the tape and mice were hung individually, on a hook. The TST immobility time was recorded for 10 minutes. Mice were considered immobile only when they hung passively and completely motionless.
Sucrose preference test (SPT). SPT was performed 2, 4, 7 days after i.c.v. infusion. Mice were exposed to water and 1% sucrose solution for 48 h, followed by 4 hours of water and food deprivation. The two identical bottles containing water and 1% sucrose were weighed before and at the end of this period (1 hour). 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 ± standard error of the mean (S.E.M.). Analysis was performed using PASW Statistics 20 (formerly SPSS Statistics; SPSS). Comparisons between groups were performed using the one-way analysis of variance (ANOVA), followed by post-hoc Tukey test. The P-values of less than 0.05 were considered statistically significant.