5-Hydroxytryptamine-Independent Antidepressant Actions of (R)-Ketamine in a Chronic Social Defeat Stress Model

5-Hydroxytryptamine-Independent Antidepressant Actions of (R)-Ketamine in a Chronic Social Defeat... Background: Previous reports suggest that 5-hydroxytryptamine might play a role in the antidepressant actions of ( R,S)- ketamine. However, its role in the antidepressant actions of ( R)-ketamine, which is more potent than (S)-ketamine, is unknown. This study was conducted to examine whether 5-hydroxytryptamine depletion affects the antidepressant actions of (R)-ketamine in a chronic social defeat stress model. Methods: An inhibitor of 5-hydroxytryptamine synthesis, par a-chlorophenylalanine methyl ester hydrochloride (300 mg/kg, twice daily for 3 consecutive days), or vehicle was administered to control and chronic social defeat stress-susceptible mice. Levels of 5-hydroxytryptamine and its metabolite, 5-hydroxyindoleacetic acid, in mouse brain regions were measured using high-performance liquid chromatography. Furthermore, antidepressant effects of ( R)-ketamine (10 mg/kg) in the vehicle- and para-chlorophenylalanine methyl ester hydrochloride-treated susceptible mice were assessed using tail suspension test and 1% sucrose preference test. Results: para-Chlorophenylalanine methyl ester hydrochloride treatment caused marked reductions of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the brain regions of control and chronic social defeat stress susceptible mice. In the tail suspension test, (R)-ketamine significantly attenuated the increased immobility time in the chronic social defeat stress- susceptible mice with or without 5-hydroxytryptamine depletion. In the sucrose preference test (2 and 5 days after a single dose), (R)-ketamine significantly enhanced reduced sucrose consumption in the chronic social defeat stress-susceptible mice with or without 5-hydroxytryptamine depletion. Conclusions: These findings show that 5-hydroxytryptamine depletion did not affect the antidepressant effects of ( R)- ketamine in a chronic social defeat stress model. Therefore, it is unlikely that 5-hydroxytryptamine plays a major role in the antidepressant actions of ( R)-ketamine. Keywords: antidepressant, ( R)-ketamine, serotonin, stress Received: September 5, 2017; Revised: October 17, 2017; Accepted: November 15, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 157 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 158 | International Journal of Neuropsychopharmacology, 2018 Significance Statement The rapid and long-lasting antidepressant effects of ( R,S)-ketamine in patients with treatment-resistant depression are the most important discovery in the field of depression research in half a century. Although previous studies suggest that 5-hydroixy - tryptamine (5-HT, serotonin) plays a role in the antidepressant effects of ketamine, the role of 5-HT in the antidepressant effects of (R)-ketamine remain unknown. Here we report that 5-HT depletion by para-chlorophenylalanine did not affect antidepressant actions of ( R)-ketamine in a chronic social defeat stress model. It is, therefore, unlikely that 5-HT may play a major role in the antidepressant actions of ( R)-ketamine. the role of 5-HT in the antidepressant effects of ( R)-ketamine Introduction in animal models such as a chronic social defeat stress (CSDS) The N-methyl-D-aspartate receptor antagonist ketamine exhibits model has not been reported. rapid and long-lasting antidepressant effects in treatment-resist - To elucidate the role of 5-HT in the antidepressant actions ant patients with major depressive disorder or bipolar disorder of (R)-ketamine, this study was undertaken to examine whether (Newport et al., 2015 ; Kishimoto et al., 2016). At present, ketamine is 5-HT depletion in the brain caused by the administration of the most attractive antidepressant for the treatment of treatment- PCPA could influence the antidepressant effects of (R)-ketamine resistant depression (Monteggia and Zarate, 2015 ; Duman et  al., in a CSDS model. 2016; Hashimoto, 2016a, 2016b, 2017), although the precise mecha- nisms underlying its antidepressant actions remain unknown. (R,S)-Ketamine is a racemic mixture containing equal parts Methods and Materials of (R)-ketamine and (S)-ketamine. (S)-Ketamine shows approxi - mately 3- to 4-fold greater anesthetic potency and greater unde - Animals sirable psychotomimetic side effects than ( R)-ketamine (Domino, Male adult C57BL/6 mice (n = 72), aged 8 weeks (body weight 2010; Hashimoto, 2016a). Meanwhile, (R)-ketamine shows greater 20–25 g, Japan SLC, Inc.) and male adult CD1 (ICR) mice (n = 20), potency and longer-lasting antidepressant effects than ( S)- aged 13 to 15 weeks (body weight >40  g, Japan SLC, Inc.) were ketamine in animal models of depression ( Zhang et al., 2014; Yang used. Animals were housed under controlled temperatures and et al., 2015, Fukumoto et al., 2017; Yang et al., 2017a, 2017b, 2017c). 12-hour-light/-dark cycles (lights on between 7:00 am and 7:00 Unlike (S)-ketamine, (R)-ketamine does not induce psychotomi - pm), with ad libitum food (CE-2; CLEA Japan, Inc) and water. The metic side effects or exhibit abuse potential in rodents ( Yang protocol was approved by the Chiba University Institutional et al., 2015, 2016). Furthermore, we reported a marked reduction Animal Care and Use Committee (permission no. 29–345). This of dopamine D receptor binding in conscious monkey striatum 2/3 study was carried out in strict accordance with the recommen - after a single infusion of ( S)-ketamine but not that of ( R)-ketamine dations in the Guide for the Care and Use of Laboratory Animals (Hashimoto et  al., 2017), suggesting that ( S)-ketamine-induced of the National Institutes of Health, USA. Animals were deeply dopamine release might be associated with acute psychotomi- anaesthetized with isoflurane before being killed by cervical dis - metic and dissociative side effects in humans (Hashimoto et al., location. All efforts were made to minimize suffering. 2017). Therefore, (R)-ketamine could be a safer antidepressant in humans than (S)-ketamine (Hashimoto, 2016a, 2016b, 2017). 5-Hydroxytryptamine (5-HT, serotonin) plays a major role Materials in the antidepressant effects of the antidepressant drugs cur - rently being used. In the forced swimming test (FST), depletion (R)-Ketamine hydrochloride was prepared by recrystallization of of brain 5-HT by treatment with an inhibitor of 5-HT synthesis, (R,S)-ketamine (Ketalar, ketamine hydrochloride, Daiichi Sankyo para-chlorophenylalanine methyl ester hydrochloride (PCPA), Pharmaceutical Ltd) and D-(-)-tartaric acid, as described previously attenuated the sustained (24 hour), but not acute (1 hour), (Zhang et al., 2014). The purity of (R)-ketamine was determined by reduction in the immobility time after a single dose of ( R,S)- a high-performance liquid chromatography (HPLC) (CHIRALPAK ketamine (25  mg/kg) ( Gigliucci et  al., 2013). Interestingly, the IA, column size: 250 x 4.6  mm, mobile phase: n-hexane/dichlo - increase in the immobility time provoked by repeated restraint romethane/diethylamine [75/25/0.1], Daicel Corporation). The stress was blocked by a single dose of ketamine (25  mg/kg, dose (10 mg/kg as hydrochloride) of (R)-ketamine dissolved in the 24 hours prior to FST), but ketamine’s effects were not abol - physiological saline was used as previously reported (Zhang et al., ished when rats were subjected to 5-HT depletion ( Gigliucci 2014; Yang et al., 2015, 2016a, 2016b, 2017a, 2017b, 2017c). The dose et  al., 2013). In contrast,Fukumoto et  al. (2016) reported that of PCPA (300 mg/kg, 5-HT synthesis inhibitor, Sigma-Aldrich Co.) the antidepressant-like effects of ( R,S)-ketamine (30  mg/kg, was used as previously reported (Fukumoto et  al., 2016). Other 30 minutes prior to FST) were attenuated by depletion of reagents were purchased commercially. 5-HT in the brain upon treatment with PCPA. MoreoverPham , et  al. (2017) also reported that pretreatment with PCPA abol - CSDS Model ished (R,S)-ketamine (10 mg/kg, 24 hours prior to FST)-induced antidepressant-like effects in the FST. Although these 3 stud - The procedure of CSDS was performed as previously reported ies suggest the role of 5-HT in R,S ( )-ketamine’s antidepressant (Zhang et al., 2015; Yang et al., 2015, 2016a, 2016b, Yang et al., 2017a, effects in control rodents ( Gigliucci et al., 2013; Fukumoto et al., 2017b, 2017c; Ren et al., 2016). The C57BL/6 mice were exposed 2016; Pham et al., 2017), it is unlikely that 5-HT plays a role in to a different CD1 aggressor mouse for 10 min/d for consecutive the antidepressant effects of ( R,S)-ketamine in rodents with a 10 days. When the social defeat session ended, the resident CD1 depression-like phenotype ( Gigliucci et  al., 2013). Thus, there mouse and the intruder mouse were housed in one-half of the appears to be variation in the role of 5-HT in the antidepres - cage separated by a perforated Plexiglas divider to allow visual, sant-like effects of ( R,S)-ketamine depending on the baseline olfactory, and auditory contact for the remainder of the 24-hour status of the rodents to which it is administered. In addition, period. At 24 hours after the last session, all mice were housed Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Zhang et al. | 159 individually. On day 11, a social interaction test was performed to (CMC)] or PCPA (300  mg/kg, twice daily (9:00 am and 7:00 pm) identify subgroups of mice that were susceptible and unsuscepti - for 3 consecutive days) was administered i.p. into the control ble to social defeat stress. This was accomplished by placing mice and CSDS susceptible mice (day 12–14). On day 15, all mice were in an interaction test box (42 × 42 cm) with an empty wire-mesh killed by decapitation after isoflurane anesthesia, and brain cage (10 × 4.5 cm) located at one end. The movement of the mice regions including prefrontal cortex (PFC), hippocampus, and was tracked for 2.5 minutes, followed by 2.5 minutes in the pres - striatum were collected. These brain samples were stored at ence of an unfamiliar aggressor confined in the wire-mesh cage. -80℃ before assay (Figure 1A). The duration of the subject’s presence in the “interaction zone” The brain samples were homogenized in 0.2 M perchloric (defined as the 8-cm-wide area surrounding the wiremesh cage) acid (HClO ) containing 100 μM disodium EDTA and 100 ng/mL was recorded by a stopwatch. The interaction ratio was calculated isoproterenol (internal standard) and were then centrifuged at as time spent in an interaction zone with an aggressor/time spent 20 000 × g for 15 minutes at 4°C. The supernatants were filtered in an interaction zone without an aggressor. An interaction ratio through a 0.45-μm-pore membrane (Millex-LH, 4 mm; Millipore) of 1 was set as the cutoff: mice with scores <1 were defined as and analyzed for 5-HT and 5-HIAA by HPLC coupled with electro - “susceptible” to social defeat stress and those with scores ≥1 were chemical detection. The HPLC system consisted of a liquid chro - defined as “resilient.” Approximately 70% to 80% of mice were sus - matograph pump (HTEC-500, Eicom), a reversed phase column ceptible after CSDS. Susceptible mice were randomly divided in (Eicompak SC-5ODS 150  × 3.0 mm; Eicom), and a data processor the subsequent experiments. Control C57BL/6 mice not exposed (EPC-500, Eicom). The mobile phase was 0.1 M acetate-citric acid CSDS were housed in the cage before the behavioral tests. buffer (pH 3.5) containing 13% methanol, 5 mg/L disodium EDTA, and 190 mg/L sodium octyl sulfate. Treatment and Measurement of 5-HT and 5-HIAA in Mouse Brain by HPLC Treatment and Behavioral Tests Control mice and CSDS susceptible mice were divided into The CSDS susceptible mice were divided into 4 groups; vehicle + saline group, vehicle + ( R)-ketamine group, PCPA + saline group, 2 groups, respectively. Vehicle [0.5% carboxymethylcellulose Figure 1. Schedule of a chronic social defeat stress (CSDS) model, treatment, and high-performance liquid chromatography (HPLC) measurement. (A) CSDS was per - formed from day 1 to day 10, and the social interaction test (SIT) was performed on day 11. Vehicle (0.5% carboxymethylcellulose [CMC]) or para-chlorophenylalanine methyl ester hydrochloride (PCPA) (300 mg/kg, twice daily [9:00 am and 7:00 pm] for 3 consecutive days) was administered i.p. in the susceptible mice from day 12 to day 14. All mice were sacrificed by decapitation, and brain samples were collected on day 15. (B) Prefrontal cortex (PFC), (C) hippocampus, (D) striatum. The values represent the mean ± SEM (n = 8). *P < .05, **P < .01, ***P < .001. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 160 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Effects of 5-hydroxytryptamine (5-HT) deletion in the antidepressant effects of ( R)-ketamine in a chronic social defeat stress (CSDS) model. (A) CSDS was per - formed from day 1 to day 10, and the social interaction test (SIT) was performed on day 11. Vehicle (0.5% carboxymethylcellulose [CMC]) or para-chlorophenylalanine methyl ester hydrochloride (PCPA) (300 mg/kg, twice daily [9:00 am and 7:00 pm] for 3 consecutive days) was administered i.p. in the susceptible mice from day 12 to day 14. Saline (10 mL/kg) or (R)-ketamine (10 mg/kg) was administered i.p. into mice on day 15. LMT and TST were performed 2 and 4 hours after a single injection of ( R)-ketamine or saline, respectively. SPT was performed 2 and 5 days after a single injection of ( R)-ketamine or saline. (B) Locomotion test (LMT) (day 15). (C) Tail suspension test (TST) (day 15). (D) Sucrose preference test (SPT) (day 17). (E) SPT (day 20). The values represent the mean ± SEM (n = 8). * P < .05, **P < .01, ***P < .001. N.S., not significant; R-KT, ( R)-ketamine. and PCPA + (R)-ketamine group. Vehicle (0.5% CMC) or PCPA Locomotion (300 mg/kg twice daily [9:00 am and 7:00 pm] for 3 consecutive The locomotor activity was measured by an animal movement days) was administered i.p. to the susceptible mice after CSDS analysis system SCANETMV-40 (MELQUEST Co., Ltd). The mice (days 12–14). Subsequently, saline (10 mL/kg) or (R)-ketamine were placed in experimental cages (length × width × height: (10 mg/kg) was administered i.p. into mice (day 15) ( Figure 2A). 560 × 560 × 330  mm). The cumulative locomotor activity counts Behavioral tests, including locomotion test (LMT), tail suspension were recorded for 60 minutes. Cages were cleaned between test - test (TST), and 1% sucrose preference test (SPT), were performed ing session. as reported previously (Zhang et al., 2015; Yang et al., 2015, 2017a, 2017b, 2017c; Ren et al., 2016). LMT and TST were performed 2 and TST 4 hours after a single injection of ( R)-ketamine or saline, respect- A small piece of adhesive tape was placed approximately 2 cm ively. SPT was performed 2 and 5 days after a single injection from the tip of the tail for mouse. A single hole was punched in of (R)-ketamine or saline. Behavioral tests were performed by 2 the tape, and mice were hung individually on a hook. The immo - observers who were blinded to the group assignment of mice. bility time was recorded for 10 minutes. Mice were considered Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Zhang et al. | 161 immobile only when they hung passively and completely (R)-ketamine-treated group (Figure 2D–E). These results suggest motionless. that depression-like phenotype in the susceptible mice after CSDS was not affected by 5-HT depletion and that depletion of SPT 5-HT in the brain by PCPA did not abolish the antidepressant Mice were exposed to water and 1% sucrose solution for 48 effects of ( R)-ketamine in a CSDS model. hours, followed by 4 hours of water and food deprivation and a 1-hour exposure to 2 identical bottles (water and 1% sucrose so - Discussion lution). The bottles containing water and sucrose were weighed before and at the end of this period. The sucrose preference was In the present study, we demonstrated that reductions of 5-HT calculated as a percentage of sucrose solution consumption to and 5-HIAA in mouse brain upon the administration of PCPA did the total liquid consumption. not affect the depression-like phenotype in the susceptible mice after CSDS and that PCPA-induced 5-HT depletion did not abolish the antidepressant effects of ( R)-ketamine in the CSDS suscepti- Statistical Analysis ble mice. These findings suggest that 5-HT does not play a major The data show as the mean ± SEM. Analysis was performed role in the antidepressant actions of ( R)-ketamine in a CSDS using PASW Statistics 20 (formerly SPSS Statistics; SPSS). The model. To our knowledge, this is the first report showing that data of 5-HT and 5-HIAA were analyzed using the 2-way ANOVA 5-HT depletion in the brain does not affect the antidepressant followed by posthoc Tukey test. The behavioral data were ana - effects of ( R)-ketamine in mice with a depression-like phenotype. lyzed using the 1-way ANOVA followed by posthoc Tukey test. A previous study showed that the depletion of 5-HT by P < .05 was considered statistically significant. PCPA did not abolish the acute antidepressant effects of ( R,S)- ketamine (25 mg/kg, 1 h prior to FST) in control SD rats ( Gigliucci et  al., 2013). In contrast, depletion of 5-HT by PCPA abolished Results the acute antidepressant effects of ( R,S)-ketamine (30 mg/kg, 30 minutes prior to FST) in control C57BL/6J mice (Fukumoto et al., Effects of PCPA on the Levels of 5-HT and 5-HIAA in 2016). The reasons underlying this discrepancy are currently the Brain Regions unknown. First, the dose (25 mg/kg for rats vs 30 mg/kg for mice) The effects of PCPA treatment on the tissue levels of 5-HT and of (R,S)-ketamine may have contributed to this discrepancy, 5-HIAA in the PFC, hippocampus, and striatum were exam - since these doses can increase locomotor activity after a single ined. PCPA treatment caused significant reduction of 5-HT administration ( Yang et al., 2015; Fukumoto et al., 2017). Second, and 5-HIAA in these 3 regions (Figure  1B–D). Two-way ANOVA the species difference (rats vs mice) may have contributed to (PCPA and stress) showed statistical results (PFC: 5-HT, PCPA, this discrepancy. Therefore, acute hyperactivity should be taken F = 452.76, P < .001, stress, F = 2.27, P = .14, interaction, into consideration to examine the acute antidepressant effects 1,32 1,32 F = 0.30, P = .59; 5-HIAA, PCPA, F = 460.75, P < .001, stress, of (R,S)-ketamine and its enantiomers. 1,32 1,32 F = 5.80, P = .02, interaction, F = 5.53, P = .03; hippocampus: Using Flinders Sensitive Line (FSL) rats (a genetic model of 1,32 1,32 5-HT, PCPA, F = 669.57, P < .001, stress, F = 3.37, P = .08, inter - depression), du Jardin et al. (2016b) examined the effects of 5-HT 1,32 1,32 action, F = 0.95, P = .34, 5-HIAA, PCPA, F = 680.08, P < .001, depletion by PCPA on the antidepressant effects of ( S)-ketamine 1,32 1,32 stress, F = 0.63, P = .44, interaction, F = 1.15, P = .29; striatum: (15  mg/kg). The depression-like phenotype in FSL rats was not 1,32 1,32 5-HT, PCPA, F = 434.70, P < .001, stress, F = 3.96, P = .06, inter - associated with 5-HT depletion. Interestingly, the acute (1 hour) 1,32 1,32 action, F = 2.56, P = .12, 5-HIAA, PCPA, F = 609.74, P < .001, stress, and sustained (24 hours) antidepressant effects of ( S)-ketamine 1,32 1,32 F = 1.49, P = .23, interaction, F = 2.20, P = .15). These data sug- (15 mg/kg) in FSL rats were abolished by 5-HT depletion, suggest- 1,32 1,32 gest that the reductions of 5-HT in the brain regions by PCPA ing that the acute and sustained antidepressant-like effects of were not affected by CSDS. (S)-ketamine in FSL rats depend on the endogenous 5-HT concen- tration ( du Jardin et al., 2016a , 2016b ). However, our present results indicate that acute and long-lasting (5  days after a single dose) Effects of 5-HT Reduction in the Antidepressant antidepressant effects of ( R)-ketamine (10 mg/kg) in a CSDS model Effect of (R)-Ketamine in a CSDS Model in male C57BL/6 mice occur independently of brain 5-HT depletion. Next, we examined the effects of PCPA-induced 5-HT reduc - Interestingly, 5-HT depletion in the brain did not affect the sus - tion in the antidepressant effects of ( R)-ketamine (10 mg/ tained antidepressant effects of ( R,S)-ketamine (25 mg/kg, 24 hours kg) in a CSDS model ( Figure 2A). Locomotion showed no dif- prior to FST) on the increased immobility time caused by repeated ference (F = 0.43, P = .79) among the 5 groups (Figure 2B). restraint stress ( Gigliucci et  al., 2013). Although the reasons for 4,40 One-way ANOVA of TST data showed a statistical significance these discrepancies are currently unknown, there are several fac - (F = 2.41, P < .01) among the 5 groups (Figure 2C). Posthoc tests tors that could have contributed to them, such as different models 4,40 showed that ( R)-ketamine (10 mg/kg) significantly attenuated (control animals vs genetic rat model vs stress model) and different the increased immobility times in susceptible mice after CSDS doses and isomers of ketamine (25 mg/kg vs 30 mg/kg vs 10 mg/ (Figure 2C). However, there were no differences between ve - kg; (R,S)-ketamine vs (S)-ketamine vs (R)-ketamine). Therefore, fur - hicle + (R)-ketamine group and PCPA + (R)-ketamine group in ther detailed studies on the role of 5-HT in the acute and sustained the TST immobility times ( Figure 2C). One-way ANOVA of SPT antidepressant effects of ( R,S)-ketamine and its enantiomers in data showed statistical significances (2 days after a single injec - rodents with a depression-like phenotype are needed. tion: F = 5.06, P = .003, 5 days after a single injection: F = 4.36, Fukumoto et  al. (2016) reported the role of 5-HT neurons in 4,40 4,40 P = .01) among 5 groups ( Figure 2D–E). Posthoc tests showed that the dorsal raphe nucleus (DRN) regulated by the medial PFC sucrose preference in vehicle + (R)-ketamine-treated group was (mPFC)–DRN projections in the acute antidepressant effects of significantly higher than in the vehicle + saline-treated group (R,S)-ketamine (30 mg/kg, 30 minutes prior to FST). In addition, (Figure 2D–E). Furthermore, sucrose preference in the vehicle + antidepressant effects of ( R,S)-ketamine were reported to be (R)-ketamine-treated group was not different from the PCPA + blocked by an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 162 | International Journal of Neuropsychopharmacology, 2018 acid receptor antagonist, NBQX (10  mg/kg). Domino EF (2010) Taming the ketamine tiger. Anesthesiology Interestingly, microinjection of ( R,S)-ketamine into mPFC sig - 113:678–684. nificantly increased the c-Fos expression in 5-HT neurons in Duman RS, Aghajanian GK, Sanacora G, Krystal JH (2016) the DRN, and the effect of ( R,S)-ketamine was attenuated by the Synaptic plasticity and depression: new insights from stress microinjection of NBQX into the mPFC. These results suggest that and rapid-acting antidepressants. Nat Med 22:238–249. the activation of 5-HT neurons in the DRN regulated by stimula - Fukumoto K, Iijima M, Chaki S (2016) The antidepressant effects of tion of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic an mGlu2/3 receptor antagonist and ketamine require AMPA acid receptor in the mPFC may be involved in the antidepres- receptor stimulation in the mPFC and subsequent activation sant effects of ( R,S)-ketamine (Fukumoto et al., 2016). Moreover, of the 5-HT neurons in the DRN. Neuropsychopharmacol Pham et al. (2017) reported an increase (144%) in the extracellu - 41:1046–1056. lar 5-HT levels in the mPFC after a single dose of R,S ( )-ketamine Fukumoto K, Toki H, Iijima M, Hashihayata T, Yamaguchi JI, (10  mg/kg). In addition, pretreatment with NBQX blunted the Hashimoto K, Chaki S (2017) Antidepressant potential of ( R)- effects of intra-mPFC ketamine on both the swimming duration ketamine in rodent models: comparison with (S)-ketamine. J in the FST and extracellular 5-HT in the mPFC, suggesting a key Pharmacol Exp Ther 361:9–16. role of cortical 5-HT release in ( R,S)-ketamine’s antidepressant- Gigliucci V, O’Dowd G, Casey S, Egan D, Gibney S, Harkin A (2013) like activity (Pham et  al., 2017). However, these 2 studies used Ketamine elicits sustained antidepressant-like activity via control animals, but not animals with a depression-like pheno- a serotonin-dependent mechanism. Psychopharmacology type. Therefore, further detailed studies on the role of 5-HT in (Berl) 228:157–166. the mPFC–DRN projections on the antidepressant effects of ( R,S)- Hashimoto K (2016a) R-ketamine: a rapid-onset and sustained ketamine and its 2 enantiomers in animals with a depression- antidepressant without risk of brain toxicity. Psychol Med like phenotype are needed. 46:2449–2451. This paper has some limitations. Although we show Hashimoto K (2016b) Ketamine’s antidepressant action: 5-HT-independent antidepressant actions of ( R)-ketamine in beyond NMDA receptor inhibition. Expert Opin Ther Targets a CSDS model, the precise molecular mechanisms underlying 20:1389–1392. (R)-ketamine’s antidepressant actions are currently unknown. Hashimoto K (2017) Chapter 4. Rapid antidepressant activity of A  recent study showed that mTORC1 signaling does not play ketamine beyond NMDA receptor. In: The NMDA receptors a role in the antidepressant actions of ( R)-ketamine, but not (Hashimoto K, ed), pp 69–81. New York: Humana Press. (S)-ketamine, in a CSDS model ( Yang et al., 2017c). In addition, it Hashimoto K, Kakiuchi T, Ohba H, Nishiyama S, Tsukada H is reported that the gut-microbiota-brain axis may play a role in (2017) Reduction of dopamine D receptor binding in the 2/3 the antidepressant actions of ( R)-ketamine (Yang et al., 2017b). striatum after a single administration of esketamine, but Further studies on molecular mechanisms of (R)-ketamine’s not R-ketamine: a PET study in conscious monkeys. Eur Arch antidepressant actions are necessary. Psychiatry Clin Neurosci 267:173–176. In conclusion, this study suggests that 5-HT depletion by Kishimoto T, Chawla JM, Hagi K, Zarate CA, Kane JM, Bauer PCPA did not affect the acute and long-lasting antidepressant M, Correll CU (2016) Single-dose infusion ketamine and effects of ( R)-ketamine in a CSDS model. Therefore, it is unlikely non-ketamine N-methyl-D-aspartate receptor antago - that 5-HT plays a major role in the antidepressant actions of nists for unipolar and bipolar depression: a meta-analy - (R)-ketamine. sis of efficacy, safety and time trajectories. Psychol Med 46:1459–1472. Monteggia LM, Zarate C Jr (2015) Antidepressant actions of keta - Acknowledgments mine: from molecular mechanisms to clinical practice. Curr This study was supported by the Strategic Research Program Opin Neurobiol 30:139–143. Newport DJ, Carpenter LL, McDonald WM, Potash JB, Tohen M, for Brain Sciences from Japan Agency for Medical Research and Development, AMED (to K.H.). Dr. Chao Dong was supported by Nemeroff CB, APA Council of Research Task Force on Novel Biomarkers and Treatments (2015) Ketamine and other the Uehara Memorial Foundation (Tokyo, Japan). NMDA antagonists: early clinical trials and possible mecha - nisms in depression. Am J Psychiatry 172:950–966. Statement of Interest Pham TH, Mendez-David I, Defaix C, Guiard BP, Tritschler L, David DJ, Gardier AM (2017) Ketamine treatment involves Dr. Hashimoto is an inventor on a filed patent application on medial prefrontal cortex serotonin to induce a rapid antide - “The use of (R)-ketamine in the treatment of psychiatric dis - pressant-like activity in BALB/cJ mice. Neuropharmacology eases” by Chiba University. Dr. Hashimoto has received research 112:198–209. support from Mochida, Otsuka, and Taisho. Other authors de - Ren Q, Ma M, Ishima T, Morisseau C, Yang J, Wagner KM, Zhang clare no conflict of interest. JC, Yang C, Yao W, Dong C, Han M, Hammock BD, Hashimoto K (2016) Gene deficiency and pharmacological inhibition of sol - References uble epoxide hydrolase confers resilience to repeated social defeat stress. 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Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Zhang et al. | 163 Yang C, Qu Y, Abe M, Nozawa D, Chaki S, Hashimoto K (2017a) Yang C, Shirayama Y, Zhang JC, Ren Q, Yao W, Ma M, Dong C, (R)-ketamine shows greater potency and longer last - Hashimoto K (2015) R-ketamine: a rapid-onset and sustained ing antidepressant effects than its metabolite (2 R,6R)- antidepressant without psychotomimetic side effects. Transl hydroxynorketamine. Biol Psychiatry 82:e43–e44. Psychiatry 5:e632. Yang C, Qu Y, Fujita Y, Ren Q, Ma M, Dong C, Hashimoto K Zhang JC, Li SX, Hashimoto K (2014) R(-)-ketamine shows greater (2017b) Possible role of gut-microbiota in the antidepressant potency and longer lasting antidepressant effects than S(+)- effects of ( R)-ketamine in a social defeat stress model. Transl ketamine. Pharmacol Biochem Behav 116:137–141. Psychiatry. In press. Zhang JC, Yao W, Dong C, Yang C, Ren Q, Ma M, Han M, Hashimoto Yang C, Ren Q, Qu Y, Zhang JC, Ma M, Dong C, Hashimoto K K (2015) Comparison of ketamine, 7,8-dihydroxyflavone, (2017c) Mechanistic target of rapamycin-independent anti - and ANA-12 antidepressant effects in the social defeat depressant effects of ( R)-ketamine in a social defeat stress stress model of depression. Psychopharmacology (Berl) model. Biol Psychiatry doi: 10.1016/j.biopsych.2017.05.016. 232:4325–4335. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Journal of Neuropsychopharmacology Oxford University Press

5-Hydroxytryptamine-Independent Antidepressant Actions of (R)-Ketamine in a Chronic Social Defeat Stress Model

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Abstract

Background: Previous reports suggest that 5-hydroxytryptamine might play a role in the antidepressant actions of ( R,S)- ketamine. However, its role in the antidepressant actions of ( R)-ketamine, which is more potent than (S)-ketamine, is unknown. This study was conducted to examine whether 5-hydroxytryptamine depletion affects the antidepressant actions of (R)-ketamine in a chronic social defeat stress model. Methods: An inhibitor of 5-hydroxytryptamine synthesis, par a-chlorophenylalanine methyl ester hydrochloride (300 mg/kg, twice daily for 3 consecutive days), or vehicle was administered to control and chronic social defeat stress-susceptible mice. Levels of 5-hydroxytryptamine and its metabolite, 5-hydroxyindoleacetic acid, in mouse brain regions were measured using high-performance liquid chromatography. Furthermore, antidepressant effects of ( R)-ketamine (10 mg/kg) in the vehicle- and para-chlorophenylalanine methyl ester hydrochloride-treated susceptible mice were assessed using tail suspension test and 1% sucrose preference test. Results: para-Chlorophenylalanine methyl ester hydrochloride treatment caused marked reductions of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the brain regions of control and chronic social defeat stress susceptible mice. In the tail suspension test, (R)-ketamine significantly attenuated the increased immobility time in the chronic social defeat stress- susceptible mice with or without 5-hydroxytryptamine depletion. In the sucrose preference test (2 and 5 days after a single dose), (R)-ketamine significantly enhanced reduced sucrose consumption in the chronic social defeat stress-susceptible mice with or without 5-hydroxytryptamine depletion. Conclusions: These findings show that 5-hydroxytryptamine depletion did not affect the antidepressant effects of ( R)- ketamine in a chronic social defeat stress model. Therefore, it is unlikely that 5-hydroxytryptamine plays a major role in the antidepressant actions of ( R)-ketamine. Keywords: antidepressant, ( R)-ketamine, serotonin, stress Received: September 5, 2017; Revised: October 17, 2017; Accepted: November 15, 2017 © The Author(s) 2017. Published by Oxford University Press on behalf of CINP. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http:// creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, 157 provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 158 | International Journal of Neuropsychopharmacology, 2018 Significance Statement The rapid and long-lasting antidepressant effects of ( R,S)-ketamine in patients with treatment-resistant depression are the most important discovery in the field of depression research in half a century. Although previous studies suggest that 5-hydroixy - tryptamine (5-HT, serotonin) plays a role in the antidepressant effects of ketamine, the role of 5-HT in the antidepressant effects of (R)-ketamine remain unknown. Here we report that 5-HT depletion by para-chlorophenylalanine did not affect antidepressant actions of ( R)-ketamine in a chronic social defeat stress model. It is, therefore, unlikely that 5-HT may play a major role in the antidepressant actions of ( R)-ketamine. the role of 5-HT in the antidepressant effects of ( R)-ketamine Introduction in animal models such as a chronic social defeat stress (CSDS) The N-methyl-D-aspartate receptor antagonist ketamine exhibits model has not been reported. rapid and long-lasting antidepressant effects in treatment-resist - To elucidate the role of 5-HT in the antidepressant actions ant patients with major depressive disorder or bipolar disorder of (R)-ketamine, this study was undertaken to examine whether (Newport et al., 2015 ; Kishimoto et al., 2016). At present, ketamine is 5-HT depletion in the brain caused by the administration of the most attractive antidepressant for the treatment of treatment- PCPA could influence the antidepressant effects of (R)-ketamine resistant depression (Monteggia and Zarate, 2015 ; Duman et  al., in a CSDS model. 2016; Hashimoto, 2016a, 2016b, 2017), although the precise mecha- nisms underlying its antidepressant actions remain unknown. (R,S)-Ketamine is a racemic mixture containing equal parts Methods and Materials of (R)-ketamine and (S)-ketamine. (S)-Ketamine shows approxi - mately 3- to 4-fold greater anesthetic potency and greater unde - Animals sirable psychotomimetic side effects than ( R)-ketamine (Domino, Male adult C57BL/6 mice (n = 72), aged 8 weeks (body weight 2010; Hashimoto, 2016a). Meanwhile, (R)-ketamine shows greater 20–25 g, Japan SLC, Inc.) and male adult CD1 (ICR) mice (n = 20), potency and longer-lasting antidepressant effects than ( S)- aged 13 to 15 weeks (body weight >40  g, Japan SLC, Inc.) were ketamine in animal models of depression ( Zhang et al., 2014; Yang used. Animals were housed under controlled temperatures and et al., 2015, Fukumoto et al., 2017; Yang et al., 2017a, 2017b, 2017c). 12-hour-light/-dark cycles (lights on between 7:00 am and 7:00 Unlike (S)-ketamine, (R)-ketamine does not induce psychotomi - pm), with ad libitum food (CE-2; CLEA Japan, Inc) and water. The metic side effects or exhibit abuse potential in rodents ( Yang protocol was approved by the Chiba University Institutional et al., 2015, 2016). Furthermore, we reported a marked reduction Animal Care and Use Committee (permission no. 29–345). This of dopamine D receptor binding in conscious monkey striatum 2/3 study was carried out in strict accordance with the recommen - after a single infusion of ( S)-ketamine but not that of ( R)-ketamine dations in the Guide for the Care and Use of Laboratory Animals (Hashimoto et  al., 2017), suggesting that ( S)-ketamine-induced of the National Institutes of Health, USA. Animals were deeply dopamine release might be associated with acute psychotomi- anaesthetized with isoflurane before being killed by cervical dis - metic and dissociative side effects in humans (Hashimoto et al., location. All efforts were made to minimize suffering. 2017). Therefore, (R)-ketamine could be a safer antidepressant in humans than (S)-ketamine (Hashimoto, 2016a, 2016b, 2017). 5-Hydroxytryptamine (5-HT, serotonin) plays a major role Materials in the antidepressant effects of the antidepressant drugs cur - rently being used. In the forced swimming test (FST), depletion (R)-Ketamine hydrochloride was prepared by recrystallization of of brain 5-HT by treatment with an inhibitor of 5-HT synthesis, (R,S)-ketamine (Ketalar, ketamine hydrochloride, Daiichi Sankyo para-chlorophenylalanine methyl ester hydrochloride (PCPA), Pharmaceutical Ltd) and D-(-)-tartaric acid, as described previously attenuated the sustained (24 hour), but not acute (1 hour), (Zhang et al., 2014). The purity of (R)-ketamine was determined by reduction in the immobility time after a single dose of ( R,S)- a high-performance liquid chromatography (HPLC) (CHIRALPAK ketamine (25  mg/kg) ( Gigliucci et  al., 2013). Interestingly, the IA, column size: 250 x 4.6  mm, mobile phase: n-hexane/dichlo - increase in the immobility time provoked by repeated restraint romethane/diethylamine [75/25/0.1], Daicel Corporation). The stress was blocked by a single dose of ketamine (25  mg/kg, dose (10 mg/kg as hydrochloride) of (R)-ketamine dissolved in the 24 hours prior to FST), but ketamine’s effects were not abol - physiological saline was used as previously reported (Zhang et al., ished when rats were subjected to 5-HT depletion ( Gigliucci 2014; Yang et al., 2015, 2016a, 2016b, 2017a, 2017b, 2017c). The dose et  al., 2013). In contrast,Fukumoto et  al. (2016) reported that of PCPA (300 mg/kg, 5-HT synthesis inhibitor, Sigma-Aldrich Co.) the antidepressant-like effects of ( R,S)-ketamine (30  mg/kg, was used as previously reported (Fukumoto et  al., 2016). Other 30 minutes prior to FST) were attenuated by depletion of reagents were purchased commercially. 5-HT in the brain upon treatment with PCPA. MoreoverPham , et  al. (2017) also reported that pretreatment with PCPA abol - CSDS Model ished (R,S)-ketamine (10 mg/kg, 24 hours prior to FST)-induced antidepressant-like effects in the FST. Although these 3 stud - The procedure of CSDS was performed as previously reported ies suggest the role of 5-HT in R,S ( )-ketamine’s antidepressant (Zhang et al., 2015; Yang et al., 2015, 2016a, 2016b, Yang et al., 2017a, effects in control rodents ( Gigliucci et al., 2013; Fukumoto et al., 2017b, 2017c; Ren et al., 2016). The C57BL/6 mice were exposed 2016; Pham et al., 2017), it is unlikely that 5-HT plays a role in to a different CD1 aggressor mouse for 10 min/d for consecutive the antidepressant effects of ( R,S)-ketamine in rodents with a 10 days. When the social defeat session ended, the resident CD1 depression-like phenotype ( Gigliucci et  al., 2013). Thus, there mouse and the intruder mouse were housed in one-half of the appears to be variation in the role of 5-HT in the antidepres - cage separated by a perforated Plexiglas divider to allow visual, sant-like effects of ( R,S)-ketamine depending on the baseline olfactory, and auditory contact for the remainder of the 24-hour status of the rodents to which it is administered. In addition, period. At 24 hours after the last session, all mice were housed Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Zhang et al. | 159 individually. On day 11, a social interaction test was performed to (CMC)] or PCPA (300  mg/kg, twice daily (9:00 am and 7:00 pm) identify subgroups of mice that were susceptible and unsuscepti - for 3 consecutive days) was administered i.p. into the control ble to social defeat stress. This was accomplished by placing mice and CSDS susceptible mice (day 12–14). On day 15, all mice were in an interaction test box (42 × 42 cm) with an empty wire-mesh killed by decapitation after isoflurane anesthesia, and brain cage (10 × 4.5 cm) located at one end. The movement of the mice regions including prefrontal cortex (PFC), hippocampus, and was tracked for 2.5 minutes, followed by 2.5 minutes in the pres - striatum were collected. These brain samples were stored at ence of an unfamiliar aggressor confined in the wire-mesh cage. -80℃ before assay (Figure 1A). The duration of the subject’s presence in the “interaction zone” The brain samples were homogenized in 0.2 M perchloric (defined as the 8-cm-wide area surrounding the wiremesh cage) acid (HClO ) containing 100 μM disodium EDTA and 100 ng/mL was recorded by a stopwatch. The interaction ratio was calculated isoproterenol (internal standard) and were then centrifuged at as time spent in an interaction zone with an aggressor/time spent 20 000 × g for 15 minutes at 4°C. The supernatants were filtered in an interaction zone without an aggressor. An interaction ratio through a 0.45-μm-pore membrane (Millex-LH, 4 mm; Millipore) of 1 was set as the cutoff: mice with scores <1 were defined as and analyzed for 5-HT and 5-HIAA by HPLC coupled with electro - “susceptible” to social defeat stress and those with scores ≥1 were chemical detection. The HPLC system consisted of a liquid chro - defined as “resilient.” Approximately 70% to 80% of mice were sus - matograph pump (HTEC-500, Eicom), a reversed phase column ceptible after CSDS. Susceptible mice were randomly divided in (Eicompak SC-5ODS 150  × 3.0 mm; Eicom), and a data processor the subsequent experiments. Control C57BL/6 mice not exposed (EPC-500, Eicom). The mobile phase was 0.1 M acetate-citric acid CSDS were housed in the cage before the behavioral tests. buffer (pH 3.5) containing 13% methanol, 5 mg/L disodium EDTA, and 190 mg/L sodium octyl sulfate. Treatment and Measurement of 5-HT and 5-HIAA in Mouse Brain by HPLC Treatment and Behavioral Tests Control mice and CSDS susceptible mice were divided into The CSDS susceptible mice were divided into 4 groups; vehicle + saline group, vehicle + ( R)-ketamine group, PCPA + saline group, 2 groups, respectively. Vehicle [0.5% carboxymethylcellulose Figure 1. Schedule of a chronic social defeat stress (CSDS) model, treatment, and high-performance liquid chromatography (HPLC) measurement. (A) CSDS was per - formed from day 1 to day 10, and the social interaction test (SIT) was performed on day 11. Vehicle (0.5% carboxymethylcellulose [CMC]) or para-chlorophenylalanine methyl ester hydrochloride (PCPA) (300 mg/kg, twice daily [9:00 am and 7:00 pm] for 3 consecutive days) was administered i.p. in the susceptible mice from day 12 to day 14. All mice were sacrificed by decapitation, and brain samples were collected on day 15. (B) Prefrontal cortex (PFC), (C) hippocampus, (D) striatum. The values represent the mean ± SEM (n = 8). *P < .05, **P < .01, ***P < .001. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 160 | International Journal of Neuropsychopharmacology, 2018 Figure 2. Effects of 5-hydroxytryptamine (5-HT) deletion in the antidepressant effects of ( R)-ketamine in a chronic social defeat stress (CSDS) model. (A) CSDS was per - formed from day 1 to day 10, and the social interaction test (SIT) was performed on day 11. Vehicle (0.5% carboxymethylcellulose [CMC]) or para-chlorophenylalanine methyl ester hydrochloride (PCPA) (300 mg/kg, twice daily [9:00 am and 7:00 pm] for 3 consecutive days) was administered i.p. in the susceptible mice from day 12 to day 14. Saline (10 mL/kg) or (R)-ketamine (10 mg/kg) was administered i.p. into mice on day 15. LMT and TST were performed 2 and 4 hours after a single injection of ( R)-ketamine or saline, respectively. SPT was performed 2 and 5 days after a single injection of ( R)-ketamine or saline. (B) Locomotion test (LMT) (day 15). (C) Tail suspension test (TST) (day 15). (D) Sucrose preference test (SPT) (day 17). (E) SPT (day 20). The values represent the mean ± SEM (n = 8). * P < .05, **P < .01, ***P < .001. N.S., not significant; R-KT, ( R)-ketamine. and PCPA + (R)-ketamine group. Vehicle (0.5% CMC) or PCPA Locomotion (300 mg/kg twice daily [9:00 am and 7:00 pm] for 3 consecutive The locomotor activity was measured by an animal movement days) was administered i.p. to the susceptible mice after CSDS analysis system SCANETMV-40 (MELQUEST Co., Ltd). The mice (days 12–14). Subsequently, saline (10 mL/kg) or (R)-ketamine were placed in experimental cages (length × width × height: (10 mg/kg) was administered i.p. into mice (day 15) ( Figure 2A). 560 × 560 × 330  mm). The cumulative locomotor activity counts Behavioral tests, including locomotion test (LMT), tail suspension were recorded for 60 minutes. Cages were cleaned between test - test (TST), and 1% sucrose preference test (SPT), were performed ing session. as reported previously (Zhang et al., 2015; Yang et al., 2015, 2017a, 2017b, 2017c; Ren et al., 2016). LMT and TST were performed 2 and TST 4 hours after a single injection of ( R)-ketamine or saline, respect- A small piece of adhesive tape was placed approximately 2 cm ively. SPT was performed 2 and 5 days after a single injection from the tip of the tail for mouse. A single hole was punched in of (R)-ketamine or saline. Behavioral tests were performed by 2 the tape, and mice were hung individually on a hook. The immo - observers who were blinded to the group assignment of mice. bility time was recorded for 10 minutes. Mice were considered Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Zhang et al. | 161 immobile only when they hung passively and completely (R)-ketamine-treated group (Figure 2D–E). These results suggest motionless. that depression-like phenotype in the susceptible mice after CSDS was not affected by 5-HT depletion and that depletion of SPT 5-HT in the brain by PCPA did not abolish the antidepressant Mice were exposed to water and 1% sucrose solution for 48 effects of ( R)-ketamine in a CSDS model. hours, followed by 4 hours of water and food deprivation and a 1-hour exposure to 2 identical bottles (water and 1% sucrose so - Discussion lution). The bottles containing water and sucrose were weighed before and at the end of this period. The sucrose preference was In the present study, we demonstrated that reductions of 5-HT calculated as a percentage of sucrose solution consumption to and 5-HIAA in mouse brain upon the administration of PCPA did the total liquid consumption. not affect the depression-like phenotype in the susceptible mice after CSDS and that PCPA-induced 5-HT depletion did not abolish the antidepressant effects of ( R)-ketamine in the CSDS suscepti- Statistical Analysis ble mice. These findings suggest that 5-HT does not play a major The data show as the mean ± SEM. Analysis was performed role in the antidepressant actions of ( R)-ketamine in a CSDS using PASW Statistics 20 (formerly SPSS Statistics; SPSS). The model. To our knowledge, this is the first report showing that data of 5-HT and 5-HIAA were analyzed using the 2-way ANOVA 5-HT depletion in the brain does not affect the antidepressant followed by posthoc Tukey test. The behavioral data were ana - effects of ( R)-ketamine in mice with a depression-like phenotype. lyzed using the 1-way ANOVA followed by posthoc Tukey test. A previous study showed that the depletion of 5-HT by P < .05 was considered statistically significant. PCPA did not abolish the acute antidepressant effects of ( R,S)- ketamine (25 mg/kg, 1 h prior to FST) in control SD rats ( Gigliucci et  al., 2013). In contrast, depletion of 5-HT by PCPA abolished Results the acute antidepressant effects of ( R,S)-ketamine (30 mg/kg, 30 minutes prior to FST) in control C57BL/6J mice (Fukumoto et al., Effects of PCPA on the Levels of 5-HT and 5-HIAA in 2016). The reasons underlying this discrepancy are currently the Brain Regions unknown. First, the dose (25 mg/kg for rats vs 30 mg/kg for mice) The effects of PCPA treatment on the tissue levels of 5-HT and of (R,S)-ketamine may have contributed to this discrepancy, 5-HIAA in the PFC, hippocampus, and striatum were exam - since these doses can increase locomotor activity after a single ined. PCPA treatment caused significant reduction of 5-HT administration ( Yang et al., 2015; Fukumoto et al., 2017). Second, and 5-HIAA in these 3 regions (Figure  1B–D). Two-way ANOVA the species difference (rats vs mice) may have contributed to (PCPA and stress) showed statistical results (PFC: 5-HT, PCPA, this discrepancy. Therefore, acute hyperactivity should be taken F = 452.76, P < .001, stress, F = 2.27, P = .14, interaction, into consideration to examine the acute antidepressant effects 1,32 1,32 F = 0.30, P = .59; 5-HIAA, PCPA, F = 460.75, P < .001, stress, of (R,S)-ketamine and its enantiomers. 1,32 1,32 F = 5.80, P = .02, interaction, F = 5.53, P = .03; hippocampus: Using Flinders Sensitive Line (FSL) rats (a genetic model of 1,32 1,32 5-HT, PCPA, F = 669.57, P < .001, stress, F = 3.37, P = .08, inter - depression), du Jardin et al. (2016b) examined the effects of 5-HT 1,32 1,32 action, F = 0.95, P = .34, 5-HIAA, PCPA, F = 680.08, P < .001, depletion by PCPA on the antidepressant effects of ( S)-ketamine 1,32 1,32 stress, F = 0.63, P = .44, interaction, F = 1.15, P = .29; striatum: (15  mg/kg). The depression-like phenotype in FSL rats was not 1,32 1,32 5-HT, PCPA, F = 434.70, P < .001, stress, F = 3.96, P = .06, inter - associated with 5-HT depletion. Interestingly, the acute (1 hour) 1,32 1,32 action, F = 2.56, P = .12, 5-HIAA, PCPA, F = 609.74, P < .001, stress, and sustained (24 hours) antidepressant effects of ( S)-ketamine 1,32 1,32 F = 1.49, P = .23, interaction, F = 2.20, P = .15). These data sug- (15 mg/kg) in FSL rats were abolished by 5-HT depletion, suggest- 1,32 1,32 gest that the reductions of 5-HT in the brain regions by PCPA ing that the acute and sustained antidepressant-like effects of were not affected by CSDS. (S)-ketamine in FSL rats depend on the endogenous 5-HT concen- tration ( du Jardin et al., 2016a , 2016b ). However, our present results indicate that acute and long-lasting (5  days after a single dose) Effects of 5-HT Reduction in the Antidepressant antidepressant effects of ( R)-ketamine (10 mg/kg) in a CSDS model Effect of (R)-Ketamine in a CSDS Model in male C57BL/6 mice occur independently of brain 5-HT depletion. Next, we examined the effects of PCPA-induced 5-HT reduc - Interestingly, 5-HT depletion in the brain did not affect the sus - tion in the antidepressant effects of ( R)-ketamine (10 mg/ tained antidepressant effects of ( R,S)-ketamine (25 mg/kg, 24 hours kg) in a CSDS model ( Figure 2A). Locomotion showed no dif- prior to FST) on the increased immobility time caused by repeated ference (F = 0.43, P = .79) among the 5 groups (Figure 2B). restraint stress ( Gigliucci et  al., 2013). Although the reasons for 4,40 One-way ANOVA of TST data showed a statistical significance these discrepancies are currently unknown, there are several fac - (F = 2.41, P < .01) among the 5 groups (Figure 2C). Posthoc tests tors that could have contributed to them, such as different models 4,40 showed that ( R)-ketamine (10 mg/kg) significantly attenuated (control animals vs genetic rat model vs stress model) and different the increased immobility times in susceptible mice after CSDS doses and isomers of ketamine (25 mg/kg vs 30 mg/kg vs 10 mg/ (Figure 2C). However, there were no differences between ve - kg; (R,S)-ketamine vs (S)-ketamine vs (R)-ketamine). Therefore, fur - hicle + (R)-ketamine group and PCPA + (R)-ketamine group in ther detailed studies on the role of 5-HT in the acute and sustained the TST immobility times ( Figure 2C). One-way ANOVA of SPT antidepressant effects of ( R,S)-ketamine and its enantiomers in data showed statistical significances (2 days after a single injec - rodents with a depression-like phenotype are needed. tion: F = 5.06, P = .003, 5 days after a single injection: F = 4.36, Fukumoto et  al. (2016) reported the role of 5-HT neurons in 4,40 4,40 P = .01) among 5 groups ( Figure 2D–E). Posthoc tests showed that the dorsal raphe nucleus (DRN) regulated by the medial PFC sucrose preference in vehicle + (R)-ketamine-treated group was (mPFC)–DRN projections in the acute antidepressant effects of significantly higher than in the vehicle + saline-treated group (R,S)-ketamine (30 mg/kg, 30 minutes prior to FST). In addition, (Figure 2D–E). Furthermore, sucrose preference in the vehicle + antidepressant effects of ( R,S)-ketamine were reported to be (R)-ketamine-treated group was not different from the PCPA + blocked by an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup 162 | International Journal of Neuropsychopharmacology, 2018 acid receptor antagonist, NBQX (10  mg/kg). Domino EF (2010) Taming the ketamine tiger. Anesthesiology Interestingly, microinjection of ( R,S)-ketamine into mPFC sig - 113:678–684. nificantly increased the c-Fos expression in 5-HT neurons in Duman RS, Aghajanian GK, Sanacora G, Krystal JH (2016) the DRN, and the effect of ( R,S)-ketamine was attenuated by the Synaptic plasticity and depression: new insights from stress microinjection of NBQX into the mPFC. These results suggest that and rapid-acting antidepressants. Nat Med 22:238–249. the activation of 5-HT neurons in the DRN regulated by stimula - Fukumoto K, Iijima M, Chaki S (2016) The antidepressant effects of tion of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic an mGlu2/3 receptor antagonist and ketamine require AMPA acid receptor in the mPFC may be involved in the antidepres- receptor stimulation in the mPFC and subsequent activation sant effects of ( R,S)-ketamine (Fukumoto et al., 2016). Moreover, of the 5-HT neurons in the DRN. Neuropsychopharmacol Pham et al. (2017) reported an increase (144%) in the extracellu - 41:1046–1056. lar 5-HT levels in the mPFC after a single dose of R,S ( )-ketamine Fukumoto K, Toki H, Iijima M, Hashihayata T, Yamaguchi JI, (10  mg/kg). In addition, pretreatment with NBQX blunted the Hashimoto K, Chaki S (2017) Antidepressant potential of ( R)- effects of intra-mPFC ketamine on both the swimming duration ketamine in rodent models: comparison with (S)-ketamine. J in the FST and extracellular 5-HT in the mPFC, suggesting a key Pharmacol Exp Ther 361:9–16. role of cortical 5-HT release in ( R,S)-ketamine’s antidepressant- Gigliucci V, O’Dowd G, Casey S, Egan D, Gibney S, Harkin A (2013) like activity (Pham et  al., 2017). However, these 2 studies used Ketamine elicits sustained antidepressant-like activity via control animals, but not animals with a depression-like pheno- a serotonin-dependent mechanism. Psychopharmacology type. Therefore, further detailed studies on the role of 5-HT in (Berl) 228:157–166. the mPFC–DRN projections on the antidepressant effects of ( R,S)- Hashimoto K (2016a) R-ketamine: a rapid-onset and sustained ketamine and its 2 enantiomers in animals with a depression- antidepressant without risk of brain toxicity. Psychol Med like phenotype are needed. 46:2449–2451. This paper has some limitations. Although we show Hashimoto K (2016b) Ketamine’s antidepressant action: 5-HT-independent antidepressant actions of ( R)-ketamine in beyond NMDA receptor inhibition. Expert Opin Ther Targets a CSDS model, the precise molecular mechanisms underlying 20:1389–1392. (R)-ketamine’s antidepressant actions are currently unknown. Hashimoto K (2017) Chapter 4. Rapid antidepressant activity of A  recent study showed that mTORC1 signaling does not play ketamine beyond NMDA receptor. In: The NMDA receptors a role in the antidepressant actions of ( R)-ketamine, but not (Hashimoto K, ed), pp 69–81. New York: Humana Press. (S)-ketamine, in a CSDS model ( Yang et al., 2017c). In addition, it Hashimoto K, Kakiuchi T, Ohba H, Nishiyama S, Tsukada H is reported that the gut-microbiota-brain axis may play a role in (2017) Reduction of dopamine D receptor binding in the 2/3 the antidepressant actions of ( R)-ketamine (Yang et al., 2017b). striatum after a single administration of esketamine, but Further studies on molecular mechanisms of (R)-ketamine’s not R-ketamine: a PET study in conscious monkeys. Eur Arch antidepressant actions are necessary. Psychiatry Clin Neurosci 267:173–176. In conclusion, this study suggests that 5-HT depletion by Kishimoto T, Chawla JM, Hagi K, Zarate CA, Kane JM, Bauer PCPA did not affect the acute and long-lasting antidepressant M, Correll CU (2016) Single-dose infusion ketamine and effects of ( R)-ketamine in a CSDS model. Therefore, it is unlikely non-ketamine N-methyl-D-aspartate receptor antago - that 5-HT plays a major role in the antidepressant actions of nists for unipolar and bipolar depression: a meta-analy - (R)-ketamine. sis of efficacy, safety and time trajectories. Psychol Med 46:1459–1472. Monteggia LM, Zarate C Jr (2015) Antidepressant actions of keta - Acknowledgments mine: from molecular mechanisms to clinical practice. Curr This study was supported by the Strategic Research Program Opin Neurobiol 30:139–143. Newport DJ, Carpenter LL, McDonald WM, Potash JB, Tohen M, for Brain Sciences from Japan Agency for Medical Research and Development, AMED (to K.H.). Dr. Chao Dong was supported by Nemeroff CB, APA Council of Research Task Force on Novel Biomarkers and Treatments (2015) Ketamine and other the Uehara Memorial Foundation (Tokyo, Japan). NMDA antagonists: early clinical trials and possible mecha - nisms in depression. Am J Psychiatry 172:950–966. Statement of Interest Pham TH, Mendez-David I, Defaix C, Guiard BP, Tritschler L, David DJ, Gardier AM (2017) Ketamine treatment involves Dr. Hashimoto is an inventor on a filed patent application on medial prefrontal cortex serotonin to induce a rapid antide - “The use of (R)-ketamine in the treatment of psychiatric dis - pressant-like activity in BALB/cJ mice. Neuropharmacology eases” by Chiba University. Dr. Hashimoto has received research 112:198–209. support from Mochida, Otsuka, and Taisho. Other authors de - Ren Q, Ma M, Ishima T, Morisseau C, Yang J, Wagner KM, Zhang clare no conflict of interest. JC, Yang C, Yao W, Dong C, Han M, Hammock BD, Hashimoto K (2016) Gene deficiency and pharmacological inhibition of sol - References uble epoxide hydrolase confers resilience to repeated social defeat stress. Proc Natl Acad Sci USA 2113:E1944–E1952. du Jardin KG, Liebenberg N, Müller HK, Elfving B, Sanchez C, Yang C, Han M, Zhang JC, Ren Q, Hashimoto K (2016a) Loss of Wegener G (2016a) Differential interaction with the ser - o parvalbumin-immunoreactivity in mouse brain regions after tonin system by S-ketamine, vortioxetine, and fluoxetine in a repeated intermittent administration of esketamine, but not genetic rat model of depression. Psychopharmacology (Berl) R-ketamine. Psychiatric Res 239:281–283. 233:2813–2825. Yang B, Zhang JC, Han M, Yao W, Yang C, Ren Q, Ma M, Chen QX, du Jardin KG, Müller HK, Elfving B, Dale E, Wegener G, Sanchez Hashimoto K (2016b) Comparison of R-ketamine and rapasti- C (2016b) Potential involvement of serotonergic signaling nel antidepressant effects in the social defeat stress model of in ketamine’s antidepressant actions: a critical review. Prog depression. Psychopharmacology (Berl) 233: 3647–3657. Neuropsychopharmacol Biol Psychiatry 71:27–38. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Copyedited by: oup Zhang et al. | 163 Yang C, Qu Y, Abe M, Nozawa D, Chaki S, Hashimoto K (2017a) Yang C, Shirayama Y, Zhang JC, Ren Q, Yao W, Ma M, Dong C, (R)-ketamine shows greater potency and longer last - Hashimoto K (2015) R-ketamine: a rapid-onset and sustained ing antidepressant effects than its metabolite (2 R,6R)- antidepressant without psychotomimetic side effects. Transl hydroxynorketamine. Biol Psychiatry 82:e43–e44. Psychiatry 5:e632. Yang C, Qu Y, Fujita Y, Ren Q, Ma M, Dong C, Hashimoto K Zhang JC, Li SX, Hashimoto K (2014) R(-)-ketamine shows greater (2017b) Possible role of gut-microbiota in the antidepressant potency and longer lasting antidepressant effects than S(+)- effects of ( R)-ketamine in a social defeat stress model. Transl ketamine. Pharmacol Biochem Behav 116:137–141. Psychiatry. In press. Zhang JC, Yao W, Dong C, Yang C, Ren Q, Ma M, Han M, Hashimoto Yang C, Ren Q, Qu Y, Zhang JC, Ma M, Dong C, Hashimoto K K (2015) Comparison of ketamine, 7,8-dihydroxyflavone, (2017c) Mechanistic target of rapamycin-independent anti - and ANA-12 antidepressant effects in the social defeat depressant effects of ( R)-ketamine in a social defeat stress stress model of depression. Psychopharmacology (Berl) model. Biol Psychiatry doi: 10.1016/j.biopsych.2017.05.016. 232:4325–4335. Downloaded from https://academic.oup.com/ijnp/article-abstract/21/2/157/4636220 by Ed 'DeepDyve' Gillespie user on 16 March 2018

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International Journal of NeuropsychopharmacologyOxford University Press

Published: Feb 1, 2018

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