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Original article

Repeated ketamine administration redeems the time lag for citalopram’s antidepressant-like effects G.-F. Zhang a,1, W.-X. Liu a,1, L.-L. Qiu a, J. Guo b, X.-M. Wang a, H.-L. Sun a, J.-J. Yang a,c,d,**, Z.-Q. Zhou a,* a

Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China Department of Anesthesiology, Affiliated Hospital of Nanjing, University of Traditional Chinese Medicine, Nanjing, China Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, China d Jiangsu Province Key Laboratory of Anesthesia and Analgesia Application Technology, Xuzhou, China b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 August 2014 Received in revised form 16 October 2014 Accepted 9 November 2014 Available online xxx

Current available antidepressants exhibit low remission rate with a long response lag time. Growing evidence has demonstrated acute sub-anesthetic dose of ketamine exerts rapid, robust, and lasting antidepressant effects. However, a long term use of ketamine tends to elicit its adverse reactions. The present study aimed to investigate the antidepressant-like effects of intermittent and consecutive administrations of ketamine on chronic unpredictable mild stress (CUMS) rats, and to determine whether ketamine can redeem the time lag for treatment response of classic antidepressants. The behavioral responses were assessed by the sucrose preference test, forced swimming test, and open field test. In the first stage of experiments, all the four treatment regimens of ketamine (10 mg/kg ip, once daily for 3 or 7 consecutive days, or once every 7 or 3 days, in a total 21 days) showed robust antidepressant-like effects, with no significant influence on locomotor activity and stereotype behavior in the CUMS rats. The intermittent administration regimens produced longer antidepressant-like effects than the consecutive administration regimens and the administration every 7 days presented similar antidepressant-like effects with less administration times compared with the administration every 3 days. In the second stage of experiments, the combination of ketamine (10 mg/kg ip, once every 7 days) and citalopram (20 mg/kg po, once daily) for 21 days caused more rapid and sustained antidepressantlike effects than citalopram administered alone. In summary, repeated sub-anesthestic doses of ketamine can redeem the time lag for the antidepressant-like effects of citalopram, suggesting the combination of ketamine and classic antidepressants is a promising regimen for depression with quick onset time and stable and lasting effects. ß 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Depression Ketamine Citalopram Behavior

1. Introduction Drugs increasing the synaptic availability of monoamines, including tricyclic antidepressants, selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), and dopamine reuptake inhibitors have been used to treat depression for more than 50 years [36,39]. However, these

* Corresponding author. Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China. Tel.: +86 25 52323834; fax: +86 25 84806839. ** Co-corresponding author. Department of Anesthesiology, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, China. E-mail addresses: [email protected] (J.-J. Yang), [email protected] (Z.-Q. Zhou). 1 The two authors contributed equally to this work.

traditional antidepressants exhibit low response rates, with about one-third of major depressive disorder (MDD) patients failing to attain remission in first course of treatment. More important, symptom relief requires a response lag time of weeks [16,38,41]. The treatment guidelines for MDD recommend the continuous use of antidepressants for 4 to 8 weeks after the initial beneficial effects due to their delayed onset of response [28]. Therefore, there is an urgent need to develop novel antidepressants with fast-acting and better rates of response especially for depressed patients who are particularly vulnerable to suicide. Recent clinical studies have shown that ketamine, a nonselective N-methyl-D-aspartate (NMDA) receptor antagonist, produces robust, rapid, and sustained antidepressant effects after the intravenous infusion of a single sub-anesthetic dose in depressed patients [6,10,26,44,45]. Preclinical studies have also

http://dx.doi.org/10.1016/j.eurpsy.2014.11.007 0924-9338/ß 2014 Elsevier Masson SAS. All rights reserved.

Please cite this article in press as: Zhang G-F, et al. Repeated ketamine administration redeems the time lag for citalopram’s antidepressant-like effects. European Psychiatry (2015), http://dx.doi.org/10.1016/j.eurpsy.2014.11.007

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demonstrated that ketamine in sub-anesthetic doses exerts rapid and long-lasting antidepressant-like effects in rodent models of depression [7,20,21,23,24,47]. Nevertheless, ketamine has been shown to induce psychosis in healthy subjects and to exacerbate psychotic symptoms in individuals affected by schizophrenia, and has also been abused as an addictive ‘‘club drug’’ [2,32], which largely limits its clinical utility as a potent antidepressant. The current research field of ketamine treating depression therefore tends to focus on seeking a reasonable clinical regimen of ketamine or exploring other NMDA receptor antagonists as ketamine-like antidepressants to reduce the occurrence of ketamine’s adverse reactions. However, preliminary studies have shown that some other NMDA receptor antagonists can provide antidepressant-like effects without psychotomimetic or addictive adverse reaction, but their antidepressant efficacies are not as robust as ketamine’s [15,33]. Several studies have been performed to testify whether repeated sub-anesthetic doses of ketamine could sustain the same antidepressant properties of ketamine [1,27,35,37]. A case study of three patients with high treatment-resistant depression via a naturalistic observation has shown that the long-term repeated intravenous ketamine infusions improve the depressive symptoms of the three cases significantly with good tolerance [37]. Three previous clinical studies have indicated that six ketamine infusions over two weeks result in a high response rate and long response time in a total of 48 treatment-resistant major depression patients [1,27,35], confirming that the initial ketamine-induced antidepressant actions can be maintained by multiple ketamine administrations. Considering the possible adverse reactions induced by a long time period of ketamine administrations and the response lag of classic antidepressants, we hypothesized the combination of several sub-anesthestic doses of ketamine infusions and consecutive orally uses of classic antidepressants can produce rapid, sustained, and safe antidepressant-like effects. However, it remains unknown either intermittent or consecutive ketamine infusions are preferable for MDD patients, moreover, the antidepressant effects of ketamine can be currently studied only in a small sample size of depressive patients after a long period of case recruitment. Therefore, we tended to testify our hypothesis in a rodent model. In this study, we aimed to determine which of the following regimens is preferable for the chronic unpredictable mild stress (CUMS) rats to elicit the antidepressantlike effects of ketamine: intermittent or consecutive repeated administrations, and then to investigate whether repeated ketamine administrations can redeem the time lag of one of the classic antidepressants, citalopram, serving as an ideal antidepressant regimen. 2. Materials and methods 2.1. Animals The present study was approved by the Ethics Committee of Jinling Hospital, and was performed in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health, USA. Male Sprague-Dawley rats, 7 weeks old, weighing 230–270 g, were purchased from the Jinling hospital, Nanjing, China. The rats were housed 5 per cage with food and water available ad libitum and were maintained on a 12 h light/ dark cycle (lights on at 7:00 am). All the rats involved in the present study were allowed 7 days to acclimate to the surroundings before the beginning of experiments. 2.2. Experimental design and drug treatment The experimental design was shown as Fig. 1A. Before the start of the CUMS protocol, the animals were weighed and tested in the

Fig. 1. The experimental design and drug treatment. A. The CUMS procedure was performed for 35 days. Drug treatment was performed for 21 days. The SIT was performed at baseline, day 21, 35 (T0), 42 (T7), 49 (T14), and 56 (T21). The OFT and FST were performed at T7, T14, and T21. B. In the first stage, rats were randomly divided into six groups (n = 8 per group): control group, control rats + saline; CS group, CUMS rats + saline; CK1 group, CUMS rats + ketamine (10 mg/kg, once daily for 7 consecutive days); CK2 group, CUMS rats + ketamine (10 mg/kg, once every 3 days in a total 21 days); CK3 group, CUMS rats + ketamine (10 mg/kg, once daily for 3 consecutive days); and CK4 group, CUMS rats + ketamine (10 mg/kg, once per week in a total 21 days). In the second stage, rats were randomly divided into four groups (n = 8 per group): CS group, CUMS rats + saline; CK group, CUMS rats + ketamine (10 mg/kg, once per week); CC group, CUMS rats + citalopram (20 mg/kg, per day); and CKC group, CUMS rats + citalopram (20 mg/kg, per day) + ketamine (10 mg/kg, once per week).

1% sucrose intake test (SIT). The impacts of CUMS on rats were evaluated by the SIT at days 21 and 35 after the start of CUMS, and the effects of antidepressant treatment were measured by the forced swimming test (FST) and SIT at days 7, 14 and 21 after the start of drug interventions. The open field test (OFT) was also performed at days 7, 14 and 21 after the start of drug interventions. Moreover, the body weight was measured every 7 days throughout the stress period and 3 or 4 days throughout the antidepressant treatment period. As shown in Fig. 1B, eighty rats were randomly and equally allocated into total ten groups (n = 8 in each group) in the present study. In the first stage of experiments, to determine which of the following four regimens of ketamine is the best for the CUMS rats, six groups were used to observe the antidepressant-like effects of ketamine regimens: control rats + saline group (control group), CUMS rats + saline group (CS group), CUMS rats + ketamine group (CK1 group, 10 mg/kg, once daily for 7 consecutive days), CUMS rats + ketamine group (CK2 group, 10 mg/kg, once every 3 days in a total 21 days), CUMS rats + ketamine group (CK3 group, 10 mg/kg, once daily for 3 consecutive days), and CUMS rats + ketamine group (CK4 group, 10 mg/kg, once per week in a total 21 days). In the second stage of experiments, to investigate whether repeated ketamine administrations can redeem the time lag of one

Please cite this article in press as: Zhang G-F, et al. Repeated ketamine administration redeems the time lag for citalopram’s antidepressant-like effects. European Psychiatry (2015), http://dx.doi.org/10.1016/j.eurpsy.2014.11.007

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of the classic antidepressants, citalopram, four groups were used to observe the antidepressant-like effects of the combination of ketamine and citalopram: CUMS rats + saline (CS group), CUMS rats + ketamine (CK group, 10 mg/kg, once per week), CUMS rats + citalopram (CC group, 20 mg/kg, per day), and CUMS rats + citalopram (20 mg/kg, per day) + ketamine (CKC group, 10 mg/kg, once per week). All the drug interventions were performed in a total 21 days. The treatment regimen of ketamine was chosen on the basis of the results of the first stage, showing that ketamine administered once per week is the best treatment regimen for the CUMS rats. Citalopram at a dose of 20 mg/kg/day was orally administered on the basis of previous studies showing that citalopram administered intraperitoneally or oral at a dose of 10 or 20 mg/kg/day during two or three weeks exerts antidepressant-like effects in rodents [3,43]. Ketamine at the dose of 10 mg/kg in the volume of 1 mL was intraperitoneally injected according to the above regimens. Citalopram at the dose 20 mg/kg was administered orally via gastric gavage for 21 days to mimic the oral route of drug administration in psychiatric patients. Normal saline was injected only on day 1 in the CS group after the start of the treatment. All the drug treatments were performed during 15:00–17:00 after the behavioral tests. Ketamine was obtained from the Gutian Pharmaceutical Company, Fujian, China, and citalopram was prepared fresh in water each day from commercial tablets from H. Lundbeck A/S Company, Copenhagen-Valby, Denmark. 2.3. Chronic unpredictable mild stress (CUMS) procedure The CUMS procedure was performed as described [22] with a slight modification. The following twelve stressors were used: cage tilting 458 for 24-h, wet bedding for 24-h, crowding for 24-h, isolation for 24-h, white noise for 30 min, cold immobilization at 4 8C for 1 h, cold swimming at 4 8C for 5 min, swimming at 22 8C for 10-min, fasting for 24-h, water deprivation for 24-h, rotation at 30 revolutions per minute for 30 min, and inversion of light/dark cycle for 24-h. These stressors were applied for 35 days and two different stressors were used per day. Rats received these stressors at different time every day and the same stressor was not applied consecutively over two days so that rats could not predict the coming stressor. Rats in the control group were undisturbed except for necessary procedures such as behavioral testing and routine cage cleaning. 2.4. Open field test (OFT) The OFT was performed as described in the previous study [9]. The OFT apparatus consists of a 100  100 cm square floor surrounded by 40 cm high wall. The base and the side walls of the open field are made of gray polyvinylchloride. The test was performed under a dark light of 50 lx. Rats were placed in the center of the open field and the activity was tested for a 10-min period. The activity and behavior during the whole session were recorded with a video camera, and the total distance was automatically registered and analyzed with the computerized tracking system. The open field arena was thoroughly cleaned after the test of every rat. 2.5. Forced swimming test (FST) The FST was performed as described in the previous studies with some modifications [20,22–24,42]. The swimming sessions were conducted by placing the rats in the cylinder that is 65 cm tall, 30 cm in diameter, and filled with water (22–23 8C) to a depth of 40 cm. All the procedures were conducted during 9:00–15:00. The FST was conducted for 6 min and the behavior of rats was

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videotaped. The immobility time during the last 5 min of the 6 min test and the latency to the first immobility were recorded. A timesampling scoring technique was used, whereby the predominant behavior-immobility or swimming-in each 5-s period of the 300-s test was recorded. Immobility was defined as that the rat remains floating in the water without struggling and makes only those movements necessary to keep its head above water. Water in the tank was changed after the test of every rat. 2.6. Sucrose intake test (SIT) The SIT was performed as described in the previous study [17]. To perform this test, 1% sucrose solution was offered in a rat’s home cage. Each rat had a free choice between a 1% sucrose solution bottle and a tap water bottle. Sucrose and tap water intakes were separately measured by weighing each bottle before and after the test (a 24-h period). The sum of sucrose and tap water intake (in grams) was calculated as the total water intake. The sucrose preference was expressed as the percentage of sucrose water intake relative to the total water intake. A decrease in sucrose intake represented a depressive response in rodents. All the rats were placed in individual cages in the test period. 2.7. Stereotype rating To study psychosis-like behavior, stereotype rating was performed as described in the previous studies [14,48]. This activity was observed for 5 min every time. For the test, the same two observers rated the stereotyped behavior every 1 min in this procedure in a blinded manner. Score 1, lying down, eyes closed (asleep); score 2, lying down, eyes opened (inactive); score 3, normal grooming or chewing cage litter (in place activities); score 4, moving about cage, sniffing, rearings (normal, alert, active); score 5, running movement (hyperactive); score 6, repetitive exploration of the cage at normal activity (slow patterned); score 7, repetitive exploration of the cage with hyperactivity (fast patterned); score 8, remaining in the same place in cage with fast repetitive head and/or foreleg movement (restricted); and score 9, backing up, jumping, seizures, abnormally maintained postures, dyskinetic movements (dyskinetic-reactive). 2.8. Statistical analysis Data are expressed as the mean  S.E.M. and were analyzed by the Statistical Package for Social Sciences (SPSS, Version 17.0, IL, USA). The data of body weight and behavioral test were analyzed by twoway analysis of variance for repeated measures to determine the differences among groups, followed by Tukey test for post-hoc comparisons. A difference was considered significant at P < 0.05.

3. Results 3.1. Effects of CUMS on rats Compared with the control, the CUMS procedure decreased the body weight on day 7 (P < 0.01), which continued to day 35 after the start the CUMS (P < 0.001) (Fig. 2A); and reduced the sucrose preference on days 21 and 35 (P < 0.001) (Fig. 2B). 3.2. Antidepressant-like effects of repeated administrations of ketamine alone The body weight was significantly different among the six groups on days 0, 3, 7, 10, 14, 17 and 21 after the start of the treatment in the first stage (P < 0.001). Compared with the CS

Please cite this article in press as: Zhang G-F, et al. Repeated ketamine administration redeems the time lag for citalopram’s antidepressant-like effects. European Psychiatry (2015), http://dx.doi.org/10.1016/j.eurpsy.2014.11.007

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Fig. 2. The effects of the CUMS procedure on weight (A) and sucrose preference (B) of the rats in the first stage. Data are expressed as the mean  S.E.M. of 8 rats per group. ** P < 0.01, ***P < 0.001 vs control group.

group, the body weight increased on day 10 after the start of the treatment (P = 0.009), which continued to day 14 (P = 0.018) in ketamine administered once daily for 7 consecutive days group (CK1 group). The body weight increased on day 10 after the start of the treatment (P = 0.004), which continued to day 21 (P = 0.008) in ketamine administered once every 3 days group (CK2 group). The body weight increased on day 7 after the start of the treatment (P = 0.036), which continued to day 14 (P = 0.021) in ketamine administered once daily for 3 consecutive days group (CK3 group). The body weight increased on day 7 after the start of the treatment (P = 0.028), which continued to day 21 (P = 0.004) in ketamine administered once per week group (CK4 group) (Fig. 3A).

The sucrose preference was significantly different among the six groups on day 7, 14 and 21 after the start of the treatment (P < 0.001). Compared with the CS group, ketamine administered once daily for 7 consecutive days (CK1 group) increased the sucrose intake on day 7 after the start of the treatment (P < 0.001), which continued to day 14 (P < 0.001). Ketamine administered once every 3 days (CK2 group) increased the sucrose intake on day 7 after the start of the treatment (P = 0.001), which continued to day 21 (P = 0.001). Ketamine administered once daily for 3 consecutive days (CK3 group) increased the sucrose intake on day 7 after the start of the treatment (P < 0.001), which continued to day 14 (P = 0.001). Ketamine administered once per week (CK4 group)

Fig. 3. The effects of different ketamine regimens on weight (A), sucrose preference (B), immobility time (C), latency (D), total distance (E) and scores of stereotypy (F) in the rats in first stage. Data are expressed as the mean  S.E.M. of 8 rats per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs CS group.

Please cite this article in press as: Zhang G-F, et al. Repeated ketamine administration redeems the time lag for citalopram’s antidepressant-like effects. European Psychiatry (2015), http://dx.doi.org/10.1016/j.eurpsy.2014.11.007

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increased the sucrose intake on day 7 after the start of the treatment (P < 0.001), which continued to day 21 (P = 0.002) (Fig. 3B). The immobility time and the latency to the first immobility were significantly different among the six groups on day 7, 14 and 21 after the start of the treatment (P < 0.01). Compared with the CS group, ketamine administered once daily for 7 consecutive days (CK1 group) decreased the immobility time (P = 0.002) and increased the latency (P = 0.031) on day 7 after the start of the treatment, which continued to day 14 (P = 0.006; P = 0.032). Ketamine administered once every 3 days (CK2 group) decreased the immobility time (P = 0.038) and increased the latency on day 7 (P = 0.035) after the start of the treatment, which continued to day 21 (P = 0.039; P = 0.002). Ketamine administered once daily for 3 consecutive days (CK3 group) decreased the immobility time (P = 0.013) and increased the latency (P = 0.004) on day 7 after the start of the treatment, and the increase of latency continued to day 14 (P = 0.029). Ketamine administered once per week (CK4 group) decreased the immobility time (P = 0.038) and increased the latency on day 7 after the start of the treatment (P = 0.036), which continued to day 21 (P = 0.011; P = 0.007) (Fig. 3C and D). The locomotor activity and stereotyped behavior of rats were evaluated in the open field. No significant difference was observed in the total distance and stereotyped behavior among the six groups in the first stage (P > 0.05) (Fig. 3E and F). 3.3. Antidepressant-like effects of the combination of ketamine and citalopram The body weight was significantly different among the four groups in the second stage on day 7, 10, 14, 17 and 21 after the start

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of the treatment (P < 0.01). Compared with the CS group, the body weight increased on day 7 after the start of the treatment (P = 0.007; P = 0.034), which continued to day 21 (P < 0.001) in ketamine administered alone once per week group (CK group) and ketamine and citalopram combined treatment group (CKC group). The body weight increased on day 14 after the start of the treatment (P = 0.024), which continued to day 21 (P = 0.004) in citalopram administered alone once daily group (CC group) (Fig. 4A). The sucrose preference was significantly different among the four groups on day 7, 14, and 21 after the start of the treatment (P < 0.001). Compared with the CS group, ketamine administered alone once per week (CK group) and ketamine and citalopram combined treatment (CKC group) increased the sucrose intake on day 7 after the start of the treatment (P < 0.001), which continued to day 21 (P < 0.001). Citalopram administered alone once daily (CC group) increased the sucrose intake on day 14 after the start of the treatment (P = 0.001), which continued to day 21 (P < 0.001) (Fig. 4B). The immobility time and the latency to the first immobility were significantly different among the four groups on day 7, 14 and 21 after the start of the treatment (P < 0.05). Compared with the CS group, ketamine administered alone once per week (CK group) and ketamine and citalopram combined treatment (CKC group) decreased the immobility time (P = 0.025; P = 0.004) and increased the latency (P = 0.036; P = 0.006) on day 7 after the start of the treatment, which continued to day 21 (P = 0.004, P = 0.002; P = 0.032, P = 0.014). Citalopram administered alone once per day (CC group) decreased the immobility time (P = 0.01) and increased the latency on day 21 after the start of the treatment (P = 0.043) (Fig. 4C and D).

Fig. 4. The effects of the combination of ketamine and citalopram on weight (A), sucrose preference (B), immobility time (C), latency (D), total distance (E) and scores of stereotypy (F) in the rats in the second stage. Data are expressed as the mean  S.E.M. of 8 rats per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs CS group.

Please cite this article in press as: Zhang G-F, et al. Repeated ketamine administration redeems the time lag for citalopram’s antidepressant-like effects. European Psychiatry (2015), http://dx.doi.org/10.1016/j.eurpsy.2014.11.007

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No significant difference was observed in the total distance or stereotyped behavior among the four groups in the second stage (P > 0.05) (Fig. 4E and F). 4. Discussion The CUMS is a widely used behavioral test for evaluating the therapeutic effects and the underlying mechanisms of antidepressant agents applied either during the stress or as a post-stress treatment [18]. In the most of previous studies, classic antidepressants, including fluoxetine, venlafaxine, citalopram and so on were given during the stress procedure to explore their antidepressant-like effects [19,22]. However, in the present study, drugs administration were performed after the stress in order to mimic the development of depression and the treatment procedure in clinical. Usually, varied types of physical stresses are applied over a relatively prolonged time period (between 1 and 7 weeks) in the CUMS procedure. In the present study, rats were daily subjected to two of twelve stressors in an unpredictable order and at an unpredictable time of the day for a period of 5 weeks, which led to decreased weight and sucrose intake and increased immobility time in the FST, suggesting these CUMS rats with stress-induced depressive features. Recent clinical studies have demonstrated that ketamine produces rapid and robust antidepressant actions for depressed patients with a high remission rate of two-thirds of these patients in contrast to the traditional antidepressants with a lower remission rate and a much longer onset time [6,10,16,26,38,41,44,45]. Therefore, how to effectively maintain the antidepressant efficacy of ketamine is an important issue for clinical psychiatrists. To answer this question, few clinical studies have reported that the initial antidepressant response of ketamine can be maintained by multiple infusions [1,27,35,37]. The study from Garcia LS et al. has shown that chronic treatment with ketamine daily for 7 days produces antidepressant-like effects in the rat model of chronic mild stress [12]. However, preferable ketamine regimen has not been validated yet. In the present study, the two intermittent ketamine regimens produced longer antidepressant-like properties than the two consecutive ketamine regimens, and the administration every 7 days presented similar antidepressant-like effects with less administration times compared with the administration every 3 days. That is, ketamine given once per week is a preferable regimen for the antidepressant treatment of the CUMS rats. In the present study, the two intermittent ketamine regimens induced similar antidepressant-like effects, suggesting the possibility of ‘‘ceiling effect’’ of intermittent ketamine regimens in the present study. Various doses of ketamine including 20, 10, 5 and even 3 mg/kg have been testified to exert antidepressant effects [8,12,21,30]. We also used the dose of 10 mg/kg to elicit ketamine’s antidepressant in our previous studies [46,47] as well as the present study, however further studies focusing on the other intermittent ketamine regimens including lower doses or less injection times seemed to be more interesting. To date, the mechanisms mediating the rapid and sustained antidepressant actions of ketamine have not been fully understood. The alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, mammalian target of rapamycin, glycogen synthase kinase-3 and eukaryotic elongation factor 2 kinase have been reported to be involved in the antidepressant actions of ketamine in previous studies [4,7,20,24,25]. Ketamine reverses the atrophy of spines in the prefrontal cortex (PFC) and leads to a deconsolidation of synaptic that underlie the various signal pathways. As we know that citalopram mainly acts on serotonergic transmission by increasing extracellular serotonin and requires several weeks to exert their therapeutic effects. Previous studies indicated that 5-HT also plays a role in the rapid antidepressant

effects of ketamine, and similar to SSRIs, ketamine increases 5-HT levels in the PFC [13,29]. In the present study, no significant synergistic effect of ketamine and citalopram was found, but the antidepressant effects were obtained after the start of this combined treatment without any time lag until citalopram exerted its therapeutic properties. In addition, we only observed the antidepressant responses in the present study, but did not evaluate any biochemical markers. Therefore, the underlying molecular mechanisms of the antidepressant effects of ketamine and citalopram are to be investigated in future studies. Ketamine (20 mg/kg, twice daily for 15 days) repeated exposure may lead to a reduced weight gain in adult rats [31], conversely ketamine administered once daily for 3 or 7 consecutive days increased the body weight 10 days after the start of treatment in the present study. This difference may be attributed to the different doses and repeated times used in the two studies, but the underlying mechanism remains unclear. Additionally, the body weight gain was significantly different in the ketamine groups compared with the CS group though no significant difference was observed among the ketamine groups. Although ketamine appears to be a very promising antidepressant agent, the adverse reactions induced after a long time use, especially the addictive and propsychotic effects, significantly limit its clinical use as an antidepressant [5]. Nevertheless, the traditional antidepressants only present a low remission rate and a delayed response time of weeks [16,38,41]. Therefore, the combination of several repeated sub-anesthetic doses of ketamine and consecutive orally administration of classic antidepressants seems to provide rapid, sustained, and potent antidepressant effects without significant adverse responses for depressed patients. To explore this possibility, we used a rodent model of depression in the present study and showed for the first time that ketamine administered once per week in the response lag of citalopram elicited rapid, lasting, and stable antidepressant-like effects in the CUMS rats. In addition, ketamine is apt to induce propsychotic-like effects, and repeated administration has been usually used to induce propsychotic-like behavior in previous studies [11,34,40]. However, we did not find significantly enhanced locomotor activity or stereotype behavior after multiple administrations of sub-anesthestic dose of ketamine, ruling out the possibility that ketamine’s propsychotic-like effects could affect the results of the present study. 5. Conclusions In conclusion, our results suggest that repeated administrations of sub-anesthestic dose of ketamine can redeem the time lag for treatment response of citalopram, suggesting that the combination of multiple doses of ketamine and a classic antidepressant may serve as a promising regimen for depression treatment in clinical practice. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements This study was supported by the grant from the National Natural Science Foundation of China (No. 81271216). References [1] aan het Rot M, Collins KA, Murrough JW, Perez AM, Reich DL, Charney DS, et al. Safety and efficacy of repeated-dose intravenous ketamine for treatmentresistant depression. Biol Psychiatry 2009;67(2):139–45.

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Please cite this article in press as: Zhang G-F, et al. Repeated ketamine administration redeems the time lag for citalopram’s antidepressant-like effects. European Psychiatry (2015), http://dx.doi.org/10.1016/j.eurpsy.2014.11.007