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Progress in Neuro-Psychopharmacology & Biological Psychiatry 32 (2008) 140 144 www.elsevier.com/locate/pnpbp

Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF levels in the rat hippocampus
Lda S.B. Garcia a , Clarissa M. Comim a , Samira S. Valvassori a , Gislaine Z. Rus a , Luciana M. Barbosa a , Ana Cristina Andreazza b , Laura Stertz b , Gabriel R. Fries b , Elaine Cristina Gavioli a , Flavio Kapczinski b , Joo Quevedo a,
b

Laboratrio de Neurocincias, Programa de Ps-Graduao em Cincias da Sade, Unidade Acadmica de Cincias da Sade, Universidade do Extremo Sul Catarinense, 88806-000 Cricima, SC, Brazil Departamento de Bioqumica, Instituto de Cincias Bsicas da Sade, Universidade Federal do Rio Grande do Sul, Bipolar Disorders Program, Centro de Pesquisas, Hospital de Clnicas. Rua Ramiro Barcelos, 2350, 90035-003 Porto Alegre, RS, Brazil Received 24 May 2007; received in revised form 31 July 2007; accepted 31 July 2007 Available online 8 August 2007

Abstract Ketamine is a non-competitive antagonist to the phencyclidine site of N-methyl-D-aspartate (NMDA) receptor. Clinical findings point to a rapid onset of action for ketamine on the treatment of major depression. Considering that classic antidepressants may take long-lasting time to exhibit their main therapeutic effects, the present study aims to compare the behavioral effects and the BDNF hippocampus levels of acute administration of ketamine and imipramine in rats. To this aim, rats were acutely treated with ketamine (5, 10 and 15 mg/kg) and imipramine (10, 20 and 30 mg/kg) and animal behavioral was assessed in the forced swimming and open-field tests. Afterwards, BDNF protein hippocampal levels were assessed in imipramine- and ketamine-treated rats by ELISA-sandwich assay. We observed that ketamine at the doses of 10 and 15 mg/kg, and imipramine at 20 and 30 mg/kg reduced immobility time compared to saline group, without affecting locomotor activity. Interesting enough, acute administration of ketamine at the higher dose, but not imipramine, increased BDNF protein levels in the rat hippocampus. In conclusion, our findings suggest that the increase of hippocampal BDNF protein levels induced by ketamine might be necessary to produce a rapid onset of antidepressant action. 2007 Elsevier Inc. All rights reserved.
Keywords: Antidepressants; BDNF; Forced swimming test; Imipramine; Ketamine; NMDA receptor

1. Introduction Depression is one of the most prevalent and costly psychopathologies and a leading cause of morbidity and mortality in the world. It is worthy of note that the pharmacotherapy of depression is costly and widely prescribed by physicians, although less than half of treated patients attain complete remission after therapy with a single antidepressant. Others exhibit partial,
Abbreviations: BDNF, brain-derived-neurotrophic factor; IP, intraperitoneal; EGTA, ethylene glycol tetraacetic acid; IMI, imipramine; KET, ketamine; NMDA, N-methyl-D-aspartate; OD, optical density; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate buffer solution. Corresponding author. Fax: +55 48 3443 4817. E-mail address: quevedo@unesc.net (J. Quevedo). 0278-5846/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.pnpbp.2007.07.027

refractory or intolerant responses to the pharmacological treatment, emphasizing the need to discover novel antidepressants (Pacher et al., 2001). The challenges for the design of new agents to treat depression are threefold: rapid onset of antidepressant response, broader efficacy, and fewer adverse effects. While progress has been made to reduce side-effects, currently available antidepressants do not show convincing evidence for a shorter delay of onset of therapeutic actions neither for improved efficacy on the treatment of major depression (Nutt, 2002). Thus, there is clearly a need to develop rapidly acting and potent treatments for major depression. Glutamate is the primary excitatory neurotransmitter in the mammalian brain. Glutamatergic neurotransmission may be modulated in the brain by different receptor types, including

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ionotropic and metabotropic receptors. Studies have pointed to the ionotropic glutamate N-methyl-D-aspartate receptor (NMDA) as an important player in the etiology of psychopathologies, such as anxiety and major depression (Javitt, 2004; Krystal et al., 1999). Several preclinical studies have demonstrated that NMDA antagonists, such as MK-801, AP7, CPP, neramexane and others, display anxiolytic- and antidepressant-like effects in rats injected into distinct brain areas and subjected to various animal models of anxiety and depression (Kos et al., 2006a; Molchanov and Guimares, 2002; Menard and Treit, 2000; Adamec et al., 1999; Matheus and Guimares, 1997; Przegalinski et al., 1997; Skolnick et al., 1996; Maj et al., 1992; Trullas and Skolnick, 1990). Ketamine is a non-competitive antagonist to the phencyclidine site of N-methyl-D-aspartate (NMDA) receptor for glutamate, but it also interacts with voltage sensitive Ca+2 channels, and opioid, monoaminergic, and muscarinic receptors (for a review see: Hirota and Lambert, 1996). Recently, clinical studies suggested that acute administration of ketamine ameliorate depressive symptoms in patients suffering from major depression (Zarate et al., 2006; Berman et al., 2000). In agreement with these clinical findings, some evidence from the literature suggests that ketamine induces anxiolytic- and antidepressant-like effects in rodents subjected to animal models of anxiety and depression (Kos et al., 2006b; Yilmaz et al., 2002; Chaturvedi et al., 2001; Silvestre et al., 1997). Brain-derived-neurotrophic factor (BDNF) is one of several endogenous proteins that play critical roles in the survival, maintenance, and growth of the brain and peripheral neurons (Lewin and Barde, 1997). A growing body of evidence suggests that BDNF could be mediating the pathophysiology of mood disorders. In fact, reduced brain BDNF levels have been found in postmortem samples from depressed patients (Karege et al., 2002), whereas brain infusion of BDNF produces antidepressantlike action in rats (Siuciak et al., 1997). In addition, exposure to stress decreases levels of BDNF in brain regions associated with depression, while antidepressant treatment produces opposite actions and blocks the effects of stress on BDNF (for a review see: Duman and Monteggia, 2006). Interestingly, chronic, but not acute, antidepressant treatment induces increasing of BDNF expression and BDNF immunoreactive fibers in the hippocampus of rodents (Nibuya et al., 1996; De Foubert et al., 2004). Thus, agents capable of rapidly enhancing BDNF levels may lead aid the development of innovative antidepressant drugs. The main aim of the present study was to compare behavioral and molecular effects induced by acute administration of ketamine and imipramine in rats. The behavioral effects of both drugs were evaluated in the forced swimming test, which is a behavioral despair assay widely used for screening antidepressant drugs (McArthur and Borsini, 2006). The BDNF protein levels were measured using an ELISA kit in the hippocampus of rats acutely treated with ketamine and imipramine. 2. Materials and methods 2.1. Animals Male Adult Wistar rats (60 days old) were obtained from UNESC (Universidade do Extremo Sul Catarinense, Cricima,

Brazil) breeding colony. They were housed five 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 experimental procedures involving animals were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care. 2.2. Drugs and treatments Ketamine was obtained from Fort Dodge (Brazil) and imipramine, the standard antidepressant, from Novartis Pharmaceutical Industry (Brazil). Different groups of rats (n = 15 each) were administered intraperitoneally (IP) with saline or different doses of ketamine (5, 10 and 15 mg/kg) or imipramine (10, 20 and 30 mg/kg) 60 minutes before the test sessions, i.e. forced swimming or open-field tests. All treatments were administered in a volume of 1 ml/kg. The range of doses of ketamine employed in this work was chosen based on a previous study, which reported an increase of spontaneous locomotion at 25 mg/kg, while no changes were observed at 10 mg/kg (Hunt et al., 2006). 2.3. Apparatus The forced swimming test was conducted according to previous reports (Porsolt et al., 1977; Detke et al., 1995). The test involves two individual exposures to a cylindrical tank with water in which rats cannot touch the bottom of the tank or escape. The tank is made of transparent Plexiglas, 80 cm tall, 30 cm in diameter, and filled with water (2223 C) to a depth of 40 cm. Water in the tank was changed after each rat. For the first exposure, rats without drug treatment were placed in the water for 15 min (pre-test session). Twenty-four hours later, rats were placed in the water again for a 5 min session (test session), and the immobility time of rats were recorded in seconds. Rats were treated with ketamine, imipramine or saline only 60 min before the second exposure to the cylindrical tank of water (test session). In a separate series of experiments, nave rats were treated with ketamine (515 mg/kg), imipramine (1030 mg/kg) and saline 60 min before the exposure to the open-field apparatus, in order to assess possible effects of drug treatment on spontaneous locomotor activity. Analysis of rat spontaneous activity was carried out in an open field apparatus, which is an arena 45 60 cm surrounded by 50 cm high walls made of brown plywood with a frontal glass wall. The floor of the open field was divided into 9 rectangles (15 20 cm each) by black lines. Animals were gently placed on the left rear quadrant, and left to explore the arena for 5 min. The number of horizontal (crossings) and vertical (rearings) activity performed by each rat during the 5-min observation period was counted by an expert observer. 2.4. Experimental procedure Immediately after the forced swimming test, acutely saline, imipramine and ketamine-treated rats were sacrificed and the skulls were removed and hippocampus was dissected and stored at 80 C for biochemical analyses. BDNF levels in hippocampus

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Fig. 1. Effects of the acute administration of ketamine (5, 10 and 15 mg/kg, i.p.) and imipramine (10, 20 and 30 mg/kg, i.p.) on the immobility time of rats subjected to the forced swimming test. Bars represent means S.E.M. of 15 rats. p b 0.05 vs. saline according to ANOVA followed by Tukey post-hoc test.

Fig. 3. Effects of the acute administration of ketamine (5, 10 and 15 mg/kg, i.p.) and imipramine (10, 20 and 30 mg/kg, i.p.) on the BDNF levels in the rat hippocampus. Bars represent means S.E.M. of 15 rats. p b 0.05 vs. saline according to ANOVA followed by Tukey post-hoc test.

were measured by anti-BDNF sandwich-ELISA, according to the manufacturer instructions (Chemicon, USA). Briefly, rat hippocampus was homogenized in phosphate buffer solution (PBS) with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 1 mM (EGTA). Microtiter plates (96-well flat-bottom) were coated for 24 h with the samples diluted 1:2 in sample diluent and standard curve ranged from 7.8 to 500 pg/ml of BNDF. The plates were then washed four times with sample diluent and a monoclonal anti-BNDF rabbit antibody diluted 1:1000 in sample diluent was added to each well and incubated for 3 h at room temperature. After washing, a peroxidase conjugated anti-rabbit antibody (diluted 1:1000) was added to each well and incubated at room temperature for 1 h. After addition of streptavidin-enzyme, substrate and stop solution, the amount of BDNF was determined by absorbance in 450 nm. The standard curve demonstrates a

direct relationship between Optical Density (OD) and BDNF concentration. Total protein was measured by Lowry's method using bovine serum albumin as a standard, as previously described by Frey et al. (2006). 2.5. Statistical analysis All data are presented as mean S.E.M. Differences among experimental groups in the forced swimming, open field test and in the assessment of BDNF levels were determined by one-way ANOVA, followed by Tukey post-hoc test when ANOVA was significant; p values less than 0.05 were considered to be statistical significant. 3. Results As depicted in Fig. 1, the acute administration of the standard antidepressant imipramine reduced, in a significant manner, the immobility time of rats at 20 and 30 mg/kg compared to saline (F(697) = 5.45; p b 0.05; Fig. 1). The intraperitoneal treatment with ketamine at the doses of 10 and 15 mg/kg decreased significantly the immobility time of rats compared to saline group (F(697) = 5.45; p b 0.05; Fig. 1). In the open-field test, the treatment with ketamine and imiprimine at all doses tested did not modify the number of crossing and rearing compared to saline treated-rats (Fig. 2A and B). Fig. 3 illustrated the effects of the acute treatment with imipramine (10, 20 and 30 mg/kg), ketamine (5, 10 and 15 mg/kg) and saline in BDNF protein hippocampus levels of rats. A statistical significant increase in BDNF protein levels in the hippocampus was observed in rats treated with ketamine only at the higher dose (15 mg/kg; F(336) = 5.73; p b 0.05), but not with imipramine, compared to saline group. 4. Discussion The present study demonstrated that: (1) the acute treatment with ketamine (10 and 15 mg/kg) and imipramine (20 and 30 mg/kg) decreased the immobility time of rats in the forced swimming test; (2) ketamine and imipramine did not affect spontaneous locomotor activity in the open-field test; and (3) the acute treatment with ketamine at the higher dose,

Fig. 2. Effects of the acute administration of ketamine (5, 10 and 15 mg/kg, i.p.) and imipramine (10, 20 and 30 mg/kg, i.p.) on the number of crossings (A) and rearings (B) of rats subjected to the open field test. Bars represent means S.E.M. of 15 rats. p b 0.05 vs. saline according to ANOVA followed by Tukey post-hoc test.

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but not imipramine, increased BDNF protein levels in the rat hippocampus. The behavioral effects induced by ketamine in rats reported in the present study are in agreement with literature data, which support an antidepressant action for ketamine in basic and clinical studies. In fact, the treatment with ketamine reversed the shockinduced increase of immobility time in the mouse forced swimming test (Chaturvedi et al., 2001). Another study demonstrated that a single injection of an anesthetic dose of ketamine (160 mg/kg) induced antidepressant-like effects in rats tested in 3, 7, or 10 days after the forced swimming test (Yilmaz et al., 2002). Additionally, in the mouse tail suspension test ketamine produced anti-immobility effects, suggesting an antidepressant-like action in mice (Kos et al., 2006b). Taken together, our findings are in agreement with these previous observations, and are strengthening the view that ketamine induces antidepressant-like effects in rodents. In 2000, a pilot study showed that a single dose of ketamine produced antidepressant effects in patients suffering from major depression (Berman et al., 2000). Recently, Zarate et al. (2006) extended this study to a higher number of patients, and they found that the acute administration of ketamine rapidly improved depressive symptoms in patients with major depression. In this study, ketamine ameliorates the symptoms of depression within 110 minutes after injection, and these effects remained significant until 7 days after ketamine injection (Zarate et al., 2006). Therefore, these data strongly suggest that ketamine can induce robust and rapid antidepressant effects in depressed patients after a single intravenous injection. Our findings also showed that acute administration of ketamine, but not imipramine, significantly increased BDNF protein levels in the rat hippocampus compared with saline group. Several studies have suggested that normal BDNF-TrkB receptor signaling is both necessary and sufficient for antidepressant drug action (for a review see: Castrn et al., 2007). Some authors have found that antidepressants acting through different mechanisms rapidly increase TrkB receptor activation and signaling within an hour after drug administration (Saarelainen et al., 2003; Rantamaki et al., 2006). Despite that, studies have shown that rats treated with fluoxetine for 4, 7, 14 and 21 days displayed unaltered hippocampal BDNF protein levels when assessed by ELISA assay (De Foubert et al., 2004), and the same holds true to 3 weeks of treatment with desipramine (Jacobsen and Mork, 2004). Thus, taken together our data are showing that acute administration of ketamine increase hippocampal BDNF protein levels, whist acute (present data) and chronic treatment with classic antidepressant did not affect it. A growing body of evidence support an important role of neurotrophic factors in mood disorders. In fact, reduced brain BDNF levels predispose to depression, whereas increases in brain BDNF levels produce an antidepressant action (for a review see: Castrn et al., 2007). Our present findings revealed that acute administration of ketamine causes an increase of BDNF hippocampal levels detected immediately after the forced swimming test. Importantly, our data did not evaluate the duration of ketamine effects on BNDF levels in rats. Further

studies aiming to establish a timeresponse curve to ketamine in behavioral and molecular assays are worthy of doing. Altogether, our findings support a quite unique effect induced by ketamine in the hippocampal BDNF protein levels, which suggests that the rapid onset of action of ketamine in the clinic might be due to the increase of hippocampal BDNF protein levels. Additionally, our findings contribute in explaining the slow onset of antidepressant activity observed with classic antidepressants. Finally, it should be noted that although ketamine is a highaffinity NMDA receptor antagonist, it has less, but potentially relevant, affinity for opiate, monoaminergic, and muscarinic receptors and also interacts with voltage sensitive Ca+2 channels (Lindefors et al., 1997; Elliott et al., 1995; Wong et al., 1996; Kapur and Seeman, 2001; Eide et al., 1997). Thus, the antidepressant-like effects of ketamine observed in the present study could be due to interactions of ketamine with several receptor systems, not only with NMDA receptors, which could produce synergic effects on the brain pathways involved in the modulation of behavioral and molecular actions of antidepressants. Lastly, basic and clinical findings suggest that brain pathways modulated by ketamine could play an important role in reducing the onset of action of antidepressants. 5. Conclusion The antidepressant-like effects of ketamine are in agreement with literature data, which support an important role played by the NMDA receptor signaling in major depression. However, it should be kept in mind that, besides NMDA receptors, ketamine interacts with distinct receptor systems, such opioid, monoaminergic, muscarinic receptors and voltage sensitive Ca+2 channels, which could produce synergic effects on the brain pathways involved in the modulation of behavioral and molecular actions of antidepressants. Interestingly enough, our study demonstrated that ketamine, but not imipramine, increased BDNF levels in rat hippocampus after one single injection. Altogether, basic and clinical findings might suggest that acute increase of BDNF protein levels in hippocampus might be critical to antidepressant drugs with rapid onset of action. Future studies need to be carried out in an attempt to further investigate pharmacological and molecular mechanism by which ketamine, and other NMDA antagonists, induce antidepressant-like effects. Acknowledgements This study was supported in part by CNPq (Brazil), UNESC (Brazil), FAPESC (Brazil). References
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