Abstract

Introduction

A growing body of evidence points to involvement of N-methyl-D-aspartate (NMDA) in the pathophysiology and treatment of psychiatric disorders.¹1. Coyle JT, Tsai G, Goff D. Converging evidence of NMDA receptor hypofunction in the pathophysiology of schizophrenia. Ann N Y Acad Sci. 2003;1003:318-27.,²2. Garcia LS, Comim CM, Valvassori SS, Réus GZ, Barbosa LM, Andreazza AC, et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF levels in the rat hippocampus. Prog Neuropsychopharmacol Biol Psychiatry. 2008;32:140-4. In fact, clinical studies indicate that administration of subanesthetic doses of ketamine, an NMDA receptor antagonist, to healthy individuals can mimic the positive and negative symptoms seen in schizophrenic patients,³3. Adler CM, Malhotra AK, Elman I, Goldberg T, Egan M, Pickar D, et al. Comparison of ketamine-induced thought disorder in healthy volunteers and thought disorder in schizophrenia. Am J Psychiatry. 1999;156:1646-9.,⁴4. Lahti AC, Weiler MA, Tamara Michaelidis BA, Parwani A, Tamminga CA. Effects of ketamine in normal and schizophrenic volunteers. Neuropsychopharmacology. 2001;25:455-67. including social withdrawal and cognitive deficits.⁵5. Astur RS, Taylor LB, Mamelak AN, Philpott L, Sutherland RJ. Humans with hippocampus damage display severe spatial memory impairments in a virtual Morris water task. Behav Brain Res. 2002;132:77-84. On the other hand, acute administration of ketamine rapidly improved depressive symptoms in patients with major depression.⁶6. Zarate CA, Jr, Singh JB, Carlson PJ, Brutsche NE, Ameli R, Luckenbaugh DA, et al. A randomized trial of an N-methyl-D-aspartate antagonist in treatment-resistant major depression. Arch Gen Psychiatry. 2006;63:856-64.

However, demonstrations of the psychotherapeutic effects of ketamine on animal models are complicated by its side effects on general activity, which have not been routinely measured or taken into account in experimental studies.⁷7. Engin E, Treit D, Dickson CT. Anxiolytic- and antidepressant-like properties of ketamine in behavioral and neurophysiological animal models. Neuroscience. 2009;161:359-69. Interestingly, glutamate is known to regulate neurogenesis, synaptogenesis, and neuron survival.⁸8. Arlotta P, Magavi SS, Macklis JD. Induction of adult neurogenesis: molecular manipulation of neural precursors in situ. Ann N Y Acad Sci. 2003;991:229-36. Ketamine plays important roles in synaptic plasticity and neuronal learning that can regulate cell proliferation and dysregulation, which are widely discussed as pathogenetic factors of neuropsychiatric disorders.⁹9. Harrison PJ. The neuropathology of schizophrenia. A critical review of the data and their interpretation. Brain. 1999;122:593-624.

It is well known that neuropsychiatric diseases are associated with alterations in nervous signal transmission or disturbances in growth factors in the brain.¹⁰10. Fisar Z, Hroudova J. Intracellular signalling pathways and mood disorders. Folia Biol (Praha). 2010;56:135-48. Brain-derived neurotrophic factor (BDNF) is one of the major neurotrophic factors that primarily support the growth and survival of cholinergic, dopaminergic, and motor neurons. BDNF is synthesized by sensory neurons and glia and may have both autocrine and paracrine functions in mediating activity-dependent plasticity.¹¹11. Ichisaka S, Katoh-Semba R, Hata Y, Ohshima M, Kameyama K, Tsumoto T. Activity-dependent change in the protein level of brain-derived neurotrophic factor but no change in other neurotrophins in the visual cortex of young and adult ferrets. Neuroscience. 2003;117:361-71. Although the mechanism of NMDA receptor blockade-mediated apoptotic neurodegeneration in the brain is not clearly defined, conflicting reports demonstrate both increases and decreases in BDNF levels after exposure to the noncompetitive NMDA receptor antagonist MK-801. A decrease in plasma levels of BDNF has been shown in animal models of schizophrenia,¹²12. Fumagalli F, Bedogni F, Perez J, Racagni G, Riva MA. Corticostriatal brain-derived neurotrophic factor dysregulation in adult rats following prenatal stress. Eur J Neurosci. 2004;20:1348-54. and decreased levels in the dorsolateral prefrontal cortex have been reported in individuals with schizophrenia.¹³13. Buckley PF, Pillai A, Evans D, Stirewalt E, Mahadik S. Brain derived neurotropic factor in first-episode psychosis. Schizophr Res. 2007;91:1-5. In contrast, other studies have demonstrated an increase in BDNF levels in the cortical areas.¹⁴14. Weickert CS, Hyde TM, Lipska BK, Herman MM, Weinberger DR, Kleinman JE. Reduced brain-derived neurotrophic factor in prefrontal cortex of patients with schizophrenia. Mol Psychiatry. 2003;8:592-610. In addition, reduced levels of BDNF have been reported in the serum, plasma, and brain tissues of animals and humans with depression, and antidepressant treatment has been shown to increase BDNF.¹⁵15. De Foubert G, Carney SL, Robinson CS, Destexhe EJ, Tomlinson R, Hicks CA, et al. Fluoxetine-induced change in rat brain expression of brain-derived neurotrophic factor varies depending on length of treatment. Neuroscience. 2004;128:597-604. These contradictory observations may account for both the neuroprotective and proapoptotic nature of MK-801.¹⁶16. Palmer GC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies. Curr Drug Targets. 2001;2:241-71.

Although there are many studies linking ketamine with neuropsychiatric diseases, behavioral parameters and BDNF levels at different times after the administration of ketamine have yet to be fully characterized. The main objective of this study was therefore to evaluate the effects of ketamine on the open-field and forced swimming tests at different times after the administration of the drug, as well as to analyze BDNF protein levels in specific rat brain areas (prefrontal cortex, hippocampus, amygdala, and striatum).

Methods

Animals

Adult male Wistar rats (60 days old) were obtained from the Central Animal Facility of Universidade do Extremo Sul Catarinense (UNESC), Criciúma, state of Santa Catarina, Brazil. They were caged in groups of five with free access to food and water and were maintained on a 12-hour light-dark cycle (lights on 7:00 a.m.), at a temperature of 22±1 °C. All experimental procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Brazilian Society for Neuroscience and Behavior (SBNeC) recommendations for animal care, with the approval of the UNESC Ethics Committee.

Drugs and treatment

A chronic subanesthetic dose (25 mg/kg) of ketamine (Fort Dodge, Brazil) was administered once a day over 7 consecutive days, intraperitoneally (i.p.), at a volume of 1 mL/100 g. The dose and protocols were as previously described.¹⁷17. Hunt MJ, Raynaud B, Garcia R. Ketamine dose-dependently induces high-frequency oscillations in the nucleus accumbens in freely moving rats. Biol Psychiatry. 2006;60:1206-14. Animals were evaluated on the 7th day of treatment using the behavioral tests described below at 1 and 6 hours after the last injection. The rats were then sacrificed by decapitation after behavioral evaluation, and the brain structures of interest (prefrontal cortex, striatum, amygdala and hippocampus) were dissected. Animals were divided into two time sets (1 and 6 hours); each set including two groups of 10 animals each.

Open-field test

We used the open-field task to assess locomotor activity as described elsewhere.¹⁸18. de Oliveira L, Fraga DB, De Luca RD, Canever L, Ghedim FV, Matos MP, et al. Behavioral changes and mitochondrial dysfunction in a rat model of schizophrenia induced by ketamine. Metab Brain Dis. 2011;26:69-77. The covered distance was assessed for 30 min in an automatic arena. Locomotor activity was constantly monitored by an automated system installed in the arena.

Forced swimming test

The forced swimming test was conducted according to previous reports.²2. Garcia LS, Comim CM, Valvassori SS, Réus GZ, Barbosa LM, Andreazza AC, et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF levels in the rat hippocampus. Prog Neuropsychopharmacol Biol Psychiatry. 2008;32:140-4.,¹⁹19. Porsolt RD, Le Pichon M, Jalfre M. Depression: a new animal model sensitive to antidepressant treatments. Nature. 1977;266:730-2. During the test session, some behavioral parameters were recorded in seconds, such as the immobility, climbing and the swimming.

BDNF measurement

BDNF levels in the hippocampus, striatum, amygdala, and prefrontal cortex were determined by sandwich-ELISA using monoclonal antibodies specific for BDNF (R&D Systems, Minneapolis, United States). Total protein was measured by Bradford's method²⁰20. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-54. using bovine serum albumin as standard.

Statistical analysis

Evaluation of study variables showed that normal-distribution parametric tests would be most appropriate. Differences among experimental groups in the forced swimming and open-field tests and in the assessment of BDNF levels were determined by one-way analysis of variance (ANOVA), followed by a Tukey post-hoc test when ANOVA findings were significant, and are presented as mean ± standard error of the mean (SEM). All analyses were performed using SPSS version 18.0.

Results

Administration of ketamine at a dose of 25 mg/kg over 7 consecutive days induced an increase of the covered distance in rats evaluated 1 hour after the last injection in all the periods studied, as compared with rats that received saline (Figure 1A); p < 0.05). On the other hand, the locomotor effect of ketamine was decreased in rats evaluated 6 hours after the last injection in 5 and 10 min, as compared with the saline group (Figure 1A); p < 0.05). The evaluation period was divided into blocks of 5 minutes, as shown in Figure 1A.

In the forced swimming test, 1 hour after the last injection of ketamine, there were no alterations in the immobility, swimming or climbing times (Figure 1B). However, 6 hours after the last injection, there was a decrease in immobility time (Figure 1B); p < 0.05) compared with the saline group. Furthermore, 6 hours after the last injection of ketamine, there was an increase in climbing time (Figure 1B); F_(2-40) = 8.69; p < 0.001) as compared with the saline and ketamine groups evaluated 1 hour after the last injection. There was no alteration in swimming time (Figure 1B); F_(2-40) = 0.46; p > 0.05).

Figure 2 illustrates the effects of ketamine administration on BDNF protein levels in the rat brain. When the animals were killed 6 hours after the last injection of ketamine, there was a significant decrease in BDNF protein levels in the prefrontal cortex (p < 0.001) and amygdala (p = 0.012), compared to the saline and ketamine groups that were killed 1 hour after the last injection. In the hippocampus, there was a decrease in BDNF protein levels when the rats were killed 1 or 6 hours after the last injection of ketamine (p = 0.007) as compared with the saline group. In the striatum, BDNF protein levels were reduced when the rats were killed 6 hours after the last injection (p = 0.048) as compared to the saline group.

Discussion

The effects of NMDA antagonists have been explored in rodents. In keeping with our results, Irifune et al.²¹21. Irifune M, Shimizu T, Nomoto M. Ketamine-induced hyperlocomotion associated with alteration of presynaptic components of dopamine neurons in the nucleus accumbens of mice. Pharmacol Biochem Behav. 1991;40:399-407. showed that ketamine induced a marked hyperlocomotion in rodents that dissipated 80 min after drug administration and was associated with an indirect dopaminergic agonist action in the nucleus accumbens. We also observed a decrease in locomotor activity 6 hours after the last injection of ketamine. The effects observed may be explained, at least in part, by the fact that repeated ketamine administration produces a reduction in motivation, suggesting that ketamine induces both positive and negative effects on locomotor activity depending on the time elapsed between the last injection and evaluation.

In the present study, we found decreased immobility time and increased climbing time 6 hours after the last injection of ketamine (but not 1 hour after last injection). In contrast, other studies have shown that chronic treatment with phencyclidine (PCP), an NMDA receptor antagonist, decreased spontaneous locomotor activity and enhanced the forced swim-induced immobility.²²22. da Silva FC, do Carmo de Oliveira Cito M, da Silva MI, Moura BA, de Aquino Neto MR, Feitosa ML, et al. Behavioral alterations and pro-oxidant effect of a single ketamine administration to mice. Brain Res Bull. 2010;83:9-15. However, in keeping with our data, another study has shown that acute treatment with ketamine at the doses of 5, 10 and 20 mg/kg increased locomotor activity and decreased immobility time in the forced swimming and tail suspension tests in mice,²³23. Reich DL, Silvay G. Ketamine: an update on the first twenty-five years of clinical experience. Can J Anaesth. 1989;36:186-97. suggesting that the effects exerted by ketamine are dependent on timing of administration and dose. In fact, ketamine has been shown to induce psychosis in an age-dependent manner.²⁴24. Chaturvedi HK, Bapna JS, Chandra D. Effect of fluvoxamine and N-methyl-D-aspartate receptor antagonists on shock-induced depression in mice. Indian J Physiol Pharmacol. 2001;45:199-207.

Preclinical studies have shown that ketamine decreases the immobility time in the forced swimming test after acute or chronic treatment.²2. Garcia LS, Comim CM, Valvassori SS, Réus GZ, Barbosa LM, Andreazza AC, et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF levels in the rat hippocampus. Prog Neuropsychopharmacol Biol Psychiatry. 2008;32:140-4. Furthermore, in rodents subjected to the animal models of depression, ketamine has shown efficacy as an antidepressant.²⁵25. Olney JW, Labruyere J, Wang G, Wozniak DF, Price MT, Sesma MA. NMDA antagonist neurotoxicity: mechanism and prevention. Science. 1991;254:1515-8. Clinical studies demonstrated that the administration of ketamine ameliorates depressive symptoms in patients with major depression.²⁶26. Berman RM, Cappiello A, Anand A, Oren DA, Heninger GR, Charney DS, et al. Antidepressant effects of ketamine in depressed patients. Biol Psychiatry. 2000;47:351-4. Interestingly, in the present study, we showed an antidepressant effect exerted by ketamine only 6 hours after the last injection, at which time motor activity was not increased. Previous studies from our laboratory evaluating the antidepressant effects of ketamine showed an antidepressant effect without affecting spontaneous locomotor activity.²2. Garcia LS, Comim CM, Valvassori SS, Réus GZ, Barbosa LM, Andreazza AC, et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF levels in the rat hippocampus. Prog Neuropsychopharmacol Biol Psychiatry. 2008;32:140-4. Moreover, acute, but not chronic treatment with ketamine at a dose of 15 mg/kg increased BDNF protein levels in the rat hippocampus,²2. Garcia LS, Comim CM, Valvassori SS, Réus GZ, Barbosa LM, Andreazza AC, et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF levels in the rat hippocampus. Prog Neuropsychopharmacol Biol Psychiatry. 2008;32:140-4. suggesting that these effects were not detectable at a later time because of adaptive mechanisms or due to the development of tolerance to the effects of ketamine on hippocampal BDNF levels.

Liu et al.²⁷27. Liu F, Zou X, Sadovova N, Zhang X, Shi L, Guo L, et al. Changes in gene expression after phencyclidine administration in developing rats: a potential animal model for schizophrenia. Int J Dev Neurosci. 2011;29:351-8. recently demonstrated an increase in the expression of NMDA receptor NR1 subunits and pro-apoptotic genes, decreased anti-apoptotic genes, as well as altered expression of BDNF and exacerbated neurodegeneration in developing brains exposed repeatedly to PCP, subsequently resulting in the abnormal development of neurons and neuronal networks, which might be critical for schizophrenia symptoms in adulthood. Complex studies have shown that BDNF plays a role in schizophrenia.²⁸28. Danielyan A, Nasrallah HA. Neurological disorders in schizophrenia. Psychiatr Clin North Am. 2009;32:719-57. In fact, the neurodevelopmental abnormalities seen in schizophrenia could arise from abnormal cell migration, disconnection, disturbances in neurotransmitter systems, and changes in neural plasticity caused by alterations in the expression and/or functioning of neurotrophins.²⁹29. Carlino D, Leone E, Di Cola F, Baj G, Marin R, Dinelli G, et al. Low serum truncated-BDNF isoform correlates with higher cognitive impairment in schizophrenia. J Psychiatr Res. 2011;45:273-9. Carlino et al.²⁹29. Carlino D, Leone E, Di Cola F, Baj G, Marin R, Dinelli G, et al. Low serum truncated-BDNF isoform correlates with higher cognitive impairment in schizophrenia. J Psychiatr Res. 2011;45:273-9. found a slight reduction in BDNF levels using ELISA assay in the serum of patients with schizophrenia, and an increase in pro-BDNF, mat-BDNF and truncated-BDNF using Western blot analysis. In addition, subjects with schizophrenia had significantly lower BDNF levels in the prefrontal cortex and cerebrospinal fluid (CSF),³⁰30. Issa G, Wilson C, Terry AV, Jr., Pillai A. An inverse relationship between cortisol and BDNF levels in schizophrenia: data from human postmortem and animal studies. Neurobiol Dis. 2010;39:327-33. suggesting that a deficiency in pro-BDNF processing may be a possible biological mechanism underlying schizophrenia. Other studies also found a significant decrease in BDNF levels in the serum of patients with schizophrenia.³¹31. Tan YL, Zhou DF, Cao LY, Zou YZ, Zhang XY. Decreased BDNF in serum of patients with chronic schizophrenia on long-term treatment with antipsychotics. Neurosci Lett. 2005;382:27-32. Conversely, another study showed an increase in BDNF levels.³²32. Gama CS, Andreazza AC, Kunz M, Berk M, Belmonte-de-Abreu PS, Kapczinski F. Serum levels of brain-derived neurotrophic factor in patients with schizophrenia and bipolar disorder. Neurosci Lett. 2007;420:45-8. In the present study, we found a decrease in BDNF protein levels in the hippocampus and amygdala in rats killed 6 hours after the last injection of ketamine as compared with the saline group and with the group that was killed 1 hour after the last injection. BDNF levels in the striatum were also decreased in comparison with the saline group among rats killed 6 hours after the last injection,, and levels in the hippocampus were decreased in the groups that were killed 1 and 6 hours after the last injection as compared with the saline group. Therefore, the effects of ketamine on BDNF levels were associated with the time at which the animals were evaluated after the last injection.

It is important to note that we chose the prefrontal cortex, hippocampus, striatum and amygdala as areas of interest in the current study because these brain areas are implicated in neuropsychiatric disorders.³³33. Benjamin S, McQuoid DR, Potter GG, Payne ME, MacFall JR, Steffens DC, et al. The brain-derived neurotrophic factor Val66Met polymorphism, hippocampal volume, and cognitive function in geriatric depression. Am J Geriatr Psychiatry. 2010;18:323-31. A clinical study showed that BDNF protein was altered in the hippocampus but unchanged in the prefrontal cortex of schizophrenia patients.³⁴34. Kang H, Schuman EM. Long-lasting neurotrophin-induced enhancement of synaptic transmission in the adult hippocampus. Science. 1995;267:1658-62. Additionally, neonatal ventral hippocampal lesion, used as an animal model of schizophrenia, caused a decrease in BDNF expression in the hippocampus and a tendency toward reduction in the prefrontal cortex.¹⁶16. Palmer GC. Neuroprotection by NMDA receptor antagonists in a variety of neuropathologies. Curr Drug Targets. 2001;2:241-71. Kang & Schuman³⁵35. Takahashi M, Shirakawa O, Toyooka K, Kitamura N, Hashimoto T, Maeda K, et al. Abnormal expression of brain-derived neurotrophic factor and its receptor in the corticolimbic system of schizophrenic patients. Mol Psychiatry. 2000;5:293-300. showed that BDNF protein levels may cause changes in the strength of synaptic transmission in the hippocampus. On the other hand, unregulated BDNF levels in the hippocampus can induce other schizophrenia-like behavior.³⁶36. Takahashi M, Kakita A, Futamura T, Watanabe Y, Mizuno M, Sakimura K, et al. Sustained brain-derived neurotrophic factor up-regulation and sensorimotor gating abnormality induced by postnatal exposure to phencyclidine: comparison with adult treatment. J Neurochem. 2006;99:770-80. Furthermore, Hasbi et al.³⁷37. Hasbi A, Fan T, Alijaniaram M, Nguyen T, Perreault ML, O'Dowd BF, et al. Calcium signaling cascade links dopamine D1-D2 receptor heteromer to striatal BDNF production and neuronal growth. Proc Natl Acad Sci U S A. 2009;106:21377-82. showed that the striatum, which is a dopaminergic area, regulated neuronal growth and maturation through BDNF. Thus, it is possible that locomotor hyperactivity be related, at least in part, with alterations in the hippocampus, striatum, prefrontal cortex, or amygdala in the current study. Studies from our group showed that acute administration of ketamine at a dose of 15 mg/kg reduced immobility time and increased BDNF protein levels in the hippocampus,²2. Garcia LS, Comim CM, Valvassori SS, Réus GZ, Barbosa LM, Andreazza AC, et al. Acute administration of ketamine induces antidepressant-like effects in the forced swimming test and increases BDNF levels in the rat hippocampus. Prog Neuropsychopharmacol Biol Psychiatry. 2008;32:140-4. but chronic administration did not exert this effect. The increase in BDNF protein levels may have been a defense mechanism. Indeed, animal models of schizophrenia induced by MK-801 administration increased BDNF levels,³⁸38. Guo C, Yang Y, Su Y, Si T. Postnatal BDNF expression profiles in prefrontal cortex and hippocampus of a rat schizophrenia model induced by MK-801 administration. J Biomed Biotechnol. 2010;2010:783297.,³⁹39. Hirota K, Lambert DG. Ketamine: its mechanism(s) of action and unusual clinical uses. Br J Anaesth. 1996;77:441-4. suggesting a protective response induced by cytotoxic insults.

In conclusion, chronic administration of ketamine altered behavioral parameters and BDNF protein levels in the rat brain, suggesting that both may be related, at least in part, to the effects of ketamine on the NMDA receptor. However, it is important to note that ketamine also acts on other systems, such as the monoaminergic and muscarinic receptors and voltage sensitive Ca²⁺ channels,⁴⁰40. Kozisek ME, Middlemas D, Bylund DB. Brain-derived neurotrophic factor and its receptor tropomyosin-related kinase B in the mechanism of action of antidepressant therapies. Pharmacol Ther. 2008;117:30-51. which are also involved in neuropsychiatric diseases; thus, the effects of ketamine could be related to synergistic effects on all of these pathways. Future studies evaluating the mechanisms by which ketamine exerts different molecular and behavioral effects in relation to the time elapsed after administration would be welcome to expand on the results reported herein.

Acknowledgements

This study was supported in part by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq - Alexandra I. Zugno, João Quevedo, and Flávio Kapczinski), from Instituto Cérebro e Mente (João Quevedo), and from UNESC (Alexandra I. Zugno and João Quevedo). Alexandra I. Zugno, João Quevedo, and Flávio Kapczinski are recipients of CNPq (Brazil) Productivity Fellowships. Daiane B. Fraga holds a studentship from Fundação de Amparo è Pesquisa e Inovação do Estado de Santa Catarina (FAPESC). Gislaine Z. Réus holds a studentship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

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