Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia

G Model BBR-8872; No. of Pages 12

ARTICLE IN PRESS Behavioural Brain Research xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Behavioural Brain Research journal homepage: www.elsevier.com/locate/bbr

Research report

Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia Brandon H. Cline a,b,c,l,1 , Daniel C. Anthony d,l,1 , Alexander Lysko e,l,1 , Oleg Dolgov f,l , Konstantin Anokhin f,l , Careen Schroeter g,l , Dmitry Malin e,h,l , Aslan Kubatiev e,l , Harry W. Steinbusch j,l , Klaus-Peter Lesch j,i,l , Tatyana Strekalova g,j,k,l,∗ a

CNRS, UMR6265, CSGA, 9E boulevard Jeanne d’Arc, 21000 Dijon, France INRA, UMR1324, CSGA, 9E boulevard Jeanne d’Arc, 21000 Dijon, France c Université de Bourgogne, CSGA, 9E boulevard Jeanne d’Arc, 21000 Dijon, France d Department of Pharmacology, Oxford University, Mansfield Road, OX1 3QT Oxford, UK e Institute of General Pathology and Pathophysiology, Baltyiskaia 8, 125315 Moscow, Russia f Institute of Normal Physiology, Baltyiskaia 8, 125315 Moscow, Russia g Maastricht Medical Centre in Annadal, Department of Preventive Medicine, Becanusstraat 17 A0, 6216 BX Maastricht, Netherlands h University of Wisconsin, Carbon Cancer Centre, WIMR 3016, 1111 Highland Avenue, Madison, WI 53705, USA i Division of Molecular Psychiatry, Laboratory of Translational Neuroscience, Department of Psychiatry, Psychosomatics and Psychotherapy, University of Wuerzburg, Germany j Department of Neuroscience, Maastricht University, Universiteitssingel 40, NL 6229 ER Maastricht, Netherlands k Institute of Physiologically Active Compounds RAS, Severnii proesd 1, 142100 Chernogolovka, Moscow Region, Russia l Institute for Hygiene and Tropical Medicine, New University of Lisbon, Lisbon, Portugal b

h i g h l i g h t s • • • • •

Chronic stress affects brain peroxidation in anhedonic but not resilient mice. Acute stress elicits opposing effects to chronic stress on peroxidation. Imipramine precludes changes in peroxidation after chronic and acute stress. Stress-induced changes in peroxidation and behaviour last after anhedonia recovery. Altered peroxidation may reflect individual susceptibility to depression.

a r t i c l e

i n f o

Article history: Received 22 February 2014 Received in revised form 18 April 2014 Accepted 21 April 2014 Available online xxx Keywords: Lipid peroxidation Chronic stress depression model Anhedonia Prefrontal cortex Imipramine Microarray

a b s t r a c t Antioxidant enzymes and lipid peroxidation in the brain are involved in neuropsychiatric pathologies, including depression. 14- or 28-day chronic stress model induced a depressive syndrome defined by lowered reward sensitivity in C57BL/6J mice and changed gene expression of peroxidation enzymes as shown in microarray assays. We studied how susceptibility or resilience to anhedonia is related to lipid peroxidation in the prefrontal cortex (PFC). With 14-day stress, a comparison of the activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX) and accumulation of malondialdehyde (MDA) revealed a decrease of the first two measures in susceptible, but not in resilient animals or in stressed mice chronically dosed with imipramine (7 mg/kg/day). Acute stress elevated activity of CAT and SOD and dynamics of MDA accumulation in the PFC that was prevented by imipramine (30 mg/kg). 28-day stress evoked anhedonia lasting two but not five weeks while behavioural invigoration was detected at the latter time point in anhedonic but not non-anhedonic mice; enhanced aggressive traits were observed in both groups. After two weeks of a stress-free period, CAT and SOD activity levels in the PFC were reduced in anhedonic animals; after five weeks, only CAT was diminished. Thus, in the present chronic

∗ Corresponding author at: School for Mental Health and Neuroscience, Department of Neuroscience, Maastricht University, Universiteitssingel 40, NL 6229 ER Maastricht, Netherlands. Tel.: +31 43 38 84 110; fax: +31 43 3671 096. E-mail address: [email protected] (T. Strekalova). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbr.2014.04.037 0166-4328/© 2014 Published by Elsevier B.V.

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

2

stress depression paradigm, lasting alterations in brain peroxidation occur not only during anhedonia but also in the recovery period and are accompanied by behavioural abnormalities in mice. This mimics behavioural and neurochemical deficits observed in depressed patients during remission which could be used to develop remedies preventing their relapse. © 2014 Published by Elsevier B.V.

1. Introduction It has been widely demonstrated that the generation of reactive oxygen species plays a critical role in the pathophysiology of several neuropsychiatric disorders [1,2]. The brain is particularly vulnerable to reactive oxygen species production because it metabolizes 20% of total body oxygen and has a limited amount of antioxidant capacity [1]. In situations where the generation of free radicals exceeds the capacity of antioxidant defence, oxidative stress may lead to membrane degradation, cellular dysfunction and apoptosis [3,4]. This might be relevant for the pathogenesis of affective disorders, because in vivo magnetic resonance spectroscopy studies have demonstrated changes in brain compounds related to oxidative phosphorylation, energy production and phospholipid metabolism [5]. In addition, it has been hypothesized that affective disorders are associated with mitochondrial dysfunction, and abnormalities in respiratory complex activity and energy production may lead to cellular degeneration [6,7]. Recent studies have consistently reported increased products of lipid peroxidation and alterations of the major antioxidant enzymes in patients with affective disorders, in particular, with depression [1,8–10]. The associations between oxidative and antioxidative systems during depressive disorder and efficacy of antidepressant therapies have been demonstrated in pharmacological studies as well [11]. Yet, the outcome so far from clinical and pre-clinical studies, particularly undertaken with various translational approaches to model depression, did not result in a clear picture of which and how lipid peroxidation enzymes are changed in the CNS during depression. Moreover, some studies reported an increase while others evidenced a decrease or no change in the main enzymes of peroxidation [9,12]. Scarce data are available concerning lipid peroxidation during a recovery phase from depression and residual behavioural abnormalities in animals and patients with preceding depressive syndrome [13,14]. Meanwhile, early relapse of depressive episodes remain a substantial medical and social problem, since about 20% of patients experience persistent subsyndromal depression during the recovery phase [15] and display aberrant behaviour including high impulsivity and aggression, as well as an increased stress response. Importantly, these mechanisms seem to differ from those of depression development and treatment resistance [16]. The identification of how lipid peroxidation enzymes in the brain is altered during depression/remission states is challenged by the limitations of the use of non-invasive research tools that are applicable in clinical studies. As for the translational approach, the investigation of these questions is greatly constrained by the use of experimental procedures of modelling depression, which per se affect brain peroxidation. As for instance, frequently used stress techniques that evoke learned helplessness, behavioural despair and/or deficient sensitivity to reward in experimental animals, can change brain peroxidation irrespectively to the induction of a depressive-like state [17–20]. On this basis, to investigate whether the activities of lipid peroxidation enzymes are changed during depressive/recovery states, we have used variants of a recently validated 14- and 28-days chronic stress depression models with internal control for stress in mice [19–21]. Since anhedonia, a decreased ability to experience pleasure from activities normally thought to be enjoyable, is a key

feature of major depressive syndrome [21], an animal paradigm that would mimic this feature to address such question [22,23], was selected. We have chosen to employ experimental protocols in which inbred C57BL/6J mice can be differentiated upon their individual vulnerability to the stress-induced anhedonic state as defined by a decrease in the preference to sucrose solution over water. Stressed mice either have shown a decrease of sucrose preference below the lowest control values (sucrose preference <65%) and were defined as susceptible to stress-induced anhedonia, or did not show such changes (sucrose preference >65%) and were considered as resilient to the induction of this deficit [24]. As such, resilient, non-anhedonic mice were used as an internal control that enables the analysis of potential peroxidation changes in stressed mice specifically in relation to anhedonia versus the consequences of stress alone. The use of a subgroup of individuals who are negative for the induction of a desired phenotype during experimental paradigms as an internal control for more refined modelling of neurobiological phenomena and medical conditions is not novel [25]. It was recently implemented in modelling depression with chronic stress, allowing the separation of resilient from susceptible to depressive-like state animals [24,26] and, this way, to increase the validity of such paradigms (for a review, see Strekalova et al. [27]). Given the fact that profound changes such as increased SERT, COX-1, TNF-␣, 5-HT2A receptor expression and increased microglia activation, seen in a mouse model with stress-induced anhedonia, were found in the prefrontal cortex [28], we have chosen this brain structure to assess the activity of peroxidation enzymes. Notably, while similar changes were observed also in the hippocampus, they were less pronounced than in the prefrontal cortex. Microarray analysis of hippocampal expression of genes of peroxidation enzymes after 14-day stress was supplementary included in the study. Additionally, we have included stressed imipramine-treated animals, as preliminary data have shown that most of the molecular changes observed in the prefrontal cortex of stressed anhedonic mice are precluded by chronic imipramine treatment in a 10day stress paradigm [29]. In order to segregate potential overlap between the effects of chronic stress and the last stressor applied in a course of the procedure, we have separately analyzed the effects of a single rat exposure in naive mice, also under conditions of a pre-treatment with bolus injection of imipramine. To relay potential changes in the brain peroxidation to behavioural signs of anhedonia and residual symptoms that are characteristic for remission periods from depression, a sensitivity to reward in sucrose test, triggered by bright illumination behavioural invigoration in locomotor tests and parameters of aggression were evaluated in mice at various time points with respect to stress. We anticipated defining whether or not behavioural and brain peroxidation measures can be specifically attributed to individual susceptibility to stress-induced anhedonia and if any of the behavioural and neurochemical deficits can be observed during remission from the anhedonia phase. 2. Materials and methods 2.1. Animals Studies were performed using 3.5-month-old male C57BL/6J mice. 3.5-month-old male CD1 mice were used as resident

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

3

Fig. 1. Schematic representation of stress paradigms and behavioural experiments. (A) 14 day chronic stress with imipramine treatment (B) 28-day chronic stress time line (C) acute stress. Legend insert states the behavioural experiments.

intruders for social stress and 2–5-month-old Wistar rats were used for predator stress. All animals were from the Gulbenkian Institute of Science, Oeiras, Portugal. C57BL/6J mice were housed individually for 10–14 days before the start of experiments; CD1 mice and rats were housed in groups of five before the experiment and then individually. All animals were under a reversed 12-h light–dark cycle (lights on: 21:00 h) starting from the day of animals’ transportation in the laboratory; with food and water ad libitum, under controllable laboratory conditions (22 ± 1 ◦ C, 55% humidity). Animals were handled weekly during the changes of cages by care takers, as well as when experimental procedures were performed. Mice in the control group had a facial tissue in their cages that was also placed on the bedding material of the stress group prior the onset of stress, but not in the period of stress or post-stress; they were housed separately from experimental groups. All experiments were carried out in accordance with the European Communities Council Directive for the care and use of laboratory animals upon approval by the local governmental bodies of animal care and welfare. 2.2. Chronic stress experiments This study uses recently validated variants of a 14-day [28] and 28-day stress protocols [24] comprising night time rat exposure and day time application of two of three stressors: a social defeat, restraint stress and tail suspension, a combination of which was applied in a semi-random manner (for details see Supplementary Material). Briefly, between the hours of 09:00 and 18:00 two stressors per day were employed in the following sequence: social defeat for 30 min, restraint stress for 2 h and tail suspension for 40 min with an inter-session interval of at least 4 h. With the 14-day stress protocol, 10 mice constituted a control group. 29 mice were subjected to stress, 9 of them have received imipramine (7 mg/kg/day) via drinking water during 7 days prior the onset of stress and during the entire stress procedure as described elsewhere [29,30] (see Supplementary data). All mice were tested in the novel cage and open field lit with bright light (200 lx), as well as for social interaction [2,28,31] on the day of the termination of chronic stress, starting 6 h after the last rat exposure stress session, with inter-test interval of 2 h, in the listed order. Sucrose preference, two-bottle test, was performed during the dark phase of the animals’ cycle between 09:00 and 17:00 on the day after the termination of the chronic stress procedure as described elsewhere [31]. Thereafter, i.e., approximately thirty-six hours after the end of the last stress session, mice were sacrificed for brain dissection and a subsequent brain peroxidation assay in the

prefrontal cortex and hippocampal gene expression with Illumina assay (Fig. 1A). In the 28-day stress experiment, 10 naive control mice were used, and 24 were subjected to stress. The latter mice were assigned to resilient and anhedonic mice according to their sucrose preference (see below) after the termination of the stress procedure. Sucrose test was repeated 2 weeks and 5 weeks after the termination of stress, where subgroups of control, resilient and anhedonic animals were sacrificed for peroxidation enzyme assay in the prefrontal cortex (the first time point: each group was comprised of 5 mice; the second time point: 5 controls, 6 resilient and 7 anhedonic mice). Subgroups of mice that were either sacrificed or kept for further testing were balanced upon sucrose preference. After the last sucrose test that was carried out 5 weeks after the termination of the chronic stress procedure, mice were tested, on the same day, in a resident intruder test [31]. On the next day, animals were consequently examined in brightly illuminated novel cage and open field (200 lx; [28]), as well as a step-down anxiety test [2], with a 2 h-inter-test protocol. In average, thirty-six hours after the onset of sucrose test, animals were sacrificed and their brains were dissected (Fig. 1B). Additionally, the remaining 5 control animals were subjected to a single rat exposure, in order to investigate potential effects of acute stress on the brain peroxidation (see below: Section 2.3). They were sacrificed 6 h after the termination of acute stress and their brains were dissected to evaluate brain peroxidation in the prefrontal cortex (Fig. 1C). For all behavioural tests, validated protocols of behavioural testing that were previously adapted to chronically stressed C57BL/6J mice were used in the study [2,27,30,32]. 2.3. Acute stress The effects of acute stress were investigated using the acute stress control group. In a separate experiment, this group underwent predator stress the night prior to their killing and dissection of the prefrontal cortex. Mice were acutely stressed (n = 8) or acutely stressed and treated with a bolus intraperitoneal injection of imipramine (30 mg/kg; [33]; see Supplementary data) 30 min prior the stress (n = 9); control animals were not treated (n = 7; Fig. 1C). 2.4. Sucrose preference test Mice were given 8 h of free choice between two bottles of either 1% sucrose or standard drinking water at day −7 and by the termination of stress. At the beginning and end of the period the

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12 4

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

bottles were weighed and consumption calculated. The beginning of the test started with the onset of the dark (active) phase of animals’ cycle, (i.e., at 9.00). To prevent the possible effects of sidepreference in drinking behaviour, the position of the bottles in the cage was switched at 4 h, halfway through testing. No previous food or water deprivation was applied before the test. To minimize the spillage of liquids during sucrose test, bottles were filled in advance and kept in the up-side-down position for at least 12 h prior to testing. In order to balance the temperature between the room and the drinking bottles, they were kept in the same room where the testing takes place. This prevents the physical effect of liquid leakage resulting from growing air temperature and pressure inside the bottles, when they are filled with liquids, which are cooler than the room air. Preliminary tests showed that with this method the error of measurement does not exceed 0.1 ml. In order to decrease variability in sucrose consumption during the very first sucrose test (baseline measurement), a day before, animals were allowed to drink 2.5% sucrose solution in a one-bottle paradigm for 2 h. Percentage preference for sucrose is calculated using the following formula: Sucrose Preference = Volume (Sucrose solution)/(Volume (Sucrose solution) + Volume (Water)) × 100. No mice from control groups ever exhibited a preference for sucrose of <65% and, accordingly, mice exhibiting a sucrose preference of <65% were defined as susceptible. Mice that had undergone stress but maintained a sucrose preference of >65% were defined as resilient. Other conditions of the test were applied as described elsewhere [2,27,30]. 2.5. Brain dissection, RNA isolation and Illumina microarray gene expression profiling Mice were sacrificed by cervical dislocation, prefrontal cortex and hippocampi were microdissected and stored at −80◦ C until use. RNA extraction was performed from hippocampi obtained in a shorter chronic stress experiment using RNeasy RNA extraction kit with DNase treatment, as previously described (Qiagen, Hilden, Germany; [33]). Gene expression profiling was performed using Illumina technology (Integragen, Evry, France and Northwestern Chicago University, USA) with the hippocampi of mice from non-stressed drug-naïve control, stressed resilient and anhedonic groups (five animals per each group were analyzed). Total RNA samples were hybridized to IlluminaBeadChips (MouseRef-8 v2 Expression BeadChip; Illumina, Inc. San Diego, CA, USA) which were prepared using the IlluminaTotalPrep RNA Amplification kit (Applied Biosystems/Ambion, Carlsbad, CA, USA); the samples were assigned to the chips in random order with the constraint that no two samples from the same group were assigned to the same chip, to avoid confounding of experimental groups with the chips. Microarray data were analyzed using standard analysis procedures, which included assessment of the overall quality of array data and statistical evaluation of differentially expressed genes (Integragen, Evry, France). Once the quality of array data was confirmed, the Gene Chip Operating System (Illumina, Inc. San Diego, CA, USA) was used to calculate signal intensities, detection calls, and their associated P values for each transcript on the array. Gene expression was normalized to the expression of the housekeeping gene, beta-actin, due to its stable expression, and calculated as percent mean of the control group. Differences in gene expression between groups were evaluated using unpaired two-tailed t-test. 2.6. Homogenate preparation and determination of protein concentration Homogenization was carried out in a smooth-walled glass tube fitted with a Teflon pestle at 1000 rpm (Laborruhrwerk MR 25) for

20 s in 0.05 M potassium phosphate buffer, pH 7.4. The tissue to medium ratio was 1:9 (g:ml). The homogenate was centrifuged for 5–10 min at 700 × g to remove nuclei and cell debris. The supernatants obtained were used for the determination of enzymatic activities of catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX) and for the quantification of the levels of malondialdehyde (MDA) and dynamics of accumulation of MDA (AcMDA). Enzymatic reactions were run at 37 ◦ C for CAT, at 25 ◦ C for SOD and GPX in the linear range of substrate utilization or product formation. The standard incubation medium was K-phosphate buffer (50 mM), pH 7.4 for CAT and GPX, and for SOD pH was 7.8. The spectrophotometer SPECORD M400 equipped with the thermostated cell holder and magnetic stirrer was used for all assays (Zeiss Industrielle Messtechnik GmbH, Jena, Germany). Each sample was evaluated in triplicate. Protein concentration was quantified according to the microbiuret method described by Bailey et al. [34], using bovine serum albumin as a standard. 2.7. Activity of antioxidant enzymes and quantification of lipid peroxidation 2.7.1. Catalase activity Catalase activity was measured by the method of Aebi [35] adapted by Cohen [36]. The reaction was started by the addition of freshly prepared 30 mM H2 O2 . The rate of H2 O2 decomposition is directly proportional to the amount of enzyme at a fixed H2 O2 concentration and obeys first-order kinetics with respect to H2 O2 concentration. The breakdown of H2 O2 was determined by following spectrophotometrically the decrease of absorbance at 240 nm. The time required (t) in minutes for A240 nm to decrease from 0.450 to 0.400 (corresponding to the decomposition of 1.15 ␮mol H2 O2 in the 1 ml assay) was used to calculate specific activity: specific activity units = (1.15 ␮mol) × (t)−1 × (mg protein)−1 . To an aliquot of supernatant fluid, ethanol was added to a final concentration of 0.17 M and samples were incubated for 30 min in ice-water bath; this procedure decomposes Complex II [37]. Catalase activity was expressed as ␮mol H2 O2 utilized/min/mg of protein. 2.7.2. Superoxide dismutase activity Superoxide dismutase (SOD) activity was assayed spectrophotometrically as described by Beauchamp and Fridovich [38] and modified by Greenwald [39]. With this method, xanthine/xanthine oxidase was used to generate O2 , which was detected by the reduction of nitrotetrazolium blue chloride (NBT) to blue formazan. Spectrophotometric measurement of the rate of blue formazan formation in the presence of increasing amounts of cellular protein was performed. Total SOD activity was determined at 560 nm by the inhibition of [hypoxantine (0.1 mM/xanthine oxidase (0.004 units)]-induced generation of superoxide radicals in the presence of 25 mM NBT (DETAPAC, Fluka, USA). The enzyme activity was expressed as U/mg of homogenate protein. 1 Unit (U) of SOD activity corresponds to a 50% inhibition of superoxide radicals generation in the above mentioned reaction. 2.7.3. Glutathione peroxidase activity Glutathione peroxidase, acting in both cytosol and mitochondria, accounts for the majority of all enzyme activity able to reduce H2 O2 and fatty acid hydroperoxide in brain [40]. Glutathione peroxidase activity (GPX) was determined based on indirect evaluation of the consumption of NADPH at 340 nm using the protocol developed by Paglia and Valentine [41] and adapted by Tappel and co-authors [42], since glutathione peroxidase uses glutathione to reduce the tert-butyl hydroperoxide. Subsequently, NADPH is involved as a reducing equivalent donor [43]. Absorbance at 340 nm was monitored for the rate of disappearance of NADPN at 25 ◦ C, the

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

extinction coefficient εNADPH = 6200 M−1 cm−1 was applied. GTA was expressed as nmol NADPH oxidized/min/mg protein. 2.7.4. Quantification of malondialdehyde Malondialdehyde (MDA) initial levels and dynamics of its formation, were determined as an indicator for the presence of thiobarbituric acid reactive species, a parameter of lipid peroxidation, according to Ohkawa et al. [44]. With this method, MDA, an end-product of lipid peroxidation, provides a reliable assessment of the general peroxidation in a tissue. The samples were incubated at 95 ◦ C for 60 min in K-phosphate buffer (50 mM), pH 7.4 (37 ◦ C). Initial levels of MDA products were determined at 0 min before initiation of lipid peroxidation by FeSO4 (3 ␮M) and ascorbate (500 ␮M) and were expressed as nmol MDA/g wet weight of tissue. The dynamics of accumulation of MDA (AcMDA), parameter that reflects an over-all test for tissue susceptibility to free-radical action in brain homogenates was observed at 5, 10 and 15 min after initiation of peroxidation and expressed as nmol MDA/g wet weight of tissue/min. 2.8. Statistics Data were analyzed with GraphPad Prism version 5.0 for Windows (San Diego, CA). One-way ANOVA was used followed by either Tukey’s or Dunnett’s post hoc comparison test (unless otherwise stated). The level of confidence was set at 95% (p < 0.05) and data are shown as mean ± SEM. 3. Results 3.1. 14-Day stress decreases sucrose preference and induces behavioural invigoration in a subgroup of mice that is counteracted by imipramine No control animals showed a preference for sucrose of <65% and, prior to stress induction, mice assigned to the distinct experimental groups displayed similar (>65%) preference for sucrose solutions. At no point did any control animal show a change in total sucrose, total water or total liquid intake (data not shown). Fourteen days of chronic stress resulted in a significant decrease (p = 0.0011, F = 8.237, R2 = 0.3139, ANOVA; **p < 0.01, q = 4.999 and # p < 0.05, q = 4.278 versus control and imipramine treated respectively, Tukey’s test, Fig. 2A) in sucrose preference for animals not treated with imipramine. Animals not treated with imipramine were further divided into resilient and anhedonic animals based upon their sucrose preference (resilient >65%; anhedonic <65%). Both resilient and anhedonic mice showed an significant increase in the number of rearings in novel cage exploration (p < 0.0001, F = 16.81, R2 = 0.5973, ANOVA) as compared to control and imipramine treated groups (p < 0.001, q = 7.692, resilient vs con; p < 0.001, q = 6.687, resilient vs imi and p < 0.001, q = 7.412 anhedonic vs con; p < 0.001, q = 6.453, anhedonic vs imi, Tukey’s test, Fig. 2B). Likewise, the total distanced travelled in open field was also significantly increased (p = 0.0008, F = 7.046, R2 = 0.3765, ANOVA, Fig. 2C). Both resilient and anhedonic animals displayed a significant increase in total distance versus control (p < 0.05, q = 4.367 and p < 0.01, q = 5.217, Tukey’s test; respectively) as well as against imipramine treated mice (p < 0.05, q = 3.880 and p < 0.05, q = 4.737, Tukeys’s test; respectively). Fourteen days of stress also induced changes in observed aggressive behaviour for the latency to attack (p = 0.0002, F = 8.900, R2 = 0.4327, ANOVA Fig. 1D) and the total number of attacks (p < 0.0001, F = 15.51, R2 = 0.5708, Fig. 2E). Resilient and anhedonic animals showed a significant decrease in the latency to attack (p < 0.01, q = 4.876 and p < 0.05, q = 4.569, Tukey’s test; respectively); again, not seen in imipramine animals

5

(p = ns, q = 0.9414, Tukey’s test) vs. control. The total number of attacks was significantly greater in the resilient and anhedonic cohorts (p < 0.001, q = 7.084 and p < 0.01, q = 5.883, Tukey’s test; respectively). 3.2. Differential hippocampal expression of genes encoding peroxidation enzymes in resilient versus animals susceptible to anhedonia in a 14-day stress study There was a trend to decreased SOD-1 gene expression for anhedonic (103.16% from control) versus resilient (113.45% from control) mice as shown by Illumina microarray analysis (p = 0.08, t = 1.351 df = 6; unpaired t-test; data not shown). Illumina microarray has demonstrated a significantly lower SOD-1 and GPX-1 gene expression in anhedonic mice (94.61% and 99.45% from control, respectively) over this measure in resilient animals (132.94% and 124.64% from control, respectively; p = 0.037, t = 1.755, df = 18 and p = 0.047, t = 1.799; t-test; data not shown). 3.3. Peroxidation activity enzymes are decreased during 14-day stress-induced anhedonia and increased after acute stress: preventive effects of imipramine Activity of antioxidative enzymes was significantly affected by the 14 days stress procedure. Superoxide dismutase and catalase were all significantly lowered (p = 0.0001, F = 7.201, R2 = 0.3479; p = 0.0003, F = 6.377, R2 = 0.3291; ANOVA, respectively, Fig. 3A) following the stress procedure whereas basal MDA levels (p = 0.9255, F = 0.2211, R2 = 0.01612) and glutathione peroxidase activity (p = 0.8588, F = 0.3268, R2 = 0.02364, ANOVA) were not altered. Post test showed that only the non-treated and anhedonic cohorts showed a change in enzyme activity. For SOD, stress and anhedonic animals had significantly lower activity (p < 0.05, q = 2.805, p < 0.01, q = 3.959, respectively, Dunnett’s test). The levels of CAT were also lower for stress animals (p < 0.01, q = 3.154, Dunnett’s test) as well as for anhedonic (p < 0.001, q = 4.451, Dunnett’s test). Conversely, acute stress resulted in an enhancement of enzyme activity for CAT and AcMDA (p = 0.0001, F = 14.52, R2 = 0.5803, and p < 0.0001, F = 16.61, R2 = 0.6127, respectively, ANOVA, Fig. 3B) but not for activity of SOD (p = 0.1980, F = 1.745, R2 = 0.1369). Under all conditions, imipramine was able to prevent any stress induced changes to antioxidative enzyme activity. 3.4. 28-Day stress results in decreased sucrose preference and lasting behavioural invigoration in a subgroup of mice Animals underwent a 28 days stress paradigm to determine the residual effects of chronic stress at 2 weeks and 5 weeks following the stress procedure. Control animals never demonstrated a sucrose preference below that of 65%. ANOVA revealed significantly lower preferences for sucrose immediately after (p < 0.0001, F = 52.12, R2 = 0.7380, Fig. 4A), two weeks after (p = 0.0004, F = 9.509, R2 = 9.509), but not 5 weeks after (p = 0.2595, F = 1.478, R2 = 0.1646) the stress protocol. Mice that showed sucrose preference below 65% were assigned to the anhedonic group; they revealed this deficit as compared to control two weeks after the stress period (immediately: p < 0.001, q = 9.853, and at 2 weeks: p < 0.001, q = 3.982, Dunnett’s test) which abated at 5 weeks (p = ns, q = 1.718, Dunnett’s test) following stress. 5 weeks after stress, animals also displayed a significant alteration in exploratory behaviour (p = 0.0017, F = 10.11, R2 = 0.5741, ANOVA, Fig. 4B) as well as locomotory (p = 0.0021, F = 10.70, R2 = 0.6408, ANOVA, Fig. 4C); again, this response was only observed in the anhedonic group (p < 0.01, q = 3.431, p < 0.01, q = 4.312, respectively, Dunnett’s test). The stress procedure also altered the latency to attack (p = 0.0083, F = 7.348,

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12 6

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

Fig. 2. 14-Day chronic stress induces physiological and behavioural changes in a subgroup of anhedonic mice, which are not evident in mice resilient to stress. (A) Animals did not show differences to sucrose preference at baseline; however, following 14 days stress, the untreated animals showed a decrease in sucrose preference and a cohort were determined to be anhedonic (preference <65%). Control and imipramine treated groups did not show a decrease in sucrose preference (p = 0.0011 ANOVA, * vs control, # vs S 2w imi, Tukey’s). (B) Resilient and anhedonic animals showed a significant increase in the number of rearings in novel cage test which was reversed with imipramine treatment (p < 0.0001, ANOVA, * vs con and S imi, Tukey’s). (C) Increased locomotion was observed in resilient and anhedonic animals determined by total distance travelled in open-field, this was not seen in the treated animals (p = 0.0008 ANOVA, * vs con and # vs S imi, Tukey’s). (D and E) Aggressive behaviour was ameliorated in imipramine treated animals but significantly altered in resilient and anhedonic mice as seen in a decreased latency to attack (p = 0.0002 ANOVA, * vs con, # vs S imi, Tukey’s) and an increase in the number of attacks (p < 0.0001 ANOVA, * vs con and # vs S imi, Tukey’s; *p < 0.05, **p < 0.01, ***p < 0.001, Tukey’s test).

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

7

Fig. 3. Effects of 14-days and acute stress on lipid peroxidation in prefrontal cortex. (A) Superoxide dismutase and catalase were down regulated in anhedonic animals subjected to 14-day stress (p = 0.0001 and p = 0.0003, p < 0.0001 respectively, ANOVA); effects of stress were prevented by imipramine treatment. There was no change for glutathione peroxidase activity or malondialdehyde (p = 0.9255 and p = 0.8588 respectively, ANOVA). (B) Acute stress up regulated catalase activity and accumulation of malondialdehyde but not activity of superoxide dismutase in stress animals which was prevented by imipramine treatment (p = 0.0001, p < 0.0001, and p = 0.1980 respectively, ANOVA) (*p < 0.05, **p < 0.01, ***p < 0.001, Dunnett’s test).

R2 = 0.5505, ANOVA, Fig. 4D) as well as the number of attacks (p = 0.0001, F = 29.51, R2 = 0.8677, ANOVA, Fig. 4E) up to 5 weeks after the last stressor. Dunnett’s post-test revealed that this effect was seen in both resilient (p < 0.05, q = 3.304 and p < 0.001, q = 7.231, latency to attack and number of attacks respectively) and anhedonic animals (p < 0.05, q = 3.336 and p < 0.001, q = 5.320, latency to attack and number of attacks respectively). No changes in baseline of stepping down, a parameter of anxiety-like behaviour [2], was found between the groups in step-down anxiety (p > 0.5, ANOVA, data not shown). 3.5. Lasting decrease in peroxidation enzymes activity in susceptible but not resilient to 28-day stress-induced anhedonia As seen in the 14 days stress procedure, oxidative enzymes were also altered following 28 days stress. Two weeks after the last

stressor, SOD (p = 0.0052, F = 8.402, R2 = 0.5834, ANOVA, Fig. 5A) and CAT (p = 0.0309, F = 4.712, R2 = 0.4399, ANOVA) were significantly decreased and only in the anhedonic cohorts (p < 0.01, q = 3.765, p < 0.05, q = 3.765, respectively, Dunnett’s test). Five weeks after the last stressor, only the level of CAT remained significantly altered in the anhedonic group (p = 0.0199, F = 5.057, R2 = 0.3873, ANOVA, Fig. 5B). 4. Discussion Here we show that exposure to either 14- or 28-day chronic stress resulted in a depressive-like syndrome, behavioural invigoration and aggression, and decreased activity of two major brain peroxidation enzymes, superoxide dismutase and catalase. Conversely, acute stress elicited the opposite effect on these enzymes; irrespectively to stress, these changes were precluded by

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12 8

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

Fig. 4. Lasting effects of 28-day stress on hedonic state and other behaviours in a subgroup of mice susceptible to stress. (A) Mice underwent a 28 day chronic stress paradigm and were then subjected to a sucrose preference test and separated into resilient and anhedonic groups (sucrose preference >65% and <65% respectively). Sucrose preference was re-tested at 2 weeks and 5 weeks following the last stress procedure. Only the anhedonic cohort showed a significantly decreased sucrose preference following the stress procedure (p < 0.0001, ANOVA) which was still present 2 weeks thereafter (p = 0.0004, ANOVA) but finally abated at 5 weeks after (p = 0.2595, ANOVA). (B) Although anhedonia abated 5 weeks following the stress procedure, only the anhedonic cohort showed disturbances in locomotory behaviour as evidenced in the number of rearings in novel cage (p = 0.0017, ANOVA) (C) as well as total desistance travelled in opened field (p = 0.0021, ANOVA). (D) Both the resilient and anhedonic groups demonstrated changes in aggressive behaviour as compared to controls in the latency to attack (p = 0.0083, ANOVA) (E) as well as the number of attacks (p = 0.0001, ANOVA) at 5 weeks following the stress procedure (*p < 0.05, **p < 0.01, ***p < 0.001, Dunnett’s test).

imipramine. The effects of chronic stress were specific with regard to the occurrence of a depressive-like state which was observed only in a cohort of mice that demonstrated a drop in sucrose preference below 65%, a sign of hedonic deficit, but not in animals resilient to anhedonia. As such, the differential changes in peroxidation parameters after acute versus chronic stress make it highly unlikely that the observed differences were evoked by the last stress session. Altered peroxidation lasted five weeks after discontinuation of the 28-day stress and was accompanied with behavioural

invigoration and aggressive behaviour despite that the normal hedonic state was restored at this time point and no changes in anxiety-like behaviour were found. Previous studies utilizing rodent chronic stress models revealed similar results, while the specificity of its effects with respect to the occurrence of depressive-like state was not reported in the available literature. Mice subjected to several weeks of stress had lowered activities of SOD and CAT, enhanced accumulation of MDA and unchanged [45] or increased [18,46] activity of GPX. Rats

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

9

Fig. 5. Peroxidation enzymes at 2 and 5 weeks after 28-day stress. Following the 28-day stress procedure, a subgroup of each cohort was sacrificed at 2 weeks and 5 weeks after the last stressor and parameters of peroxidation were measured. (A) Only the anhedonic group showed a significant reduction in enzyme activity for SOD and CAT (p = 0.0052 and p = 0.0309, respectively, ANOVA) while there were no changes to GPX and MDA at 2 weeks. (B) 5 weeks after the procedure, only the levels of CAT were still reduced and only in the anhedonic cohort (p = 0.0199, ANOVA), there were no other differences (*p < 0.05, **p < 0.01, ***p < 0.001, Dunnett’s test).

exposed to several weeks of stress revealed decreased sucrose preference and diminished SOD activity in the cortex, hippocampus and cerebellum [47]; other study showed decreased GPX activity in the cortex [17]. Additionally, a 14-day unpredictable chronic stress in mice was found to decrease the activity of CAT in the cerebral cortex and hippocampus, GPX in the hippocampus, and increased the dynamics of lipid peroxidation in both structures [19]. As for acute stress effects, in several studies it was shown that single stressors, e.g., restraint stress, induced activity of SOD and GPX in the hippocampus and accelerated lipid peroxidation in mice [48–50]. It is

believed that the activation of peroxidation enzymes is implicated in the adaptive response of tissues to stress, while a decrease in these functions below what is considered normal levels may reflect an insufficient compensatory response of anti-oxidant functions during a state of distress [51]. Importantly, we have shown that the effects of acute and chronic stress exposures on brain peroxidation were precluded by imipramine administration. Chronic administration of imipramine completely ameliorated the changes observed in peroxidation enzymes as well as behavioural abnormalities induced by the 14

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12 10

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

days stress procedure. Animals treated with imipramine also did not show any susceptibility to depression as measured in the sucrose preference test (preference >65%). In agreement with the chronic stress procedure and treatment, acute imipramine administration followed by an acute stressor, also prevented the effects of stress on markers for lipid peroxidation. Changes in peroxidative markers in the 28 days stress protocol were again only observed in the anhedonic cohort indicating that susceptible individuals to depression have a disruption in the oxidative/antioxidative system. Similar results with antidepressant treatment were reported earlier. For example, a bolus administration of the antidepressant fluoxetine (5 mg/kg), 30 min prior to a single restraint stress in mice, prevented a hyperactivation of lipid peroxidation enzymes [48]; studies with a comparable design confirmed these findings [49,50]. Studies with chronic stressed induced depressive syndrome in mice and rats have demonstrated that various antidepressants reversed changes observed in lipid peroxidation, e.g., for imipramine [46,52,53], desipramine or citalopram [54], fluoxetine [19,46], clomipramine [45], venlafaxin [17,46] and lamotrigine [53]. Particularly, these studies showed that antidepressants rescue stressed-induced reduction of SOD, CAT and GPX and normalize levels of malondialdehyde in several brain structures, including prefrontal cortex [46,53]. These data from animal studies agree with reported changes in lipid peroxidation and pro-inflammatory cytokines measured in patients with major depression [5,10,55–58]. Notably, our experiments have revealed the opposite effects of imipramine on the lipid peroxidation in chronically versus acutely stressed mice, suggesting that they could not be fully attributed to general anti-oxidant effects of imipramine. Moreover, since the current literature suggests activating effects of imipramine and other antidepressants on the main peroxidation enzymes of the brain in naive mice under certain dosing conditions [59,60], we have chosen to apply imipramine doses in our study that are unlikely to evoke such effects. Specifically, the dose applied here, 7 mg/kg/day for chronic treatment, is even lower than the dose range (10–20 mg/kg/kg) at which this drug still had no effect on activities of CAT and SOD in the prefrontal cortex of chronically dosed naive mice [59]. Interestingly, imipramine, chronically delivered to mice at the dose of 7 mg/kg/day, unlikely to its higher dose (15 mg/kg/day), does not induce noticeable behavioural abnormalities in mice [29,30]. As for the bolus imipramine administered dose, consisting of 30 mg/kg/day, it was reported to increase not decrease the CAT activity in the prefrontal cortex of naive mice (while SOD activity was unaltered), suggesting that the mechanisms of these effects of imipramine in naive and stressed animals were likely to be distinct. This normalization of, altered by stress, activity of brain peroxidation enzymes was paralleled by a normalization of behavioural read-outs of hedonic sensitivity, impulsivity and aggression in chronically stressed mice (Fig. 2, [29]) and of consummator and locomotor behaviour in acutely stressed mice (Strekalova, unpublished results) suggesting a link between ameliorative effects of imipramine on neurochemical and behavioural levels. Together, the positive effects of chronic and acute administration of imipramine on peroxidative markers in our study could be attributed to possible antioxidative properties of imipramine. In addition to changes in the activity of lipid peroxidation enzymes, which were revealed in the prefrontal cortex, microarray assay pointed to similar changes in the hippocampal formation, another brain structure implicated in the stress response [61,62]. The expression of genes encoding SOD and GPX is significantly higher in resilient versus anhedonic animals; however, anhedonic mice showed no significant decreases in gene expression of peroxidation enzymes. Given that most studies report similar changes in lipid peroxidation during stress and

depressive-like state in the hippocampus and prefrontal cortex [19,63,64], these data can be interpreted as indirect support of the above-described findings with the enzymatic activity of peroxidation enzymes. This interpretation is further supported by gene expression data that point to inhibition of the mitochondrial complex I and II–III in the hippocampus of mice [27] or rats [65] exposed to paradigms of stress-induced depressive syndrome. Increased production of reactive oxygen species is known to evoke metabolic deficiency and contribute to the pathogenesis of depressive syndrome [20,43,44,66]. As such, the up regulation of antioxidative enzymes in resilient individuals might provide better coping when challenged with stress induced oxidative stress and perhaps partially explains their stress resilience. The detected changes in lipid peroxidation described here parallel a previously reported aberrant expression of pro-inflammatory factors in the prefrontal cortex and hippocampus using a similar model of stress-induced anhedonia [28]. We showed that chronic stress up-regulated gene expression of TNF-␣ in the prefrontal cortex was accompanied with microglial activation, in susceptible, but not resilient mice. Up-regulation of IL-6 and COX-1 in anhedonic animals was also shown in the hippocampal formation [28]. This association further supports a functional relationship between changes in lipid peroxidation, activation of arachidonic acid metabolism and cytokine production, whose importance in the pathogenesis of depression is ever increasingly recognized [66–69]. At the moment, there is no agreement to changes seen in peroxidation enzymes in relation to remission [9,12,39]. In this connection, following 28 days of stress, we looked for residual effects of stress on the levels of peroxidative markers at two weeks and 5 weeks following the last stressor. At two weeks post stress, the anhedonic cohort continued to show a hedonic deficit. Interestingly, 5 weeks post stress, the hedonic deficit abated, yet the anhedonic cohort still showed increased invigoration with enhanced aggressive traits, as well as significantly reduced levels in SOD and CAT indicating that although the key indicator for depression, anhedonia, was in remission, this group of susceptible animals continued to display abnormal behaviour as well as reduction in peroxidative enzymes (Figs. 4 and 5). This observation is particularly intriguing as clinically depressed patients in remission commonly continue to display abnormal behaviour [40,58]. Also noteworthy, patients in remittance for major depressive disorder following treatment with fluoxetine continue to show disturbances in the balance between pro- and antioxidative processes [41]. To summarize, our data supports previous studies implicating brain oxidative processes as part of the mechanisms for depressive syndrome and its therapy. Lasting reduction in the activity of peroxidative enzymes in susceptible individuals may underlie behavioural abnormalities during recovery from anhedonia causing increased vulnerability for relapse to a depressive-like state which may be of clinical relevance. A detailed characterization of these factors and related downstream mechanisms could open new possibilities for pharmacological management of depressed patients during remission and prevent a relapse of the disease. On a general note, our findings further suggest the spectrum nature of neuropsychiatric syndromes and pathogenetic linkage between various disordered domains, such as hedonic deficit, impulsivity, over-expression of inflammatory factors and mitochondrial dysfunction and others that emphasizes the importance of integrated cross-domain concept as a good empowering approach for translational biological psychiatry [70,71].

Acknowledgements We would like to acknowledge the important contribution of Drs. Dolores Bonaparte and Dinora Fereira, as well as the

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

technical help of Mr. João Costa-Nunes and Mrs. Julie Vignisse. This study was supported by the European Community (EC: AGGRESSOTYPE FP7/No. 602805), NARSAD Brain and Behaviour Research Foundation, USA (17611 to T.S.) and RFBR 11-04-01411. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2014.04.037. References [1] Floyd RA. Antioxidants, oxidative stress, and degenerative neurological disorders. Proc Soc Exp Biol Med 1999;222:236–45. [2] Strekalova T, Steinbusch HW. Measuring behavior in mice with chronic stress depression paradigm. Prog Neuropsychopharmacol Biol Psychiatry 2010;34:348–61. [3] Ott M, Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria, oxidative stress and cell death. Apoptosis 2007;12:913–22. [4] Richter C, Kass GE. Oxidative stress in mitochondria: its relationship to cellular Ca2+ homeostasis, cell death, proliferation, and differentiation. Chem Biol Interact 1991;77:1–23. [5] Stork C, Renshaw PF. Mitochondrial dysfunction in bipolar disorder: evidence from magnetic resonance spectroscopy research. Mol Psychiatry 2005;10:900–19. [6] Rezin GT, Amboni G, Zugno AI, Quevedo J, Streck EL. Mitochondrial dysfunction and psychiatric disorders. Neurochem Res 2009;34:1021–9. [7] Hroudova J, Fisar Z. Activities of respiratory chain complexes and citrate synthase influenced by pharmacologically different antidepressants and mood stabilizers. Neuro Endocrinol Lett 2010;31:336–42. [8] Bilici M, Efe H, Koroglu MA, Uydu HA, Bekaroglu M, Deger O. Antioxidative enzyme activities and lipid peroxidation in major depression: alterations by antidepressant treatments. J Affect Disord 2001;64:43–51. [9] Ozcan ME, Gulec M, Ozerol E, Polat R, Akyol O. Antioxidant enzyme activities and oxidative stress in affective disorders. Int Clin Psychopharmacol 2004;19:89–95. [10] Sarandol A, Sarandol E, Eker SS, Erdinc S, Vatansever E, Kirli S. Major depressive disorder is accompanied with oxidative stress: short-term antidepressant treatment does not alter oxidative–antioxidative systems. Hum Psychopharmacol 2007;22:67–73. [11] Machado-Vieira R, Andreazza AC, Viale CI, Zanatto V, Cereser Jr V, da Silva Vargas R, et al. Oxidative stress parameters in unmedicated and treated bipolar subjects during initial manic episode: a possible role for lithium antioxidant effects. Neurosci Lett 2007;421:33–6. [12] Frey BN, Valvassori SS, Reus GZ, Martins MR, Petronilho FC, Bardini K, et al. Effects of lithium and valproate on amphetamine-induced oxidative stress generation in an animal model of mania. J Psychiatry Neurosci 2006;31:326–32. [13] Stefanescu C, Ciobica A. The relevance of oxidative stress status in first episode and recurrent depression. J Affect Disord 2012;143:34–8. [14] Vargas HO, Nunes SO, Pizzo de Castro M, Cristina Bortolasci C, Sabbatini Barbosa D, Kaminami Morimoto H, et al. Oxidative stress and lowered total antioxidant status are associated with a history of suicide attempts. J Affect Disord 2013;150:923–30. [15] Fekadu A, Rane LJ, Wooderson SC, Markopoulou K, Poon L, Cleare AJ. Prediction of longer-term outcome of treatment-resistant depression in tertiary care. Br J Psychiatry 2012;201:369–75. [16] Christensen T, Bisgaard CF, Wiborg O. Biomarkers of anhedonic-like behavior, antidepressant drug refraction, and stress resilience in a rat model of depression. Neuroscience 2011;196:66–79. [17] Eren I, Naziroglu M, Demirdas A. Protective effects of lamotrigine, aripiprazole and escitalopram on depression-induced oxidative stress in rat brain. Neurochem Res 2007;32:1188–95. [18] Kurhe Y, Radhakrishnan M, Gupta D, Devadoss T. QCM-4 a novel 5-HT3 antagonist attenuates the behavioral and biochemical alterations on chronic unpredictable mild stress model of depression in Swiss albino mice. J Pharm Pharmacol 2014;66:122–32. [19] Moretti M, Colla A, de Oliveira Balen G, dos Santos DB, Budni J, de Freitas AE, et al. Ascorbic acid treatment, similarly to fluoxetine, reverses depressive-like behavior and brain oxidative damage induced by chronic unpredictable stress. J Psychiatr Res 2012;46:331–40. [20] Seo JS, Park JY, Choi J, Kim TK, Shin JH, Lee JK, et al. NADPH oxidase mediates depressive behavior induced by chronic stress in mice. J Neurosci 2012;32:9690–9. [21] Hamilton M. Development of a rating scale for primary depressive illness. Br J Soc Clin Psychol 1967;6:278–96. [22] Katz RJ, Roth KA, Carroll BJ. Acute and chronic stress effects on open field activity in the rat: implications for a model of depression. Neurosci Biobehav Rev 1981;5:247–51. [23] Willner P, Towell A, Sampson D, Sophokleous S, Muscat R. Reduction of sucrose preference by chronic unpredictable mild stress, and its restoration by a tricyclic antidepressant. Psychopharmacology (Berl) 1987;93:358–64.

11

[24] Strekalova T, Spanagel R, Bartsch D, Henn FA, Gass P. Stress-induced anhedonia in mice is associated with deficits in forced swimming and exploration. Neuropsychopharmacology 2004;29:2007–17. [25] Piazza PV, Maccari S, Deminiere JM, Le Moal M, Mormede P, Simon H. Corticosterone levels determine individual vulnerability to amphetamine selfadministration. Proc Natl Acad Sci USA 1991;88:2088–92. [26] Krishnan V, Han MH, Graham DL, Berton O, Renthal W, Russo SJ, et al. Molecular adaptations underlying susceptibility and resistance to social defeat in brain reward regions. Cell 2007;131:391–404. [27] Strekalova T, Couch Y, Kholod N, Boyks M, Malin D, Leprince P, et al. Update in the methodology of the chronic stress paradigm: internal control matters. Behav Brain Funct 2011;7:9. [28] Couch Y, Anthony DC, Dolgov O, Revischin A, Festoff B, Santos AI, et al. Microglial activation, increased TNF and SERT expression in the prefrontal cortex define stress-altered behaviour in mice susceptible to anhedonia. Brain Behav Immun 2013;29:136–46. [29] Cline BH, Steinbusch HW, Malin D, Revishchin AV, Pavlova GV, Cespuglio R, et al. The neuronal insulin sensitizer dicholine succinate reduces stress-induced depressive traits and memory deficit: possible role of insulin-like growth factor 2. BMC Neurosci 2012;13:110. [30] Strekalova T, Anthony DC, Dolgov O, Anokhin K, Kubatiev A, Steinbusch HM, et al. The differential effects of chronic imipramine or citalopram administration on physiological and behavioral outcomes in naive mice. Behav Brain Res 2013;245:101–6. [31] Costa-Nunes J, Zubareva O, Araujo-Correia M, Valenca A, Schroeter CA, Pawluski JL, et al. Altered emotionality, hippocampus-dependent performance and expression of NMDA receptor subunit mRNAs in chronically stressed mice. Stress 2014;17:108–16. [32] Strekalova T, Spanagel R, Dolgov O, Bartsch D. Stress-induced hyperlocomotion as a confounding factor in anxiety and depression models in mice. Behav Pharmacol 2005;16:171–80. [33] Markova N, Chernopiatko A, Schroeter CA, Malin D, Kubatiev A, Bachurin S, et al. Hippocampal gene expression of deiodinases 2 and 3 and effects of 3,5-diiodo-l-thyronine T2 in mouse depression paradigms. BioMed Res Int 2013;2013:565218. [34] Bailey JL. Techniques in protein chemistry. Amsterdam, New York: Elsevier Pub. Co.; 1962. [35] Aebi H. Catalase in vitro. Methods Enzymol 1984;105:121–6. [36] Cohen G, Dembiec D, Marcus J. Measurement of catalase activity in tissue extracts. Anal Biochem 1970;34:30–8. [37] Claiborne A. CRC handbook of methods for oxygen radical research. Boca Raton, Florida, USA: CRC Press; 1985. [38] Beauchamp C, Fridovich I. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 1971;44:276–87. [39] Greenwald RA. CRC handbook of methods for oxygen radical research. Boca Raton, Florida, USA: CRC Press; 1985. [40] Sies H. Oxidative stress: oxidants and antioxidants. London, UK: Academic Press; 1991. [41] Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70: 158–69. [42] Tappel ME, Chaudiere J, Tappel AL. Glutathione peroxidase activities of animal tissues. Comp Biochem Physiol B 1982;73:945–9. [43] Beutler E, Blume KG, Kaplan JC, Lohr GW, Ramot B, Valentine WN. International Committee for Standardization in Haematology: recommended methods for red-cell enzyme analysis. Br J Haematol 1977;35:331–40. [44] Ohkawa H, Ohishi N, Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal Biochem 1979;95:351–8. [45] Zhang D, Wen XS, Wang XY, Shi M, Zhao Y. Antidepressant effect of Shudihuang on mice exposed to unpredictable chronic mild stress. J Ethnopharmacol 2009;123:55–60. [46] Zafir A, Ara A, Banu N. Invivo antioxidant status: a putative target of antidepressant action. Prog Neuropsychopharmacol Biol Psychiatry 2009;33:220–8. [47] Wang C, Wu HM, Jing XR, Meng Q, Liu B, Zhang H, et al. Oxidative parameters in the rat brain of chronic mild stress model for depression: relation to anhedonialike responses. J Membr Biol 2012;245:675–81. [48] Freitas AE, Bettio LE, Neis VB, Santos DB, Ribeiro CM, Rosa PB, et al. Agmatine abolishes restraint stress-induced depressive-like behavior and hippocampal antioxidant imbalance in mice. Prog Neuropsychopharmacol Biol Psychiatry 2014;50:143–50. [49] Novio S, Nunez MJ, Amigo G, Freire-Garabal M. Effects of fluoxetine on the oxidative status of peripheral blood leucocytes of restraint-stressed mice. Basic Clin Pharmacol Toxicol 2011;109:365–71. [50] Budni J, Zomkowski AD, Engel D, Santos DB, dos Santos AA, Moretti M, et al. Folic acid prevents depressive-like behavior and hippocampal antioxidant imbalance induced by restraint stress in mice. Exp Neurol 2013;240:112–21. [51] Popa-Wagner A, Mitran S, Sivanesan S, Chang E, Buga AM. ROS and brain diseases: the good, the bad, and the ugly. Oxid Med Cell Longev 2013;2013:963520. [52] Mello BS, Monte AS, McIntyre RS, Soczynska JK, Custodio CS, Cordeiro RC, et al. Effects of doxycycline on depressive-like behavior in mice after lipopolysaccharide (LPS) administration. J Psychiatr Res 2013;47:1521–9. [53] Abelaira HM, Reus GZ, Ribeiro KF, Steckert AV, Mina F, Rosa DV, et al. Effects of lamotrigine on behavior, oxidative parameters and signaling cascades in rats exposed to the chronic mild stress model. Neurosci Res 2013;75: 324–30.

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037

G Model BBR-8872; No. of Pages 12 12

ARTICLE IN PRESS B.H. Cline et al. / Behavioural Brain Research xxx (2014) xxx–xxx

[54] Garg R, Kumar A. Possible role of citalopram and desipramine against sleep deprivation-induced anxiety like-behavior alterations and oxidative damage in mice. Indian J Exp Biol 2008;46:770–6. [55] Alcocer-Gomez E, de Miguel M, Casas-Barquero N, Nunez-Vasco J, SanchezAlcazar JA, Fernandez-Rodriguez A, et al. NLRP3 inflammasome is activated in mononuclear blood cells from patients with major depressive disorder. Brain Behav Immun 2014;36:111–7. [56] Rawdin BJ, Mellon SH, Dhabhar FS, Epel ES, Puterman E, Su Y, et al. Dysregulated relationship of inflammation and oxidative stress in major depression. Brain Behav Immun 2013;31:143–52. [57] Kim HK, Andreazza AC, Yeung PY, Isaacs-Trepanier C, Young LT. Oxidation and nitration in dopaminergic areas of the prefrontal cortex from patients with bipolar disorder and schizophrenia. J Psychiatry Neurosci 2014;39: 130155. [58] Steckert AV, Valvassori SS, Moretti M, Dal-Pizzol F, Quevedo J. Role of oxidative stress in the pathophysiology of bipolar disorder. Neurochem Res 2010;35:1295–301. [59] Reus GZ, Stringari RB, de Souza B, Petronilho F, Dal-Pizzol F, Hallak JE, et al. Harmine and imipramine promote antioxidant activities in prefrontal cortex and hippocampus. Oxid Med Cell Longev 2010;3:325–31. [60] Abdel-Salam OM, Salem NA, Hussein JS. Effect of aspartame on oxidative stress and monoamine neurotransmitter levels in lipopolysaccharide-treated mice. Neurotox Res 2012;21:245–55. [61] Anisman H, Du L, Palkovits M, Faludi G, Kovacs GG, Szontagh-Kishazi P, et al. Serotonin receptor subtype and p11 mRNA expression in stress-relevant brain regions of suicide and control subjects. J Psychiatry Neurosci 2008;33:131–41. [62] Stanley M, Mann JJ. Increased serotonin-2 binding sites in frontal cortex of suicide victims. Lancet 1983;1:214–6. [63] de Morais H, de Souza CP, da Silva LM, Ferreira DM, Werner MF, Andreatini R, et al. Increased oxidative stress in prefrontal cortex and hippocampus is

[64]

[65]

[66]

[67] [68]

[69]

[70] [71]

related to depressive-like behavior in streptozotocin-diabetic rats. Behav Brain Res 2014;258:52–64. Liu Y, Jia G, Gou L, Sun L, Fu X, Lan N, et al. Antidepressant-like effects of tea polyphenols on mouse model of chronic unpredictable mild stress. Pharmacol Biochem Behav 2013;104:27–32. Della FP, Abelaira HM, Reus GZ, Santos MA, Tomaz DB, Antunes AR, et al. Treatment with tianeptine induces antidepressive-like effects and alters the neurotrophin levels, mitochondrial respiratory chain and cycle Krebs enzymes in the brain of maternally deprived adult rats. Metab Brain Dis 2013;28: 93–105. Maes M, Galecki P, Chang YS, Berk M. A review on the oxidative and nitrosative stress (O&NS) pathways in major depression and their possible contribution to the (neuro)degenerative processes in that illness. Prog Neuropsychopharmacol Biol Psychiatry 2011;35:676–92. Dantzer R. Cytokine, sickness behavior, and depression. Immunol Allergy Clin N Am 2009;29:247–64. Campbell SJ, Perry VH, Pitossi FJ, Butchart AG, Chertoff M, Waters S, et al. Central nervous system injury triggers hepatic CC and CXC chemokine expression that is associated with leukocyte mobilization and recruitment to both the central nervous system and the liver. Am J Pathol 2005;166:1487–97. Zoppi S, Madrigal JL, Caso JR, Garcia-Gutierrez MS, Manzanares J, Leza JC, et al. Regulatory role of the cannabinoid-2 receptor in stress-induced neuroinflammation in mice. Br J Pharmacol 2014, http://dx.doi.org/10.1111/bph.12607 [Epub ahead of print]. Kalueff AV, Stewart AM. Modeling neuropsychiatric spectra to empower translational biological psychiatry. Behav Brain Res 2014. Cross-Disorder Group of the Psychiatric Genomics Consortium; Genetic Risk Outcome of Psychosis (GROUP) Consortium. Identification of risk loci with shared effects on five major psychiatric disorders: a genome-wide analysis. Lancet 2013;381:1371–9.

Please cite this article in press as: Cline BH, et al. Lasting downregulation of the lipid peroxidation enzymes in the prefrontal cortex of mice susceptible to stress-induced anhedonia. Behav Brain Res (2014), http://dx.doi.org/10.1016/j.bbr.2014.04.037