Chronic mild stress damages mitochondrial ultrastructure and function in mouse brain

Neuroscience Letters 488 (2011) 76–80

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Chronic mild stress damages mitochondrial ultrastructure and function in mouse brain Yu Gong, Yi Chai, Jian-Hua Ding, Xiu-Lan Sun ∗ , Gang Hu Jiangsu Key Laboratory of Neurodegeneration, Department of Pharmacology, Nanjing Medical University, 140 Hanzhong Road, Nanjing, Jiangsu 210029, PR China

a r t i c l e

i n f o

Article history: Received 14 September 2010 Received in revised form 22 October 2010 Accepted 2 November 2010 Keywords: Chronic mild stress Mitochondria Depressive disorder

a b s t r a c t Increasing evidence implicates mitochondrial failure as a crucial factor in the pathogenesis of mental disorders, such as depression. The aim of the present study was to investigate the effects of exposure to chronic mild stress (CMS), a paradigm developed in the late 1980s as an animal model of depression, on the mitochondrial function and mitochondrial ultrastructure in the mouse brain. The results showed that the CMS regime induced depressive-like symptoms in mice characterized by reduced sucrose preference and body weight. Moreover, CMS exposure was associated with a significant increase in immobility time in the tail suspension test. Exposure to the CMS paradigm inhibited mitochondrial respiration rates and dissipated mitochondrial membrane potential in hippocampus, cortex and hypothalamus of mice. In addition, we found a damaged mitochondrial ultrastructure in brains of mice exposed to CMS. These findings provide evidence for brain mitochondrial dysfunction and ultrastructural damage in a mouse model of depression. Moreover, these findings suggest that mitochondrial malfunction-induced oxidative injury could play a role in stress-related disorders such as depression. © 2010 Elsevier Ireland Ltd. All rights reserved.

Major depression is a serious and recurrent disorder manifested with symptoms at the psychological, behavioral and physiological levels. Depression affects 17–20% of the population of the world and may result in premature death, major social and economic consequences [10,24]. Among persons with major depression, 75–85% has recurrent episodes and 10–30% recovery incompletely and has persistent, residual depressive symptoms [14,30]. Increasing evidence implicates mitochondrial failure as a crucial factor in the pathogenesis of depressive disorder [26]. Mitochondria are the main site of energy production in eukaryotic cells. However, mitochondria are also a major source of reactive oxygen species (ROS) [25]. Under physiological conditions, ROS from mitochondrial respiratory chain could be reduced by intracellular antioxidant enzymes including superoxide dismutase, glutathione peroxidase and catalase as well as some antioxidant molecules such as glutathione and vitamin E [22]. Low levels of ROS are required for normal cell functions, i.e., cell signaling [15]. However, under pathological conditions, because of the elevated production, these antioxidants may fail to eliminate the large amount

Abbreviations: ROS, reactive oxygen species; CMS, chronic mild stress; TST, tail suspension test; ST3, state 3 respiration; ST4, state 4 respiration; RCR, respiratory control ratio. ∗ Corresponding author. Tel.: +86 25 86863169; fax: +86 25 86863108. E-mail address: [email protected] (X.-L. Sun). 0304-3940/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2010.11.006

of ROS generated, leading to oxidative stress and cellular damage [16,32]. Thus, mitochondria are not only an important producer of ROS, but also a sensitive target for ROS [25]. Excessive generation of ROS could result in mitochondria dysfunction and activate mitochondria-dependent apoptotic pathway [7]. The brain is particularly vulnerable to reactive ROS production because it metabolizes 20% of total body oxygen and has a limited amount of antioxidant capacity [23]. Oxidative stress is well known to contribute to neuronal degeneration in brain during the aging process as well as in neurodegenerative diseases such as amyotrophic lateral sclerosis, Alzheimer’s dementia and Parkinson’s disease [23,31,32]. Recently, several studies have reported that ROS increased in the plasma of patients with major depression [19,26], suggesting oxidative stress-induced injury, especially mitochondrial dysfunction, is implicated in the pathogenesis of depression [26]. Numerous attempts have been made to set up animal models of depression or at least of some disease aspects. The chronic mild stress (CMS) model has been shown to induce lower consumption of sucrose (sweet food) postulated to reflect an hedonia (the loss of interest or pleasure) in animals, one of the two core symptoms required for diagnosis of a major depressive episode in humans [13,33]. The exposure of mice to CMS also induces changes in hypothalamic–pituitary–adrenal axis, body weight and adrenal glands all consistent with human depression [6,9]. Therefore, in the present study, we investigated the effects of CMS paradigm on mitochondrial function and mitochondrial ultrastructure in mouse

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brain to provide the direct evidence for mitochondrial dysfunction in depressive disorder. C57 male mice (8–12 weeks, 25–32 g) were bred and maintained 4–5 mice per cage with a 12 h light/dark cycle at ambient temperature (22 ◦ C) and relative humidity (55 ± 5%). All experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Guidelines for the Care and Use of Animals in Neuroscience Research by the Society for Neuroscience and approved by IACUC (Institutional Animal Care and Use Committee of Nanjing Medical University). CMS was achieved as described previously [6]. Briefly, individually housed mice (n = 16–20 for each groups) were allowed to acclimate for 1 week and then were subjected to 6 weeks of stressors, which were mild and unpredictable in nature, duration, and frequency. Stressors included inversion of day/night light cycle, 45◦ tilted cage, restraint, overnight food and water deprivation, and pairing with another stressed animal. Sucrose preference was weekly monitored consistently throughout the course of experiments. Before CMS procedure, animals were first trained to consume a sucrose solution. Mice were submitted to 3 days of continuous exposure to pipettes of water and of a 1% sucrose solution. After consumption stabilization, mice were divided in two groups, matched for their sucrose consumption baseline and their body weight baseline (n = 18). When perform sucrose preference test, mice were given the choice to drink from two bottles for 10 h; one contained a sucrose solution (1%) and the other contained only tap water. To prevent possible effects of side-preference in drinking behavior, the positions of the bottles in the cage were switched after 5 h. The animals were not deprived of food or water before the test. The consumption of tap water, sucrose solution and total intake of liquids was estimated simultaneously in the control and experimental groups by weighing the bottles. The mice whose sucrose consumption were high or low obviously than the others in the same groups were discarded in the later experiment. The preference for sucrose was measured as a percentage of the consumed sucrose solution relative to the total amount of liquid intake. The TST is one of the most widely used models for assessing antidepressant-like activity in mice [27]. In this experiment, mice were individually suspended by the distal portion of their tails with adhesive tape for a period of 6 min (30 cm from the floor) in a visually isolated area. The time of immobility of the tail-suspended mice during the last 4 min was measured with a stopwatch. Mitochondria were isolated by using a differential centrifugation method that retains mitochondrial structure and respiratory function [8]. Mitochondrial protein concentration was quantified according to Bradford using BSA as standard, and the mitochondria were diluted in isolation buffer to yield a fixed concentration according to the requirement of measurement. The mitochondrial membrane potential ( m ) was assessed with the fluorescent probe JC-1 (Molecular Probes). JC-1 is a cationic dye that exhibits potential-dependent accumulation in mitochondria, indicated by a fluorescence emission shift from green (∼525 nm) to red (∼590 nm). Mitochondria depolarization is indicated by a decrease in the red to green fluorescence intensity ratio. The green JC-1 signals were measured at Ex 485 nm (20 nm BW)/Em 535 nm (25 nm BW), the red signals at Ex 535 nm (25 nm BW)/Em 590 nm (20 nm BW). The ratio of red fluorescence signal (RFU at 590 nm) of JC-1 was recorded with a varioskan flash (Thermo scientific, USA). The initial protein concentration of isolated mitochondria for the staining procedure was 1 mg/ml. The detection was performed in 96 well plates. Respiratory activities of mitochondrial preparations were measured by determining oxygen consumption using a Clark Electrode provided by Hansatech (King’s Lynn, UK). The incubation medium was constantly stirred using an electromagnetic stirrer and bar

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flea. The oxygen consumption studies were conducted at 30 ◦ C in respiration medium consisting of 25 mM sucrose, 75 mM mannitol, 95 mM KCl, 5 mM KH2 PO4 , 20 mM Tris–HCl, 1 mM EGTA, pH 7.4. The concentrations of substrates used were glutamate (5 mM) and malate (5 mM), which were added to the respiration media before the mitochondria were added. Approximately 0.5 mg of mitochondrial protein was preincubated in the oxygen electrode in a total volume of 1 ml with substrates for 5 min. State 3 respiration (ST3) was induced by the addition of adenosine diphosphate (ADP) (1 mM). Approximately 3 min later, ST3 was terminated and state 4 respiration (ST4) (resting) was detected. And the respiratory control ratio (RCR) was calculated from the ratio of the ST3/ST4 oxygen consumption rates. After perfusion, cortex, hippocampus and hypothalamus of mice were microdissected from each mouse and were post-fixed in 2% Osmium tetraoxide at 0.1 M, pH 7.4 phosphate buffers at 48 ◦ C for 1 h, and stained with uranyl acetate during 2 h. Later the sections were flat-embedded in Durcupan. Semi-thin (1 ␮m) sections were first stained with CFV and screened. Ultrathin sections of three regions were cut and placed on single-hole grids. After staining with uranyl acetate and lead citrate, the sections were examined by EM (Zeiss EM-9S). All values are reported as means ± S.D. The significance of the difference between controls and samples treated with various drugs was determined by Student’s t-test. Differences were considered significant at P < 0.05. Stress-induced decrease in sucrose preference in rodents is regarded as an analog of anhedonia, a key symptom of depression. No significant difference of sucrose preference was found among unstressed mice. The CMS procedure significantly decreased the sucrose preference to 85 ± 7% compared to control mice (n = 18, P < 0.05) at the end of study (Fig. 1a). TST—the behavioral models of antidepressant activity was to assess depression-related behaviors of mice. In the behavioral tests, immobility is interpreted as a “behavioral despair”. The CMS procedure increased the immobility time in TST (137 ± 20 s), compared to control mice (114 ± 23 s, n = 18, P < 0.05) (Fig. 1b). The effects of CMS on body weight are shown in Fig. 1c. At week 6, average body weight of control mice and CMS-treated mice were 27.6 ± 1.7 g and 21.6 ± 1.5 g, respectively. The body weight of stressed mice was reduced 2.6 g (11%) after 6-week-stress (n = 18, P < 0.05). The control mice gained weight over time, body weight of control mice increased 2.3 g (8%) at week 6 (n = 18, P < 0.01). RCR is the major indices of mitochondrial function, which are determined based on measurements of oxygen utilization by mitochondria in vitro. Expose to CMS procedure reduced mitochondrial ST3 respiration rates in cortex, hippocampus and hypothalamus (n = 6, P < 0.05) (Fig. 2a) when the electron transport chain COX I substrates malate and glutamate were present, but failed to affect ST4 respiration rates (Fig. 2b). ST4 and ST3 respiration rates were used to calculate RCR. RCR of the mitochondria in the three brain regions were also remarkably decreased in CMS-treated mice (n = 6, P < 0.01) (Fig. 2c). JC-1 can be used as an indicator of mitochondrial potential in a variety of cell types as well as in the isolated mitochondria. The most widely application of JC-1 is for detection of mitochondrial depolarization when occurring mitochondrial dysfunction or in the early stages of apoptosis. In the present study, we used the molecular probe JC-1 to detect the effects of CMS on  m . The results showed that CMS expose resulted in a loss of  m , as indicated by decreases in the red fluorescence signal (the value of RFU at 590 nm was shown in Table 1). As shown in Fig. 3, swollen and vacuolated mitochondria were increased in the cortex, hypothalamus and hippocampus of mice exposed to CMS. The damaged mitochondria were markedly swollen, with broken or disrupted cristae or incomplete mem-

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Fig. 1. CMS procedure induced depressive-like behavioral syndrome. (a) CMS paradigm significantly decreased the sucrose preference of mice. (b) CMS paradigm increased the immobility time in tail suspension test. (c) CMS paradigm reduced body weight of mice. Values were presented as means ± S.D. n = 18, *P < 0.05 vs. control groups at 0 W; ## P < 0.01 vs. CMS groups at 0 W.

branes. Some mitochondria exhibited a condensed or polymorphic matrix. Moreover, some degenerated mitochondria presented myelin-like formations and monstrous in the stressed mice. The present study demonstrated that CMS mice displayed: (1) reduced sucrose preference; decreased body weight compared to non-stressed mice; significant increases in immobility time of TST; (2) reduced mitochondrial respiration rate and dissipated mitochondrial membrane potential; (3) damaged mitochondrial ultrastructure of hippocampus, cortex and hypothalamus in mice. The CMS paradigm is a model of depression obtained by using chronic unpredictable mild stressors [33]. Sucrose preference and the TST are regarded as useful and valid behavioral markers of chronic stress in an animal paradigm. And the lost of sucrose preference, which shows anhedonia, a core symptom of cliniTable 1 CMS paradigm dissipated mitochondrial membrane potential isolated from cortex, hippocampus and hypothalamus of mice. Values were presented as means ± S.D. of five individual determinations. Brain region

Cortex Hippocampus Hypothalamus

RFU at 590 nm

P-Value

Control groups

CMS groups

2.64 ± 0.22 2.98 ± 0.68 3.54 ± 0.09

2.10 ± 0.29 2.09 ± 0.42 2.70 ± 0.61

0.01 0.03 0.02

Fig. 2. CMS induced mitochondria oxidative respiratory dysfunction. CMS induced decreases in ST3 (a) and RCR (c), but failed to affect ST4 respiration (b). Values were presented as means ± S.D. of five individual determinations. *P < 0.05, **P < 0.01 vs. respective control group.

cal depression [33]. In the CMS model, both consumption of and preference for sucrose intake as well as decreased intracranial self-stimulation behavior have served as markers of generalized decrease in sensitivity to reward, which behavior is quite related to anhedonia [2,6]. In accordance with the reports, the present data confirmed that the mice exposed to CMS procedure reduced sucrose preference and increased the immobility time in TST, compared to non-stressed groups. In addition, our findings demonstrated a lack of body weight gain in the mice expose to CMS, which was consistent with major literatures data that stressed mice had a significant loss of body weight as well as behavioral alterations [2]. However, there were reports showed that 5-week CMS procedure caused no significant alterations in animals’ body weight [18]. The reason for these disparate results on body weight gain might be due to the variety in animal species, stress schedule and stimuli intensity. Oxidative stress has been implicated in the pathogenesis of various neurodegenerative diseases and mental disorders, includ-

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Fig. 3. CMS paradigm damaged mitochondrial ultrastructure of cortex, hippocampus and hypothalamus. The damaged mitochondria were markedly swollen, with broken or disrupted cristae or incomplete membranes (indicated as ). Some mitochondria exhibited a condensed or polymorphic matrix (indicated as 夽). Moreover, some degenerated mitochondria presented myelin-like formations (indicated as →) and monstrous (indicated as *). Left column presented electron microscopic photogram of control groups, right column presented electron microscopic photogram of CMS groups. 20,000×.

ing Alzheimer’s disease, Parkinson’s disease, and depression [1,11,17,21]. Previous studies reported that animal model induced by repeated restraint or immobilization stress showed remarkable increases in the levels of lipid peroxide, protein oxidation and lipid per-oxidation in the cerebral cortex, cerebellum and hippocampus [5]. Elevated ROS was also found in plasma of patients with major depression, especially in those with melancholic type [3]. These findings suggest that oxidative stress-induced injury contributes to depressive disorder. Mitochondria have a double membrane structure: an outer membrane and a folded inner membrane. Across the inner membrane of intact mitochondria, there is a voltage gradient (membrane potential) with the inside negative and the outside positive. Mitochondria membrane potential ( m ) loss would induce the releases of cytochrome c or apoptosis inducing factor (AIF) from mitochondria. Thus, mitochondrial membrane potential dissipation is known to be an early event in apoptosis [28]. We demonstrated in this study that CMS procedure significantly decreased  m , in the cortex, hippocampus as well

as in the hypothalamus, indicating that CMS induced mitochondrial malfunction and caused damage in mouse brain. Moreover, we found that CMS inhibited the respiration rate of mitochondria isolated from the three brain regions. The decreases of ST3 are usually companied by lower ATP production [29]. RCR indicates the tightness of the coupling between respiration and phosphorylation. The reduced RCR implicated that the coupling was not perfect and thereby ATP synthesis was injured [26]. So, our results revealed the functional impairment of oxidative phosphorylation and less efficient utilization of oxygen in CMS-treated mice. Insufficiency of ATP synthesized by mitochondria might contribute to the disturbance of brain function in depressive disorder. Oxidative stress also would impair the mitochondrial structure, often accompanied by mitochondrial dysfunction. Mitochondrial swelling, as first described 80 years ago, remains one of the most universal ultrastructural changes after brain injury such as ischemia [4,12,20]. However, there is no information about the ultrastructural changes of mitochondria in depressive disorder.

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The present study showed that the damaged mitochondria characterized by swelling, vacuolization, or condensation, and even myelin-like formations were significantly increased in the cortex, hypothalamus and hippocampus of CMS-treated mice. The results suggested that ultrastructural damage was responsible for the malfunction of mitochondria in depression. Our results revealed that CMS paradigm induced the collapse of mitochondrial membrane potential, inhibited mitochondrial respiration rates as well as destroyed mitochondrial ultrastructure. The present study, for the first time, provides evidence for brain mitochondrial dysfunction and ultrastructural damage in a mouse model of depression. Moreover, these findings suggest that mitochondrial malfunction-induced oxidative injury could play a role in stress-related disorders such as depression. Acknowledgements This study was supported by the grants from the National Natural Science Foundation of China (No. 30625038) and the National Key Basic Research Program of China (No. 2009CB521906 and No. 2006CB500706). References [1] G. Aliev, H.H. Palacios, B. Walrafen, A.E. Lipsitt, M.E. Obrenovich, L. Morales, Brain mitochondria as a primary target in the development of treatment strategies for Alzheimer disease, Int. J. Biochem. Cell. Biol. 41 (2009) 1989–2004. [2] S. Bekris, K. Antoniou, S. Daskas, Z. Papadopoulou-Daifoti, Behavioural and neurochemical effects induced by chronic mild stress applied to two different rat strains, Behav. Brain Res. 161 (2005) 45–59. [3] M. Bilici, H. Efe, M.A. Koroglu, H.A. Uydu, M. Bekaroglu, O. Deger, Antioxidative enzyme activities and lipid peroxidation in major depression: alterations by antidepressant treatments, J. Affect. Disord. 64 (2001) 43–51. [4] L.V. Cherkasov, D.R.F. Avletchina, Ultrastructure of the cerebral cortical neurons in hypoxic hypoxia, Zh. Nevropatol. Psikhiatr. Im. S.S. Korsakova 88 (1988) 16–19. [5] F.U. Fontella, I.R. Siqueira, A.P. Vasconcellos, A.S. Tabajara, C.A. Netto, C. Dalmaz, Repeated restraint stress induces oxidative damage in rat hippocampus, Neurochem. Res. 30 (2005) 105–111. [6] A.L. Garcia-Garcia, N. Elizalde, D. Matrov, J. Harro, S.M. Wojcik, E. Venzala, M.J. Ramirez, J. Del Rio, R.M. Tordera, Increased vulnerability to depressive-like behavior of mice with decreased expression of VGLUT1, Biol. Psychiatry 66 (2009) 275–282. [7] R.A. Gottlieb, Role of mitochondria in apoptosis, Crit. Rev. Eukaryot. Gene Expr. 10 (2000) 231–239. [8] R.A. Gottlieb, S. Adachi, Nitrogen cavitation for cell disruption to obtain mitochondria from cultured cells, Methods Enzymol. 322 (2000) 213–221. [9] J. Harro, R. Haidkind, M. Harro, A.R. Modiri, P.G. Gillberg, R. Pahkla, V. Matto, L. Oreland, Chronic mild unpredictable stress after noradrenergic denervation: attenuation of behavioural and biochemical effects of DSP-4 treatment, Eur. Neuropsychopharmacol. 10 (1999) 5–16. [10] R. Jain, The epidemiology and recognition of pain and physical symptoms in depression, J. Clin. Psychiatry 70 (2009) e04, LID—doi:10.4088/JCP.8001tx1c.e04. [11] P. Jenner, Oxidative stress in Parkinson’s disease, Ann. Neurol. 53 (Suppl. 3) (2003) S26–S36 (Discussion S36-28).

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