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Glucocorticoid effects on oxidative stress and mitochondrial dysfunction Tang, Victor Mark 2012

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GLUCOCORTICOID EFFECTS ON OXIDATIVE STRESS AND MITOCHONDRIAL DYSFUNCTION by Victor Mark Tang BSc, The University of British Columbia, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Neuroscience)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) May 2012 © Victor Mark Tang, 2012  Abstract Background: Many major psychiatric illnesses have been associated with aberrant stress response systems. In particular, those with mood disorders have been found to have an excessive and prolonged release of glucocorticoid stress hormones. Studies have also begun to reveal increased oxidative damage through mitochondrial dysfunction as part of the pathophysiology of such disorders. As both behaviorally induced stress and glucocorticoid treatment can increase the production of reactive oxygen species, this study aims to look at the effects of glucocorticoids on protein oxidative damage, mitochondrial function, and antioxidant activity. Methods: The effects of chronic treatment with corticosterone on cultured rat PC12 cells were examined. Protein oxidative damage was measured using both a spectrophotometric and immunoblotting method for protein carbonylation. Activity of the mitochondrial electron transport chain protein was examined through spectrophotometric techniques that measured complex I by quantifying NADH oxidation and complex III by cytochrome C reduction. Total antioxidant capacity (TAC) was measured to assess intracellular antioxidant capacity using a spectrophotometric assay. Results: It was found that chronic treatment with corticosterone was able to increase the amount of protein carbonylation in PC12 cells. Complex I activity, but not complex III, was decreased with drug treatment. TAC did not show any significant differences at doses which affected carbonylation or complex I activity. Limitations: The effects found with the time course and dosage of glucocorticoid treatment used here in cultured cells may be different then those found in normal or pathophysiological human conditions. Further research should also determine whether there are significant effects on neuronal function. Conclusions: Excessive glucocorticoid activity can decrease mitochondrial activity, leading to oxidative damage. Thus, over-active stress response systems and oxidative stress may be interrelated in the neurobiology of mood disorders, and may underlie neuronal pathophysiology in associated diseases. Moreover, chronic psychological stress could lead to detrimental effects on the brain through protein oxidation and mitochondrial dysfunction. Elucidation of these processes may open new possibilities for psychiatric treatments.  	
    Table of Contents Abstract……………………………………………………………………………............ii Table of Contents…………………………………………………………………………iii List of Figures……………………………………………………………………………..v Acknowledgements……………………………………………………………………….vi 1. Introduction…………………………………………………………………………...1 1.1 Stress responses, hypothalamic-pituitary-adrenal axis, and glucocorticoids……...1 1.2 Mitochondrial dysfunction and oxidative stress………..…………………………9 1.3 Glucocorticoid effects on oxidative stress and mitochondrial dysfunction…..….14 1.4 Glucocorticoids, oxidative stress, and bipolar disorder………………...………..17 1.5 Objectives and hypotheses………………………………………….……………20 2. Methods……………………………………………………………………………...22 2.1 PC12 cell cultures……………….………………………………………………22 2.1.1 Culturing cells……………………………………………………………...22 2.1.2 Bradford assay for determination of protein concentration………………..22 2.2 Measurement of carbonyl content……………………..………………………...23 2.2.1 Colorimetric analysis………………………………………………………23 2.2.2 Immunobotting analysis……………………………………………………24 2.3 Mitochondrial complex activity………………………………………………..26 2.3.1 Extraction of mitochondria………………………………………………...26 2.3.2 Complex I activity………………………………………………………….26 2.3.3 Complex III activity………………………………………………………..28 2.4 Total antioxidant capacity……………………………………………………...30  	
    2.4.1 Cell culture sample preparation……………………………………………31 2.4.2 Preparation of uric acid standard curve…………………………………….31 2.4.3 Assay protocol……………………………………………………………...32 2.5 Data analysis……………………………………………………………………32 3. Results……………………………………………………………………….………...34 3.1 Dose dependent glucocorticoid effects on protein carbonylation……………….34 3.2 The effect of corticosterone on activities of mitochondrial complex I and III….39 3.3 The effect of corticosterone on total antioxidant capacity………………………41 4 Discussion……………………………………………………………………………...44 References………………………………………………………………………………..60  	
    List of Figures Figure 1. Hypothalamic-pituitary-adrenal axis………..…………………………………..2 Figure 2. Glucocorticoid pathways in the cell…………………………………………….8 Figure 3. Production of oxidative stress in the cell …...…………………………………13 Figure 4. Protein carbonylation by spectrophotometer colorimetric assay. ……………..35 Figure 5. Pilot tests for immunobotting experiments…………………………………….37 Figure 6. Protein carbonylation by immunoblotting analysis. …………………………..38 Figure 7. Mitochondrial complex I and III activity. ….…………………………………40 Figure 8. Uric acid standard curve for total antioxidant capacity assay..………………..42 Figure 9. Total antioxidant capacity………………..……………………………………43 Figure 10. Glucocorticoids, oxidative stress, and mitochondrial dysfunction…………...59  	
    List of Abbreviations 4-HNE; 4-hydroxy-2-nonenal ACTH; Adrenocorticotrophic hormone AMPA; α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor BD; Bipolar Disorder BSA; Bovine serum albumin BDNF; Brain derived neurotrophic factor CRH; Corticotropin releasing hormone ETC; Electron transport chain GC; Glucocorticoid GR; Glucocorticoid receptor GRE; Glucocorticoid response element H2O2; Hydrogen peroxide HPA; Hypothalamic pituitary adrenal O2-; Superoxide radical MRS; Magnetic Resonance Spectroscopy MR; Mineralocorticoid receptor MRE; Mineralocorticoid response element mtDNA; Mitochondrial DNA NAC; N-acetyl-cysteine NMDA; Ν-methyl-D-aspartate NO; Nitric oxide NOS; Nitric oxide synthase  	
    PVN; Paraventricular nucleus PFC; Prefrontal cortex ROS; Reactive oxygen species SOD; Superoxide dismutase TAC; Total antioxidant capacity  	
    Acknowledgements  The duration of my time working on this project has been brilliantly enriched and made deeply enjoyable due to the people who have shared this experience with me. First, many thanks go towards Drs. Jun-Feng Wang and Allan Young for being extremely generous and supportive in their supervision. Both have kept an encouraging learning environment, been kind in their provision of time and resources, and under their mentorship I have broadened the scope of my knowledge in the field of neuroscience and psychiatry far greater than I would have anticipated. Second, I would like to extend my gratitude to the supporting cast of faculty - Drs. Ric Procyshyn, Alasdair Barr, Donna Lang, and Clare Beasley - that have been a valuable source of advice, dialogue, and discussion, and have set great examples as researchers. Thanks goes to the fellow students that have worked closed to me; the camaraderie we’ve shared has been a very memorable experience.  None of the experiments conducted would have been possible without the invaluable help in the lab from Drs. Hua Tan and Li Shao. I am grateful that they have met every question, inquiry, and request with patience and kindness. Outside of the academic setting, I am lucky to have such deeply supportive people in my personal life. Thanks to every one of my friends and family for the good times, the encouragement, the personal inspiration, and motivating me to follow through with my interests and ambitions.  	
    1. Introduction  1.1 Stress responses, hypothalamic-pituitary-adrenal axis, and glucocorticoids  The hypothalamic-pituitary-adrenal (HPA) axis is activated during the experience of stressful situations. Starting in the hypothalamus, the parvocellular neurons in the paraventricular nucleus (PVN) release two neuropeptides: the corticotropin releasing hormone (CRH) and vasopressin (AVP). These are both transported through the hypothalamic-hypophyseal blood vessel portal system to the anterior lobe of the pituitary gland. This in turn stimulates the release of adrenocorticotrophic hormone (ACTH) from corticotropic cells into the general circulation. From here ACTH travel to receptors in the cortex of the adrenal glands, causing a systemic release of glucocorticoids (GCs), cortisol in humans and corticosterone in rodents. These released glucocorticoids target organs throughout the body. Glucocorticoid levels are controlled through a negative feedback reaction when it binds to receptors in the PVN, the anterior pituitary gland, and the hippocampus, causing an attenuation of HPA axis activation (Figure 1).  	
    Figure 1. Hypothalamic-pituitary-adrenal axis  The hypothalamic-pituitary-adrenal axis is the central stress regulating pathway of the body. Upon the appraisal of threat, the hypothalamus will release corticotropin releasing hormone (CRH) to stimulate the pituitary glands to release adrenocorticotropic hormone (ACTH), which flows to the adrenal glands to release glucocorticoids. Elevation of glucocorticoids will feed back to glucocorticoid receptors in the brain, particularly the hypothalamus, which will attenuate the HPA axis activity.  Within the limits of physiological and psychological homeostasis, stress responses are adaptive activations of coping mechanisms towards threat. Generally these responses are observed to be surges in arousal, alertness, vigilance, attention, and cognitive efficiency. These adaptive responses become compromised when they are prolonged or 	
    excessive, and the physiological responses that occur from HPA axis activation become overloaded and lead to deleterious effects on the brain and the body, leading to disease susceptibility. In research of both chronically stressed animals and humans, this can have lasting consequences on brain and behavior. In the brain, structural abnormalities have been well documented in hippocampus, amygdala, and PFC (McEwen, 2007). On the cellular level, this has been observed as reduction of dendritic complexity and synaptic contacts (Wellman, 2001, Sandi et al., 2003), and altered expression of neurotransmitter receptors (Froger et al., 2004). These effects are thought to be mainly due to the heightened levels of GCs. Excessive amounts of GCs may lead to increases in oxidative stress in the cell, which may be one avenue through which HPA axis hyperactivity induces its damaging effects. Oxidative stress occurs when levels of reactive oxygen species (ROS) are too high. This can occur through the overproduction of ROS, deficiencies in cellular antioxidant capacity, or both. As mitochondria are the primary contributors to ROS generation, it has been long suggested in the literature that mitochondrial dysfunction is a central part of oxidative stress pathophysiology. Taken together, this project proposes that excessive levels of GCs can lead to increases in oxidative stress by causing mitochondrial dysfunction and decreases in antioxidant capacity. This increased oxidative stress results in higher amounts of oxidative damage to cellular macromolecules such as proteins. The sum of these effects thus appears to underlie a number of stress-related neuropsychiatric disorders, and HPA axis abnormalities confer risk towards developing such illnesses (de Kloet et al., 2005). A prominent example in the literature is the diminished suppression of cortisol in response to a low dose injection of the synthetic GC dexamethasone in those with bipolar disorder  	
    (BD; Watson et al., 2004), as well as in healthy subjects with a high familial risk for mood disorder (Holsboer et al., 1995).  Cellular effects from glucocorticoid signaling are best understood through their activation of glucocorticoid receptors (GRs) and mineralocorticoid receptors (MRs). The affinity of MRs for glucocorticoid is greater than the affinity of GRs by a factor of 10. Thus, at basal concentrations, MRs are typically occupied, and GRs become activated primarily during the elevated levels that occur in stress responses (de Kloet et al., 1998). As such, GRs have received an extensive amount of attention in research on stress responses, as well as its involvement in stress related disease of the brain (de Kloet et al., 2005). GRs and MRs are typically understood to be intracellular receptors that have genomic effects as transcription factors upon activation. Upon binding of corticosterone, activated receptors will homodimerize before translocating to the cell nucleus. Evidence is also available to suggest that in certain brain structures in which GRs and MRs colocalize, such as the hippocampus, heterodimerisation may occur (Liu et al., 1995). From there, they bind to mineralocorticoid response elements (MRE) or glucocorticoid response elements (GRE) in order to modulate gene transcription (Figure 2). This occurs either through transactivation or transrepression. Transactivation induces an increase in transcription, and typically occurs in promoter regions by recruiting relevant co-factors (Datson et al., 2008). Alternatively, transrepression results in the inhibition of gene expression, and occurs when monomers of the GR or MR interact with other transcription factor such as nuclear factor κB, activator protein 1, or interferon regulatory factor-3 (Prager and Johnson, 2009). The availability of different co-factors and associated  	
    proteins in these processes is likely to provide diversity of GC action on gene transcription in response to various cellular environments and varying GC levels (Nishi and Kawata, 2006). The details of the response may be influenced from cross talk with other activated signaling cascades, such as those triggered by monoaminergic or glutamatergic neurotransmission. It is well known that HPA axis responses are activated in concert with other sympathetic, parasympathetic, and limbic circuits in the nervous system (Kolber et al., 2008). Versatility on the cellular and network levels of stress response signaling allows for the orchestration of the appropriate behavioral response depending on the context (Datson et al., 2005). Importantly for our study, activated GRs have also been found to translocate to the mitochondria and affect mitochondrial gene expression (Demonacos et al., 1996).  The brain is a major target for GCs, and distribution of MRs and GRs vary throughout the brain, further expanding the heterogeneity of responses depending on the subregional GR/MR balance and circulation of hormones. GRs have been observed to have widespread expression throughout the brain, with densities highest in the PVN, neurons of ascending aminergic pathways, and in limbic areas that modulate the hypothalamus (de Kloet et al., 2005). Distributions of MRs are restricted to limbic structures such as the hippocampus, amygdala, lateral septal nuclei, and some cortical areas (Datson et al., 2005). Gene targets for GCs are diverse and encompass a wide range of functional classes, such as growth factors, signaling cascades, neuronal structure, synaptic proteins, energy metabolism, and neurotransmitter catabolism. It is important which receptors are available within a given cell type, as GC-responsive genes can be GR  	
    activated, MR activated, or requiring both (Datson et al., 2001). Moreover, as MRs have a much higher affinity for GCs, circulating concentrations of GCs determine which genes may be activated. Indeed, it has been generally proposed that MRs modulate the basal influences of GCs to maintain neuronal integrity and a stable excitatory tone, whereas GRs modulate responses to increased glucocorticoid levels during stress, with functions in recovery or neuroplasticity from stress responses and encoding and storage of information (Joels et al., 2008).  Research has advanced in more recent years to uncover GC signaling mechanisms other than the well-established genomic effects of cytosolic receptors (Figure 2). One such mechanism is through membrane associated MRs and GRs, which can have membrane to gene signaling either though activation of intracellular signaling cascades or through direct translocation to the nucleus to bind to GREs or MREs. The signaling cascades appear to be mediated by a variety of second messenger molecules such as protein kinase C, cRaf-1, and extracellular signal-regulated kinases (ERK1/2), the last of which translocates to the nucleus and elicits genomic modifications (Qiu et al., 2001). Other membrane bound GRs are located in a receptor complex that binds to GREs. A complex of Gβ, Gγ, and receptor activated C kinase 1 was found to co-migrate with membrane GRs to the nucleus (Kino et al., 2005). GCs have also been found to have nongenomic action on cells, which generate faster signaling responses and influences a wide range of behaviors and endocrine outputs in a matter of minutes, much too rapid to be explained by genomic effects (de Kloet et al., 1999). Studies of various brain structures such as the hippocampus, amygdala, and PFC have demonstrated rapid  	
    modulation of excitatory post-synaptic curren frequency by GCs (Groeneweg et al., 2011). On a cellular level, these effects may be largely due to activation of ion channels in the brain by GCs or through other neurosteroids that are products of GC metabolism (Evanson et al., 2010). Applications of corticosterone have been shown to change the excitability of α-amino-3-hydrox-5-methyl-4-isoxazolepropionic acid (AMPA) and Nmethyl-D-aspartate (NMDA) receptors (Karst et al., 2005; Sato et al., 2004). Importantly, there are differential effects depending on dose and receptor type. For example, lower concentrations of corticosterone appear to increase neuronal excitability dependent on MRs, whereas higher doses can result in GR dependent decreases in excitability (Prager and Johnson, 2009). Such influences on ion channels result in rapid changes to synaptic transmission and neuronal excitability, with subsequent downstream effects on activity dependent processes such as long-term potentiation and modeling of dendritic architecture (Krugers et al., 2005). Lastly, GCs have a significant bidirectional influence on other signaling systems. For example, GC treatment can decrease glutamatergic activity on CRH neurons, and antagonizing endocannabinoid receptors can block these effects (Herman et al., 2003). Another example is found in the amygdala, where GC modulation of this structure is dependent on adrenergic signaling, and treatment with GCs can increase levels of noradrenaline (McReynolds et al., 2010).  	
    Figure 2. Glucocorticoid pathways in the cell  Glucocorticoids have various signaling pathways in cells of the brain. When bound to cytosolic GRs, they will translocate to the nucleus and bind to glucocorticoid response elements (GRE) in order to modulate transcription (1). These cytosolic receptors can also translocate to the mitochondria to influence mitochondrial genome transcription (2). Membrane bound GRs can influence second messenger cascades that continue down to regulate transcription factors to modulate gene expression (3), or be with other membrane proteins to form a complex that translocates together to the nucleus in order to bind to the GRE (4). Activated GRs in the cytosol or membrane can also modulate the activity of various ion channels such as AMPA or NMDA receptors in order to influence cellular membrane excitability (5). GRs and GREs can be interchangeable for MRs (Mineralocorticoid receptors) and MREs (Mineralocorticoid Response Elements)  	
    1.2 Mitochondrial dysfunction and oxidative stress  Mitochondria are membrane bound organelles with the primary function of energy production. The mitochondria have an outer membrane that encloses on an inner membrane. An intermembrane space exists in between, and the inner membrane surrounds the mitochondrial matrix. Energy production occurs through the process of oxidative phosphorylation, where electrons derived from the oxidation of nutrients are taken through the mitochondrial electron transport chain (ETC) in the inner mitochondrial membrane to create an electrochemical gradient that drives a process generating the energy-rich molecule adenosine triphosphate (ATP) and consuming molecular oxygen (O2). The ETC consists of a series of protein complexes imbedded within the inner mitochondrial membrane, and serve as specialized electron donor and acceptor molecules. As electrons are passed through the series of protein complexes, energy is released in order to pump protons (H+ ions) from the inner mitochondrial matrix to the intermembrane space. The resulting H+ gradient serves as an energy source to phosphorylate adenosine diphosphate (ADP) to produce ATP. This occurs through the membrane bound enzyme ATP synthase, which provides a pathway for protons to flow down the electrochemical gradient in order to drive the reaction of ADP + Pi to ATP.  The process of oxidative phosphorylation begins with the transfer of electrons to coenzyme Q (or ubiquinone) through Complex I on the ETC. Nicotinamide adenine dinucleotide (NAD+) becomes reduced to NADH through the earlier oxidation of nutrient molecules and is the electron carrier that donates to this initial reaction. Complex I,  	
    otherwise known as NADH dehydrogenase, is the largest of the ETC complexes, consisting of more than 40 polypeptide subunits. It accepts electrons from NADH through the reduction of the flavin mononucleotide (FMN) molecule, which then passes them through at least seven iron-sulfur centers to ubiquinone, a lipid soluble electron carrier independent of the protein complexes. Alternatively, ubiquinone can accept electrons from complex II, or succinate dehydrogenase, which receives its electrons from the oxidization of succinate from the citric acid cycle by flavin adenine dinucleotide (FAD). Ubiquinone transfers its electrons to the ETC complex III, or the cytochrome bc1 complex. This dimeric protein complex consists of at least 11 polypeptide subunits, and accepts electrons from ubiquinone in order to pass them onto the electron carrier cytochrome c. This electron carrier in the intermembrane space passes the electrons to Complex IV, or the cytochrome oxidase complex, consisting of 13 polypeptide subunits and also functions as a dimer. Complex IV completes the chain of reactions when electrons are transferred to reduce O2 to water. The O2 is held by complex IV at a special bimetallic center, enclosed by a heme-linked iron atom and a copper atom, and released upon reaction to produce water. This reaction is estimated to account for close to 90% of all oxygen uptakes in most cells. Importantly, O2 can also pick up free electrons, leading to the formation of the superoxide radical (O2-), a reactive oxygen species (ROS). The O2anion can lead to the production of other highly reactive free radicals through various chemical cascades (Figure 3).  As oxygen-containing free radicals, ROS have unpaired electrons that are highly reactive as strong oxidizing agents (very inductive to absorbing electrons from other  	
    molecules). These free radicals can cause substantial damage to macromolecules through the generation of adducts, destruction of unsaturated C-C bonds, and oxidation of disulfides (Maher & Schubert, 2000). More generally, these are damaging effects to proteins, lipids, and nucleic acids. The main source of ROS production is known to be due to oxidative phosphorylation in the ETC. Under physiological conditions, roughly 15% of all oxygen consumed by the cell is converted into ROS (Lee and Wei, 2005), and occurs when electrons are prematurely released from the ETC. This occurs when electrons are leaked as they are being passed through the chain, most commonly at complexes I and III (Halliwell, 1997). As these free electrons interact with molecular oxygen, they form O2-•. These free radicals can then be neutralized by antioxidant defenses: either by dismutation to hydrogen peroxide (H2O2) by superoxide dismutase (SOD), or to water by glutathione peroxidases or catalase (Wallace, 2005). In the presence of ferrous iron (Fe), H2O2 is converted into the highly reactive hydroxyl radical in what is described as the Fenton reaction: H2O2 + Fe2+ à Fe3+ + OH- + OH•. The OH• generated from this reaction can go on to react with proteins in order to introduce carbonyl groups into lysine, proline, arginine, threonine and other amino acid residues. Measurement of carbonyl content in proteins is the standard method for measuring oxidative stress mediated protein oxidation, since the generation of carbonyl derivatives is orders of magnitude greater than other forms of protein oxidation (Dean et al., 1997). The effects of oxidation to proteins can range from alteration of function, formation of deleterious intermolecular aggregates, or complete inactivation or degradation. Moreover, this may lead to downstream effects on gene expression through abnormal transcription  	
    factor functioning, alterations in signaling pathways, and dysfunction on cell-to-cell communication, which is of particular importance in the nervous system.  Under controlled conditions by antioxidant systems, free radicals can serve important roles in physiological processes (Halliwell, 1997). Thus, pathological states arise when the ROS are produced in excess and overwhelm cellular antioxidant capacity, and/or these antioxidant defenses are not functioning adequately. As such, dysfunction in the mitochondrial ETC could lend to greater inefficiencies in oxidative phosphorylation and increased loss of electrons most likely through complexes I and III. This in turn would increase the amount of ROS produced and form a state of oxidative stress in the cell. Interestingly, mitochondria may not only be the major intracellular source of ROS, it may also be a main target for oxidant attack (Harmin, 1956). Free radicals can damage mitochondria-related macromolecules, including those of the ETC (Radi, 1994), with the iron-sulfur clusters that mediate electron transport in complexes I and III as the most susceptible (Wallace, 2005). Also, mtDNA is considered to be more sensitive than nuclear DNA to oxidative stress due to the proximity of the oxidant generation source, as well as lacking protective histones and having a more limited DNA repair system (Miquel, 1992).  	
    Figure 3. Production of oxidative stress in the cell.  Electrons that are lost from the electron transport chain of the mitochondria (1.) can react with molecular oxygen to form reactive oxygen species (2.), which can accumulate to oxidative damage to cellular macromolecules (3.). Complex I/II/III/IV (CI, CII, CIII, CIV), Superoxide (O2-• ), superoxide dismutase (SOD), hydrogen peroxide (H2O2), Iron (Fe), hydroxyl radical (OH•), carbonyl group (C=O), Lys (Lysine), Arginine (Arg), Proline (Pro), Threonine (Thr).  Importantly, there may be a greater propensity towards ROS production in the brain, as it is known to metabolize 20% of total body oxygen while only being 2% of total body weight. As rising O2 concentration is associated with an increased rate of electron leakage (Fridovich, 1978), the great amounts of oxygen consumption by the brain may result in high O2-• formation. Furthermore, it has been widely argued that the  	
    brain is especially prone to oxidative damage, as attributable by the brain (1) having an enrichment of peroxidizable fatty acids, (2) being poor in catalase activity and only moderate amounts of SOD and glutathione peroxidase, and (3) being rich in iron in several areas (Halliwell, 1992; Floyd, 1999). To this end, the effects of free radicals have been widely implicated in the pathogenesis of many brain-based neurological and psychiatric disorders. In BD research, reports of both mitochondrial dysfunction and increased oxidative stress in the neurobiology of the disease process have been mounting steadily within recent years.  1.3 Glucocorticoid effects on oxidative stress and mitochondrial dysfunction  In recognizing the widespread influences of both GC signaling, mitochondrial dysfunction, and oxidative stress in the brain, it is well within reason to assume there are many points of overlap and bi-directional effects between these processes. Beginning with mitochondria, it is known that these organelles have their own independent genome in the form of a circular double stranded mtDNA molecule located in the mitochondrial matrix (Iborra et al., 2004). With GCs and their conventional mode of transcriptional regulation through GREs, it has been proposed that the reach of their genomic effects may include the mitochondria. Indeed, GRs have been observed to be localized in the mitochondrial matrix of rat brain cells (Moutsatsou et al., 2001), as well as humanderived HeLa and Hep-2 cell cultures (Scheller et al., 2000). Moreover, there exists the presence of nucleotide sequences in mtDNA that resemble nuclear GREs (Demonacos et al., 1995). GCs have been also been known to influence gene expression of the mtDNA  	
    (Mansour and Nass, 1970), which influences mitochondria function and code for proteins of oxidative phosphorylation (Psarra & Sekeris, 2009; Zhang et al., 2006). A recent in vitro study using murine neural stem cells showed that dexamethasone treatment resulted in altered expression of genes encoding for the ETC and antioxidant enzymes (Mutsaers and Tofighi, 2012). Mitochondrial function as measured by mitochondrial calcium levels and membrane potential was found to be influenced by GCs in a concentration dependent manner (Du et al., 2009), suggesting a possible relationship to the elevated GC amounts seen in stress responses. Indeed, it has been demonstrated in animals that chronic stress is able to induce decrease mitochondrial complex I-III activity (Madrigal et al., 2001).  Research on the effects of chronic stress paradigms on animals has provided evidence of a link to increased oxidative stress. Perez-Nievas et al. (2007) reported that 6h of immobilization restraint stress was able to induce upregulation of lipid peroxidation markers and calcium dependent nitric oxide (NO) activity, a precursor to the reactive nitrogen species peroxynitrite. In accordance with this, a chronic 21-day stress paradigm was able to increase levels of nitric oxide synthase (NOS), which may infer a larger production of NO amounts (Harvey et al., 2004). Abidin et al. (2004) reported a correlation between lipid peroxidation and corticosterone levels in the chronically stressed rats of their study. To this end, on the behavioral level there is accumulating evidence that stress is associated with oxidative stress. There are some reports studies looking specifically at the effects of excess GCs. In hippocampal slice cultures, GCs treatment led to higher levels of a fluorescent measure of ROS production (McIntosh and Sapolsky, 1996), which also appears to be dependent on GR activation (You et al., 2009).  	
    Cerebral cortical neuron cultures given corticosterone treatment for 1-3 days showed increases in oxidative damage (Lee et al., 2009). In vivo, corticosterone has been shown to increase NOS levels (Pinnock et al., 2007), and rats given high doses of orally ingested corticosterone show increases in oxidative damage (Zafir and Banu, 2009). Interestingly, prenatal exposure to GCs showed increases in ROS-induced cell death, decreases in the antioxidant catalase, and abnormalities in mitochondrial function pertaining to calcium accumulation and oxygen consumption (Ahlbom et al., 2000), suggesting an effect of maternal stress on oxidative stress and mitochondrial dysfunction of their offspring. Antioxidant capacity is important to address in determining the causes of oxidative stress. A study by Filipovic and Pajovic (2009) demonstrated that while acute stress leads to increases in expression of the copper-zinc SOD antioxidant, these effects were abolished in rats that were chronically stressed. Rats directly treated with chronic injections of corticosterone show decreased levels of copper-zinc SOD in the hippocampus, cortex, and cerebellum (McIntosh et al., 1998a), and decreases in catalase as well (Cviljic et al., 1995)  Lastly, GCs may increase oxidative damage if they are exacerbating existing oxidative stress processes. For example, Andriamycin is an oxygen radical generator, and treatment with GCs can increase its ability to generate ROS (McIntosh and Sapolsky, 1996). Lipid peroxidation induced by FeSO4 and amyloid β-peptide was further increased upon corticosterone administration (Goodman et al., 1996). GCs are also known to increase Ca2+ through its effects on ion channels as described earlier, and this can lead to the excitotoxic effects of increased free radical generation (Lee et al., 2002) and  	
    depolarization of mitochondrial membrane potential (Takahashi et al., 2002). With regard to antioxidants, McIntosh et al. (1998b) showed that GCs blocked the induction of antioxidant defenses that would normally upregulate in response to the ROS generating excitotoxin kainic acid. Therefore it appears that pre-existing states of oxidative stress may be further worsened in the presence of elevated GC levels.  1.4 Glucocorticoid, oxidative stress and bipolar disorder  HPA axis abnormalities and an exaggerated stress response have been implicated in BD. Stress is one of the most commonly associated factors with affective illnesses, and animal studies have consistently demonstrated that chronic stress can induce maladaptive changes to the HPA axis. In these pathological states, HPA axis function is over-activated and gives rise to chronic and prolonged release of GC steroid hormones, including cortisol, which in turn induces a number of deleterious effects on the brain (Daban et al., 2005). In particular, BD has been found to be associated with enlarged pituitary glands (Takahashi et al., 2009), increases in circadian secretion of cortisol (Cervantes et al., 2001), and higher adrenocorticotropic hormone release upon stimulation (Vieta et al., 1999). GRs are responsible for negative feedback on the HPA axis, which serves to attenuate GC levels after stress induction, but this ability appears to be impaired in those with BD (Watson et al., 2004). Importantly, it has been demonstrated that the GR system may be a potential therapeutic target for novel BD treatments, as the GR antagonist RU486 was shown to alleviate mood and cognitive symptoms in patients (Young et al., 2004). A more recent meta-analysis pooled data from all studies available at the time on  	
    antiglucocorticoid interventions in BD and revealed promising results in some diagnostic subtypes, particularly non-psychotic depression (Gallagher et al., 2009).  Recently, research has emerged to increasingly suggest mitochondrial dysfunction and oxidative stress in the pathophysiology of BD. Imaging studies through magnetic resonance spectroscopy (MRS) have demonstrated that energy metabolism within the brains of BD patients may be abnormal, inferring mitochondrial dysfunction (Kato et al., 1994; Deicken Feln, & Welner, 1995). Alongside these studies have been the genetic research highlighting increases in mitochondrial DNA (mtDNA) deletions and mutations (Kato et al., 1997) and altered expression of mitochondrial protein coding mRNAs associated with the disease (Konradi et al., 2004). Furthermore, specific abnormalities have been found in the mitochondrial ETC, where DNA microarray analyses have identified decreased expression of mRNAs that code for ETC complexes (Sun et al, 2006), postmortem brain tissue studies showing decreased levels of ETC complex I subunit expression and impaired functionality (Andreazza et al., 2010). ETC deficits have been proposed to be associated to findings of increased oxidative stress in BD, where overproduction of ROS overwhelms cellular antioxidant capacity to lead to oxidative damage to cellular macromolecules. Studies with postmortem brain tissue have revealed evidence of increased oxidative damage to proteins, nucleic acids, and lipids (Andreazza et al., 2010; Che et al., 2010; Wang et al., 2009). Evidence of compromised antioxidant capacity in BD comes from reports of decreased glutathione, the major antioxidant in the brain (Gawryluk et al., 2010), as well as expression down regulation of a number of antioxidant enzymes. Taken together, increased oxidative stress may damage the  	
    biochemical properties of proteins, lipids, and nucleic acids in order to cause functional abnormalities in the brain and impairments in neuroplasticity.	
   The current project seeks to extend our knowledge of the effects of GC on oxidative stress and mitochondrial dysfunction. Using an in vitro cell culture experiment, we will examine if GCs are able to increase levels of protein oxidation, which would demonstrate a closer link to functional abnormalities in neurons due to increased oxidative stress. Moreover, many studies use the synthetic GR agonist dexamethasone to study GC effects on oxidative stress. The current study uses corticosterone which is the endogenous, naturally occurring GC released in rodents, which may closer resemble the cellular effects of stress in vivo. Furthermore, while GCs have been found to mediate and influence various measures of mitochondrial function such as membrane potential, ATP production, and gene expression, there have been no direct demonstrations of changes in activities of ETC complexes. Thus, another important aim of the current proposal is to examine whether GCs have effects on ETC complex I and III activity, the two main sites of electron leakage leading to production of free radicals. As antioxidant defenses make up the other side of the equation in determination of oxidative stress, we will also determine the effects on cellular antioxidant capacity with GC treatment and how this corresponds with the changes in protein oxidation and mitochondrial dysfunction. Combined, these results will allow comparisons of corticosterone induced oxidative damage to corresponding changes in mitochondrial function and antioxidant capacity. Indeed, we will be able to expand on the previous literature by examining the effects of a wider range of GC concentrations compared to any of the other studies in the past. This will be useful to in finding the adaptive and deleterious ranges of the stress response  	
    hormone on pro-oxidant, antioxidant, and mitochondrial activities in vitro. This study will contribute to an important understanding of the effects of HPA axis hyperactivity on a cellular level, as mitochondrial dysfunction and increased oxidative stress are becoming increasingly highlighted as sources of aberrant neuronal processing and plasticity in brain diseases such as BD. As both antiglucocorticoid and antioxidant treatments have recently become promising avenues for research into novel drug development for BD, further elucidating the mechanisms and pathways between these neurobiological systems may prove useful in advancing our treatment of this devastating illness. 1.5 Objective and hypotheses  To examine the effects of glucocorticoids on oxidative stress and mitochondrial dysfunction in order to gain insight on these important cellular and molecular mechanisms that are implicated in BD.  1. To determine the effects of glucocorticoid treatments on protein oxidative damage by testing for levels of carbonylated proteins. It is hypothesized that relatively higher concentrations of corticosterone will lead to higher levels of protein carbonylation. 2. To examine the effects of glucocorticoids on mitochondrial function through the measurement of ETC complex I and III activity. We hypothesized an impairment of complex I and III activity due to corticosterone treatment.  	
    3. To study the effect of glucocorticoids on the total antioxidant capacity of cells. We expect that glucocorticoids may impair antioxidant responses and decrease cellular antioxidant capacity.  	
    2. Methods  2.1 PC12 cell cultures  2.1.1. Culturing cells For these experiments we are using the PC12 cell line as a neuronal cell model. PC12 cells are clonal cell lines derived from rat adrenal pheochromocytoma and express neuronal properties, thus making useful model systems for studying the nervous system at the single cell and molecular levels. PC12 cells have also been used extensively throughout the literature to study processes of oxidative stress and glucocorticoid signaling in neurons. Cells are grown in DMEM medium containing 10% fetal bovine serum, grown to 80% confluence before harvested for experiments.  2.1.2. Bradford assay for determination of protein concentration The Bradford assay is a colorimetric assay based on the absorbance shift of protein samples reacting with the Coomassie Brilliant Blue G-250 dye (Bradford, 1976). Under acidic conditions, increasing protein concentrations result in color changes from a red/brown to blue. This reaction occurs when the red form of the dye donates a free electron to ionisable groups of a protein, which disrupts protein conformation in order to expose its hydrophilic pockets. When these hydrophilic pockets become available they will interact non-covalently through van der Waals forces to non-polar regions of the dye. This repositions the positive amine groups in closer proximity with the negative charge of the dye, strengthening their ionic interaction. The result is a conformational  	
    stabilization of the blue form of the Coomassie dye. Protein samples are diluted 20x in PBS and added to each well of a 96-well microplate at volumes of 10µL. A series of Bovine serum albumin (BSA) diltutions were used as a protein standard. The reaction is started by the addition of 200µL of the dye to each well, and incubated on an orbital shaker for 5 minutes. The plate is read at an absorbance wavelength of 595nm and protein concentrations are determined through comparisons with the BSA standard curve.  2.2 Measurement of protein carbonyl content  2.2.1 Colorimetric analysis Cells were treated with corticosterone diluted in DMSO for 24h at various concentrations, with confluence targeted at around 80% prior to harvesting. Following treatment, media is removed and cells are washed in ice-cold PBS two times. Cells are extracted in 10mL PBS with a cells scraper and spun down at 1,000g for 3 minutes. The supernatant is discarded and remaining cell pellet suspended in 150µL PBS. Cells were lysed by homogenization with a hand-operated, motor driven pellet –pestle grinder, and then centrifuged at 10,000g for 10 minutes at 4oC. The supernatant was aliquoted for protein determination, storage at -20oC and subsequent carbonyl content assay.  The 2,4 dinitrophenyl hydrazones formed from the reaction between carbonyl groups and 2,4-dinitrophenylhydrazines (DNPH) can be quantified spectrophotometrically at an absorbance of 370nm. Derivitization reactions were performed with the 1:1 v/v addition of DNPH to protein samples for 1h and vortexed briefly every 15 minutes. Following  	
    this, derivitized proteins were precipitated with the addition of 20% trichloroacetic acid (TCA) and centrifuged at 11,000g for 10 minutes at 4oC. Supernatants were removed and cells washed once more in TCA. This was followed by washes of 1mL of ethanol/ethyl acetate (1:1 v/v) for three times, each time spinning at 11,000g for 10 min at 4oC to remove supernatants. Finally, the protein pellet was solubilized in 0.5mL of 6M guanidine-HCl and centrifuged for 10min at 11,000g and 4oC to remove insoluble materials. Carbonyl content of the protein samples were determined by measuring absorbance at 370nm using a plate reader.  2.2.2 Immunoblotting analysis Cells were treated with corticosterone diluted in DMSO for 24h at various concentrations with confluence targeted at around 80% prior to harvesting. Following treatment, media are removed and cells are washed in ice-cold PBS two times. Cells are extracted in 10mL PBS with a cells scraper and spun down at 1,000g for 3 minutes. The supernatant was discarded and the remaining pellet was suspended in 80µL of lysis buffer (20mM HEPES, 250mM NaCl, 20% glycerol, 30mM MgCl2, 0.5mM EDTA, 100mM EGTA, 1M DTT, 1% NP-40. 1% protease inhibitor cocktail) and incubated for 1 hour on ice, with mixing by pipetting several times every 10 minutes. Cell lysates were then centrifuged for 10 minutes at 10,000g and supernatants collected, aliquoted, and stored at -20oC in between experiments.  Levels of protein carbonyl content in protein residues are determined by the derivitization of the carbonyl group when reacted with DNPH to produce 2,4-  	
    dinitrophenol hydrazone, which can be immunodetected, by a specific antibody. The derivitization protocol begins with the transfer of 4µg/µL protein sample to individual Eppendorf tubes. The proteins are denatured by the addition of 5µL 12% sodium dodecyl sulphate (SDS) for a final concentration of 6% SDS. Each sample was derivitized by the addition of 10µL of 10mM DNPH and incubated for 15 minutes. The reaction was stopped with the addition of 7.5µL of neutralization solution.  The resulting protein solution was loaded into the wells of a 1% SDS polyacrylamide gels for at 110V or until they migrated to the bottom of the gel. A pilot experiment with varying concentrations to identify which concentrations would give the best detection of immunoreactivity determined that 20µg of each sample should be used in each test. Afterwards, the gel was transferred to polyvinylidene fluoride membranes at 110V for 1.5h. The membranes are then blocked by incubation of 5% milk in Trisbuffered saline (TBS) for 1h at room temperature. The blots were then probed with a 1:1,000 dilution of primary antibody, the rabbit anti-DNP antibody (No. 90451), which is specific to the DNP moiety of proteins overnight at 4oC. Membranes were then washed in tris-buffered saline with 0.05% tween (TBS-T) for 3 times, 15 minutes each, before being incubated with a 1:2,000 dilution of the secondary antibody, the goat anti-rabbit horseradish peroxidase-antibody (No. 90452), for 1 hour at room temperature. Membranes were then washed in TBS-T for 6 times, 10 minutes each wash, and a final wash in TBS for 10 minutes. Immunoreactive bands were detected with the enhanced chemiluminescence reagent (PerkinElmer, Canada) containing luminol, which is converted to a light-emitting form at 428nm by the antigen/primary antibody/secondary  	
    antibody/peroxidase complex in an H2O2 catalized oxidation reaction. The light is then detected by the Fujifilm LAS-3000 system (Fujifilm Medical Systems USA Inc., USA) by short exposure to blue-light sensitive films. Protein bands were then analyzed densitometrically by Gene Tool software (PerkinElmer).  2.3 Mitochondrial complex activity  2.3.1 Extraction of mitochondria Cells were treated with corticosterone diluted in DMSO for 24h at 0.125 mM, with confluence targeted at around 80% prior to harvesting. This was the concentration determined from the previous experiments to induce significant increases in protein carbonylation. Cells are first centrifuged from culture at 1,000g for 3 minutes. The supernatant is removed and the remaining cell pellet is suspended in 3ml of ice-cold IBc. Cells are then homogenized with a Douce grinder for 30 seconds on ice. After homogenization, samples are centrifuged at 1,000g for 3 minutes in order to remove cell debris. The supernatant was collected and centrifuged again at 30,000g for 30 min at 4oC. The supernatant was discarded and the remaining precipitate is the mitochondrial samples and suspended in 150µL storage buffer. A separate amount was taken for determination of protein concentration and stored in aliquots at -20oC.  2.3.2 Complex I activity Preparation of Reagents: Isolation buffer: 0.32M sucrose, 5mM HEPES.  	
    Storage buffer: 0.21M mannitol, 0.07M sucrose, 10mM Tris-HCl, pH adjusted to 7.4. Assay buffer: 50mM potassium chloride, 10mM Tris-HCl, 1mM EDTA, 2mM potassium cyanide, pH adjusted to 7.4.  NADH: 500µM dissolved in distilled water, stored in -80oC. Ubiquinone (CoQ1): 500µM dissolved in 100% ethanol, stored in -20oC. Rotenone: 100µM dissolved in 100% ethanol, to be prepared fresh on the day of experiments.  The current protocol was adopted from Andreazza et al. (2010), using a spectrophotometric technique to measure complex I activity. This measurement detects NADH oxidation, the role of Complex I in transferring electrons from NADH to ubiquinone on the ETC. Complex I will convert NADH into NAD+ in the presence of the electron acceptor ubiquinone, and so the rate of disappearance of NADH is obtained from reading the absorbance values of NADH over a period of time.  Before use in the assay, mitochondrial extracts underwent three cycles of freeze and thaw in order to expose the inner mitochondrial membrane where the ETC protein complexes are located. Each mitochondrial sample was tested in triplicates for overall rate of complex I activity, rate of activity in the presence of the complex I inhibitor rotenone, and for baseline activity before the addition of ubiquinone, the electron acceptor necessary for the reaction. Complex I inhibition by rotenone is used as a control to parse out the activity of other enzymes and agents present in the samples that may be  	
    oxidizing NADH in the presence of ubiquinone. Each sample tested contained 20µg of the mitochondrial extract, brought to a total volume of 65µL with assay buffer. NADH, ubiquinone, and rotenone were placed in a water bath at a temperature of 30oC for 5-10 minutes. Overall rate of samples was measured with the addition of 15µL NADH, 10µL ubiquinone, and 10µ of assay buffer for a final volume of 100µL. Rate in the presence of an inhibitor is measured with the addition of 15µL NADH, 10µL ubiquinone, and 10µL rotenone, with a final reaction volume of 100µL. The reaction is started upon addition of ubiquinone, so it is the last reagent to be added before the plate is immediately read at 340nm, the absorbance wavelength of NADH, in 20-second intervals for a total period of 5 minutes. Baseline or blank absorbance values were obtained by reading the plate before the addition of ubiquinone. Readings are plotted as absorbance over time, forming a linear equation (y=mx+b) in which “m” is the slope of the line and represents the rate of NADH oxidation by complex I. Measuring the disappearance of NADH results in a negative value, so the absolute value of the slope is used for all calculations. In the end, complex I activity is calculated as [(Overall rate – blank) – (Inhibited rate – blank)]*1000.  2.3.3 Complex III activity The current protocol was adopted from Luo et al. (2008), using a spectrophotometric technique to measure complex III activity. Ubuquinone accepts electrons from complex I in order to be reduced to ubiquinol. This technique measures the activity of Complex III in transferring electrons from ubiquinol to cytrochrome c by quantifying cytochrome c reduction. This assay uses decylubiquinone, an analogue of  	
    ubiquinone, which has been shown to be effective in activating complex III activity in synaptosomes (Telford et al., 2010). Decylubiquinone is used to prepare decylubiquinol, which is the necessary enzyme added to start the reaction mixture in the experiment. Importantly, decylubiquinol has been shown to inhibit Complex I activity (Benit et al., 2008), thus can be used as a good substrate for specifically examining complex III.  Preparation of decyclubiquinol: First, 100µL of 500mmol/L decylubiquinone was taken and diluted to 25mmol/L in ethanol. This was then added to 6mL of a solution consisting of 0.1mol/L potassium phosphate buffer (pH 7.4) and 0.25mol/L sucrose. Following this, 1mL of cyclohexane and a pinch of solid sodium dithionite were added, and the mixture was shaken vigorously until colorless. The upper organic phase layer of cyclohexane contained the decylubiquinol and was transferred to another tube. This was repeated 2 more times with 1mL of cyclohexane, and the extracted organic phases combined organic phase combined in one tube. This was taken to be evaporated in vacuo, after which a light-yellow syrup remained at the bottom of the tube. This syrup was dissolved in 900µL ethanol and 100µL of 0.1mol/L HCl, before being stored at -80oC in aliquots.  Assay procedure for Complex III activity: Isolation of mitochondria samples was done as described for the complex I experiments. Each mitochondrial sample was tested in triplicates for overall rate of complex III activity, rate of activity in the presence of the complex III inhibitor antimycin A, and for baseline activity before the addition of decylubiquinol, the electron donor  	
    necessary for the reaction. Complex III inhibition by antimycin A is used as a control to parse out the activity of other enzymes and agents present in the samples that may be reducing cytochrome C.  The enzyme assays were performed in a 96-well plate. Each had 40µL of 0.25mmol/L cytochrome c, 80µL of ddH2O, and 40µL of 10µg mitochondria sample diluted in the same assay buffer prepared for Complex I experiments. The reaction mixture was prepared containing 250mmol/L Tris-HCl (pH 7.4), 20mmol/L NaN3, 0.24mmol/L decylubiquinol. A separate reaction mixture for the activity in the presence of the complex III inhibitor included 10µmol/L antimycin A. Upon the addition of 40µL of the reaction mixture, the plate was read for 4minutes at 35second intervals, at an absorbance wavelength of 550nm. Baseline or blank absorbance values were obtained by reading the plate before the addition of the reaction mixture. Readings are plotted as absorbance over time, forming a linear equation (y=mx+b) in which “m” is the slope of the line and represents the rate of cytochrome c reduction by complex III. As with the complex I activity, blank values were subtracted from all readings, and inhibited rate subtracted from the overall rate.  2.4 Total antioxidant activity  Antioxidant activity of PC12 cells was assessed using the Cell Biolabs’ OxiSelect Total Antioxidant Capacity (TAC) assay kit (Cell Biolabs, Inc., San Diego, California). As antioxidants typically neutralize radicals via a single electron transfer, this assay is  	
    based on measurement of the reduction of copper (II) to copper (I) by viable antioxidants in a sample within a 96-well microtiter plate. Upon reduction, the copper (I) ion will react with a coupling chromogenic reagent that produces a color with a maximum absorbance at 490 and read in a spectrophotometer. The absorbance values are then compared to a standard curve of uric acid antioxidant activity. Antioxidant capacity was determined for cells treated with corticosterone for 24h at concentrations of 0.031, 0.063, and 0.125mM compared with controls treated for 24h with the vehicle (DMSO).  2.4.1. Cell culture sample preparation  Media from plates is discarded and cells are washed 2 times in cold PBS. Cells are then scraped from the plate in 10mL PBS and spun down at 1,000g for 3 minutes. The supernatant is discarded and remaining cell pellet suspended in 150µL PBS. Cells were lysed by homogenization with a hand-operated, motor driven pellet –pestle grinder, and then centrifuged at 10,000g for 10 minutes at 4oC. The supernatant was aliquoted for protein determination, storage at -20oC and subsequent TAC assay.  2.4.2 Preparation of uric acid standard curve Uric acid standard stock solutions were prepared by weighing out uric acid powder for a 10mg/mL or 60mM solution in 1N NaOH, and stored in -80oC for up to one week. The stock solution is used to prepare serial dilutions of uric acid in deionized water, with highest concentration at 1mM diluted to 9 difference concentrations with 0.0039mM as the lowest, and the 10th sample as a blank sample containing only water.  	
    2.4.3 Assay protocol Each uric acid or cell culture sample is tested in triplicate, at 20µL volumes. From the cell extract samples, it was determined in a preliminary experiment that 20µg of protein should be used for each test. 180µL of the supplied reaction buffer was added to each sample and mixed with the pipette. An initial, baseline absorbance was read at 490nm. The reaction was started with 50µL of the supplied copper ion reagent was added into each well, and incubated for 5 minutes on an orbital shaker. Following this, the reaction was terminated with the addition of 50µL of the supplied stop solution. The plate was then read at 490nm for copper reduction absorbance. The baseline readings were subtracted from these values to produce the corrected net absorbance values. Antioxidant capacities of the samples were calculated first into “mM uric acid equivalents” by comparison against the uric acid standard concentration curve. This value is then used to obtain the “copper reducing equivalents” by multiplying the uric acid equivalent concentrations by 2189(µM Cu2+/mM uric acid). This final value is proportional to the TAC of the sample and used for data analysis.  2.5 Data analysis  All of the results will be expressed as the mean ± S.E.M. Statistics will be performed using SPSS 16 software (SPSS Inc., USA). Difference between means will be determined by independent t-test for mitochondrial complex activity. Difference between groups for protein carbonylation and TAC will be subjected to one-way analysis of  	
    variance and covariance (ANOVA), with statistical significance set as p<0.05. Significant treatments detected (p<0.05) are compared by mean values using post hoc comparisons.  	
    3. Results  3.1 Dose dependent glucocorticoid effects on protein carbonylation Protein carbonylation was measured to quantify oxidative damage to proteins. First, this was done through a colorimetric assay by spectrophotometer for samples treated with 24h corticosterone at 0.031, 0.063, 0.125, 0.25mM concentrations and a control group. Significant differences were found between groups (F4,44= 2.600; p<0.05). Post-hoc analyses showed significant increases compared to controls at 0.063 (107%, p<0.05) and 0.125mM (126%, p<0.05). Doses at 0.031 and 0.25mM did not reach significance (p>0.05). These results are shown in Figure 4.  	
    Figure 4. Protein carbonylation by spectrophotometer colorimetric assay.  PC12 cells were treated for 24h at varying doses of corticosterone. Values are shown as mean ± SE. Differences among groups were determined using a one-way ANOVA, followed by post hoc analysis comparing control groups with drug conditions. *Indicates p<0.05.  Next, we sought to confirm these findings in another commonly used assay for protein carbonylation done with immunoblotting analysis. First, western blots were needed to determine the signals of loading different amounts of protein; it was determined that 20µg would be optimal for each sample (Figure 5A). Also, a testing of  	
    positive controls was done to determine our ability for protein carbonylation immnodetection. For this we used H2O2 treatment for 30 minutes at 0.30 or 0.60M. As expected, H2O2 treatment increased protein carbonylation (Figure 5B). For corticosterone treatment, the doses tested were 0.125mM, the concentration showing the strongest effects in the previous experiments, and 0.031, which was the low dose that showed the least difference from controls. There was a significant difference found between these groups (F2,9= 4.785; p<0.05). Post-hoc analyses revealed significant increases in cells treated with 0.125mM corticosterone (159%, p<0.05), while the lower 0.031mM concentration showed no changes from the control condition (Figure 6). Together, these results show that corticosterone had significant oxidative damage effects at 0.125mM and not at lower concentrations at 0.031mM.  	
    Figure 5. Pilot tests for immunoblotting experiments.  (A) Loading control signals for DNP antibody immunofluorescence. (B) Positive controls with H2O2 treated for 30 minutes.  	
    Figure 6. Protein carbonylation by immunoblotting analysis.  PC12 cells were treated with corticosterone at 0.031 and 0.125mM corticosterone for 24h. Values are shown as mean ± SE. A representative blot is shown on the right side. Differences among groups were determined using a one-way ANOVA, followed by post hoc analysis comparing control groups with drug conditions. *Indicates p<0.05.  	
    3.2 The effect of corticosterone on activities of mitochondrial complex I and III  Complex I and III are the main sites where electrons are leaked to oxygen, resulting in production of ROS that cause oxidative stress. To determine if corticosterone-induced oxidative damage results from dysfunctional mitochondrial complex I and III, we further analyze the effect of corticosterone on activities of complex I and III in cultured PC12 cells. Cells were treated with vehicle (control) or corticosterone at 0.125mM for 24 hours. We found a significant difference between complex I, but not complex III, and controls. Complex I showed a decrease of 33.3% in cells treated with corticosterone (T(8)=2.549, p<0.05), whereas complex III had a non-significant decrease of 7.3%. Results are shown in Figure 7.  	
    Figure 7. Mitochondrial complex I and III activity.  PC12 cells treated with 0.125mM corticosterone for 24h (CORT) or a control (CTL) condition with only DMSO vehicle. (A) Complex I is a multi-subunit membrane protein of the mitochondrial electron transport chain which transfers electrons from NADH to unbiquinone. Activity is measured by quantifying the oxidation of NADH to NAD+ spectrophotometrically at 340nm. (B) Complex III is a multi-subunit membrane protein of the mitochondrial electron transport chain which transfers electrons from ubiquinone to cytochrome c. Activity is measured by quantifying the reduction of cytrochrome c spectrophotometrically at 550nm. Results are reported as the mean ± SE, and compared by independent t-test. *Indicates p<0.05.  	
    3.3. The effect of corticosterone on total antioxidant capacity Deficient endogenous antioxidant defense can also cause oxidative stress. In order to understand if impaired antioxidant defense contributes to corticosterone-induced oxidative damage, we further analyzed the effect of corticosterone on total antioxidant capacity in PC12 cells.  The antioxidant activity of uric acid was used as standard. As shown in Figure 8, uric acid dose-dependently reduced copper (II) to copper (I). Cells were treated with vehicle (control) or corticosterone at concentrations of 0.031, 0.063, and 0.125 mM for 24 hours. There was a significant difference between groups on TAC in our study (F3,16= 3.437, p<0.05. Post-hoc analyses showed that TAC was significantly increased for cells having corticosterone at 0.031mM (17.6%; p<0.05) compared to controls (Figure 9). There were no significant differences for doses of 0.063 or 0.125mM. Spectrophotometer readings were first converted to Uric Acid equivalents by the standard curve and then converted to copper reducing equivalents, as the assay measured antioxidant capacity by the ability to reduce a copper containing solution.  	
    Figure 8. Uric acid standard curve for total antioxidant capacity assay.  This assay is based on measurement of the reduction of copper (II) to copper (I) by antioxidant uric acid. Upon reduction, the copper (I) ion will react with a coupling chromogenic reagent that produces a color with a maximum absorbance at 490 and read in a spectrophotometer.  	
    Figure 9. Total antioxidant capacity.  PC12 cells were treated with 0.031, 0.063, 0.125 mM corticosterone for 24 hours or with vehicle (CTL). Total antioxidant capacity was measured by ability to reduce copper (II) to copper (I) in a copper solution. Values are shown as mean ± SE. Differences among groups were determined using a one-way ANOVA, followed by post hoc analysis comparing control groups with drug conditions. *Indicates p<0.05.  	
    4. Discussion  The present study sought to elucidate the effects of GCs on oxidative stress and mitochondrial dysfunction. We determined that at certain concentrations of GCs, protein oxidative damage was increased, mitochondrial ETC complex I activity was decreased, complex III was unchanged, and antioxidant defenses did not show an adequate capacity to neutralize high concentrations of GC-induced oxidative stress. These results suggest some of the negative cellular consequences of excessive stress responses, and these mechanisms may underlie the neurobiology of brain diseases such as BD.  Measurement of changes in protein carbonyl content was used to determine oxidative stress mediated protein oxidation. Within the range of concentrations used in our experiments, it was determined in vitro that 0.063mM and 0.125mM concentrations of corticosterone was able to induce significant increases in protein oxidative damage. As corticosterone is a primary output of the HPA axis during a stress response, these data suggest that stress can have deleterious effects on the cell through oxidative damage to proteins. This represents a downstream effect on cellular macromolecules as a result of increased ROS, and indeed, previous studies have demonstrated the ability of GCs to increase the production of ROS (You et al., 2009). These oxidative modifications are likely to infer abnormal expression or function of afflicted proteins, and suggest that increases in ROS do indeed have lasting consequences on cells. In neurons, this could have a myriad of effects on signal transduction, plasticity, synaptic function that are important in BD and other neuropsychiatric diseases depending on the types of proteins  	
    that are being carbonylated to a greater extent. As such, the next steps in future research should be to elucidate proteins being targeted by oxidative stress, either by a comprehensive screening of the changes in protein expression or function of neurons, or by specifically examining the important molecular players implicated in given diseases. Indeed, a recent experiment by Tan et al. (2011) showed that 4-hydroxy-2-nonenal (4HNE), a lipid peroxidation product, can form protein adducts with vesicular monoamine transporter 2, which is crucial in monoamine neurotransmitter packaging and extensively proposed to be involved in the aetiology and pathology of psychiatric disorders. The relationship between high GCs and oxidative stress is revealing for our understanding of BD, as both increases in GCs and oxidative damage have been identified in patients. In comparing cortisol secretion profiles of those with BD, patients in both depressed and manic episodes showed increases compared to controls (Cervantes et al. 2001). A recent study of postmortem frontal cortex tissue of subjects with BD showed increases in protein carbonyl content (Andreazza et al., 2010). To this end, it is interesting that the relationship between GC levels and oxidative stress demonstrated here may help elucidate a link between these separate bodies of research.  Increases in protein oxidation have been observed in previous studies using of GC treatment in cultured cells (Lee et al., 2002) and in rats (You et al., 2009). The data here provide important replications of these reports, but also give an extended examination of different dose responses on protein oxidation, which was not done in those studies. Identifying dose dependent stress responses are important as GC signaling is known to have dose dependent effects on gene modulation and neuronal function (Prager and  	
    Johnson, 2009), stress responses are not entirely deleterious and are supposed to be adaptive mechanisms until they are activated in excess (de Kloet et al., 1999), and magnitude of GC release is an important function of disease, as many neuropsychiatric disorders including BD are characterized by hypercortisolemia (Daban et al., 2005). Our results show that at 0.031mM corticosterone there is a small but insignificant increase in protein oxidation, demonstrating that this dose is below the threshold for disease related processes. The important adjunct to these results is the experiments of TAC, in which we showed that there were significant increases in antioxidant activity at the same dose. This suggests that cellular antioxidant capacity increases to neutralize GC-induced ROS production at manageable levels and was able to prevent increases in protein oxidation. Conversely, at 0.063 and 0.125mM concentrations, oxidative damage was significantly increased, whereas antioxidant capacity had no significant differences from controls. Oxidative stress is defined as the state where free radical production overwhelms antioxidant capacity; thus in the presence of excessively higher GC levels, as observed in vitro at 0.063 or 0.125mM, antioxidant defenses are inadequate or may be quickly exhausted, and significant increases in protein oxidation occur, which may be analogous to what occurs during disease states. Concentrations around 0.031mM may be at a level that is physiologically controllable by the cell, and thus antioxidant capacity remains robust and keeps down the level of protein carbonylation. This concept of an adaptive limit to stress responses was demonstrated by a previous study showing that acute stressors can trigger increases in SOD antioxidant activity, but animals that were chronically stressed had an impaired ability to do the same (Filipovic and Pajovic, 2009).  	
    Surprisingly, at a higher dose of 0.25mM levels of protein carbonylation did not show increases from control. In the colorimetric assay, this dose appeared to be nonsignificantly increased to a lesser extent than the smaller concentration behind it. While this might not be readily expected, comparisons with other studies of GC effects in vitro do suggest that concentrations around this range significantly induce cytotoxicity and cell death. In one such study, Zhao et al. (2009) reported that at doses around 0.3mM, corticosterone treatment for 24h reduced cell viability measures of around 50%. Thus, in the present experiments, in which carbonylation can only be measured in intact and viable cells, these extremely high doses are killing many of the cells within the 24 hours before they are harvested for testing. It is therefore possible that the high amounts of GCs in these treatments are activating necrotic or apoptotic cell death mechanisms aside from the oxidative stress pathways that we are currently interested in. They may be overwhelming a myriad of other cellular functions such as ion channel excitability, calcium storages, and second messenger systems, therefore producing toxic effects that supersede the accumulation of ROS and protein carbonylation. This is in contrast to the 0.125mM concentration, in which GC levels are below cytotoxic levels but generated ROS still cause substantial protein oxidation.  Increases in levels of oxidative stress are likely due in large part to mitochondrial dysfunction. To determine if GCs can impair ETC function, we measured the activity of complex I and III. We found that complex I, but not complex III, activity was decreased by corticosterone. This indicates that corticosterone-induced oxidative stress is mainly caused by dysfunctional complex I. While previous studies have demonstrated GC-  	
    induced abnormalities of mitochondrial functions such as ATP production and maintenance of membrane potential (Du et al, 2009), our results are the first to show specific impairment of the ETC complex known to contribute to ROS production. Mitochondrial complexes I and III are main sites where electrons are leaked to generate ROS as they are being passed through the ETC (Halliwell, 1997). It has been well documented that decreases in Complex I activity leads to increase in superoxide production, which goes on to form other ROS that induce oxidative damage to proteins (Lenaz, 2001). A previous study has already shown that behaviorally induced chronic stress can decrease combined activity of complexes I and III (Madrigal et al., 2001), which compliments our findings of a direct link between the stress hormone and complex I dysfunction. Interestingly, it has been found that complex III only contributes to the formation of superoxide radical when the mitochondrial membrane potential is high (Nohl et al., 2005). Since previous studies have shown that GCs decrease mitochondrial membrane potential (Seo et al., 2011, Takahashi et al., 2002), this suggests that under stress and high GC conditions, increases in oxidative stress may not involve contributions from complex III. Importantly, it is understood that the production of superoxide radicals is far lower compared to the rates produced by complex I (Murphy, 2009), perhaps meaning that impairment of complex III may be less important in oxidative stress as well as in disease conditions. As GCs have a variety of signaling mechanisms, there are different possibilities for how they may be impairing complex I activity. One major possibility is through genomic regulation, as GC’s have been shown to downregulate expression of mitochondrial genes encoding ETC proteins (Mutsaers and Tofighi et al., 2012). Using quantitative polymerase chain reaction (Mutsaers and Tofighi (2012)  	
    showed that dexamethasone altered gene expression of two central subunits ND1 and ND4. As the central subunits are sufficient to perform all of the bioenergetics functions of the complex (Brandt, 2006), it is very likely that these two particular subunits are contributing to the loss of electrons as they are being passed along. While they also showed altered expression of the cyt b gene in complex III, the current data do not suggest that it significantly impairs enzyme function. It is possible that since they used dexamethasone, a GR agonist, the difference may be that our study used corticosterone, which binds to both the GR and the MR. Also, oxidative modifications to proteins of the ETC may increase the amount of electron leakage to molecular oxygen and ROS formation. A study by Naoi et al. (2005) showed increases in 3-nitrotyrosine for complex I subunits compared to other ETC proteins when the SH-SY5Y neuroblastoma cell line was incubated with peroxynitrite, suggesting that oxidative stress from reactive nitrogen species may result in the inhibition of complex I activity. It is also possible that increases in oxidative stress is damaging mtDNA in order to impair ETC gene expression and function, as ROS are known to be able to damage nucleic acids as well (Imlay and Linn, 1988). Oxidative damage on mtDNA leads to mispairing and point mutations, and deletions in mtDNA, all of which are linked to etiology of many diseases (Richter 1992). Mitochondrial DNA are considered to be more sensitive than nuclear DNA to oxidative damage, perhaps because of proximity to main source of oxidant generation, or its limited DNA repair system. Increasing damage to mitochondrial DNA leads to more release of ROS, and a vicious cycle is set into motion. (Finkel & Holbrook 2000).	
    GC effects on glutamate gated calcium ion channels have been well documented, and this may have implications on oxidative stress through excess glutamatergic activity and calcium influx. Glutamate excitotoxicity occurs when heightened glutamate signaling on its receptors causes elevated or prolonged calcium release into the cell, which is known to increase the production of ROS (Wolkowitz et al., 2010). In neurons, there is a clear increase in ROS levels following application of glutamate, and this effect is abolished in calcium free buffers (Kahlert et al., 2005). This is thought to occur through the destabilization of mitochondrial membrane potential (Schinder et al., 2006). Although details on this mechanism remain unclear, mitochondria are sites of calcium storage in the cell, so one way may be through the accumulation of calcium above sequestration levels resulting in respiratory inhibition and ROS production (Leist & Nicotera, 1998). Increasing GC levels lead to glutamate accumulation (Stein-Behrens et al., 1994), and appear to modulate NMDA receptor activity to facilitate calcium influx (Xiao et al., 2010). GCs may have genomic effects that downregulate antioxidant enzyme expression, which can predispose the cell to oxidative injury. Indeed, Mutsaers and Tofighi (2012) estimated that dexamethasone treatment induced altered expression in 43% of the antioxidant genes of the murine-derived multipotent neural stem cell line C17.2. It is entirely possible that GCs increase oxidative stress through the increase of oxidative damage to antioxidant enzymes, altering their function to further increase susceptibility to ROS accumulation. Our current data do not support the notion that GCs decreases antioxidant expression or activity, although it does appear as though antioxidant increases in response to ROS overproduction become impaired at high concentrations of  	
    corticosterone. Our data show that at higher concentrations, 0.063 and 0.125mM in PC12 cells, antioxidant capacity is unchanged from control levels, while protein carbonylation is significantly increased. This is in contrast to a lower dose of corticosterone at 0.031mM, which showed increases in antioxidant capacity, appearing as though it is responding to the threat of increasing ROS. To this end, it may be that high levels of corticosterone impairs or inhibits the recruitment of antioxidant defenses to incoming oxidative insult. Conversely, it is possible that antioxidant capacity may be exhausted after a period of time, with an early increase upon GC treatment, but over the 24 hour chronic drug treatment it becomes exhausted and overwhelmed. Moreover, antioxidant enzymes may be undergoing oxidative damage, which renders them inactive or inefficient in function. Future research will be required to elucidate the nature of this lack of antioxidant response to higher concentrations of GCs. This may be through monitoring antioxidant capacity over time after immediate corticosterone application to see if there are early increases that may have gone undetected in the current experiments. Taken together, it becomes clear that there is a vastly complex interplay between ROS generation, antioxidant activity, and mitochondrial function, and thus none of the GC effects on these parameters are likely to occur in isolation.  The implications of these findings are important for our understanding of BD and other psychiatric disorders. Studies presenting DNA microarray data point to decreased expression of many mRNAs coding for subunits in mitochondrial ETC complexes I – V. The group by Konradi et al. (2004) found decreased expression of genes for Complex I, IV, and V subunits in postmortem hippocampal tissue of BD patients. Decreases in PFC  	
    mRNA for complex I, III, and IV subunits were reported by Iwamoto et al. (2004). Sun et al. (2006) found that 8 genes associated with ETC complexes I, III, IV, and V are downregulated in PFC of BD patients. While there are separately occurring lines of evidence suggesting oxidative stress and mitochondrial dysfunction in BD, there are also many reports of these two processes being linked (Clay et al., 2011). For example, in the study by Andreazza et al. (2010), decreases in complex I subunit NDUFS7 were correlated with increasing protein carbonylation. Moreover, increases in 4-HNE levels of postmortem brain tissue of BD patients were negatively correlated with pH, which may be indicative of a relationship between oxidative stress and mitochondrial dysfunction. Specifically, mitochondrial dysfunction may be leading to both an increase in ROS production and a shift from energy production in the mitochondrial ETC towards glycolysis and lactic acid production.  The effects of oxidative stress and mitochondrial dysfunction from high levels of stress hormones may have widespread deleterious effects on the brain over time, leading to a myriad of pathophysiological consequences that will predispose towards disease. Mounting evidence suggests that mitochondria are directly involved in processes of synaptic plasticity. Mitochondria are enriched in presynaptic terminals, and play important roles in neurotransmission via energy production. In addition, mitochondria buffer the levels of calcium (Billups and Forsythe, 2002). Calcium influx precedes neurotransmitter release and is necessary for vesicle docking and exocytosis. Mitochondrial dysfunction may be responsible for more pronounced calcium spikes and abnormal neurotransmitter release (Quiroz et al., 2008). Deficiencies in the ETC will lead  	
    to impairments in oxidative phosphorylation and subsequent ATP production, and can impair the energy demanding processes of vesicle docking, fusion, and endocytosis. Moreover, GC treatment was shown to decrease ATP production in hypothalamic neurons (Fujita et al., 2009) as well as decreasing activity of the ATP-ase in the ETC (Pandya et al., 2007). The sum of these abnormalities on mitochondria energy metabolism in the brain has been identified in humans using MRS. Energy related metabolite levels and pH values can be visualized using MRS, and these studies have shown that high-energy phosphates such as phosphocreatine and ATP are reduced in BD (Kato et al., 1994; Deicken Feln, & Welner, 1995). There is also evidence to suggest that oxidative stress may be related to impairments in neuroplasticity. For example, in an animal study by Wu et al. (2004), oxidative stress was shown to correlate with brain derived neutrotrophic factor (BDNF) reductions, as well as reductions in CREB and Synapsin I, molecules that are involved in cellular plasticity cascades. Such research suggests that oxidative stress may play a role in the abnormal neuroplastic processes found in BD. Recently, research on the neurobiology of BD has shifted focus towards changes in cellular structure, function, growth, and development, together characterized as mechanisms of neuroplasticity (Schloesser et al., 2008). Interestingly, Kapczinski et al. (2008) showed that serum lipid peroxidation levels were negatively correlated with BDNF levels. Another study using peripheral biomarkers showed that BD patients showed increased oxidative stress and decreased neuron-specific endolase, a neuronal glycolytic enzyme known to mediate neuroplastic pathways and cell survival (MachadoVieira et al., 2007), although the relationship between these variables were not analyzed.  	
    With increasing understanding of how oxidative stress, mitochondrial dysfunction, and glucocorticoids are involved in the pathophysiology of BD, there is the hope that this knowledge will fuel the development of novel therapeutics. Existing mood stabilizer treatments can have severe and debilitating side effects, and the efficacy of these drugs remain limited (Wang, 2007). The possibility of novel drug treatments for BD through the alleviation of oxidative stress has been supported by some human studies. In a double-blind randomized placebo-controlled trial, Berk et al. (2008) used treatment with N-Acetyl Cysteine (NAC) in BD patients in their maintenance phase. NAC is an acetylated derivative of cysteine, which is the rate-limiting precursor in GSH production (Dodd et al., 2008). This study showed that NAC caused a significant improvement in assessments of depression and global occupational and social functioning. More recent reports showed that in a small sample of bipolar II disorder patients, full remission of symptoms was significantly higher in the group given NAC in addition to their regular treatment compared to the placebo group (Magalhaes et al., 2011). Using NAC as adjunctive treatment for BD lead to reductions of depression scores in patients currently suffering from depressive phases (Berk et al., 2011). These studies are consistent with animal studies showing that NAC can reduce amphetamine induced hyperactivity maniclike behaviour, behavioral sensitization and striatal dopamine depletion (Fukami et al., 2004), and can boost GSH levels as well (Dean et al., 2009). With regard to existing mood stabilizing drugs, Machedo-Vieira et al. (2007) monitored the blood samples of unmedicated manic patients given lithium, the current gold standard treatment for BD, and found normalization of the SOD/catalase ratio and a decrease in lipid peroxidation levels. Moreover, lithium alone or in combination with olanzapine appears to relieve lipid  	
    peroxidation and increase total antioxidant status (Aliyazicioglu et al., 2007). In a study of healthy volunteers given therapeutic doses of lithium for 2-4 weeks, results showed decreases in H2O2 levels (Khairova et al., 2012). These studies are useful in providing evidence for the therapeutic effect of antioxidant properties in current mood stabilizer treatments. Similar conclusions can be made about the productivity of HPA axis intervention. RU486 in BD patients alleviated mood and neurocognitive symptoms (Young, et al. 2004). To summarize most of the clinical randomized control and crossover trial studies of anti-glucocorticoid agents, Gallagher et al. (2009) conducted a review and suggested that GR receptor antagonists and cortisol production inhibitors were able to alleviate depressive symptoms in BD. Moreover, it has been shown that RU-486 does specifically have the ability to attenuate cortisol levels in patients up to 21 days after being on the drug (Gallagher et al., 2008). The mineralocorticoid receptor antagonist spironolactone was able to alleviate residual symptoms and improve stress responses in a small preliminary study (Juruena et al., 2009).  While the current study provides evidence of increases in oxidative stress and mitochondrial dysfunction from GCs, there are limitations in the interpretations of our results. First, although in vitro studies provide tightly controlled study of cellular and molecular mechanisms of interest, they provide a very limited scope in extrapolating these conclusions to full-fledged physiological systems in vivo. The stress response system as regulated by the HPA axis is a process that involves the entire body, through a wide involvement of many organs, cell types, and neurobiological systems. The incredible versatility of GC signaling through MRs, GRs, genomic, and non-genomic  	
    mechanisms indicates the possible diversity of different stressors and the contexts with which they occur, both externally and internally. All of these important details are not included when using a very reductionist method of modeling the stress response by incubating cells in corticosterone dissolved in culture media. When released into the bloodstream, GCs are able to affect all areas of the brain, and the immense heterogeneity of neuronal cell types makes it difficult to make simple generalizations on the effects of oxidative stress and mitochondrial dysfunction. Moreover, the functional consequences of protein oxidation can essentially be limitless, as free radical induced damage is largely non-specific, and each protein with their individual sequence and structure could be affected differently by the addition of carbonyl groups. Perhaps most importantly, both the stress response and mental illnesses such as BD are processes of mind and behavior, which cannot be studied in cell cultures. Despite the limitations on interpreting the present data, a more comprehensive picture can be gained when these results are compared with other studies using animals and humans. Indeed, the GC induced changes in protein carbonylation, complex I activity, and TAC are comparable to other studies using chronically stressed animal models, in vivo GC administration, and experiments on relevant biomarkers in human populations. In attempting to better understand BD, the current study is also limited in modeling the pathophysiological state of the illness. Indeed, the dosages and time course of the GC treatments used here cannot be validated to be analogous in the human conditions of stress.  To this end, further research will be required to address the limits of the findings offered in the present study. While previous studies have used behaviorally induced stress  	
    or corticosteroid injections to increase oxidative stress, it remains to be seen if the same paradigms will result in mitochondrial dysfunction, particularly in complex I activity as shown here. It would also be valuable if the same dose dependent changes in protein carbonylation and antioxidant capacity can be reproduced in animals. To further understand the consequences of oxidative stress and mitochondrial dysfunction, it will be important for future research to show that these abnormalities can correlate with or cause the functional impairments characteristic of neuropsychiatric disease processes such as dysfunction in monoamine neurotransmission, reductions in dendritic morphology, loss of neurogenesis, or decreases in long term potentiation (LTP), a form of synaptic memory. For example, integral players of LTP such as NMDA and AMPA receptors could be examined for increased carbonylation. Moreover, in order to further demonstrate the link between GCs and oxidative stress, it would be interesting to see if manipulating one of them would change the pathophysiological effects of the other. For example, GCs and oxidative stress have both separately been shown to reduce dendritic spine morphology, so a possible study could be to test the effects of an antioxidant on GCinduced spine reductions, or the effects of a GR-antagonist on ROS-induced spine reductions. Lastly, it would be interesting to link oxidative stress and GCs in clinical populations as to better understand diseases such as BD. Such studies could be correlating biomarkers of HPA axis abnormalities with those for oxidative stress and mitochondrial dysfunction. For example, is there a relationship between deficiencies in the dexamethasone suppression test and increased peripheral or post mortem markers for protein or lipid oxidation? Other such research approaches could be to determine if antiglucocorticoid drugs in patients can normalize oxidative stress parameters (such as  	
    blood biomarkers) or mitochondrial function (such as energy metabolism through investigations with MRS). The same could be shown of antioxidant treatments for normalization of HPA axis activity. These studies would be important steps in examining candidate drugs in the development of novel therapeutics for disease.  In conclusion, the data here provide some novel insights into mechanisms that are important in understanding BD and other brain-based disorders. GCs were demonstrated to change levels of protein carbonylation depending on the dose, and at concentrations resulting in increased oxidative damage, mitochondrial complex I activity was decreased and cellular antioxidant capacity was not able to upregulate in response to the stress (Figure 10). These findings extend the current scope of the literature by identifying complex I dysfunction and blunted antioxidant defenses as mediating factors in the oxidative stress caused by GCs. This study was also able to show adaptive coping states of stress responses that were not deleterious to the cell, as lower concentrations of corticosterone application did not significantly increase carbonylation and antioxidant defenses showed adequate increases to neutralize the oxidative threat. The research done here sets the stage for further characterization of the functional consequences of oxidative damage and mitochondrial dysfunction on the level of the individual neuron, neural networks, and the overlying behavioral changes. This progress in understanding is important for continuing to elucidate the neurobiology of brain disorders such as BD, which can hopefully drive the development of better treatments for those afflicted.  	
    Figure 10. Glucocorticoids, oxidative stress, and mitochondrial dysfunction.  Under hyperactive stress responses due to excessive activity of the HPA axis, there is an increase in the glucocorticoid (GC) concentrations released compared to normal (A). Increases in GCs can induce mitochondrial electron transport chain (ETC) dysfunction, as demonstrated in our findings showing decreased complex I activity (B). When the ETC is not functioning properly, there will be an increased leakage of electrons to form reactive oxygen species (ROS), which in accumulation leads to oxidative stress (C). Typically, ROS is neutralized by endogenous antioxidant defense systems, but our results show that high levels of GCs impair the antioxidant increase in response to oxidative stress (D). Combined, this results in oxidative damage to cellular macromolecules in the brain, with our current data highlighting increases in protein carbonylation (E).  	
    References Abidin, I, Yargiçoglu, P, Agar, A, Gümüslü, S, Aydin, S, Oztürk, O, Sahin, E. 2004. The effect of chronic restraint stress on spatial learning and memory: relation to oxidant stress. The International journal of neuroscience 114: 683-99. Ahlbom, E, Gogvadze, V, Chen, M, Celsi, G, Ceccatelli, S. 2000. Prenatal exposure to high levels of glucocorticoids increases the susceptibility of cerebellar granule cells to oxidative stress-induced cell death. Proceedings of the National Academy of Sciences of the United States of America 97: 14726-30. Aliyazicioğlu, R, Kural, B, Colak, M, Karahan, SC, Ayvaz, S, Değer, O. 2007. Treatment with lithium, alone or in combination with olanzapine, relieves oxidative stress but increases atherogenic lipids in bipolar disorder. The Tohoku journal of experimental medicine 213: 79-87. Andreazza, AC, Shao, L, Wang, J-F, Young, LT. 2010. Mitochondrial complex I activity and oxidative damage to mitochondrial proteins in the prefrontal cortex of patients with bipolar disorder. Archives of general psychiatry 67: 360-8. Berk, M, Copolov, DL, Dean, O, Lu, K, Jeavons, S, Schapkaitz, I, Anderson-Hunt, M, Bush, AI. 2008. N-acetyl cysteine for depressive symptoms in bipolar disorder--a double-blind randomized placebo-controlled trial. Biological psychiatry 64: 468-75. Berk, M, Dean, O, Cotton, SM, Gama, CS, Kapczinski, F, Fernandes, BS, Kohlmann, K, Jeavons, S, Hewitt, K, Allwang, C, Cobb, H, Bush, AI, Schapkaitz, I, Dodd, S,  	
    Malhi, GS. 2011. The efficacy of N-acetylcysteine as an adjunctive treatment in bipolar depression: An open label trial. Journal of affective disorders. Billups, B, Forsythe, ID. 2002. Presynaptic mitochondrial calcium sequestration influences transmission at mammalian central synapses. The Journal of neuroscience  : the official journal of the Society for Neuroscience 22: 5840-7. Bradford, MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical biochemistry 72: 248-54. Brandt, U. 2006. Energy converting NADH:quinone oxidoreductase (complex I). Annual review of biochemistry 75: 69-92. Bénit, P, Slama, A, Rustin, P. 2008. Decylubiquinol impedes mitochondrial respiratory chain complex I activity. Molecular and cellular biochemistry 314: 45-50. Cervantes, P, Gelber, S, Kin, FN, Nair, VN, Schwartz, G. 2001. Circadian secretion of cortisol in bipolar disorder. Journal of psychiatry & neuroscience  : JPN 26: 411-6. Che, Y, Wang, J-F, Shao, L, Young, T. 2010. Oxidative damage to RNA but not DNA in the hippocampus of patients with major mental illness. Journal of psychiatry & neuroscience  : JPN 35: 296-302. Clay, HB, Sillivan, S, Konradi, C. 2011. Mitochondrial dysfunction and pathology in bipolar disorder and schizophrenia. International journal of developmental  	
    neuroscience  : the official journal of the International Society for Developmental Neuroscience 29: 311-24. Cvijić, G, Radojicić, R, Djordjević, J, Davidović, V. 1995. The effect of glucocorticoids on the activity of monoamine oxidase, copper-zinc superoxide dismutase and catalase in the rat hypothalamus. Functional neurology 10: 175-81. Daban, C, Vieta, E, Mackin, P, Young, a H. 2005. Hypothalamic-pituitary-adrenal axis and bipolar disorder. The Psychiatric clinics of North America 28: 469-80. Datson, NA, Morsink, MC, Meijer, OC, de Kloet, ER. 2008. Central corticosteroid actions: Search for gene targets. European journal of pharmacology 583: 272-89. Datson, NA, van der Perk, J, de Kloet, ER, Vreugdenhil, E. 2001. Identification of corticosteroid-responsive genes in rat hippocampus using serial analysis of gene expression. The European journal of neuroscience 14: 675-89. Dean, OM, van den Buuse, M, Bush, a I, Copolov, DL, Ng, F, Dodd, S, Berk, M. 2009. A role for glutathione in the pathophysiology of bipolar disorder and schizophrenia? Animal models and relevance to clinical practice. Current medicinal chemistry 16: 2965-76. Dean, RT, Fu, S, Stocker, R, Davies, MJ. 1997. Biochemistry and pathology of radicalmediated protein oxidation. The Biochemical journal 324 ( Pt 1: 1-18. Deicken, RF, Weiner, MW, Fein, G. 1995. Decreased temporal lobe phosphomonoesters in bipolar disorder. Journal of affective disorders 33: 195-9.  	
    Demonacos, C, Djordjevic-Markovic, R, Tsawdaroglou, N, Sekeris, CE. 1995. The mitochondrion as a primary site of action of glucocorticoids: the interaction of the glucocorticoid receptor with mitochondrial DNA sequences showing partial similarity to the nuclear glucocorticoid responsive elements. The Journal of steroid biochemistry and molecular biology 55: 43-55. Dodd, S, Dean, O, Copolov, DL, Malhi, GS, Berk, M. 2008. N-acetylcysteine for antioxidant therapy: pharmacology and clinical utility. Expert opinion on biological therapy 8: 1955-62. Du, J, McEwen, B, Manji, HK. 2009. Glucocorticoid receptors modulate mitochondrial function: A novel mechanism for neuroprotection. Communicative & integrative biology 2: 350-2. Einat, H, Manji, HK. 2006. Cellular plasticity cascades: genes-to-behavior pathways in animal models of bipolar disorder. Biological psychiatry 59: 1160-71. Evanson, NK, Herman, JP, Sakai, RR, Krause, EG. 2010. Nongenomic actions of adrenal steroids in the central nervous system. Journal of neuroendocrinology 22: 846-61. Filipović, D, Pajović, SB. 2009. Differential regulation of CuZnSOD expression in rat brain by acute and/or chronic stress. Cellular and molecular neurobiology 29: 67381. Finkel, T, Holbrook, NJ. 2000. Oxidants, oxidative stress and the biology of ageing. Nature 408: 239-47.  	
    Floyd, RA. 1999. Antioxidants, oxidative stress, and degenerative neurological disorders. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.) 222: 236-45. Fridovich, I. 1978. The biology of oxygen radicals. Science (New York, N.Y.) 201: 87580. Froger, N, Palazzo, E, Boni, C, Hanoun, N, Saurini, F, Joubert, C, Dutriez-Casteloot, I, Enache, M, Maccari, S, Barden, N, Cohen-Salmon, C, Hamon, M, Lanfumey, L. 2004. Neurochemical and behavioral alterations in glucocorticoid receptor-impaired transgenic mice after chronic mild stress. The Journal of neuroscience  : the official journal of the Society for Neuroscience 24: 2787-96. Fujita, C, Ichikawa, F, Teratani, T, Murakami, G, Okada, T, Shinohara, M, Kawato, S, Ohta, Y. 2009. Direct effects of corticosterone on ATP production by mitochondria from immortalized hypothalamic GT1-7 neurons. The Journal of steroid biochemistry and molecular biology 117: 50-5. Fukami, G, Hashimoto, K, Koike, K, Okamura, N, Shimizu, E, Iyo, M. 2004. Effect of antioxidant N-acetyl-L-cysteine on behavioral changes and neurotoxicity in rats after administration of methamphetamine. Brain research 1016: 90-5. Gallagher, P, Malik, N, Newham, J, Ah, Y, In, F, Mackin, P. 2009. Antiglucocorticoid treatments for mood disorders ( Review ). Library.  	
    Gallagher, P, Watson, S, Elizabeth Dye, C, Young, AH, Nicol Ferrier, I. 2008. Persistent effects of mifepristone (RU-486) on cortisol levels in bipolar disorder and schizophrenia. Journal of psychiatric research 42: 1037-41. Gawryluk, JW, Wang, J-F, Andreazza, AC, Shao, L, Young, LT. 2011. Decreased levels of glutathione, the major brain antioxidant, in post-mortem prefrontal cortex from patients with psychiatric disorders. The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP) 14: 123-30. Goodman, Y, Bruce, a J, Cheng, B, Mattson, MP. 1996. Estrogens attenuate and corticosterone exacerbates excitotoxicity, oxidative injury, and amyloid beta-peptide toxicity in hippocampal neurons. Journal of neurochemistry 66: 1836-44. Groeneweg, FL, Karst, H, de Kloet, ER, Joëls, M. 2011. Rapid non-genomic effects of corticosteroids and their role in the central stress response. The Journal of endocrinology 209: 153-67. Halliwell, B. 1992. Reactive oxygen species and the central nervous system. Journal of neurochemistry 59: 1609-23. Halliwell, B. 1997. What nitrates tyrosine? Is nitrotyrosine specific as a biomarker of peroxynitrite formation in vivo? FEBS letters 411: 157-60. Harman, D. 1956. Aging: a theory based on free radical and radiation chemistry. Journal of gerontology 11: 298-300.  	
    Harvey, BH, Oosthuizen, F, Brand, L, Wegener, G, Stein, DJ. 2004. Stress-restress evokes sustained iNOS activity and altered GABA levels and NMDA receptors in rat hippocampus. Psychopharmacology 175: 494-502. Herman, JP, Figueiredo, H, Mueller, NK, Ulrich-Lai, Y, Ostrander, MM, Choi, DC, Cullinan, WE. 2003. Central mechanisms of stress integration: hierarchical circuitry controlling hypothalamo-pituitary-adrenocortical responsiveness. Frontiers in neuroendocrinology 24: 151-80. Holsboer, F, Lauer, CJ, Schreiber, W, Krieg, J-C. 1995. Altered Hypothalamic-PituitaryAdrenocortical Regulation in Healthy Subjects at High Familial Risk for Affective Disorders. Neuroendocrinology 62: 340-347. Iborra, FJ, Kimura, H, Cook, PR. 2004. The functional organization of mitochondrial genomes in human cells. BMC biology 2: 9. Imlay, JA, Linn, S. 1988. DNA damage and oxygen radical toxicity. Science (New York, N.Y.) 240: 1302-9. Iwamoto, K, Bundo, M, Kato, T. 2005. Altered expression of mitochondria-related genes in postmortem brains of patients with bipolar disorder or schizophrenia, as revealed by large-scale DNA microarray analysis. Human molecular genetics 14: 241-53. Joëls, M, Karst, H, DeRijk, R, de Kloet, ER. 2008. The coming out of the brain mineralocorticoid receptor. Trends in neurosciences 31: 1-7.  	
    Juruena, MF, Gama, CS, Berk, M, Belmonte-de-Abreu, PS. 2009. Improved stress response in bipolar affective disorder with adjunctive spironolactone (mineralocorticoid receptor antagonist): case series. Journal of psychopharmacology (Oxford, England) 23: 985-7. Kahlert, S, Zündorf, G, Reiser, G. Glutamate-mediated influx of extracellular Ca2+ is coupled with reactive oxygen species generation in cultured hippocampal neurons but not in astrocytes. Journal of neuroscience research 79: 262-71. Kapczinski, F, Frey, BN, Andreazza, AC, Kauer-Sant’Anna, M, Cunha, ABM, Post, RM. 2008. Increased oxidative stress as a mechanism for decreased BDNF levels in acute manic episodes. Revista brasileira de psiquiatria (São Paulo, Brazil  : 1999) 30: 2435. Karst, H, Berger, S, Turiault, M, Tronche, F, Schütz, G, Joëls, M. 2005. Mineralocorticoid receptors are indispensable for nongenomic modulation of hippocampal glutamate transmission by corticosterone. Proceedings of the National Academy of Sciences of the United States of America 102: 19204-7. Kato, T, Shioiri, T, Murashita, J, Hamakawa, H, Inubushi, T, Takahashi, S. 1994. Phosphorus-31 magnetic resonance spectroscopy and ventricular enlargement in bipolar disorder. Psychiatry research 55: 41-50. Kato, T, Stine, OC, McMahon, FJ, Crowe, RR. 1997. Increased levels of a mitochondrial DNA deletion in the brain of patients with bipolar disorder. Biological psychiatry 42: 871-5.  	
    Khairova, R, Pawar, R, Salvadore, G, Juruena, MF, de Sousa, RT, Soeiro-de-Souza, MG, Salvador, M, Zarate, CA, Gattaz, WF, Machado-Vieira, R. 2012. Effects of lithium on oxidative stress parameters in healthy subjects. Molecular medicine reports 5: 680-2. Kino, T, Tiulpakov, A, Ichijo, T, Chheng, L, Kozasa, T, Chrousos, GP. 2005. G protein beta interacts with the glucocorticoid receptor and suppresses its transcriptional activity in the nucleus. The Journal of cell biology 169: 885-96. De Kloet, ER, Vreugdenhil, E, Oitzl, MS, Joëls, M. 1998. Brain corticosteroid receptor balance in health and disease. Endocrine reviews 19: 269-301. de Kloet, ER, Joëls, M, Holsboer, F. 2005. Stress and the brain: from adaptation to disease. Nature reviews. Neuroscience 6: 463-75. de Kloet, ER, Oitzl, MS, Joëls, M. 1999. Stress and cognition: are corticosteroids good or bad guys? Trends in neurosciences 22: 422-6. Kolber, BJ, Wieczorek, L, Muglia, LJ. 2008. Hypothalamic-pituitary-adrenal axis dysregulation and behavioral analysis of mouse mutants with altered glucocorticoid or mineralocorticoid receptor function. Stress (Amsterdam, Netherlands) 11: 321-38. Konradi, C, Eaton, M, MacDonald, ML, Walsh, J, Benes, FM, Heckers, S. 2004. Molecular evidence for mitochondrial dysfunction in bipolar disorder. Archives of general psychiatry 61: 300-8.  	
    Krugers, HJ, Alfarez, DN, Karst, H, Parashkouhi, K, van Gemert, N, Joëls, M. 2005. Corticosterone shifts different forms of synaptic potentiation in opposite directions. Hippocampus 15: 697-703. Lee, AL, Ogle, WO, Sapolsky, RM. 2002. Stress and depression: possible links to neuron death in the hippocampus. Bipolar disorders 4: 117-28. Lee, H-C, Wei, Y-H. 2005. Mitochondrial biogenesis and mitochondrial DNA maintenance of mammalian cells under oxidative stress. The international journal of biochemistry & cell biology 37: 822-34. Lee, K-W, Kim, J-B, Seo, J-S, Kim, T-K, Im, J-Y, Baek, I-S, Kim, K-S, Lee, J-K, Han, P-L. 2009. Behavioral stress accelerates plaque pathogenesis in the brain of Tg2576 mice via generation of metabolic oxidative stress. Journal of neurochemistry 108: 165-75. Leist, M, Nicotera, P. 1998. Apoptosis, excitotoxicity, and neuropathology. Experimental cell research 239: 183-201. Lenaz, G. 2001. The mitochondrial production of reactive oxygen species: mechanisms and implications in human pathology. IUBMB life 52: 159-64. Liu, W, Wang, J, Sauter, NK, Pearce, D. 1995. Steroid receptor heterodimerization demonstrated in vitro and in vivo. Proceedings of the National Academy of Sciences of the United States of America 92: 12480-4.  	
    Luo, C, Long, J, Liu, J. 2008. An improved spectrophotometric method for a more specific and accurate assay of mitochondrial complex III activity. Clinica chimica acta; international journal of clinical chemistry 395: 38-41. Machado-Vieira, R, Andreazza, AC, Viale, CI, Zanatto, V, Cereser, V, da Silva Vargas, R, Kapczinski, F, Portela, LV, Souza, DO, Salvador, M, Gentil, V. 2007. Oxidative stress parameters in unmedicated and treated bipolar subjects during initial manic episode: a possible role for lithium antioxidant effects. Neuroscience letters 421: 336. Madrigal, JL, Olivenza, R, Moro, M a, Lizasoain, I, Lorenzo, P, Rodrigo, J, Leza, JC. 2001. Glutathione depletion, lipid peroxidation and mitochondrial dysfunction are induced by chronic stress in rat brain. Neuropsychopharmacology  : official publication of the American College of Neuropsychopharmacology 24: 420-9. Maher, P, Schubert, D. 2000. Signaling by reactive oxygen species in the nervous system. Cellular and molecular life sciences  : CMLS 57: 1287-305. Mansour, AM, Nass, S. 1970. In vivo cortisol action on RNA synthesis in rat liver nuclei and mitochondria. Nature 228: 665-7. McEwen, BS. 2007. Physiology and neurobiology of stress and adaptation: central role of the brain. Physiological reviews 87: 873-904.  	
    McIntosh, LJ, Cortopassi, KM, Sapolsky, RM. 1998b. Glucocorticoids may alter antioxidant enzyme capacity in the brain: kainic acid studies. Brain research 791: 215-22. McIntosh, LJ, Hong, KE, Sapolsky, RM. 1998a. Glucocorticoids may alter antioxidant enzyme capacity in the brain: baseline studies. Brain research 791: 209-14. McIntosh, LJ, Sapolsky, RM. 1996. Glucocorticoids increase the accumulation of reactive oxygen species and enhance adriamycin-induced toxicity in neuronal culture. Experimental neurology 141: 201-6. McReynolds, JR, Donowho, K, Abdi, A, McGaugh, JL, Roozendaal, B, McIntyre, CK. 2010. Memory-enhancing corticosterone treatment increases amygdala norepinephrine and Arc protein expression in hippocampal synaptic fractions. Neurobiology of learning and memory 93: 312-21. Miquel, J. 1992. An update on the mitochondrial-DNA mutation hypothesis of cell aging. Mutation research 275: 209-16. Moutsatsou, P, Psarra, a M, Tsiapara, a, Paraskevakou, H, Davaris, P, Sekeris, CE. 2001. Localization of the glucocorticoid receptor in rat brain mitochondria. Archives of biochemistry and biophysics 386: 69-78. Murphy, MP. 2009. How mitochondria produce reactive oxygen species. The Biochemical journal 417: 1-13.  	
    Mutsaers, HAM, Tofighi, R. 2012. Dexamethasone Enhances Oxidative Stress-Induced Cell Death in Murine Neural Stem Cells. Neurotoxicity research. Naoi, M, Maruyama, W, Shamoto-Nagai, M, Yi, H, Akao, Y, Tanaka, M. 2005. Oxidative stress in mitochondria: decision to survival and death of neurons in neurodegenerative disorders. Molecular neurobiology 31: 81-93. Nishi, M, Kawata, M. 2006. Brain corticosteroid receptor dynamics and trafficking: Implications from live cell imaging. The Neuroscientist  : a review journal bringing neurobiology, neurology and psychiatry 12: 119-33. Nohl, H, Gille, L, Staniek, K. 2005. Intracellular generation of reactive oxygen species by mitochondria. Biochemical pharmacology 69: 719-23. Pandya, JD, Agarwal, NA, Katyare, SS. 2007. Dexamethasone treatment differentially affects the oxidative energy metabolism of rat brain mitochondria in developing and adult animals. International journal of developmental neuroscience  : the official journal of the International Society for Developmental Neuroscience 25: 309-16. Pinnock, SB, Balendra, R, Chan, M, Hunt, LT, Hunt, LT, Turner-Stokes, T, Herbert, J. 2007. Interactions between nitric oxide and corticosterone in the regulation of progenitor cell proliferation in the dentate gyrus of the adult rat. Neuropsychopharmacology  : official publication of the American College of Neuropsychopharmacology 32: 493-504.  	
    Prager, EM, Johnson, LR. 2009. Stress at the synapse: signal transduction mechanisms of adrenal steroids at neuronal membranes. Science signaling 2: re5. Psarra, A-MG, Sekeris, CE. 2009. Glucocorticoid receptors and other nuclear transcription factors in mitochondria and possible functions. Biochimica et biophysica acta 1787: 431-6. Pérez-Nievas, BG, García-Bueno, B, Caso, JR, Menchén, L, Leza, JC. 2007. Corticosterone as a marker of susceptibility to oxidative/nitrosative cerebral damage after stress exposure in rats. Psychoneuroendocrinology 32: 703-11. Qiu, J, Wang, P, Jing, Q, Zhang, W, Li, X, Zhong, Y, Sun, G, Pei, G, Chen, Y. 2001. Rapid activation of ERK1/2 mitogen-activated protein kinase by corticosterone in PC12 cells. Biochemical and biophysical research communications 287: 1017-24. Quiroz, JA, Gray, NA, Kato, T, Manji, HK. 2008. Mitochondrially mediated plasticity in the pathophysiology and treatment of bipolar disorder. Neuropsychopharmacology  : official publication of the American College of Neuropsychopharmacology 33: 2551-65. Radi, R, Rodriguez, M, Castro, L, Telleri, R. 1994. Inhibition of mitochondrial electron transport by peroxynitrite. Archives of biochemistry and biophysics 308: 89-95. Richter, C. 1992. Reactive oxygen and DNA damage in mitochondria. Mutation research 275: 249-55.  	
    Sandi, C, Davies, HA, Cordero, MI, Rodriguez, JJ, Popov, VI, Stewart, MG. 2003. Rapid reversal of stress induced loss of synapses in CA3 of rat hippocampus following water maze training. The European journal of neuroscience 17: 2447-56. Sato, S, Osanai, H, Monma, T, Harada, T, Hirano, A, Saito, M, Kawato, S. 2004. Acute effect of corticosterone on N-methyl-D-aspartate receptor-mediated Ca2+ elevation in mouse hippocampal slices. Biochemical and biophysical research communications 321: 510-3. Scheller, K, Sekeris, CE, Krohne, G, Hock, R, Hansen, I a, Scheer, U. 2000. Localization of glucocorticoid hormone receptors in mitochondria of human cells. European journal of cell biology 79: 299-307. Schinder, AF, Olson, EC, Spitzer, NC, Montal, M. 1996. Mitochondrial dysfunction is a primary event in glutamate neurotoxicity. The Journal of neuroscience  : the official journal of the Society for Neuroscience 16: 6125-33. Schloesser, RJ, Huang, J, Klein, PS, Manji, HK. 2008. Cellular plasticity cascades in the pathophysiology and treatment of bipolar disorder. Neuropsychopharmacology  : official publication of the American College of Neuropsychopharmacology 33: 11033. Seo, J-S, Lee, K-W, Kim, T-K, Baek, I-S, Im, J-Y, Han, P-L. 2011. Behavioral stress causes mitochondrial dysfunction via ABAD up-regulation and aggravates plaque pathology in the brain of a mouse model of Alzheimer disease. Free radical biology & medicine 50: 1526-35.  	
    Stein-Behrens, B a, Lin, WJ, Sapolsky, RM. 1994. Physiological elevations of glucocorticoids potentiate glutamate accumulation in the hippocampus. Journal of neurochemistry 63: 596-602. Sun, X, Wang, J-F, Tseng, M, Young, LT. 2006. Downregulation in components of the mitochondrial electron transport chain in the postmortem frontal cortex of subjects with bipolar disorder. Journal of psychiatry & neuroscience  : JPN 31: 189-96. Takahashi, T, Kimoto, T, Tanabe, N, Hattori, T-aki, Yasumatsu, N, Kawato, S. 2002. Corticosterone acutely prolonged N-methyl-d-aspartate receptor-mediated Ca2+ elevation in cultured rat hippocampal neurons. Journal of neurochemistry 83: 144151. Takahashi, T, Malhi, GS, Wood, SJ, Walterfang, M, Yücel, M, Lorenzetti, V, Soulsby, B, Suzuki, M, Velakoulis, D, Pantelis, C. 2009. Increased pituitary volume in patients with established bipolar affective disorder. Progress in neuro-psychopharmacology & biological psychiatry 33: 1245-9. Tan, H, Young, LT, Shao, L, Che, Y, Honer, WG, Wang, J-F. 2011. Mood stabilizer lithium inhibits amphetamine-increased 4-hydroxynonenal-protein adducts in rat frontal cortex. The international journal of neuropsychopharmacology / official scientific journal of the Collegium Internationale Neuropsychopharmacologicum (CINP): 1-11. Telford, JE, Kilbride, SM, Davey, GP. 2010. Decylubiquinone increases mitochondrial function in synaptosomes. The Journal of biological chemistry 285: 8639-45.  	
    Vieta, E, Martínez-De-Osaba, MJ, Colom, F, Martínez-Arán, a, Benabarre, a, Gastó, C. 1999. Enhanced corticotropin response to corticotropin-releasing hormone as a predictor of mania in euthymic bipolar patients. Psychological medicine 29: 971-8. Wallace, DC. 2005. A mitochondrial paradigm of metabolic and degenerative diseases, aging, and cancer: a dawn for evolutionary medicine. Annual review of genetics 39: 359-407. Wang, J-F. 2007. Defects of mitochondrial electron transport chain in bipolar disorder: implications for mood-stabilizing treatment. Canadian journal of psychiatry. Revue canadienne de psychiatrie 52: 753-62. Wang, J-F, Shao, L, Sun, X, Young, LT. 2009. Increased oxidative stress in the anterior cingulate cortex of subjects with bipolar disorder and schizophrenia. Bipolar disorders 11: 523-9. Watson, S, Gallagher, P, Ritchie, JC, Ferrier, IN, Young, AH. 2004. Hypothalamicpituitary-adrenal axis function in patients with bipolar disorder. The British journal of psychiatry  : the journal of mental science 184: 496-502. Wellman, CL. 2001. Dendritic reorganization in pyramidal neurons in medial prefrontal cortex after chronic corticosterone administration. Journal of neurobiology 49: 24553. Wolkowitz, OM, Epel, ES, Reus, VI, Mellon, SH. 2010. Depression gets old fast: do stress and depression accelerate cell aging? Depression and anxiety 27: 327-38.  	
    Wu, A, Ying, Z, Gomez-Pinilla, F. 2004. The interplay between oxidative stress and brain-derived neurotrophic factor modulates the outcome of a saturated fat diet on synaptic plasticity and cognition. The European journal of neuroscience 19: 1699707. Xiao, L, Feng, C, Chen, Y. 2010. Glucocorticoid rapidly enhances NMDA-evoked neurotoxicity by attenuating the NR2A-containing NMDA receptor-mediated ERK1/2 activation. Molecular endocrinology (Baltimore, Md.) 24: 497-510. You, J-M, Yun, S-J, Nam, KN, Kang, C, Won, R, Lee, EH. 2009. Mechanism of glucocorticoid-induced oxidative stress in rat hippocampal slice cultures. Canadian journal of physiology and pharmacology 87: 440-7. Young, AH, Gallagher, P, Watson, S, Del-Estal, D, Owen, BM, Ferrier, IN. 2004. Improvements in neurocognitive function and mood following adjunctive treatment with mifepristone (RU-486) in bipolar disorder. Neuropsychopharmacology  : official publication of the American College of Neuropsychopharmacology 29: 1538-45. Zafir, A, Banu, N. 2009. Modulation of in vivo oxidative status by exogenous corticosterone and restraint stress in rats. Stress (Amsterdam, Netherlands) 12: 16777. Zhang, L, Zhou, R, Li, X, Ursano, RJ, Li, H. 2006. Stress-induced change of mitochondria membrane potential regulated by genomic and non-genomic GR  	
    signaling: a possible mechanism for hippocampus atrophy in PTSD. Medical hypotheses 66: 1205-8. Zhou, H, Li, X, Gao, M. 2009. Curcumin protects PC12 cells from corticosteroneinduced cytotoxicity: possible involvement of the ERK1/2 pathway. Basic & clinical pharmacology & toxicology 104: 236-40.  	


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