Microglia contribute to cognitive decline in hypercholesterolemic LDLr-/- mice.

Familial hypercholesterolemia (FH) is caused by mutations in the gene that encodes the low-density lipoprotein (LDL) receptor, which leads to an excessive increase in plasma LDL cholesterol levels. Previous studies have shown that FH is associated with gliosis, blood-brain barrier dysfunction, and memory impairment, but the mechanisms associated with these events are still not fully understood. Therefore, we aimed to investigate the role of microgliosis in the neurochemical and behavioral changes associated with FH using LDL receptor knockout (LDLr-/- ) mice. We noticed that microgliosis was more severe in the hippocampus of middle-aged LDLr-/- mice, which was accompanied by microglial morphological changes and alterations in the immunocontent of synaptic protein markers. At three months of age, the LDLr-/- mice already showed increased microgliosis and decreased immunocontent of claudin-5 in the prefrontal cortex (PFC). Subsequently, 6-month-old male C57BL/6 wild-type and LDLr-/- mice were treated once daily for 30 days with minocycline (a pharmacological inhibitor of microglial cell reactivity) or vehicle (saline). Adult LDLr-/- mice displayed significant hippocampal memory impairment, which was ameliorated by minocycline treatment. Non-treated LDLr-/- mice showed increased microglial density in all hippocampal regions analyzed, a process that was not altered by minocycline treatment. Region-specific microglial morphological analysis revealed different effects of genotype or minocycline treatment on microglial morphology, depending on the hippocampal subregion analyzed. Moreover, 6-month-old LDLr-/- mice exhibited a slight but not significant increase in IBA-1 immunoreactivity in the PFC, which was reduced by minocycline treatment without altering microglial morphology. Minocycline treatment also reduced the presence of microglia within the perivascular area in both the PFC and hippocampus of LDLr-/- mice. However, no significant effects of either genotype or minocycline treatment were observed regarding the phagocytic activity of microglia in the PFC and hippocampus. Our results demonstrate that hippocampal microgliosis, microglial morphological changes, and the presence of these glial cells in the perivascular area, but not increased microglial phagocytic activity, are associated with cognitive deficits in a mouse model of FH.

Role of Oxidative Stress on Insulin Resistance in Diet-Induced Obesity Mice.

Insulin resistance is the link between obesity and type 2 diabetes mellitus. The molecular mechanism by which obese individuals develop insulin resistance has not yet been fully elucidated; however, inconclusive and contradictory studies have shown that oxidative stress may be involved in the process. Thus, this study aimed to evaluate the effect of reactive species on the mechanism of insulin resistance in diet-induced obese mice. Obese insulin-resistant mice were treated with N-acetylcysteine (NAC; 50 mg/kg per day, for 15 days) by means of oral gavage. Twenty-four hours after the last NAC administration, the animals were euthanized and their tissues were extracted for biochemical and molecular analyses. NAC supplementation induced improved insulin resistance and fasting glycemia, without modifications in food intake, body weight, and adiposity. Obese mice showed increased dichlorofluorescein (DCF) oxidation, reduced catalase (CAT) activity, and reduced glutathione levels (GSH). However, treatment with NAC increased GSH and CAT activity and reduced DCF oxidation. The gastrocnemius muscle of obese mice showed an increase in nuclear factor kappa B (NFκB) and protein tyrosine phosphatase (PTP1B) levels, as well as c-Jun N-terminal kinase (JNK) phosphorylation compared to the control group; however, NAC treatment reversed these changes. Considering the molecules involved in insulin signaling, there was a reduction in insulin receptor substrate (IRS) and protein kinase B (Akt) phosphorylation. However, NAC administration increased IRS and Akt phosphorylation and IRS/PI3k (phosphoinositide 3-kinase) association. The results demonstrated that oxidative stress-associated obesity could be a mechanism involved in insulin resistance, at least in this animal model.

The striatum drives the ergogenic effects of caffeine.

Caffeine is one of the main ergogenic resources used in exercise and sports. Previously, we reported the ergogenic mechanism of caffeine through neuronal A2AR antagonism in the central nervous system [1]. We now demonstrate that the striatum rules the ergogenic effects of caffeine through neuroplasticity changes. Thirty-four Swiss (8-10 weeks, 47 ± 1.5 g) and twenty-four C57BL/6J (8-10 weeks, 23.9 ± 0.4 g) adult male mice were studied behaviorly and electrophysiologically using caffeine and energy metabolism was studied in SH-SY5Y cells. Systemic (15 mg/kg, i.p.) or striatal (bilateral, 15 µg) caffeine was psychostimulant in the open field (p < 0.05) and increased grip efficiency (p < 0.05). Caffeine also shifted long-term depression (LTD) to potentiation (LTP) in striatal slices and increased the mitochondrial mass (p < 0.05) and membrane potential (p < 0.05) in SH-SY5Y dopaminergic cells. Our results demonstrate the role of the striatum in the ergogenic effects of caffeine, with changes in neuroplasticity and mitochondrial metabolism.

Branched-chain amino acids (BCAA) administration increases autophagy and the autophagic pathway in brain tissue of rats submitted to a Maple Syrup Urine Disease (MSUD) protocol.

Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism (EIM) biochemically characterized by the tissue accumulation of branched-chain amino acids (BCAA) and their branched-chain alpha-keto acids. The mechanisms by which BCAA and their branched-chain alpha-keto acids lead to the neurological damage observed in MSUD are poorly understood. Mounting evidence has demonstrated that BCAA induce the overproduction of reactive oxygen species, which may modulate several important signaling pathways necessary for cellular homeostasis maintenance, such as autophagy. Taking this into account, we evaluated the effects of BCAA on the autophagic pathway in brain structures of rats submitted to the administration of these amino acids (animal model of MSUD). Our findings showed that BCAA significantly increased the levels of Beclin-1, ATG7, and ATG5 in the cerebral cortex of rats. In addition, BCAA augmented ATG12 levels in the striatum and ATG5 and LC3 I-II in the hippocampus. Therefore, our work demonstrates that the administration of BCAA increases autophagy and autophagic cell death, possibly mediated by the elevated levels of reactive species generated by BCAA.

Experimental evidence of tyrosine neurotoxicity: focus on mitochondrial dysfunction.

Tissue exposure to high levels of tyrosine, which is characteristic of an inborn error of metabolism named Tyrosinemia, is related to severe symptoms, including neurological alterations. The clinical manifestations and pathogenesis of tyrosine neurotoxicity can be recapitulated in experimental models in vivo and in vitro. A widely used experimental model to study brain tyrosine damage is the chronic and acute administration of this amino acid in infant rats. Other research groups and we have extensively studied the pathogenic events in the brain structures of rats exposed to high tyrosine levels. Rats administered acutely and chronically with tyrosine presented decreased and inhibition of the essential metabolism enzymes, e.g., Krebs cycle enzymes and mitochondrial respiratory complexes in the brain structures. These alterations induced by tyrosine toxicity were associated with brain oxidative stress, astrocytes, and, ultimately, cognitive impairments. Notably, in vivo data were corroborated by in vitro studies using cerebral regions homogenates incubated with tyrosine excess. Considering metabolism's importance to brain functioning, we hypothesized that mitochondrial and metabolic dysfunctions are closely related to neurological alterations induced by tyrosine neurotoxicity. Herein, we reviewed the main mechanisms associated with tyrosine neurotoxicity in experimental models, emphasizing the role of mitochondrial dysfunction.

Nanotechnology as a therapeutic strategy to prevent neuropsychomotor alterations associated with hypercholesterolemia.

Hypercholesterolemia has been linked to neurodegenerative disease development. Previously others and we demonstrated that high levels of plasma cholesterol-induced memory impairments and depressive-like behavior in mice. More recently, some evidence reported that a hypercholesterolemic diet led to motor alterations in rodents. Peripheral inflammation, blood-brain barrier (BBB) dysfunction, and neuroinflammation seem to be the connective factors between hypercholesterolemia and brain disorders. Herein, we aimed to investigate whether treatment with gold nanoparticles (GNPs) can prevent the inflammation, BBB disruption, and behavioral changes related to neurodegenerative diseases and depression, induced by hypercholesterolemic diet intake in mice. Adult Swiss mice were fed a standard or a high cholesterol diet for eight weeks and concomitantly treated with either vehicle or GNPs by the oral route. At the end of treatments, mice were subjected to behavioral tests. After that, the blood, liver, and brain structures were collected for biochemical analysis. The high cholesterol diet-induced an increase in the plasma cholesterol levels and body weight of mice, which were not modified by GNPs treatment. Hypercholesterolemia was associated with enhanced liver tumor necrosis factor- α (TNF-α), BBB dysfunction in the hippocampus and olfactory bulb, memory impairment, cataleptic posture, and depressive-like behavior. Notably, GNPs administration attenuated liver inflammation, BBB dysfunction, and improved behavioral and memory deficits in hypercholesterolemic mice. Also, GNPs increased mitochondrial complex I activity in the prefrontal cortex of mice. It is worth highlight that GNPs' administration did not cause toxic effects in the liver and kidney of mice. Overall, our results indicated that GNPs treatment potentially mitigated peripheral, brain, and memory impairments related to hypercholesterolemia.

The metabolic effect of α-ketoisocaproic acid: in vivo and in vitro studies.

Maple syrup urine disease (MSUD) is characterized by a deficiency in the mitochondrial branched-chain α-keto acid dehydrogenase complex activity and, consequently, accumulation of the branched-chain amino acids and their respective branched-chain α-keto acids in fluids and the tissue. MSUD clinical symptoms include neurological alterations. KIC is considered one of the significant neurotoxic metabolites since its increased plasma concentrations are associated with neurological symptoms. We evaluated the effect of KIC intracerebroventricular (ICV) injection in hippocampal mitochondria function in rats. We also investigated the impact of KIC in cells' metabolic activity (using MTT assay) and reactive species (RS) production in HT-22 cells. For this, thirty-day-old male rats were bilaterally ICV injected with KIC or aCSF. Thus, 1 hour after the administration, animals were euthanized, and the hippocampus was harvested for measured the activities of mitochondrial respiratory chain enzymes and RS production. Furthermore, HT-22 cells were incubated with KIC (1-10 mM) in 6, 12, and 24 h. Mitochondrial complexes activities were reduced, and the formation of RS was increased in the hippocampus of rats after KIC administration. Moreover, KIC reduced the cells' metabolic ability to reduce MTT and increased RS production in hippocampal neurons. Impairment in hippocampal mitochondrial function seems to be involved in the neurotoxicity induced by KIC.

NLRP3 Activation Contributes to Acute Brain Damage Leading to Memory Impairment in Sepsis-Surviving Rats.

Sepsis survivors present acute and long-term cognitive impairment and the pathophysiology of neurological dysfunction in sepsis involves microglial activation. Recently, the involvement of cytosolic receptors capable of forming protein complexes called inflammasomes have been demonstrated to perpetuate neuroinflammation. Thus, we investigated the involvement of the NLRP3 inflammasome activation on early and late brain changes in experimental sepsis. Two-month-old male Wistar rats were submitted to the sepsis model by cecal ligation and perforation (CLP group) or laparotomy only (sham group). Immediately after surgery, the animals received saline or NLRP3 inflammasome formation inhibitor (MCC950, 140 ng/kg) intracerebroventricularly. Prefrontal cortex and hippocampus were isolated for cytokine analysis, microglial and astrocyte activation, oxidative stress measurements, nitric oxide formation, and mitochondrial respiratory chain activity at 24 h after CLP. A subset of animals was followed for 10 days for survival assessment, and then behavioral tests were performed. The administration of MCC950 restored the elevation of IL-1ß, TNF-α, IL-6, and IL-10 cytokine levels in the hippocampus. NLRP3 receptor levels increased in the prefrontal cortex and hippocampus at 24 h after sepsis, associated with microglial, but not astrocyte, activation. MCC950 reduced oxidative damage to lipids and proteins as well as preserved the activity of the enzyme SOD in the hippocampus. Mitochondrial respiratory chain activity presented variations in both structures studied. MCC950 reduced microglial activation, decreased acute neurochemical and behavioral alteration, and increased survival after experimental sepsis.

2-methoxy-isobutyl-isonitrile-conjugated gold nanoparticles improves redox and inflammatory profile in infarcted rats.

The tissue response to acute myocardial infarction (AMI) is key to avoiding heart complications due to inflammation, mitochondrial dysfunction, and oxidative stress. Antioxidant and anti-inflammatory agents can minimize the effects of AMI. This study investigated the role of 2-methoxy-isobutyl-isonitrile (MIBI)-associated gold nanoparticles (AuNP) on reperfusion injury after ischemia and its effect on cardiac remodeling in an experimental AMI model. Three-month-old Wistar rats were subjected to a temporary blockade of the anterior descending artery for 30 min followed by reperfusion after 24 h and 7 days by intraventricularly administering 0.4, 1.3, and 3 mg/kg AuNP-MIBI. The cardiac toxicity and renal and hepatic function levels were determined, and the infarct and peri-infarct regions were surgically removed for histopathology, analysis of inflammation from oxidative stress, and echocardiography. MIBI-conjugated AuNP promoted changes in oxidative stress and inflammation depending on the concentrations used, suggesting promising applicability for therapeutic purposes.

Pulmonary oxidative stress in wild bats exposed to coal dust: A model to evaluate the impact of coal mining on health.

This study aimed to verify possible alterations involving histological and oxidative stress parameters in the lungs of wild bats in the Carboniferous Basin of Santa Catarina (CBSC) state, Southern Brazil, as a means to evaluate the impact of coal dust on the health of wildlife. Specimens of frugivorous bat species Artibeus lituratus and Sturnira lilium were collected from an area free of coal dust contamination and from coal mining areas. Chemical composition, histological parameters, synthesis of oxidants and antioxidant enzymes, and oxidative damage in the lungs of bats were analyzed. Levels of Na, Cl, Cu, and Br were higher in both species collected in the CBSC than in the controls. Levels of K and Rb were higher in A. lituratus, and levels of Si, Ca, and Fe were higher in S. lilium collected in the carboniferous basin. Both bat species inhabiting the CBSC areas exhibited an increase in the degree of pulmonary emphysema compared to their counterparts collected from control areas. Sturnira lilium showed increased reactive oxygen species (ROS) and 2',7'-dichlorofluorescein (DCF) levels, while A. lituratus showed a significant decrease in nitrite levels in the CBSC samples. Superoxide dismutase (SOD) activity did not change significantly; however, the activity of catalase (CAT) and levels of glutathione (GSH) decreased in the A. lituratus group from CBSC compared to those in the controls. There were no differences in NAD(P)H quinone dehydrogenase 1 protein (NQO1) abundance or nitrotyrosine expression among the different groups of bats. Total thiol levels showed a significant reduction in A. lituratus from CBSC, while the amount of malondialdehyde (MDA) was higher in both A. lituratus and S. lilium groups from coal mining areas. Our results suggested that bats, especially A. lituratus, living in the CBSC could be used as sentinel species for harmful effects of coal dust on the lungs.

Animal Models of Metabolic Disorders in the Study of Neurodegenerative Diseases: An Overview.

The incidence of metabolic disorders, as well as of neurodegenerative diseases-mainly the sporadic forms of Alzheimer's and Parkinson's disease-are increasing worldwide. Notably, obesity, diabetes, and hypercholesterolemia have been indicated as early risk factors for sporadic forms of Alzheimer's and Parkinson's disease. These conditions share a range of molecular and cellular features, including protein aggregation, oxidative stress, neuroinflammation, and blood-brain barrier dysfunction, all of which contribute to neuronal death and cognitive impairment. Rodent models of obesity, diabetes, and hypercholesterolemia exhibit all the hallmarks of these degenerative diseases, and represent an interesting approach to the study of the phenotypic features and pathogenic mechanisms of neurodegenerative disorders. We review the main pathological aspects of Alzheimer's and Parkinson's disease as summarized in rodent models of obesity, diabetes, and hypercholesterolemia.

Acute exposure to leucine modifies behavioral parameters and cholinergic activity in zebrafish.

Maple Syrup Urine Disease (MSUD) is an autosomal recessive inherited disorder, caused by a deficiency on branched chain α-ketoacid dehydrogenase complex activity, resulting in accumulation of branched-chain amino acids (BCAA) (e.g. leucine). The treatment of MSDU patients increases survival time and quality of life. Thus, nowadays there are a crescent number of adolescents and adults with MSUD. Relevant studies have been reported behavioral alterations in these patients, i.e. high risk of chronic neuropsychiatric problems, such as attention deficit disorder, depression and anxiety. Moreover, MSUD is associated to neurotransmitters deficiency. Herein, we aimed to investigate whether the toxicity of leucine is associated to anxiety-like behavioral, using zebrafish acutely exposed to leucine as experimental model of MSUD. In addition, we evaluated the effects of high levels of leucine in the acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) activities, components of cholinergic neurotransmission system. Young zebrafish were exposed to 2 mM and 5 mM concentration of leucine for 24 h. After that, the animals were submitted to the Novel Tank test, having the brain collected to enzymatic determination. The exposure to both concentrations of leucine caused behavioral and brain cholinergic activity alterations in young zebrafish, indicating an anxiety-like behavior and cholinergic dysfunction. Therefore, this animal could be considered a promising organism to study the BCAA neurotoxic effects, which could help to a better comprehension of the behavioral and neurochemical alterations present in patients with MSUD.

Aerobic and strength training induce changes in oxidative stress parameters and elicit modifications of various cellular components in skeletal muscle of aged rats.

Skeletal muscle aging is associated with loss of mass, function, and strength-a condition known as sarcopenia. It has been reported that sarcopenia can be attenuated by physical exercise. Therefore, we investigated whether 2 different physical exercise protocols could modulate and induce changes in oxidative and inflammatory parameters, as well as in BDNF and DNA repair enzyme levels in skeletal muscle tissue of aged rats. Aging Wistar rats performed treadmill or strength training for 50 min 3 to 4 times a week for 8 weeks. Strength training decreased 2',7'-dichlorofluorescein (DCFH) oxidation (P = 0.0062); however, nitric oxide, protein deglycase DJ-1, and tumor necrosis factor alpha (TNF-α) levels increased after aerobic training (P = 0.04, P = 0.027 and P = 0.009, respectively). Both exercise protocols increased superoxide dismutase (SOD) and catalase (CAT) activity (P = 0.0017 and P = 0.0326) whereas the activity of glutathione (GSH) (P = 0.0001) was decreased. Brain-derived neurotropic factor (BDNF) levels were not affected by exercise, but 8-oxoguanine glycosylase (OGG1) decreased after strength training (P = 0.0007). In conclusion, oxidative parameters showed that skeletal muscle adapt to increased ROS levels, reducing the risk of free radical damage to the tissue after both exercise protocols. These results show that the effects of physical exercise on skeletal muscle are mediated in an exercise type-dependent manner.

Prior Exercise Training Prevent Hyperglycemia in STZ Mice by Increasing Hepatic Glycogen and Mitochondrial Function on Skeletal Muscle.

Diabetes mellitus is a metabolic disorder characterized by hyperglycemia. We investigated the effect of a prior 30 days voluntary exercise protocol on STZ-diabetic CF1 mice. Glycemia, and the liver and skeletal muscle glycogen, mitochondrial function, and redox status were analyzed up to 5 days after STZ injection. Animals were engaged in the following groups: Sedentary vehicle (Sed Veh), Sedentary STZ (Sed STZ), Exercise Vehicle (Ex Veh), and Exercise STZ (Ex STZ). Exercise prevented fasting hyperglycemia in the Ex STZ group. In the liver, there was decreased on glycogen level in Sed STZ group but not in EX STZ group. STZ groups showed decreased mitochondrial oxygen consumption compared to vehicle groups, whereas mitochondrial H2 O2 production was not different between groups. Addition of ADP to the medium did not decrease H2 O2 production in Sed STZ mice. Exercise increased GSH level. Sed STZ group increased nitrite levels compared to other groups. In quadriceps muscle, glycogen level was similar between groups. The Sed STZ group displayed decreased O2 consumption, and exercise prevented this reduction. The H2 O2 production was higher in Ex STZ when compared to other groups. Also, GSH level decreased whereas nitrite levels increased in the Sed STZ compared to other groups. The PGC1 α levels increased in Sed STZ, Ex Veh, and Ex STZ groups. In summary, prior exercise training prevents hyperglycemia in STZ-mice diabetic associated with increased liver glycogen storage, and oxygen consumption by the mitochondria of skeletal muscle implying in increased oxidative/biogenesis capacity, and improved redox status of both tissues. J. Cell. Biochem. 118: 678-685, 2017. © 2016 Wiley Periodicals, Inc.

Physical Exercise Attenuates Experimental Autoimmune Encephalomyelitis by Inhibiting Peripheral Immune Response and Blood-Brain Barrier Disruption.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) caused by demyelination, immune cell infiltration, and axonal damage. Herein, we sought to investigate the influence of physical exercise on mice experimental autoimmune encephalomyelitis (EAE), a reported MS model. Data show that both strength and endurance training protocols consistently prevented clinical signs of EAE and decreased oxidative stress, an effect which was likely due to improving genomic antioxidant defense-nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response elements (ARE) pathway-in the CNS. In addition, physical exercise inhibited the production of pro-inflammatory cytokines interferon (IFN)-γ, interleukin (IL)-17, and IL-1ß in the spinal cord of mice with EAE. Of note, spleen cells obtained from strength training group incubated with MOG35-55 showed a significant upregulation of CD25 and IL-10 levels, with a decrease of IL-6, MCP-1, and tumor necrosis factor (TNF)-α production, mainly, during acute and chronic phase of EAE. Moreover, these immunomodulatory effects of exercise were associated with reduced expression of adhesion molecules, especially of platelet and endothelial cell adhesion molecule 1 (PECAM-1). Finally, physical exercise also restored the expression of tight junctions in spinal cord. Together, these results demonstrate that mild/moderate physical exercise, when performed regularly in mice, consistently attenuates the progression and pathological hallmarks of EAE, thereby representing an important non-pharmacological intervention for the improvement of immune-mediated diseases such as MS. Graphical Abstract Schematic diagram illustrating the beneficial effects of physical exercise during experimental model of MS. Physical exercise, especially strength (ST) and endurance (ET) training protocols, inhibits the development and progression of disease, measured by the mean maximal clinical score (1.5 and 1.0, respectively), with inhibition of 30 % and 50 %, respectively, based on the AUC, compared with EAEuntreated group. In addition, ST and ET decreased oxidative stress, possibly, through genomic antioxidant defense, Nrf2-Keap1 signaling pathway, in the CNS. Physical exercise inhibited the production of inflammatory cytokines, such as IFN-γ, IL-17 and IL-1ß in the spinal cord after EAE induction, as well as spleen cells obtained from ST group showed a significant upregulation of regulatory T cell markers, such as CD25 and IL-10 levels, and blocked IL-6, MCP-1 and TNF-α production, mainly, during acute and chronic phase of EAE. Finally, these immunomodulatory effects of exercise were associated with inhibition of adhesion molecules and reestablishment of tight junctions expression in spinal cord tissue, thereby limiting BBB permeability and transmigration of autoreactive T cells to the CNS. NO, nitric oxide; GPx, glutathione peroxidase, GSH, glutathione; Nrf2, nuclear factor (erythroid-derived 2)-like 2; CNS, central nervous system; BBB, blood-brain barrier; IFN-g, interferon-gamma; IL-17, interleukin 17; IL-1b, interleukin-1beta.

Lithium and valproate act on the GSK-3ß signaling pathway to reverse manic-like behavior in an animal model of mania induced by ouabain.

The present study aimed to investigate the effects of mood stabilizers, specifically lithium (Li) and valproate (VPA), on the PI3K/Akt signaling pathway in the brains of rats subjected to the ouabain (OUA)-induced animal model of mania. In addition, the effects of AR-A014418, a GSK-3ß inhibitor, on manic-like behavior induced by OUA were evaluated. In the first experimental protocol Wistar rats received a single ICV injection of OUA or artificial cerebrospinal fluid (aCSF). From the day following ICV injection, the rats were treated for 6 days with intraperitoneal injections of saline, Li or VPA twice a day. In the second experimental protocol, rats received OUA, aCSF, OUA plus AR-A014418, or aCSF plus AR-A014418. On the 7th day after OUA injection, locomotor activity was measured using the open-field test. In addition, we analyzed the levels of p-PI3K, p-MAPK, p-Akt, and p-GSK-3ß in the brain of rats by immunoblot. Li and VPA reversed OUA-related hyperactivity. OUA decreased p-PI3K, p-Akt and p-GSK-3ß levels. Li and VPA improved these OUA-induced cellular dysfunctions; however, the effects of the mood stabilizers were dependent on the protein and brain region analyzed. In addition, AR-A014418 reversed the manic-like behavior induced by OUA. These findings suggest that the manic-like effects of ouabain are associated with the activation of GSK-3ß, and that Li and VPA exert protective effects against OUA-induced inhibition of the GSK-3ß pathway.