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The GLP-1 receptor agonist liraglutide reverses mania-like alterations and memory deficits induced by D-amphetamine and augments lithium effects in mice: Relevance for bipolar disorder
Journal Pre-proof The GLP-1 receptor agonist liraglutide reverses mania-like alterations and memory deficits induced by D-amphetamine and augments lithium effects in mice: Relevance for bipolar disorder
Adriano José Maia Chaves Filho, Natássia Lopes Cunha, Alana Gomes de Souza, Michele Verde-Ramo Soares, Paloma Marinho Jucá, Tatiana de Queiroz, João Victor Souza Oliveira, Samira S. Valvassori, Tatiana Barichello, Joao Quevedo, David de Lucena, Danielle S. Macedo PII:
S0278-5846(19)30595-0
DOI:
https://doi.org/10.1016/j.pnpbp.2020.109872
Reference:
PNP 109872
To appear in:
Progress in Neuropsychopharmacology & Biological Psychiatry
Received date:
18 July 2019
Revised date:
31 December 2019
Accepted date:
15 January 2020
Please cite this article as: A.J.M.C. Filho, N.L. Cunha, A.G. de Souza, et al., The GLP-1 receptor agonist liraglutide reverses mania-like alterations and memory deficits induced by D-amphetamine and augments lithium effects in mice: Relevance for bipolar disorder, Progress in Neuropsychopharmacology & Biological Psychiatry(2019), https://doi.org/ 10.1016/j.pnpbp.2020.109872
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© 2019 Published by Elsevier.
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The GLP-1 receptor agonist liraglutide reverses mania-like alterations and memory deficits induced by D-amphetamine and augments lithium effects in mice: relevance for bipolar disorder Adriano José Maia Chaves Filho1 , Natássia Lopes Cunha1 , Alana Gomes de Souza1 , Michele Verde-Ramo Soares1 , Paloma Marinho Jucá1 , Tatiana de Queiroz1 , João Victor Souza Oliveira1 ,
Samira S Valvassori2 , Tatiana Barichello2,3,4,5 , Joao Quevedo2,3,4,5 ,
Neuropharmacology Laboratory, Drug Research and Development Center, Department of Physiology
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1.
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David de Lucena1 , Danielle S Macedo1,6,*
2.
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and Pharmacology, Faculty of Medicine, Federal University of Ceara, Fortaleza, CE, Brazil Laboratory of Neurosciences, Graduate Program in Health Sciences, Health Sciences Unit,
3.
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University of Southern Santa Catarina (UNESC), Criciúma, SC, Brazil Translational Psychiatry Program, Department of Psychiatry and Behavioral Sciences, McGovern
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Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston,
4.
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TX, USA
Center of Excellence on Mood Disorders, Department of Psychiatry and Behavioral Sciences,
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McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston, TX, USA 5.
Neuroscience Graduate Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA
6.
National Institute for Translational Medicine (INCT-TM, CNPq)
*Corresponding author: *Danielle S. Macedo. Department of Physiology and Pharmacology, Federal University of Ceara, Rua Cel. Nunes de Melo 1000, zip code 60430-275, Fortaleza, CE, Brazil. Phone:
+55-85-3366-8337/+55-85-3366-8036;
Fax:
[email protected]; [email protected]
+55-85-3366-8333;
E-mail:
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Abstract
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Metabolic and psychiatric disorders present a bidirectional relationship. GLP-1 system, known for its insulinotropic effects, has also been associated with numerous regulatory effects in cognitive and emotional processing. GLP-1 receptors (GLP-1R) agonists present neuroprotective and antidepressant/anxiolytic properties. However, the effects of GLP-1R agonism in bipolar disorder (BD) mania and the related cognitive disturbances remains unknown. Here, we investigated the effects of the GLP-1R agonist liraglutide (LIRA) at monotherapy or combined with lithium (Li) against Damphetamine (AMPH)-induced mania-like symptoms, brain oxidative and BDNF alterations in mice. Swiss mice received AMPH 2 mg/kg or saline for 14 days. Between days 8-14, they received LIRA 120 or 240 µg/kg, Li 47.5 mg/kg or the combination Li+LIRA, on both doses. After behavioral evaluation the brain areas prefrontal cortex (PFC), hippocampus and amygdala were collected. AMPH induced hyperlocomotion, risk-taking behavior and multiple cognitive deficits which resemble mania. LIRA reversed AMPH-induced hyperlocomotion, working and recognition memory impairments, while Li+LIRA240 rescued all behavioral changes induced by AMPH. LIRA reversed AMPH-induced hippocampal oxidative and neurotrophic changes. Li+LIRA240 augmented Li antioxidant effects and greatly reversed AMPH-induced BDNF changes in PFC and hippocampus. LIRA rescued the weight gain induced by Li in the course of mania model. Therefore, LIRA can reverse some mania-like behavioral alterations and combined with Li augmented the mood stabilizing and neuroprotective properties of Li. This study points to LIRA as a promising adjunctive tool for BD treatment and provides the first rationale for the design of clinical trials investigating its possible antimanic effect.
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Keywords: Bipolar disorder; mania; GLP-1; Liraglutide; D-amphetamine; cognitive impairment; oxidative stress; brain-derived neurotrophic factor (BDNF).
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1. Introduction
Bipolar disorder (BD) is a chronic psychiatric disorder that follows a progressive course of mood and cognitive manifestations. Manic or hypomanic episodes are a distinctive psychopathological feature of BD. These episodes are characterized by elevated mood, impulsivity, irritability, hypersexual behavior, psychomotor agitation,
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circadian alterations and cognitive deficits (Hilty et al. 2006; Tondo et al. 2017). Mania-
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depression cyclic alternation represent a key factor for BD neuroprogression (Berk et al.
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2011b). Indeed, progressive functional impairment is observed from BD clinical stage I to stage IV, with cognitive and functional decline being observed in stages III and IV
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(Cardoso et al. 2015). Furthermore, mania seems to have more significant impact on
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cognition. In other words, manic patients usually present poorer cognitive abilities in
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2004; Gruber et al. 2007).
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several domains when compared to depressed and euthymic BD patients (Dixon et al.
Despite the pathophysiology of BD is not fully understood, several lines of
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evidence indicate that oxidative and nitrosative stress (O&NS) as well as abnormalities in brain neurotrophins,
mainly represented
by brain-derived neurotrophic factor
(BDNF), are importantly involved in BD neurobiology (Post 2010; Fernandes et al. 2011). In this context, animal models can represent useful tools to elucidate underlying biological pathways as well as to discover new therapeutic agents for this condition (Sharma et al. 2016). Regarding mania models, psychostimulants-induced manic-like symptoms have been classically used as models to predict new anti-maniac/mood stabilizing agents. The most studied of these pharmacological models is D-amphetamine (AMPH)-induced mania model (Frey et al., 2006; Macedo et al., 2013; Martins et al., 2006; Valvassori et
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al., 2019). In this model, hyperactivity has been traditionally used as main feature to measure manic-like symptoms and mood stabilizing response (Lan and Einat 2019). In this context, some studies have deeply investigated the AMPH model and demonstrated that AMPH repeated exposure induces a broader spectrum of behavioral changes
than
aggressiveness,
just
hyperlocomotion,
impulsivity/risk-taking
including behavior
enhanced and
sexual
cognitive
behavior, dysfunction
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(deCatanzaro and Griffiths 1996; Van Enkhuizen et al. 2013; Zhou et al. 2015). More
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recently, some authors also showed that, during the withdraw period after AMPH
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exposure, a depression-like phenotype associated to an increased sensitization to subsequent low doses of AMPH emerges, resembling the cyclic nature of mood changes
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in BD (Pathak et al. 2015; Valvassori et al. 2019). Although the potential overlapping
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with drug addiction, AMPH model is relevant to study the pathophysiological processes
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underlying mood disturbances in BD (Dencker and Husum 2010; Pathak et al. 2015).
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Cognition is a core domain of psychopathology in BD. At least 25% of BD patients exhibit pronounced deficits in one or more domain of function (Gualtieri and
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Morgan 2008). Cognitive dysfunction also seems to be the major responsible factor for disability and social burden independently of concurrent mood state (Gualtieri and Morgan 2008; Simonsen et al. 2008). Additionally, current mood stabilizing and antidepressant drugs cannot efficiently restore or treat cognitive impairment associated to BD (Wingo et al. 2009; Mora et al. 2013). In the last decades, a clear bidirectional relationship between BD and metabolic disorders has emerged. BD individuals with metabolic comorbidities develop an unfavorable course of disease with increased refractoriness, atypical features and disability (McIntyre et al. 2010). Metabolic disorders seem to mainly impact cognition. Metabolic abnormalities, such as insulin resistance (Hajek et al. 2014; Cairns et al.
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2018), visceral adiposity, dyslipidemia (Fleet-Michaliszyn et al. 2008; Elmslie et al. 2009) and high blood pressure (Brown et al. 2009; Depp et al. 2014) have been negatively associated to neurocognitive function in BD. These alterations, mainly insulin resistance, are frequently present in BD even in newly-diagnosed patients (Coello et al. 2019). Furthermore, mood stabilizers and antipsychotic drugs used for long-term BD management, frequently cause metabolic iatrogenic effects and increase the risk for weight gain and metabolic syndrome (McIntyre et al. 2011; Kocakaya et al.
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2018).
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Glucagon-like peptide 1 (GLP-1) is a secreted incretin that facilitates the glucose uptake by peripheral organs after feeding. Also, GLP-1 (and its synthetic analogues) can
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easily cross the blood-brain barrier and centrally regulate energy expenditure, food
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intake and hypothalamic responses (López-Ferreras et al. 2018). GLP-1 receptors (GLP1Rs) are widely distributed in brain structures, including not only hypothalamus and
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al. 2015).
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pituitary, but also in PFC, hippocampus, amygdala and other limbic structures (Cork et
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Besides its metabolic effects, compelling evidence have reported significant neuroprotective effects of GLP-1 and its analogues. In fact, GLP-1R agonists protects against cognitive damage associated to several neurodegenerative models, as well as increase neurotrophin expression and neuronal proliferation and differentiation (Porter et al. 2012; McClean and Hölscher 2014; Li et al. 2015). GLP-1R agonists also prevented depression- and anxious-like behavior associated to stress and metabolic conditions (Weina et al. 2018; de Souza et al. 2019). More recently, two open-label clinical trials have reported that the GLP-1R agonist liraglutide (LIRA) can improve cognitive function in stable BD and major depression patients (Mansur et al. 2017b, 2017a). However, at the present time there is no direct clinical or preclinical evidence
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have investigated the GLP-1R agonists effects on behavioral features related to BD mania, in order to further support the potential use of these drugs as new tools for mania treatment. Therefore, our first objective in this study was to investigate the effects of the GLP-1R agonist LIRA at monotherapy or combined with lithium (Li) against maniclike behavioral alterations induced by AMPH in mice. Our second objective was to
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verify the possible protective effects of LIRA against oxidative damage and brain-
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derived neurotrophic factor (BDNF) changes induced by AMPH in mood-regulating
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brain areas, namely prefrontal cortex (PFC), hippocampus and amygdala. In the present study, we hypothesized that LIRA alone could display protective actions against
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behavioral and neurochemical changes induced by AMPH mania model by mechanisms
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related to neurotrophic changes and oxidative homeostasis and when combined with Li
2.1 Animals
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2. METHODS
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could improve the effects of this mood stabilizing drug.
Male adult (postnatal day 70) Swiss mice, weighing 25-30 g, provided from the Animal House of the Federal University of Ceara, were used. The animals were maintained at a controlled temperature (22±2°C) with a 12-h dark/light cycle and food and water ad libitum. Mice were maintained in groups of 8 animals in 41 × 34 × 16 cm open-topped cages. The animals were manipulated according to the NIH Guide for the Care and Use of Laboratory Animals (NIH 2011). The experiments were performed in accordance with the ethical principles adopted by the Brazilian College of Animal
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Experimentation (COBEA) and approved by the Ethics Committee on Animal Research of the Federal University of Ceara with the protocol number (97/15). 2.2 Drugs Liraglutide
(Novo
Nordisk),
D-amphetamine
(AMPH)
and
lithium
carbonate (Li) (Sigma-Aldrich, Brazil) were directly dissolved in sterile saline solution (NaCl 0.9%, w/v). The drugs were made up freshly within 1–2 h of dosing and were
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administered intraperitoneally (IP) in a volume of 0.1 ml/10 g body weight. All other
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chemicals used were of analytical grade.
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2.3 Experimental design
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A reversal protocol was used as previously described (Frey et al. 2006b). By the conduction of the reversal model, we aimed to simulate the treatment of an acute manic
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state. For this end, 144 animals were used. Each group consisted of 16 randomly
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allocated animals. Mice were habituated for 7 days in their home cages before the beginning of the treatment. After this, each animal received for 14 days one daily IP
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administration of AMPH (2.0 mg/kg) or 0.9 % saline. Between the 8th and 14th days of treatment, AMPH and saline-treated animals received LIRA (120 and 240 µg/kg, SC), Li (47.5 mg/kg, IP) or saline solution once a day. For combined treatment, Li-treated groups received an additional subcutaneous injection of LIRA (120 and 240 µg/kg, SC) or saline. All other groups received a third saline injection. The time interval between each drug administration in all situations was 30 min. The selected doses of LIRA and the treatment duration were based on previous studies reporting LIRA’s neuroprotective and pro-cognitive effects in Alzheimer’s (Yang et al. 2013) and Parkinson’s disease (Zhang et al. 2018) models, as well as the
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selected doses are within the dose range capable to reverse weight gain (WG) and glucose intolerance associated to metabolic syndrome in mice (100-300 µg/kg) (Knudsen 2010; Fransson et al. 2014). Deserves mention the fact that the only difference in LIRA effects observed between humans and rodents is the much longer half-life of LIRA in humans than in rodents (Knudsen 2010). Despite this, we observed significant results of LIRA in the reversal of AMPH-induced alterations with a single daily dose. Additionally, the doses of LIRA used in this study were calculated to mice
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based on the human doses of 0.6 and 1.2 mg/day used for obesity and weight loss treatment. This calculation was performed by Reagan-Shaw dose translation algorithm
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using human-mouse body surface area (BSA) for mice (Reagan-Shaw et al. 2008). In
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the case of Li, the dosage regimens were based on previous preclinical reports of anti-
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manic-like effect of this drug (Macêdo et al. 2012; De Souza et al. 2015; Queiroz et al. 2015). Control animals received only saline. The animal weight was measured daily
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before each drug administration and showed as % WG. The behavioral determinations
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were conducted at the 14th day of treatment, 2 h after the last drug injection.
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To avoid bias related to the excessive exposure to behavioral apparatus, each experimental group assigned for behavioral determinations was divided into two subgroups of 8 animals. In this regard, the subgroup one (N = 8/group) was subjected to the open field (OFT), elevated plus maze (EPM) and passive shock avoidance test, in this order. Subgroup two (N = 8/ group) was subjected to y-maze and novel object recognition (NOR) task, in this order. A 1-hour interval was adopted between behavioral tests. The order of behavioral tests in each experimental set was defined from the least to the most stressful (Paylor et al. 2006). The tests were conducted by two independent trained observers blinded to treatment groups.
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Also, to avoid the stress associated to exposure of behavioral testing on neurochemical parameters, a third subgroup (N = 7-8/group) of different animals was used for biochemical analyses. For these assays, we selected the dose of LIRA, 240 µg/kg, SC, which showed the best effects on behavioral testing. The decision of continuing the study with the best dose of LIRA was based on the principle of the 3Rs (Replacement, Reduction and Refinement) for ethical use of animals in testing (Flecknell 2002). Afterwards, mice were sacrificed by decapitation and the prefrontal
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cortex (PFC), hippocampus, and amygdala were carefully dissected according to the method previously described (Iversen and Glowinski 1966). The brain samples were
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rapidly frozen and stored at -80º C until neurochemical determinations. To a graphical
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2.4. Serum lithium measurement
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view of the experimental design, please see Fig. 1.
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For the determination of lithium levels, the sera of lithium-treated animals were
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separated by centrifugation. Assays were performed with a digital flame photometer (Labnova AP- 1500 series, São Paulo, Brazil). The serum levels of lithium in each animal ranged from 0.8 to 1.1 mEq/L, as recommended for the routine treatment of BD (Hopkins and Gelenberg 2000).
2.5 Behavioral tests
Open Field Test (OFT) This test primarily evaluates the influence of drugs on the locomotor activity and exploratory behavior (Archer 1973). An acrylic apparatus (transparent walls and
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black background, dimensions 30x30x15cm), divided into nine squares, was used. The central zone was defined as the central square with dimensions 10x10 cm. The animal was placed in the center of the apparatus and was observed over five minutes after 1 minute of habituation. Each mouse was tested once and between two mice, the apparatus was cleaned with 10% ethanol solution. The parameters analyzed were: total distance traveled by the animal (cm2 ), the number of squares crossed by the mice with four legs, the number of entries in the central zone and the time spent in the central zone
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(exploratory behavior). The test was automatically recorded and analyzed using Panlab
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Elevated Plus Maze Test (EPM)
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Harvard Apparatus® SMART video tracking version 3.0.03 software.
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This test was carried out as previously described (Pellow and File 1986). The apparatus has two perpendicular open arms (30x5cm) and two perpendicular closed
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arms (30x5x25cm). The open and closed arms were connected by a central platform
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(5x5cm). The platform and the lateral walls of the closed arms were made of transparent
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acrylic. The floor was made of black acrylic. The maze was 45 cm above the floor. After the respective treatment, each animal was placed at the center of the EPM with its nose in the direction of one of the closed arms and was observed for 5 min according to the following parameters: number of entries into open and closed arms and the amount of time spent by in open and closed arms of the maze. These data were used to calculate: the total number of entries, % of open entries (open entries/total entries x 100) and % of time in open arms (time spent in open arms/total time x 100).
Y-maze Test
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The apparatus consisted of a Y-shaped maze with three white, opaque plastic arms (length 35, width 5, wall height 10) at a 120 angle from each other arms. Each mouse was allowed to freely move through the maze during 8 min. The series of arm entries was recorded visually. A correct alternation was defined as entries in all three arms on consecutive occasions. The percentage of correct alternations was calculated as follows: total of alternations/(total arm entries x 2), as described in detail elsewhere
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(Dall’Igna et al. 2007).
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Novel Object Recognition Test (NOR)
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Novel object recognition is a widely used task to evaluate spatial
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recognition memory. This test assesses the mouse’s ability to discriminate between familiar and novel objects (Li et al. 2017). Firstly, mice were individually habituated to
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an open field plexiglas box (30x30x15 cm size) for 5 min. Twenty-four hours after
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habituation, each mouse was subjected to a 10 min acquisition trial, during which they
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were placed in the same arena in presence of two identical objects situated at 15 cm from the arena wall. After 1 h of retention interval, the mice were placed back into the arena and exposed to the familiar object and to a novel object (different color and shape) for a further 10 min. Total time spent exploring each of the two objects (when the animal's snout was directly toward the object at a distance ≤2 cm) was recorded. Recognition index was used as direct measure of recognition memory and was calculated as follows: (time exploring new object - time exploring familiar object) / (time exploring new object + time exploring familiar object) (Ennaceur and Delacour 1988; Li et al. 2015).
Passive Shock Avoidance Test
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The apparatus consisted of an acrylic box (48x22x22cm), with an electrified grid floor, having a shock-free wooden raised zone. In the first step, the animal was allowed to freely explore the apparatus for 90s. After this, the animals were submitted to the training session that consisted in placing the mice on a shock free zone and, when stepped down with all paws on the grid floor, an electric shock (0.5mA) was delivered for 1s. The animal was removed and 1 hour later, it was placed back in the raised area and the latency of step-down to grid floor was recorded (Cammarota et al. 2003;
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Kazlauckas et al. 2005). In this task, the animal learns that a specific place should be avoided since it is associated with an aversive event (Khurana et al. 2011). Thus, a
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decrease in the step-down latency indicates impairment in aversive/affective learning.
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2.6 Neurochemical assays
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Oxidative stress parameters
GSH levels were determined
to estimate the endogenous
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Reduced
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Determination of reduced GSH concentrations
antioxidant defenses (Sedlak and Lindsay 1968). The method was based on the Ellman's reagent (DTNB) reaction with free thiol groups. Briefly, the samples were homogenized in 0.4 M Tris–HCl buffer, pH 8.9 and 0.01 M DTNB. Reduced GSH levels were determined using a microplate reader set at 412 nm. The results are expressed as µg of reduced GSH/g tissue.
Measurement of lipid peroxidation The rate of lipid peroxidation was estimated by the determination of malondialdehyde
(MDA)
equivalent
concentrations
using
the
thiobarbituric-acid
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reactive substances (TBARS) (Huong et al. 1998). The samples were mixed with 100 μl of 35% perchloric acid and were centrifuged at 5000 rpm for 10 min. 150 μl of the supernatants were removed, mixed with 50 μl of 1.2% thiobarbituric acid, and then heated in a boiling water bath for 30 min. After cooling, the MDA levels were determined using a microplate reader set at 535 nm and expressed as μg MDA/g tissue.
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Determination of BDNF The samples were homogenized in phosphate buffered saline (pH 7.4) with
quantified
by
immunoenzymatic
assay
determination
according
to
the
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were
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protease and phosphatase inhibitor cocktails (Sigma St. Louis, USA). BDNF levels
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manufacturer′s instructions (EMD Millipore, USA). The results are expressed as pg of
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2.7 Statistical Analysis
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BDNF/g of tissue.
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Statistical analysis was performed with GraphPad Prism for Windows (version 7.0, San Diego, USA). Kolmogorov-Smirnov test was used for the evaluation of the normal distribution of the data. All results are expressed as means S.E.M. and the statistical analysis of the data was performed by regular one-way ANOVA, followed by Tukey’s test as post-hoc test. The significance level was set at p < 0.05.
3. Results
Reversal of AMPH-induced hyperlocomotion by LIRA alone and in combination with Li
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Psychomotor agitation, a core behavioral alteration observed in mania, is evaluated in animal models by the increased number of crossings in the open field (Boulle et al. 2014). In the present study, we were able to replicate these findings by showing that AMPH significantly increased the zone transitions number and the total distance travelled in the open field test when compared with SAL-treated mice [(zone transitions number: SAL+SAL vs. AMPH+SAL, P= 0.004, F(8, 54)= 7.206; total distance travelled: SAL+SAL vs. AMPH+SAL, P= 0.003), F(8, 49)= 4.582]. The
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administration of LIRA120, LIRA240, Li, and Li+LIRA240 significantly reversed the number of zone transitions in relation to SAL-treated mice [(AMPH+LIRA120 vs.
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AMPH+SAL, P= 0.0265; AMPH+LIRA240 vs. AMPH+SAL, P< 0.001; AMPH+Li vs.
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AMPH+SAL, P=0.0031; AMPH+Li+LIRA240 vs. AMPH+SAL, P= 0.0027); F(8, 54)=
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7.206] (Fig. 2A). Considering the total distance travelled, LIRA240 (P= 0.003), Li (P< 0.05) and the combination LIRA240+Li (P= 0.031) significantly reversed AMPH-
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induced increase in this parameter (Fig. 2B).
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Regarding the number of entries in the center of the open field (Fig. 2C) and the
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time spent in the central square (Fig. 2D), two parameters that may be regarded as impulsive behavior, we observed no alterations in the former one, while in the latter we observed a 1.7-fold increase in AMPH-treated mice when compared to SAL group, despite this difference was
statistically non-significant.
The group treated with
AMPH+Li+LIRA240 presented a significant decrease in the time spent in the central area when compared with AMPH+SAL-treated mice [(P = 0.014), F (8, 53) = 2.71] being the results of AMPH+Li+LIRA240 group akin to SAL-treated animals. Effects of LIRA alone and in combination with Li in the reversal of risk-taking behavior in the elevated plus maze test
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It was previously reported that spontaneously high impulsive rats enter the open arms of the EPM significantly more quickly than spontaneously low impulsive rats. (Molander et al. 2011). Based on observations such as that, in the present study EPM was used to evaluate impulsive behavior. In this regard, as depicted in Fig. 3, AMPH+SAL mice presented a marked increase in the percent of open entries [(P< 0.001), F (8, 52) = 7.99] and percent of time spent in the open arms [(P< 0.001), F (8, 54) = 6.267] in comparison with SAL-treated animals. Considering the % of open
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entries (Fig. 3B) we observed that LIRA240 (P = 0.0117), Li (P = 0.0079), Li+LIRA120 (P = 0.0002) and Li+LIRA240 (P< 0.001) reversed the increase in this
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parameter induced by AMPH+SAL (F (8, 52) = 7.99). In the evaluation of the % of time
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spent in the open arms (Fig. 3C), we noticed a significant reversal of AMPH effects
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only in the animals treated with Li alone or in combination with LIRA on both doses [(AMPH+Li vs. AMPH+SAL, P = 0.0042; AMPH+Li+LIRA120 vs. AMPH+SAL, P =
P
= 0.1273; AMPH+Li+LIRA240 vs. AMPH+LIRA240, P
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AMPH+LIRA120,
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0.0014; AMPH+Li+LIRA240 vs. AMPH+SAL, P = 0.001; AMPH+Li+LIRA120 vs.
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=0.0465), F (8, 54) = 6.267]. We observed a significant decrease in the number of total entries in AMPH+Li+LIRA240 group in relation to AMPH+SAL [(P = 0.038), F (8, 55) = 3.305] (Fig. 3A).
Effects of LIRA alone and in combination with Li on memory alterations induced by AMPH Over the last years, the studies evaluating memory deficits in bipolar disorder have been increasing, since memory deficits persists in remission periods as well as neurocognitive dysfunction may significantly influence patients’ psychosocial outcomes
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(Solé et al. 2017). This is of great importance since there is not a consensus for the benefits of Li in protecting patients from memory deficits (Wingo et al. 2009). Our study revealed that in the evaluation of the percent of correct alternations in the Y maze task (Fig. 4A), there was a significant decrease in this parameter in the AMPH+SAL group compared with SAL-treated mice [(P = 0.0003), F (8, 53) = 13.38] . The treatment
with
LIRA
at
both
doses,
AMPH+LIRA120
(P
=
0.0208) and
AMPH+LIRA240 (P = 0.0058) and in combination with Li, AMPH+Li+LIRA120 (P =
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0.0009) and AMPH+Li+LIRA240 (P<0.001) significantly increased this percentual compared with AMPH+SAL group (F (8, 53) = 13.38). Additionally, a significant
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increase in this parameter was observed in AMPH+Li+LIRA240 group compared with
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AMPH+ Li [(P = 0.0182), F (8, 53) = 13.38)]. Regarding total entries in the Y maze
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apparatus (Fig. 4B), the treatment with LIRA240 (AMPH+LIRA240 vs. AMPH+SAL, P = 0.0009) and Li (AMPH+Li vs. AMPH+SAL, P = 0.0045) or their combination
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(AMPH+Li+LIRA240 vs. AMPH+SAL, P = 0.0005) significantly reduced total entries
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in relation to AMPH+SAL-treated animals [ F (8, 57) = 8.221 ].
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In the evaluation of declarative memory by the novel object recognition test (Fig.4C), AMPH+SAL treatment induced a marked decrease in recognition index (%) (P = 0.0056) when compared with SAL group, which was reversed by LIRA240 (AMPH+LIRA240 vs. AMPH+SAL, P = 0.0087) and by both doses of LIRA combined with
Li [AMPH+Li+LIRA120
AMPH+SAL,
(P
=
0.0006),
(P
=
0.0007) and
AMPH+
Li+LIRA240
vs.
F
(8,
52) = 8.32]. Further, the combination
AMPH+Li+LIRA240 significantly increased this index compared with AMPH+Li group [(P= 0.0397), F (8, 52) = 8.32] . In the passive avoidance test (Fig.4D), there was a marked reduction in stepdown latency (s) in the group AMPH+SAL (P = 0.0044) and AMPH+LIRA120 (P =
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0.0111) compared with SAL-controls [F (8, 53) = 6.364]. Li treatment alone (AMPH+Li, P = 0.0014) or combined with LIRA120 (AMPH+Li+LIRA120, P = 0.0251) and 240 (AMPH+Li+LIRA240, P = 0.0022) significantly increased this parameter compared with AMPH+SAL group. Also, the groups in combination AMPH+Li+LIRA120 and AMPH+Li+LIRA240 also presented a significant increase in this step-down latency compared with AMPH+LIRA120 and AMPH+LIRA240 groups
f
(P = 0.0379), respectively [F (8, 53) = 6.364].
oo
Evaluation of weight gain (WG) alterations
pr
Regarding the % WG, AMPH repeated administration caused no significant alteration in this parameter in relation to SAL+SAL mice (19.31% WG and 21.77%
e-
WG, respectively). As expected, AMPH+Li-treated animals presented a marked
Pr
increase in % WG compared with AMPH+SAL [AMPH+Li (47.84% WG) vs. AMPH+SAL (19.31% WG), P < 0.001] and with SAL+SAL groups [AMPH+Li
al
(47.84% WG) vs. SAL+SAL (21.77% WG), P < 0.001]. AMPH+LIRA treatment did
rn
not significantly alter % WG (AMPH+LIRA240 (14.78% WG) vs. AMPH+SAL
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(19.31% WG), non-significant; AMPH+LIRA120 (18.89% WG) vs. AMPH+SAL (19.31% WG), non-significant). Under control conditions, LIRA treatment significantly reduced % WG compared to SAL+SAL animals: [SAL+LIRA240 (9.482% WG) vs. SAL+SAL (21.77% WG), P< 0.001; and SAL+LIRA120 (13.98% WG) vs. SAL+SAL (21.77% WG),
P = 0.0379, F (8, 63) = 44.85]. Also, Li+LIRA combination groups
presented a significant reduction in weight gain when compared with AMPH+Li group: [AMPH+Li+LIRA240
(19.49% WG) vs.
AMPH+Li (47.84% WG),
P<0.001;
AMPH+Li+LIRA120 (27.47% WG) vs. AMPH+Li (47.84% WG), P<0.0001, F (8, 63) = 44.85] (Fig. 5).
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Neurochemical Determinations Reversal of AMPH-induced brain oxidative alterations by the administration of LIRA alone and in combination with Li Mitochondrial dysfunction and the resulting oxidative imbalance are core alterations observed in BD patients (Berk et al. 2011a). Our results revealed that in the PFC, AMPH+SAL group showed a significant reduction in GSH levels when compared
AMPH+Li,
P<
oo
f
with SAL+SAL (P = 0.0013). LIRA240 and Li alone (AMPH+LIRA240 and 0.0001) or in combination (AMPH+Li+LIRA240, P< 0.0001) AMPH-induced GSH deficits. Additionally, the combination
pr
significantly reversed
e-
group (AMPH+Li+LIRA240) significantly increased GSH contents compared with
Pr
AMPH+Li-treated animals (P< 0.001) [F (5, 35) = 35.24] (Fig.6A). In the hippocampus, AMPH+SAL group also presented a significant reduction in
al
GSH levels compared to SAL controls (P< 0.001). LIRA240 (AMPH+LIRA240, P<
rn
0.001) and in combination with Li (AMPH+Li+LIRA240, P< 0.0001) significantly
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reversed AMPH-induced GSH deficits. The combination AMPH+Li+LIRA240 also significantly increased GSH contents related to AMPH+Li (P < 0.0001) [F (5, 35) = 48.53] (Fig.6B).
In the amygdala, AMPH+SAL caused a significant reduction in GSH compared to SAL+SAL (P < 0.001). LIRA alone and combined with Li as well as Li alone significantly
reversed
AMPH-induced
GSH
deficits
(AMPH+Li+LIRA240
vs.
AMPH+SAL, P< 0.001; AMPH+Li vs. AMPH+SAL, P = 0.0015). LIRA alone did not reversed
AMPH-induced
alterations
in
amygdala.
Finally,
the
combination
AMPH+Li+LIRA240 induced an additional increase in GSH levels compared to Li+AMPH group (P = 0.0051) [F (5, 39) = 15.12] (Fig.6C).
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Considering lipid peroxidation, in the PFC, AMPH+SAL caused a marked increase
(3.64-fold increase) in MDA levels compared to SAL+SAL group (P < 0.001). LIRA240 and Li alone or in combination reversed AMPH-induced MDA increase [AMPH+LIRA240 vs. AMPH+SAL, P = 0.0087; AMPH+Li vs. AMPH+SAL, P< 0.0001; AMPH+Li+ LIRA240 vs. AMPH+SAL, P< 0.0001, F (5, 39) = 25.98]. (Fig.6D) In the hippocampus AMPH+SAL also induced a great increase in MDA
f
compared to SAL+SAL (P < 0.001). LIRA240 (AMPH+LIRA240 vs. AMPH+SAL, P
oo
< 0.001) and Li (AMPH+Li vs. AMPH+SAL, P< 0.0001) and their combination
pr
(AMPH+Li+ LIRA240 vs. AMPH+SAL, P< 0.0001) significantly reversed AMPH-
e-
induced MDA alterations [F (5, 39) = 23.85] (Fig.6E).
In the amygdala, AMPH treatment significantly increased MDA contents in
Pr
relation to SAL controls (P< 0.001), and LIRA240 administration did not reverse this
al
alteartion. On the other hand, Li alone or in combination with LIRA reversed AMPH-
rn
induced MDA rise (P< 0.0001). Additionally, the combination AMPH+Li+LIRA240 induced a significant decrease in MDA levels when compared to AMPH+LIRA240
-
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group (P< 0.001) [F (5, 39) = 22.76] (Fig.6F). Reversal of AMPH-induced brain BDNF deficits by LIRA and Li Based on the importance of neurotrophic mechanisms in the neurobiology of BD (Grande et al. 2010), we decided to evaluate brain levels of BDNF. In the PFC, AMPH+SAL group presented a significant reduction in BDNF levels compared to SAL+SAL (P< 0.001). The treatment with LIRA240 or Li did not reverse this AMPHinduced BDNF changes. Contrarily, the combination AMPH+Li+LIRA240 significantly increased BDNF levels compared to AMPH+SAL (P = 0.0015) and AMPH+Li (P=0.0070) [F (5, 37) = 22.09] (Fig.7A).
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In the hippocampus, AMPH administration caused a marked decrease (0.39-fold changes, MD:-4440) in BDNF levels compared to SAL-SAL controls (P < 0.0001). The treatment with LIRA240 (AMPH+LIRA240 vs. AMPH+SAL, P = 0.0156) or combined with Li (AMPH+Li+LIRA240 vs. AMPH+SAL, P < 0.001) significantly reversed AMPH-induced
BDNF
deficits.
Additionally,
in
control
conditions,
LIRA
(SAL+LIRA240) significantly increased BDNF levels in comparison with SAL+SAL
f
group (P =0.0030) [F (5, 37) = 19.55] (Fig.7B).
oo
In the amygdala, AMPH treatment induced a marked increase (2.21-fold
pr
increase, MD:+3580) in BDNF levels related to SAL-controls (P < 0.001). Also, the group treated with LIRA240 (AMPH+LIRA240) similarly increased BDNF levels
e-
compared to controls (P = 0.0034) [F (5, 33) = 7.051]. There was no additional
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4. Discussion
al
Pr
significant difference between the tested groups (Fig.7C).
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In the present study, we bring the first evidence of the potential antimanic/mood stabilizing effects of the GLP-1R agonist LIRA. According to our results, LIRA rescued hyperlocomotion, executive and spatial memory deficits induced by AMPH, but failed to reverse risk-taking behavior and fear learning impairment associated to this model. Combined with Li, LIRA reversed the most behavioral changes induced by AMPH even with a superior effect than Li itself. Also, LIRA alone displayed an important hippocampal antioxidant and neurotrophic effect, while Li showed its protective effect mainly in the PFC and amygdala. Hence, the combination of LIRA and Li fully reversed the pro-oxidative changes in all tested brain areas, and markedly counteracted BDNF impairment in the PFC and hippocampus revealing a possible superior effect in
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relation to standard Li treatment. Additionally, LIRA rescued the increased % WG induced by Li in the course of mania model. Considering that LIRA can improve not only the efficacy, but also some side effects associated to standard Li therapy, it has a great potential for translation application in clinical setting for BD. The hyperdopaminergic state that takes place in mania may be mimicked in animals by the repeated administration of AMPH and evaluated by the measure of
f
hyperlocomotion in the OFT (Sharma et al. 2016). The mood stabilizing drugs, Li and
oo
valproate, classically suppress these alterations, supporting the predictive validity of
pr
these models (Frey et al. 2006b; Macedo et al. 2013). However, mania is a complex syndrome with multifaceted symptoms and the investigation of multiple affective and
e-
cognitive domains should be performed to adequately evaluate mania (Logan and
Pr
McClung 2016). In this regard, besides hyperlocomotion, reduced anxiety/increased risk-taking behavior, hypersexual behavior and multiple cognitive deficits, for example
al
in executive, spatial and affective domains, were previously observed in AMPH model
rn
(Fries et al., 2015; Zhou et al., 2015). Hence, this model can induce other features of
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mania than only the behavioral activation. GLP-1R signaling is being related
to
the regulation of neural/cognitive
processes. Regarding mood disorders, a recent study showed, in corticosterone induceddepression model, that repeated LIRA treatment has antidepressant- and anxiolytic-like effects (Weina et al. 2018). Also, GLP-1R agonists can protect against the comorbid depression- and anxiety-like behavior associated to several conditions, such as diabetes (Mansur et al. 2018) and epilepsy (de Souza et al. 2019). Also, the effects of GLP-1R agonists in emotional behavior seems to be dependent of the duration of treatment and route of administration. Acute IP and central (intra-dorsal raphe) injections of GLP-1 analogue induced anxiety-like behavior in
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rodents, while subchronic central administration (for 9 days) promoted anxiolytic- and antidepressant-like effects (Anderberg et al. 2016). Based on this observation, in our study we used a protocol of 7 days of LIRA administration. To date, no study has evaluated the effects of GLP-1R agonists in BD mania. Here, we showed that LIRA 7 days administration at the doses of 120 and 240 ug/kg alone and in combination with Li presents antimanic-like effects based on the reversal
f
of AMPH-induced hyperlocomotion. In order to better characterize the antimanic
oo
effects of LIRA, we evaluated risk-taking behavior and impulsivity, by the evaluation of
pr
central square exploration of the OFT and open arms occupancy in EPM test (Macr et al. 2002; Tillmann and Wegener 2019) since they are key features of mania that usually
e-
have difficult management and are strongly associated to high suicide rates (Izci et al.
Pr
2016; Pawlak et al. 2016).
al
In our experimental conditions, LIRA showed a small effect against AMPH-
rn
induced risk-taking behavior. However, when combined with Li, LIRA at both doses reversed AMPH-induced center exploration and the increase in % of open arms entries
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and duration. It is worth to mention that Li alone counteracted only AMPH-induced changes in EPM test but did not reverse the increase in center occupancy in the OFT. Hence, these results suggest an augmentation of Li effects by LIRA. Li is recognized as a standard therapeutic option to control impulsivity/risktaking behavior in BD, an effect that does not appear to be solely associated to its preventive effects on mood changes (Cipriani et al. 2013). Despite this, there is some controversy in this effect of Li. In preclinical settings, while considerable evidence has showed an effect of Li in the control of impulsive and risky behavior, some authors also reported Li inability to reverse risk-taking behavior in some conditions, such as median raphe nucleus lesion and glutamate receptor deletion (Shaltiel et al. 2008; Pezzato et al.
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2015). In this regard, a meta-analysis study failed to find strong evidence for Li use as anti-impulsivity therapy in BD and concluded that more studies are necessary to firmly propose this indication (Jones et al. 2011). Our results highlight the potential of LIRA and mainly of its association with Li to control risky and impulsive behavior. GLP-1Rs are widely distributed in the CNS and present a great influence on cognition. Convergent evidence have demonstrated that the stimulation of GLP-1Rs
f
(through GLP-1 or synthetic ligands) is associated to pro-cognitive effects (McIntyre et
oo
al. 2013). In fact, a single GLP-1 injection improved spatial memory, as well as
pr
increased cognitive flexibility and executive function in naïve animals (During et al. 2003). GLP-1R agonists, such as LIRA, can protect against the cognitive deficit
e-
associated to several models of neurodegeneration such as Alzheimer’ and Parkinson’
Pr
Disease (Yang et al. 2013; McClean and Hölscher 2014; Zhang et al. 2018). Additionally, GLP-1R agonists also prevented the cognitive impairment induced by the
rn
al
high-fat diet in mice (Porter et al. 2012).
Here, we observed that AMPH-induced mania markedly compromised the
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performance of mice in the Y maze and NOR tasks. It is well described that impairments in executive function and declarative memory are frequently found in BD patients (Venza et al. 2016). In accordance with our results on memory impairment in AMPH mania model, previous studies have already demonstrated that AMPH administration can mimic several aspects of BD cognitive disability,
such as
dysfunction in executive and recognition tasks (de Souza Gomes et al. 2015; Fries et al. 2015). Thus, considering the strong relationship between metabolic and cognitive functions in BD, GLP-1R system has emerging as a promising target to modulate both functions. In our study, the standard mood stabilizer Li could not reverse AMPH-
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induced deficits in executive and spatial memory, but LIRA alone mainly at the higher tested
dose
combination
considerably improved of
Li+LIRA
mice performance on both domains.
completely
reversed
this
alteration,
overcoming
The the
limitations of Li treatment at monotherapy. Despite being the first-line maintenance treatment for BD, Li presents severe adverse effects, mainly of metabolic nature (Vendsborg et al. 1976). One important
f
adverse effect of Li is diabetes insipidus and WG, which are among the top five clinical
oo
causes for drug discontinuation (Öhlund et al. 2018). These adverse events affect up to
pr
61% of patients and a WG over than 10 kg have been described in 20% of Li-treated
e-
subjects (Chengappa et al. 2002; Praharaj 2016).
In our study, Li treatment promoted a marked increase in the % WG along the
Pr
induction of the mania model. Interestingly, LIRA in combination with Li prevented the
al
weight changes induced by Li. In naïve animals, LIRA also induced weight loss,
rn
confirming its anorexic effect. Indeed, a previous report showed that a single LIRA injection (100 µg/kg) is able to induce anorexic effects in mice and a weight loss that
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persisted for 4 days (Nonogaki and Kaji 2018). Notably, a recent study reported LIRA metabolic effects on BD patients. Despite the limited number of patients (n=29), the authors showed LIRA’s efficacy to induce weight loss even when other multiple lifestyle interventions, such as exercise and diet programs, have been failed. Also, LIRA was well-accepted and well-tolerated in stable BD patients, as well as did not compromised their psychiatric condition (Cuomo et al., 2018). Therefore, taking these evidence together, LIRA can represent a useful strategy for counteracting the weight disturbances in BD, however larger, longer and well-controlled clinical studies should be performed to make reliable conclusions about this indication.
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In the last decades, several evidences point towards the presence of systemic pro-oxidative changes in all phases of BD, including euthymic states (Kuloglu et al. 2002; Kunz et al. 2008; Steckert et al. 2010). Also, markers of oxidative damage have been consistently found in post-mortem brain of BD patients, mainly in mood-regulated brain areas (GAWRYLUK et al. 2011). In the context of DA dysregulation hypothesis, the hyperdopaminergic state involved in mania compose a strong basis for the oxidative imbalance (Berk et al. 2007). Indeed, dopamine is metabolized by monoamine oxidase
oo
f
to hydrogen peroxide (H2 O2 ). In the presence of Fe2+ and H2O2, the formation of 6hydroxydopamine (6-OHDA) takes place (Stokes et al. 1999; Yamato et al. 2010). 6-
e-
pro-apoptotic pathways (Blum et al. 2001).
pr
OHDA is a potent neurotoxin capable to impair mitochondrial functioning and activate
Pr
Here, as expected, AMPH treatment caused a marked increase in lipid peroxidation and compromised the endogenous antioxidant GSH in all studied brain
al
areas. Regarding AMPH mania model, several authors have already demonstrated
rn
oxidative damage and impairment in endogenous antioxidants in limbic brain areas
Jo u
(Frey et al. 2006b; Macêdo et al. 2012; Macedo et al. 2013). TBARS levels in BD patients presented the larger effect size compared to the other enzymatic and non-enzymatic markers of oxidative stress (Andreazza et al. 2008). Still in BD patients, increased lipid peroxidation also negatively correlates with cognitive performance, mainly executive functioning (Newton et al. 2015). Serum TBARS levels are increased in all BD phases, but reaches the higher levels in the manic episodes, indicating the occurrence of a greatest oxidative stress in mania (Kunz et al., 2008). GSH is the main non-protein thiol in cells acting as a cofactor for antioxidant and detoxifying reactions (Marí et al. 2009; Ribas et al. 2014). Reduced GSH levels and
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abnormalities in GSH synthesizing enzymes were reported in brain and serum of BD patients. Indeed, mice with genetic deletion of glutamate cysteine ligase modulatory (GCLM) gene (a key GSH synthesizing enzyme) (Yang et al., 2002) presents a complex behavior resembling features of schizophrenia and BD mania. These animals showed novelty-induced hyperlocomotion and an exacerbated response to low dose of AMPH (Kulak et al., 2012). Other models of GSH deficiency in adult mice resulted in marked cognitive alterations, such as impairment in object recognition and spatial working
oo
f
memory (Cabungcal et al. 2007; das Neves Duarte et al. 2012).
pr
The neuroprotective effects of mood stabilizing drugs have been strongly associated to its antioxidant properties (Niciu et al. 2013). Lithium at therapeutically
protein
oxidation
and
DNA
Pr
peroxidation,
e-
relevant concentrations prevented glutamate- and H2 O 2 -induced cell death, lipid fragmentation
(Shao
et
al.
2005).
Interestingly, in early-diagnosed BD patients, reduction in serum TBARS levels only
al
occurs in Li responder patients, thus, reinforcing the involvement of antioxidant
rn
properties to Li mood stabilizer efficacy (De Souza et al., 2014). In animal models of
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mania, Li protected against brain pro-oxidative changes (lipid peroxidation, DNA oxidative damage, GSH depletion) induced by AMPH (Frey et al. 2006b) and sleep deprivation (Valvassori et al. 2017). In our results, Li completely reversed the AMPH-induced increase in lipid peroxidation in all tested brain areas, as well as fully restored GSH contents in the PFC and amygdala. For the first time, we showed LIRA ability to counteract lipid peroxidation and GSH impairment induced by AMPH mania model. Interestingly, this antioxidant effect of LIRA showed a brain area-selective pattern, occurring in the PFC and hippocampus, and not in amygdala. Notably, Li+LIRA240 in combination showed
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an increased antioxidant potency, fully reversing lipid peroxidation and GSH depletion induced by AMPH in all brain areas. GLP-1Rs are molecularly coupled to cell antioxidant defenses. When activated, GLP-1Rs via Gα-protein stimulates adenyl cyclase, and subsequently Akt (also known as protein kinase B – PKB). Activated Akt phosphorylates multiple targets in the cell to regulate adaptive responses to diverse insults, including mitochondrial
GSK3β,
a
major
protein
kinase
involved
oo
inhibits
f
dysfunction and oxidative stress. Importantly, Akt phosphorylates and, consequently in
pro-apoptotic
and
pr
neurodegenerative events, as well as in mood stabilizing Li mechanism (Freyberg et al. 2010). Accordingly, Li directly and indirectly inhibits GSK3β (Freland et al.,
e-
2012).
Pr
The antioxidant effects of LIRA may be related to a stimulation of nuclear
al
factor erythroid 2-related factor (Nrf2) pathway (Caihong et al., 2018). When
involved
in
rn
activated, Nrf2 migrates to nucleus and induces the expression of several genes antioxidant
response,
including
superoxide
dismutase,
catalase
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glutathione peroxidase, glutathione reductase and γ-glutamate cysteine ligase (Hayes and Dinkova-Kostova 2014). Similarly, Li display neuroprotective actions against excitotoxicity and pro-oxidant insults through Nrf2 pathway induction (Rojo et al., 2008; Cunha et al., 2016, Pan et al., 2019). Brain-derived neurotrophic factor (BDNF) plays a major role in synaptic plasticity and
neurogenesis.
Plasma BDNF negatively correlates with symptoms
severity as well as increases with mood stabilizer treatment (Fernandes et al., 2015). Here, we observed that AMPH (for 14 days) caused a marked decrease in BDNF expression in the PFC and hippocampus, while increased its levels in the amygdala. Li induced a discrete rise in this neurotrophin levels in PFC and hippocampus, while
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LIRA restored BDNF levels in the hippocampus. The combination Li+LIRA240 markedly restored BDNF levels in the PFC and hippocampus. However, similar to Li alone, this association demonstrated just a modest effect in amygdala. BDNF impairment has been importantly associated to AMPH-induced deficits in working memory and spatial recognition in mice. In this regard, Fries et al. 2015 reported that AMPH caused time-related changes in BDNF expression (mRNA and
f
protein levels) in the PFC, hippocampus and amygdala of mice. In their study,
oo
chronic AMPH treatment (for 35 days) induced a progressive decrease in BDNF
pr
expression in the hippocampus, while increased BDNF mRNA levels in amygdala (Frey et al., 2015). Other authors also showed that repeated AMPH increased BDNF
e-
levels in the amygdala of rodents. Regarding PFC, there are discrepancies in the
Pr
literature with some studies reporting decreased BDNF in the PFC after AMPH treatment (Valvassori et al., 2018), while others reported increased (Shen et al., 2014)
rn
al
or even no change in this neurotrophin levels (Stertz et al., 2013). Amygdala is the central core for affective learning, ensuring the subject
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capacity to adequately respond to sources of danger (Paré et al., 2004). Neuroimaging studies have reported an overactivated amygdala in the brain of BD patients, especially in manic episodes (Bermpohl et al., 2009), with subsequent volume reduction with disease progression. These findings have been coupled to decrements in the activity of some PFC subregions, such as ventrolateral PFC (Altshuler et al., 2005). Further, the connectivity between amygdala and PFC, namely ventrolateral prefrontal-amygdala emotional pathway, which is responsible for tuning control of amygdala reactivity, is disrupted in BD and accounts for the abnormal responses to emotional cues and impaired attentional shift in these patients (Strakowski et al., 2011).
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Here, we found that AMPH treatment markedly impaired fear learning in mice as demonstrated by the premature stepdown latency. Interestingly, this cognitive profile was followed by increased markers of oxidative stress and abnormal increased levels of BDNF
in the amygdala. This result points to a possible ongoing
neurodegenerative process in the amygdala potentially responsible for the disrupted emotional learning induced by AMPH mania model. Lithium treatment efficiently prevented this impairment in affective memory, but only partially rescued AMPH-
oo
f
induced neurochemical changes.
pr
Here, LIRA showed a considerable ability to upregulate hippocampal BDNF expression, and when in combination with Li, also reversed PFC BDNF changes.
e-
This represents a potential mechanism for LIRA protective effects against AMPH-
Pr
induced cognitive dysfunction. In fact, LIRA and other GLP-1R agonists can increase the expression of BDNF and other neurotrophins in mice brain exposed to several
al
neuropathology conditions, such as aging (Bomba et al., 2018), epilepsy (De Souza et
rn
al., 2019) and diabetes (Gumuslu et al., 2016). This LIRA effect can be explained
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through its stimulatory effect on protein kinase A (PKA) and Akt (Deng et al. 2018). Each of these kinases can mutually phosphorylate and, thereby, activate cAMP response element-binding protein (CREB). CREB activation can subsequently upregulate BDNF expression (Yossifoff et al. 2008; Zheng et al. 2012). Recently, it was also demonstrated that LIRA can restore spine density and dendrite outgrowth in the hippocampus of dexamethasone-treated rats through activation of mTOR1 signaling and induction of BDNF expression (Park et al. 2018). These pathways represent potential underlying mechanisms for the observed pro-cognitive and BDNF upregulating effects observed here, but needs further investigation. Limitations and Perspectives
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This study has some limitations. First, no existing animal model can fully resemble the complex psychopathological expression of BD mania. Second, our study evaluated a correlation between intervention and outcome and was not delineated to clarify possible mechanistic pathways, and, with our results, we cannot propose the precise mechanisms involved in the LIRA behavioral effects observed here. Therefore, investigating the possible molecular targets of LIRA effects against neurobehavioral features of BD mania, such as Akt activation, GSK3β inhibition and induction of Nrf2
oo
f
antioxidant cell response, remains as an interesting question for future research in the field. Finally, we did not measure plasma glucose levels in animals treated with Li and
e-
pr
LIRA, this is important since both drugs can interfere with glucose metabolism.
Pr
Conclusions
al
The present study demonstrated the potential antimanic/mood stabilizing effects
rn
of the GLP-1R agonist LIRA in AMPH mania model. Of note, LIRA alone was hyperlocomotion, executive and spatial memory
Jo u
effective against AMPH-induced
dysfunction. This LIRA effect was associated to a notorious antioxidant action in hippocampus and PFC and up-regulating effects on hippocampal BDNF expression. Also, LIRA improved the profile of Li behavioral effects, thereby overcoming the inability of Li to reverse the main cognitive changes induced by AMPH, as well as augmenting its antioxidant and BNDF modulatory actions. Finally, LIRA prevented the weight gain associated to Li exposure in mice. Taking together, our results suggest the possible involvement of GLP-1 system in the pathophysiology of BD and highlight this system as a promising target to development of new therapies able to counteract both metabolic and cognitive changes involved in this disease. In this context, LIRA can
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represent useful adjunctive tool for BD treatment with a great potential for translation application.
Acknowledgements
f
The authors thank the Brazilian Institutions CAPES, FUNCAP and CNPq for the
pr
oo
financial support of this study.
e-
Author’s disclosures
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rn
al
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The authors declare no conflict of interests.
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Figure Legends Figure 1. Experimental design timeline. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium. Figure 2. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone and associated with Lithium (Li) in the reversal of amphetamine 2.0 mg/kg (AMPH)induced changes in open field test parameters: A- zone transition number (n); B- total
oo
f
distance (cm2 ), C- entries in central area (n), D- time in central area (s). N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01,
pr
***P< 0.001, ****P< 0.0001 according to one-way ANOVA followed by Tukey׳s post-
e-
hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li:
Pr
lithium.
Figure 3. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone
al
and associated with Lithium (Li) in the reversal of amphetamine 2.0 mg/kg (AMPH)-
rn
induced changes in elevated plus maze test parameters: A- total entries (n); B- open
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entries/total entries (%), C- time in open arms/total duration (%). N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 according to one-way ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium. Figure 4. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone and associated with Lithium (Li) in the reversal of amphetamine 2.0 mg/kg (AMPH)induced changes in mice cognitive performance on y-maze test (A - correct alternations %, B- total entries); novel object recognition test (C- recognition index %), and passive avoidance test (D-step-down latency in seconds). N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001
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according to one-way ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium. Figure 5. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) in Lithium (Li)-induced weight gain (grams) in the amphetamine 2.0 mg/kg (AMPH) mania model. The graphic plot represents the weight gain in percentage (%) comparing the last day of treatment (14th day) with the first one (1st day) of the experimental and
f
control groups. N=8 animals per group. Bars represent mean ± standard error media
oo
(SEM). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 according to one-way
e-
liraglutide; AMPH: amphetamine; Li: lithium.
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ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA:
Figure 6. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone
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and associated with Lithium (Li) in the amphetamine 2.0 mg/kg (AMPH)-induced
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changes in oxidative stress markers in the mice brain. Graphic plots represent reduced glutathione (GSH) concentrations in the prefrontal cortex (PFC)- A, hippocampus (HC)
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– B and amygdala (AM) – C, and malondialdehyde (MDA) concentrations in the same
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brain areas: PFC – D, HC – E and AM – F. N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 according to one-way ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium; GSH: reduced glutathione; MDA: malondialdehyde; PFC: prefrontal cortex; HC: hippocampus; AM: amygdala. Figure 7. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone and associated with Lithium (Li) in the amphetamine 2.0 mg/kg (AMPH)-induced changes in brain-derived neurotrophic factor (BDNF) concentrations in the mice brain: A - prefrontal cortex, B – hippocampus and C - amygdala. N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01, ***P< 0.001,
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****P< 0.0001 according to one-way ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium; BDNF: brain-derived neurotrophic factor; PFC: prefrontal cortex; HC: hippocampus; AM: amygdala. Figure 8. Summarizing representation of the experimental findings and possible intracellular pathways underlying the antimanic effects of Liraglutide (LIRA) treatment.
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Blue arrows represent downstream stimulatory pathways and red lines represent
neurotrophic factor; GSH: reduced glutathione; GLP-1RA:
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BDNF: brain-derived
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downstream inhibitory pathways. Abbreviations: AMPH: amphetamine; Li: lithium;
glucagon-like peptide-1 receptor agonist ; AC: adenylyl cyclase; PKA: protein kinase
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A; p-CREB: phospho- cAMP response element-binding protein; PI3K: phosphoinositide
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3-kinase; p-AKT (PKB): phosphor-protein kinase B; GSK3β: glycogen synthase kinase
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related factor 2.
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3 beta; KEAP1: kelch-like ECH associated protein 1; NRF2: nuclear factor erythroid -
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Weina H, Yuhu N, Christian H, Birong L, Feiyu S, Le W. Liraglutide attenuates the
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depressive- and anxiety- like behaviour in the corticosterone induced depression model via improving hippocampal neural plasticity. Brain Res. 2018;1694:55–62. Wingo AP, Wingo TS, Harvey PD, Baldessarini RJ. Effects of lithium on cognitive performance: a meta-analysis. J Clin Psychiatry. 2009 Nov;70(11):1588–97. Yamato M, Kudo W, Shiba T, Yamada K, Watanabe T, Utsumi H. Determination of reactive oxygen species associated with the degeneration of dopaminergic neurons during dopamine metabolism. Free Radic Res. 2010 Mar;44(3):249–57. Yang Y, Zhang J, Ma D, Zhang M, Hu S, Shao S, et al. Subcutaneous administration of liraglutide ameliorates Alzheimer-associated tau hyperphosphorylation in rats with
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Zhou Z, Wang Y, Tan H, Bharti V, Che Y, Wang JF. Chronic treatment with mood stabilizer lithium inhibits amphetamine- induced risk-taking manic-like behaviors.
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AJMCF, DSM – designed the study and wrote the first draft of the manuscript; NLC, AGS, MVRS – performed the behavioral determinations; AJMCF, PMJ, TQ, JVSO – performed the neurochemical determinations; DSM, SSV, TB – performed the statistical analysis, JQ, DL – contributed to the study design and revised the last version of the manuscript.
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The authors declare no conflict of interests
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Highlights:
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AMPH induced risk-taking behavior and cognitive deficits that resemble mania LIRA reversed AMPH-induced hyperlocomotion, working and recognition memory impairments Li+LIRA240 rescued all behavioral changes induced by AMPH LIRA reversed AMPH-induced hippocampal oxidative and neurotrophic changes Li+LIRA240 augmented Li antioxidant effects and reversed AMPHinduced BDNF changes
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Adriano José Maia Chaves Filho, Natássia Lopes Cunha, Alana Gomes de Souza, Michele Verde-Ramo Soares, Paloma Marinho Jucá, Tatiana de Queiroz, João Victor Souza Oliveira, Samira S. Valvassori, Tatiana Barichello, Joao Quevedo, David de Lucena, Danielle S. Macedo PII:
S0278-5846(19)30595-0
DOI:
https://doi.org/10.1016/j.pnpbp.2020.109872
Reference:
PNP 109872
To appear in:
Progress in Neuropsychopharmacology & Biological Psychiatry
Received date:
18 July 2019
Revised date:
31 December 2019
Accepted date:
15 January 2020
Please cite this article as: A.J.M.C. Filho, N.L. Cunha, A.G. de Souza, et al., The GLP-1 receptor agonist liraglutide reverses mania-like alterations and memory deficits induced by D-amphetamine and augments lithium effects in mice: Relevance for bipolar disorder, Progress in Neuropsychopharmacology & Biological Psychiatry(2019), https://doi.org/ 10.1016/j.pnpbp.2020.109872
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© 2019 Published by Elsevier.
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The GLP-1 receptor agonist liraglutide reverses mania-like alterations and memory deficits induced by D-amphetamine and augments lithium effects in mice: relevance for bipolar disorder Adriano José Maia Chaves Filho1 , Natássia Lopes Cunha1 , Alana Gomes de Souza1 , Michele Verde-Ramo Soares1 , Paloma Marinho Jucá1 , Tatiana de Queiroz1 , João Victor Souza Oliveira1 ,
Samira S Valvassori2 , Tatiana Barichello2,3,4,5 , Joao Quevedo2,3,4,5 ,
Neuropharmacology Laboratory, Drug Research and Development Center, Department of Physiology
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1.
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David de Lucena1 , Danielle S Macedo1,6,*
2.
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and Pharmacology, Faculty of Medicine, Federal University of Ceara, Fortaleza, CE, Brazil Laboratory of Neurosciences, Graduate Program in Health Sciences, Health Sciences Unit,
3.
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University of Southern Santa Catarina (UNESC), Criciúma, SC, Brazil Translational Psychiatry Program, Department of Psychiatry and Behavioral Sciences, McGovern
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Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston,
4.
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TX, USA
Center of Excellence on Mood Disorders, Department of Psychiatry and Behavioral Sciences,
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McGovern Medical School, The University of Texas Health Science Center at Houston (UTHealth), Houston, TX, USA 5.
Neuroscience Graduate Program, The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX, USA
6.
National Institute for Translational Medicine (INCT-TM, CNPq)
*Corresponding author: *Danielle S. Macedo. Department of Physiology and Pharmacology, Federal University of Ceara, Rua Cel. Nunes de Melo 1000, zip code 60430-275, Fortaleza, CE, Brazil. Phone:
+55-85-3366-8337/+55-85-3366-8036;
Fax:
[email protected]; [email protected]
+55-85-3366-8333;
E-mail:
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Abstract
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Metabolic and psychiatric disorders present a bidirectional relationship. GLP-1 system, known for its insulinotropic effects, has also been associated with numerous regulatory effects in cognitive and emotional processing. GLP-1 receptors (GLP-1R) agonists present neuroprotective and antidepressant/anxiolytic properties. However, the effects of GLP-1R agonism in bipolar disorder (BD) mania and the related cognitive disturbances remains unknown. Here, we investigated the effects of the GLP-1R agonist liraglutide (LIRA) at monotherapy or combined with lithium (Li) against Damphetamine (AMPH)-induced mania-like symptoms, brain oxidative and BDNF alterations in mice. Swiss mice received AMPH 2 mg/kg or saline for 14 days. Between days 8-14, they received LIRA 120 or 240 µg/kg, Li 47.5 mg/kg or the combination Li+LIRA, on both doses. After behavioral evaluation the brain areas prefrontal cortex (PFC), hippocampus and amygdala were collected. AMPH induced hyperlocomotion, risk-taking behavior and multiple cognitive deficits which resemble mania. LIRA reversed AMPH-induced hyperlocomotion, working and recognition memory impairments, while Li+LIRA240 rescued all behavioral changes induced by AMPH. LIRA reversed AMPH-induced hippocampal oxidative and neurotrophic changes. Li+LIRA240 augmented Li antioxidant effects and greatly reversed AMPH-induced BDNF changes in PFC and hippocampus. LIRA rescued the weight gain induced by Li in the course of mania model. Therefore, LIRA can reverse some mania-like behavioral alterations and combined with Li augmented the mood stabilizing and neuroprotective properties of Li. This study points to LIRA as a promising adjunctive tool for BD treatment and provides the first rationale for the design of clinical trials investigating its possible antimanic effect.
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Keywords: Bipolar disorder; mania; GLP-1; Liraglutide; D-amphetamine; cognitive impairment; oxidative stress; brain-derived neurotrophic factor (BDNF).
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1. Introduction
Bipolar disorder (BD) is a chronic psychiatric disorder that follows a progressive course of mood and cognitive manifestations. Manic or hypomanic episodes are a distinctive psychopathological feature of BD. These episodes are characterized by elevated mood, impulsivity, irritability, hypersexual behavior, psychomotor agitation,
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circadian alterations and cognitive deficits (Hilty et al. 2006; Tondo et al. 2017). Mania-
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depression cyclic alternation represent a key factor for BD neuroprogression (Berk et al.
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2011b). Indeed, progressive functional impairment is observed from BD clinical stage I to stage IV, with cognitive and functional decline being observed in stages III and IV
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(Cardoso et al. 2015). Furthermore, mania seems to have more significant impact on
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cognition. In other words, manic patients usually present poorer cognitive abilities in
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2004; Gruber et al. 2007).
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several domains when compared to depressed and euthymic BD patients (Dixon et al.
Despite the pathophysiology of BD is not fully understood, several lines of
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evidence indicate that oxidative and nitrosative stress (O&NS) as well as abnormalities in brain neurotrophins,
mainly represented
by brain-derived neurotrophic factor
(BDNF), are importantly involved in BD neurobiology (Post 2010; Fernandes et al. 2011). In this context, animal models can represent useful tools to elucidate underlying biological pathways as well as to discover new therapeutic agents for this condition (Sharma et al. 2016). Regarding mania models, psychostimulants-induced manic-like symptoms have been classically used as models to predict new anti-maniac/mood stabilizing agents. The most studied of these pharmacological models is D-amphetamine (AMPH)-induced mania model (Frey et al., 2006; Macedo et al., 2013; Martins et al., 2006; Valvassori et
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al., 2019). In this model, hyperactivity has been traditionally used as main feature to measure manic-like symptoms and mood stabilizing response (Lan and Einat 2019). In this context, some studies have deeply investigated the AMPH model and demonstrated that AMPH repeated exposure induces a broader spectrum of behavioral changes
than
aggressiveness,
just
hyperlocomotion,
impulsivity/risk-taking
including behavior
enhanced and
sexual
cognitive
behavior, dysfunction
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(deCatanzaro and Griffiths 1996; Van Enkhuizen et al. 2013; Zhou et al. 2015). More
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recently, some authors also showed that, during the withdraw period after AMPH
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exposure, a depression-like phenotype associated to an increased sensitization to subsequent low doses of AMPH emerges, resembling the cyclic nature of mood changes
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in BD (Pathak et al. 2015; Valvassori et al. 2019). Although the potential overlapping
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with drug addiction, AMPH model is relevant to study the pathophysiological processes
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underlying mood disturbances in BD (Dencker and Husum 2010; Pathak et al. 2015).
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Cognition is a core domain of psychopathology in BD. At least 25% of BD patients exhibit pronounced deficits in one or more domain of function (Gualtieri and
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Morgan 2008). Cognitive dysfunction also seems to be the major responsible factor for disability and social burden independently of concurrent mood state (Gualtieri and Morgan 2008; Simonsen et al. 2008). Additionally, current mood stabilizing and antidepressant drugs cannot efficiently restore or treat cognitive impairment associated to BD (Wingo et al. 2009; Mora et al. 2013). In the last decades, a clear bidirectional relationship between BD and metabolic disorders has emerged. BD individuals with metabolic comorbidities develop an unfavorable course of disease with increased refractoriness, atypical features and disability (McIntyre et al. 2010). Metabolic disorders seem to mainly impact cognition. Metabolic abnormalities, such as insulin resistance (Hajek et al. 2014; Cairns et al.
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2018), visceral adiposity, dyslipidemia (Fleet-Michaliszyn et al. 2008; Elmslie et al. 2009) and high blood pressure (Brown et al. 2009; Depp et al. 2014) have been negatively associated to neurocognitive function in BD. These alterations, mainly insulin resistance, are frequently present in BD even in newly-diagnosed patients (Coello et al. 2019). Furthermore, mood stabilizers and antipsychotic drugs used for long-term BD management, frequently cause metabolic iatrogenic effects and increase the risk for weight gain and metabolic syndrome (McIntyre et al. 2011; Kocakaya et al.
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2018).
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Glucagon-like peptide 1 (GLP-1) is a secreted incretin that facilitates the glucose uptake by peripheral organs after feeding. Also, GLP-1 (and its synthetic analogues) can
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easily cross the blood-brain barrier and centrally regulate energy expenditure, food
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intake and hypothalamic responses (López-Ferreras et al. 2018). GLP-1 receptors (GLP1Rs) are widely distributed in brain structures, including not only hypothalamus and
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al. 2015).
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pituitary, but also in PFC, hippocampus, amygdala and other limbic structures (Cork et
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Besides its metabolic effects, compelling evidence have reported significant neuroprotective effects of GLP-1 and its analogues. In fact, GLP-1R agonists protects against cognitive damage associated to several neurodegenerative models, as well as increase neurotrophin expression and neuronal proliferation and differentiation (Porter et al. 2012; McClean and Hölscher 2014; Li et al. 2015). GLP-1R agonists also prevented depression- and anxious-like behavior associated to stress and metabolic conditions (Weina et al. 2018; de Souza et al. 2019). More recently, two open-label clinical trials have reported that the GLP-1R agonist liraglutide (LIRA) can improve cognitive function in stable BD and major depression patients (Mansur et al. 2017b, 2017a). However, at the present time there is no direct clinical or preclinical evidence
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have investigated the GLP-1R agonists effects on behavioral features related to BD mania, in order to further support the potential use of these drugs as new tools for mania treatment. Therefore, our first objective in this study was to investigate the effects of the GLP-1R agonist LIRA at monotherapy or combined with lithium (Li) against maniclike behavioral alterations induced by AMPH in mice. Our second objective was to
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verify the possible protective effects of LIRA against oxidative damage and brain-
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derived neurotrophic factor (BDNF) changes induced by AMPH in mood-regulating
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brain areas, namely prefrontal cortex (PFC), hippocampus and amygdala. In the present study, we hypothesized that LIRA alone could display protective actions against
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behavioral and neurochemical changes induced by AMPH mania model by mechanisms
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related to neurotrophic changes and oxidative homeostasis and when combined with Li
2.1 Animals
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2. METHODS
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could improve the effects of this mood stabilizing drug.
Male adult (postnatal day 70) Swiss mice, weighing 25-30 g, provided from the Animal House of the Federal University of Ceara, were used. The animals were maintained at a controlled temperature (22±2°C) with a 12-h dark/light cycle and food and water ad libitum. Mice were maintained in groups of 8 animals in 41 × 34 × 16 cm open-topped cages. The animals were manipulated according to the NIH Guide for the Care and Use of Laboratory Animals (NIH 2011). The experiments were performed in accordance with the ethical principles adopted by the Brazilian College of Animal
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Experimentation (COBEA) and approved by the Ethics Committee on Animal Research of the Federal University of Ceara with the protocol number (97/15). 2.2 Drugs Liraglutide
(Novo
Nordisk),
D-amphetamine
(AMPH)
and
lithium
carbonate (Li) (Sigma-Aldrich, Brazil) were directly dissolved in sterile saline solution (NaCl 0.9%, w/v). The drugs were made up freshly within 1–2 h of dosing and were
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administered intraperitoneally (IP) in a volume of 0.1 ml/10 g body weight. All other
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chemicals used were of analytical grade.
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2.3 Experimental design
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A reversal protocol was used as previously described (Frey et al. 2006b). By the conduction of the reversal model, we aimed to simulate the treatment of an acute manic
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state. For this end, 144 animals were used. Each group consisted of 16 randomly
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allocated animals. Mice were habituated for 7 days in their home cages before the beginning of the treatment. After this, each animal received for 14 days one daily IP
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administration of AMPH (2.0 mg/kg) or 0.9 % saline. Between the 8th and 14th days of treatment, AMPH and saline-treated animals received LIRA (120 and 240 µg/kg, SC), Li (47.5 mg/kg, IP) or saline solution once a day. For combined treatment, Li-treated groups received an additional subcutaneous injection of LIRA (120 and 240 µg/kg, SC) or saline. All other groups received a third saline injection. The time interval between each drug administration in all situations was 30 min. The selected doses of LIRA and the treatment duration were based on previous studies reporting LIRA’s neuroprotective and pro-cognitive effects in Alzheimer’s (Yang et al. 2013) and Parkinson’s disease (Zhang et al. 2018) models, as well as the
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selected doses are within the dose range capable to reverse weight gain (WG) and glucose intolerance associated to metabolic syndrome in mice (100-300 µg/kg) (Knudsen 2010; Fransson et al. 2014). Deserves mention the fact that the only difference in LIRA effects observed between humans and rodents is the much longer half-life of LIRA in humans than in rodents (Knudsen 2010). Despite this, we observed significant results of LIRA in the reversal of AMPH-induced alterations with a single daily dose. Additionally, the doses of LIRA used in this study were calculated to mice
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based on the human doses of 0.6 and 1.2 mg/day used for obesity and weight loss treatment. This calculation was performed by Reagan-Shaw dose translation algorithm
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using human-mouse body surface area (BSA) for mice (Reagan-Shaw et al. 2008). In
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the case of Li, the dosage regimens were based on previous preclinical reports of anti-
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manic-like effect of this drug (Macêdo et al. 2012; De Souza et al. 2015; Queiroz et al. 2015). Control animals received only saline. The animal weight was measured daily
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before each drug administration and showed as % WG. The behavioral determinations
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were conducted at the 14th day of treatment, 2 h after the last drug injection.
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To avoid bias related to the excessive exposure to behavioral apparatus, each experimental group assigned for behavioral determinations was divided into two subgroups of 8 animals. In this regard, the subgroup one (N = 8/group) was subjected to the open field (OFT), elevated plus maze (EPM) and passive shock avoidance test, in this order. Subgroup two (N = 8/ group) was subjected to y-maze and novel object recognition (NOR) task, in this order. A 1-hour interval was adopted between behavioral tests. The order of behavioral tests in each experimental set was defined from the least to the most stressful (Paylor et al. 2006). The tests were conducted by two independent trained observers blinded to treatment groups.
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Also, to avoid the stress associated to exposure of behavioral testing on neurochemical parameters, a third subgroup (N = 7-8/group) of different animals was used for biochemical analyses. For these assays, we selected the dose of LIRA, 240 µg/kg, SC, which showed the best effects on behavioral testing. The decision of continuing the study with the best dose of LIRA was based on the principle of the 3Rs (Replacement, Reduction and Refinement) for ethical use of animals in testing (Flecknell 2002). Afterwards, mice were sacrificed by decapitation and the prefrontal
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cortex (PFC), hippocampus, and amygdala were carefully dissected according to the method previously described (Iversen and Glowinski 1966). The brain samples were
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rapidly frozen and stored at -80º C until neurochemical determinations. To a graphical
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2.4. Serum lithium measurement
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view of the experimental design, please see Fig. 1.
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For the determination of lithium levels, the sera of lithium-treated animals were
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separated by centrifugation. Assays were performed with a digital flame photometer (Labnova AP- 1500 series, São Paulo, Brazil). The serum levels of lithium in each animal ranged from 0.8 to 1.1 mEq/L, as recommended for the routine treatment of BD (Hopkins and Gelenberg 2000).
2.5 Behavioral tests
Open Field Test (OFT) This test primarily evaluates the influence of drugs on the locomotor activity and exploratory behavior (Archer 1973). An acrylic apparatus (transparent walls and
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black background, dimensions 30x30x15cm), divided into nine squares, was used. The central zone was defined as the central square with dimensions 10x10 cm. The animal was placed in the center of the apparatus and was observed over five minutes after 1 minute of habituation. Each mouse was tested once and between two mice, the apparatus was cleaned with 10% ethanol solution. The parameters analyzed were: total distance traveled by the animal (cm2 ), the number of squares crossed by the mice with four legs, the number of entries in the central zone and the time spent in the central zone
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(exploratory behavior). The test was automatically recorded and analyzed using Panlab
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Elevated Plus Maze Test (EPM)
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Harvard Apparatus® SMART video tracking version 3.0.03 software.
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This test was carried out as previously described (Pellow and File 1986). The apparatus has two perpendicular open arms (30x5cm) and two perpendicular closed
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arms (30x5x25cm). The open and closed arms were connected by a central platform
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(5x5cm). The platform and the lateral walls of the closed arms were made of transparent
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acrylic. The floor was made of black acrylic. The maze was 45 cm above the floor. After the respective treatment, each animal was placed at the center of the EPM with its nose in the direction of one of the closed arms and was observed for 5 min according to the following parameters: number of entries into open and closed arms and the amount of time spent by in open and closed arms of the maze. These data were used to calculate: the total number of entries, % of open entries (open entries/total entries x 100) and % of time in open arms (time spent in open arms/total time x 100).
Y-maze Test
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The apparatus consisted of a Y-shaped maze with three white, opaque plastic arms (length 35, width 5, wall height 10) at a 120 angle from each other arms. Each mouse was allowed to freely move through the maze during 8 min. The series of arm entries was recorded visually. A correct alternation was defined as entries in all three arms on consecutive occasions. The percentage of correct alternations was calculated as follows: total of alternations/(total arm entries x 2), as described in detail elsewhere
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(Dall’Igna et al. 2007).
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Novel Object Recognition Test (NOR)
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recognition memory. This test assesses the mouse’s ability to discriminate between familiar and novel objects (Li et al. 2017). Firstly, mice were individually habituated to
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an open field plexiglas box (30x30x15 cm size) for 5 min. Twenty-four hours after
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habituation, each mouse was subjected to a 10 min acquisition trial, during which they
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were placed in the same arena in presence of two identical objects situated at 15 cm from the arena wall. After 1 h of retention interval, the mice were placed back into the arena and exposed to the familiar object and to a novel object (different color and shape) for a further 10 min. Total time spent exploring each of the two objects (when the animal's snout was directly toward the object at a distance ≤2 cm) was recorded. Recognition index was used as direct measure of recognition memory and was calculated as follows: (time exploring new object - time exploring familiar object) / (time exploring new object + time exploring familiar object) (Ennaceur and Delacour 1988; Li et al. 2015).
Passive Shock Avoidance Test
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The apparatus consisted of an acrylic box (48x22x22cm), with an electrified grid floor, having a shock-free wooden raised zone. In the first step, the animal was allowed to freely explore the apparatus for 90s. After this, the animals were submitted to the training session that consisted in placing the mice on a shock free zone and, when stepped down with all paws on the grid floor, an electric shock (0.5mA) was delivered for 1s. The animal was removed and 1 hour later, it was placed back in the raised area and the latency of step-down to grid floor was recorded (Cammarota et al. 2003;
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Kazlauckas et al. 2005). In this task, the animal learns that a specific place should be avoided since it is associated with an aversive event (Khurana et al. 2011). Thus, a
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decrease in the step-down latency indicates impairment in aversive/affective learning.
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2.6 Neurochemical assays
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Oxidative stress parameters
GSH levels were determined
to estimate the endogenous
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Reduced
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Determination of reduced GSH concentrations
antioxidant defenses (Sedlak and Lindsay 1968). The method was based on the Ellman's reagent (DTNB) reaction with free thiol groups. Briefly, the samples were homogenized in 0.4 M Tris–HCl buffer, pH 8.9 and 0.01 M DTNB. Reduced GSH levels were determined using a microplate reader set at 412 nm. The results are expressed as µg of reduced GSH/g tissue.
Measurement of lipid peroxidation The rate of lipid peroxidation was estimated by the determination of malondialdehyde
(MDA)
equivalent
concentrations
using
the
thiobarbituric-acid
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reactive substances (TBARS) (Huong et al. 1998). The samples were mixed with 100 μl of 35% perchloric acid and were centrifuged at 5000 rpm for 10 min. 150 μl of the supernatants were removed, mixed with 50 μl of 1.2% thiobarbituric acid, and then heated in a boiling water bath for 30 min. After cooling, the MDA levels were determined using a microplate reader set at 535 nm and expressed as μg MDA/g tissue.
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Determination of BDNF The samples were homogenized in phosphate buffered saline (pH 7.4) with
quantified
by
immunoenzymatic
assay
determination
according
to
the
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protease and phosphatase inhibitor cocktails (Sigma St. Louis, USA). BDNF levels
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manufacturer′s instructions (EMD Millipore, USA). The results are expressed as pg of
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2.7 Statistical Analysis
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BDNF/g of tissue.
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Statistical analysis was performed with GraphPad Prism for Windows (version 7.0, San Diego, USA). Kolmogorov-Smirnov test was used for the evaluation of the normal distribution of the data. All results are expressed as means S.E.M. and the statistical analysis of the data was performed by regular one-way ANOVA, followed by Tukey’s test as post-hoc test. The significance level was set at p < 0.05.
3. Results
Reversal of AMPH-induced hyperlocomotion by LIRA alone and in combination with Li
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Psychomotor agitation, a core behavioral alteration observed in mania, is evaluated in animal models by the increased number of crossings in the open field (Boulle et al. 2014). In the present study, we were able to replicate these findings by showing that AMPH significantly increased the zone transitions number and the total distance travelled in the open field test when compared with SAL-treated mice [(zone transitions number: SAL+SAL vs. AMPH+SAL, P= 0.004, F(8, 54)= 7.206; total distance travelled: SAL+SAL vs. AMPH+SAL, P= 0.003), F(8, 49)= 4.582]. The
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administration of LIRA120, LIRA240, Li, and Li+LIRA240 significantly reversed the number of zone transitions in relation to SAL-treated mice [(AMPH+LIRA120 vs.
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AMPH+SAL, P= 0.0265; AMPH+LIRA240 vs. AMPH+SAL, P< 0.001; AMPH+Li vs.
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AMPH+SAL, P=0.0031; AMPH+Li+LIRA240 vs. AMPH+SAL, P= 0.0027); F(8, 54)=
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7.206] (Fig. 2A). Considering the total distance travelled, LIRA240 (P= 0.003), Li (P< 0.05) and the combination LIRA240+Li (P= 0.031) significantly reversed AMPH-
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induced increase in this parameter (Fig. 2B).
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Regarding the number of entries in the center of the open field (Fig. 2C) and the
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time spent in the central square (Fig. 2D), two parameters that may be regarded as impulsive behavior, we observed no alterations in the former one, while in the latter we observed a 1.7-fold increase in AMPH-treated mice when compared to SAL group, despite this difference was
statistically non-significant.
The group treated with
AMPH+Li+LIRA240 presented a significant decrease in the time spent in the central area when compared with AMPH+SAL-treated mice [(P = 0.014), F (8, 53) = 2.71] being the results of AMPH+Li+LIRA240 group akin to SAL-treated animals. Effects of LIRA alone and in combination with Li in the reversal of risk-taking behavior in the elevated plus maze test
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It was previously reported that spontaneously high impulsive rats enter the open arms of the EPM significantly more quickly than spontaneously low impulsive rats. (Molander et al. 2011). Based on observations such as that, in the present study EPM was used to evaluate impulsive behavior. In this regard, as depicted in Fig. 3, AMPH+SAL mice presented a marked increase in the percent of open entries [(P< 0.001), F (8, 52) = 7.99] and percent of time spent in the open arms [(P< 0.001), F (8, 54) = 6.267] in comparison with SAL-treated animals. Considering the % of open
oo
f
entries (Fig. 3B) we observed that LIRA240 (P = 0.0117), Li (P = 0.0079), Li+LIRA120 (P = 0.0002) and Li+LIRA240 (P< 0.001) reversed the increase in this
pr
parameter induced by AMPH+SAL (F (8, 52) = 7.99). In the evaluation of the % of time
e-
spent in the open arms (Fig. 3C), we noticed a significant reversal of AMPH effects
Pr
only in the animals treated with Li alone or in combination with LIRA on both doses [(AMPH+Li vs. AMPH+SAL, P = 0.0042; AMPH+Li+LIRA120 vs. AMPH+SAL, P =
P
= 0.1273; AMPH+Li+LIRA240 vs. AMPH+LIRA240, P
rn
AMPH+LIRA120,
al
0.0014; AMPH+Li+LIRA240 vs. AMPH+SAL, P = 0.001; AMPH+Li+LIRA120 vs.
Jo u
=0.0465), F (8, 54) = 6.267]. We observed a significant decrease in the number of total entries in AMPH+Li+LIRA240 group in relation to AMPH+SAL [(P = 0.038), F (8, 55) = 3.305] (Fig. 3A).
Effects of LIRA alone and in combination with Li on memory alterations induced by AMPH Over the last years, the studies evaluating memory deficits in bipolar disorder have been increasing, since memory deficits persists in remission periods as well as neurocognitive dysfunction may significantly influence patients’ psychosocial outcomes
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(Solé et al. 2017). This is of great importance since there is not a consensus for the benefits of Li in protecting patients from memory deficits (Wingo et al. 2009). Our study revealed that in the evaluation of the percent of correct alternations in the Y maze task (Fig. 4A), there was a significant decrease in this parameter in the AMPH+SAL group compared with SAL-treated mice [(P = 0.0003), F (8, 53) = 13.38] . The treatment
with
LIRA
at
both
doses,
AMPH+LIRA120
(P
=
0.0208) and
AMPH+LIRA240 (P = 0.0058) and in combination with Li, AMPH+Li+LIRA120 (P =
oo
f
0.0009) and AMPH+Li+LIRA240 (P<0.001) significantly increased this percentual compared with AMPH+SAL group (F (8, 53) = 13.38). Additionally, a significant
pr
increase in this parameter was observed in AMPH+Li+LIRA240 group compared with
e-
AMPH+ Li [(P = 0.0182), F (8, 53) = 13.38)]. Regarding total entries in the Y maze
Pr
apparatus (Fig. 4B), the treatment with LIRA240 (AMPH+LIRA240 vs. AMPH+SAL, P = 0.0009) and Li (AMPH+Li vs. AMPH+SAL, P = 0.0045) or their combination
al
(AMPH+Li+LIRA240 vs. AMPH+SAL, P = 0.0005) significantly reduced total entries
rn
in relation to AMPH+SAL-treated animals [ F (8, 57) = 8.221 ].
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In the evaluation of declarative memory by the novel object recognition test (Fig.4C), AMPH+SAL treatment induced a marked decrease in recognition index (%) (P = 0.0056) when compared with SAL group, which was reversed by LIRA240 (AMPH+LIRA240 vs. AMPH+SAL, P = 0.0087) and by both doses of LIRA combined with
Li [AMPH+Li+LIRA120
AMPH+SAL,
(P
=
0.0006),
(P
=
0.0007) and
AMPH+
Li+LIRA240
vs.
F
(8,
52) = 8.32]. Further, the combination
AMPH+Li+LIRA240 significantly increased this index compared with AMPH+Li group [(P= 0.0397), F (8, 52) = 8.32] . In the passive avoidance test (Fig.4D), there was a marked reduction in stepdown latency (s) in the group AMPH+SAL (P = 0.0044) and AMPH+LIRA120 (P =
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0.0111) compared with SAL-controls [F (8, 53) = 6.364]. Li treatment alone (AMPH+Li, P = 0.0014) or combined with LIRA120 (AMPH+Li+LIRA120, P = 0.0251) and 240 (AMPH+Li+LIRA240, P = 0.0022) significantly increased this parameter compared with AMPH+SAL group. Also, the groups in combination AMPH+Li+LIRA120 and AMPH+Li+LIRA240 also presented a significant increase in this step-down latency compared with AMPH+LIRA120 and AMPH+LIRA240 groups
f
(P = 0.0379), respectively [F (8, 53) = 6.364].
oo
Evaluation of weight gain (WG) alterations
pr
Regarding the % WG, AMPH repeated administration caused no significant alteration in this parameter in relation to SAL+SAL mice (19.31% WG and 21.77%
e-
WG, respectively). As expected, AMPH+Li-treated animals presented a marked
Pr
increase in % WG compared with AMPH+SAL [AMPH+Li (47.84% WG) vs. AMPH+SAL (19.31% WG), P < 0.001] and with SAL+SAL groups [AMPH+Li
al
(47.84% WG) vs. SAL+SAL (21.77% WG), P < 0.001]. AMPH+LIRA treatment did
rn
not significantly alter % WG (AMPH+LIRA240 (14.78% WG) vs. AMPH+SAL
Jo u
(19.31% WG), non-significant; AMPH+LIRA120 (18.89% WG) vs. AMPH+SAL (19.31% WG), non-significant). Under control conditions, LIRA treatment significantly reduced % WG compared to SAL+SAL animals: [SAL+LIRA240 (9.482% WG) vs. SAL+SAL (21.77% WG), P< 0.001; and SAL+LIRA120 (13.98% WG) vs. SAL+SAL (21.77% WG),
P = 0.0379, F (8, 63) = 44.85]. Also, Li+LIRA combination groups
presented a significant reduction in weight gain when compared with AMPH+Li group: [AMPH+Li+LIRA240
(19.49% WG) vs.
AMPH+Li (47.84% WG),
P<0.001;
AMPH+Li+LIRA120 (27.47% WG) vs. AMPH+Li (47.84% WG), P<0.0001, F (8, 63) = 44.85] (Fig. 5).
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Neurochemical Determinations Reversal of AMPH-induced brain oxidative alterations by the administration of LIRA alone and in combination with Li Mitochondrial dysfunction and the resulting oxidative imbalance are core alterations observed in BD patients (Berk et al. 2011a). Our results revealed that in the PFC, AMPH+SAL group showed a significant reduction in GSH levels when compared
AMPH+Li,
P<
oo
f
with SAL+SAL (P = 0.0013). LIRA240 and Li alone (AMPH+LIRA240 and 0.0001) or in combination (AMPH+Li+LIRA240, P< 0.0001) AMPH-induced GSH deficits. Additionally, the combination
pr
significantly reversed
e-
group (AMPH+Li+LIRA240) significantly increased GSH contents compared with
Pr
AMPH+Li-treated animals (P< 0.001) [F (5, 35) = 35.24] (Fig.6A). In the hippocampus, AMPH+SAL group also presented a significant reduction in
al
GSH levels compared to SAL controls (P< 0.001). LIRA240 (AMPH+LIRA240, P<
rn
0.001) and in combination with Li (AMPH+Li+LIRA240, P< 0.0001) significantly
Jo u
reversed AMPH-induced GSH deficits. The combination AMPH+Li+LIRA240 also significantly increased GSH contents related to AMPH+Li (P < 0.0001) [F (5, 35) = 48.53] (Fig.6B).
In the amygdala, AMPH+SAL caused a significant reduction in GSH compared to SAL+SAL (P < 0.001). LIRA alone and combined with Li as well as Li alone significantly
reversed
AMPH-induced
GSH
deficits
(AMPH+Li+LIRA240
vs.
AMPH+SAL, P< 0.001; AMPH+Li vs. AMPH+SAL, P = 0.0015). LIRA alone did not reversed
AMPH-induced
alterations
in
amygdala.
Finally,
the
combination
AMPH+Li+LIRA240 induced an additional increase in GSH levels compared to Li+AMPH group (P = 0.0051) [F (5, 39) = 15.12] (Fig.6C).
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Considering lipid peroxidation, in the PFC, AMPH+SAL caused a marked increase
(3.64-fold increase) in MDA levels compared to SAL+SAL group (P < 0.001). LIRA240 and Li alone or in combination reversed AMPH-induced MDA increase [AMPH+LIRA240 vs. AMPH+SAL, P = 0.0087; AMPH+Li vs. AMPH+SAL, P< 0.0001; AMPH+Li+ LIRA240 vs. AMPH+SAL, P< 0.0001, F (5, 39) = 25.98]. (Fig.6D) In the hippocampus AMPH+SAL also induced a great increase in MDA
f
compared to SAL+SAL (P < 0.001). LIRA240 (AMPH+LIRA240 vs. AMPH+SAL, P
oo
< 0.001) and Li (AMPH+Li vs. AMPH+SAL, P< 0.0001) and their combination
pr
(AMPH+Li+ LIRA240 vs. AMPH+SAL, P< 0.0001) significantly reversed AMPH-
e-
induced MDA alterations [F (5, 39) = 23.85] (Fig.6E).
In the amygdala, AMPH treatment significantly increased MDA contents in
Pr
relation to SAL controls (P< 0.001), and LIRA240 administration did not reverse this
al
alteartion. On the other hand, Li alone or in combination with LIRA reversed AMPH-
rn
induced MDA rise (P< 0.0001). Additionally, the combination AMPH+Li+LIRA240 induced a significant decrease in MDA levels when compared to AMPH+LIRA240
-
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group (P< 0.001) [F (5, 39) = 22.76] (Fig.6F). Reversal of AMPH-induced brain BDNF deficits by LIRA and Li Based on the importance of neurotrophic mechanisms in the neurobiology of BD (Grande et al. 2010), we decided to evaluate brain levels of BDNF. In the PFC, AMPH+SAL group presented a significant reduction in BDNF levels compared to SAL+SAL (P< 0.001). The treatment with LIRA240 or Li did not reverse this AMPHinduced BDNF changes. Contrarily, the combination AMPH+Li+LIRA240 significantly increased BDNF levels compared to AMPH+SAL (P = 0.0015) and AMPH+Li (P=0.0070) [F (5, 37) = 22.09] (Fig.7A).
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In the hippocampus, AMPH administration caused a marked decrease (0.39-fold changes, MD:-4440) in BDNF levels compared to SAL-SAL controls (P < 0.0001). The treatment with LIRA240 (AMPH+LIRA240 vs. AMPH+SAL, P = 0.0156) or combined with Li (AMPH+Li+LIRA240 vs. AMPH+SAL, P < 0.001) significantly reversed AMPH-induced
BDNF
deficits.
Additionally,
in
control
conditions,
LIRA
(SAL+LIRA240) significantly increased BDNF levels in comparison with SAL+SAL
f
group (P =0.0030) [F (5, 37) = 19.55] (Fig.7B).
oo
In the amygdala, AMPH treatment induced a marked increase (2.21-fold
pr
increase, MD:+3580) in BDNF levels related to SAL-controls (P < 0.001). Also, the group treated with LIRA240 (AMPH+LIRA240) similarly increased BDNF levels
e-
compared to controls (P = 0.0034) [F (5, 33) = 7.051]. There was no additional
rn
4. Discussion
al
Pr
significant difference between the tested groups (Fig.7C).
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In the present study, we bring the first evidence of the potential antimanic/mood stabilizing effects of the GLP-1R agonist LIRA. According to our results, LIRA rescued hyperlocomotion, executive and spatial memory deficits induced by AMPH, but failed to reverse risk-taking behavior and fear learning impairment associated to this model. Combined with Li, LIRA reversed the most behavioral changes induced by AMPH even with a superior effect than Li itself. Also, LIRA alone displayed an important hippocampal antioxidant and neurotrophic effect, while Li showed its protective effect mainly in the PFC and amygdala. Hence, the combination of LIRA and Li fully reversed the pro-oxidative changes in all tested brain areas, and markedly counteracted BDNF impairment in the PFC and hippocampus revealing a possible superior effect in
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relation to standard Li treatment. Additionally, LIRA rescued the increased % WG induced by Li in the course of mania model. Considering that LIRA can improve not only the efficacy, but also some side effects associated to standard Li therapy, it has a great potential for translation application in clinical setting for BD. The hyperdopaminergic state that takes place in mania may be mimicked in animals by the repeated administration of AMPH and evaluated by the measure of
f
hyperlocomotion in the OFT (Sharma et al. 2016). The mood stabilizing drugs, Li and
oo
valproate, classically suppress these alterations, supporting the predictive validity of
pr
these models (Frey et al. 2006b; Macedo et al. 2013). However, mania is a complex syndrome with multifaceted symptoms and the investigation of multiple affective and
e-
cognitive domains should be performed to adequately evaluate mania (Logan and
Pr
McClung 2016). In this regard, besides hyperlocomotion, reduced anxiety/increased risk-taking behavior, hypersexual behavior and multiple cognitive deficits, for example
al
in executive, spatial and affective domains, were previously observed in AMPH model
rn
(Fries et al., 2015; Zhou et al., 2015). Hence, this model can induce other features of
Jo u
mania than only the behavioral activation. GLP-1R signaling is being related
to
the regulation of neural/cognitive
processes. Regarding mood disorders, a recent study showed, in corticosterone induceddepression model, that repeated LIRA treatment has antidepressant- and anxiolytic-like effects (Weina et al. 2018). Also, GLP-1R agonists can protect against the comorbid depression- and anxiety-like behavior associated to several conditions, such as diabetes (Mansur et al. 2018) and epilepsy (de Souza et al. 2019). Also, the effects of GLP-1R agonists in emotional behavior seems to be dependent of the duration of treatment and route of administration. Acute IP and central (intra-dorsal raphe) injections of GLP-1 analogue induced anxiety-like behavior in
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rodents, while subchronic central administration (for 9 days) promoted anxiolytic- and antidepressant-like effects (Anderberg et al. 2016). Based on this observation, in our study we used a protocol of 7 days of LIRA administration. To date, no study has evaluated the effects of GLP-1R agonists in BD mania. Here, we showed that LIRA 7 days administration at the doses of 120 and 240 ug/kg alone and in combination with Li presents antimanic-like effects based on the reversal
f
of AMPH-induced hyperlocomotion. In order to better characterize the antimanic
oo
effects of LIRA, we evaluated risk-taking behavior and impulsivity, by the evaluation of
pr
central square exploration of the OFT and open arms occupancy in EPM test (Macr et al. 2002; Tillmann and Wegener 2019) since they are key features of mania that usually
e-
have difficult management and are strongly associated to high suicide rates (Izci et al.
Pr
2016; Pawlak et al. 2016).
al
In our experimental conditions, LIRA showed a small effect against AMPH-
rn
induced risk-taking behavior. However, when combined with Li, LIRA at both doses reversed AMPH-induced center exploration and the increase in % of open arms entries
Jo u
and duration. It is worth to mention that Li alone counteracted only AMPH-induced changes in EPM test but did not reverse the increase in center occupancy in the OFT. Hence, these results suggest an augmentation of Li effects by LIRA. Li is recognized as a standard therapeutic option to control impulsivity/risktaking behavior in BD, an effect that does not appear to be solely associated to its preventive effects on mood changes (Cipriani et al. 2013). Despite this, there is some controversy in this effect of Li. In preclinical settings, while considerable evidence has showed an effect of Li in the control of impulsive and risky behavior, some authors also reported Li inability to reverse risk-taking behavior in some conditions, such as median raphe nucleus lesion and glutamate receptor deletion (Shaltiel et al. 2008; Pezzato et al.
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2015). In this regard, a meta-analysis study failed to find strong evidence for Li use as anti-impulsivity therapy in BD and concluded that more studies are necessary to firmly propose this indication (Jones et al. 2011). Our results highlight the potential of LIRA and mainly of its association with Li to control risky and impulsive behavior. GLP-1Rs are widely distributed in the CNS and present a great influence on cognition. Convergent evidence have demonstrated that the stimulation of GLP-1Rs
f
(through GLP-1 or synthetic ligands) is associated to pro-cognitive effects (McIntyre et
oo
al. 2013). In fact, a single GLP-1 injection improved spatial memory, as well as
pr
increased cognitive flexibility and executive function in naïve animals (During et al. 2003). GLP-1R agonists, such as LIRA, can protect against the cognitive deficit
e-
associated to several models of neurodegeneration such as Alzheimer’ and Parkinson’
Pr
Disease (Yang et al. 2013; McClean and Hölscher 2014; Zhang et al. 2018). Additionally, GLP-1R agonists also prevented the cognitive impairment induced by the
rn
al
high-fat diet in mice (Porter et al. 2012).
Here, we observed that AMPH-induced mania markedly compromised the
Jo u
performance of mice in the Y maze and NOR tasks. It is well described that impairments in executive function and declarative memory are frequently found in BD patients (Venza et al. 2016). In accordance with our results on memory impairment in AMPH mania model, previous studies have already demonstrated that AMPH administration can mimic several aspects of BD cognitive disability,
such as
dysfunction in executive and recognition tasks (de Souza Gomes et al. 2015; Fries et al. 2015). Thus, considering the strong relationship between metabolic and cognitive functions in BD, GLP-1R system has emerging as a promising target to modulate both functions. In our study, the standard mood stabilizer Li could not reverse AMPH-
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induced deficits in executive and spatial memory, but LIRA alone mainly at the higher tested
dose
combination
considerably improved of
Li+LIRA
mice performance on both domains.
completely
reversed
this
alteration,
overcoming
The the
limitations of Li treatment at monotherapy. Despite being the first-line maintenance treatment for BD, Li presents severe adverse effects, mainly of metabolic nature (Vendsborg et al. 1976). One important
f
adverse effect of Li is diabetes insipidus and WG, which are among the top five clinical
oo
causes for drug discontinuation (Öhlund et al. 2018). These adverse events affect up to
pr
61% of patients and a WG over than 10 kg have been described in 20% of Li-treated
e-
subjects (Chengappa et al. 2002; Praharaj 2016).
In our study, Li treatment promoted a marked increase in the % WG along the
Pr
induction of the mania model. Interestingly, LIRA in combination with Li prevented the
al
weight changes induced by Li. In naïve animals, LIRA also induced weight loss,
rn
confirming its anorexic effect. Indeed, a previous report showed that a single LIRA injection (100 µg/kg) is able to induce anorexic effects in mice and a weight loss that
Jo u
persisted for 4 days (Nonogaki and Kaji 2018). Notably, a recent study reported LIRA metabolic effects on BD patients. Despite the limited number of patients (n=29), the authors showed LIRA’s efficacy to induce weight loss even when other multiple lifestyle interventions, such as exercise and diet programs, have been failed. Also, LIRA was well-accepted and well-tolerated in stable BD patients, as well as did not compromised their psychiatric condition (Cuomo et al., 2018). Therefore, taking these evidence together, LIRA can represent a useful strategy for counteracting the weight disturbances in BD, however larger, longer and well-controlled clinical studies should be performed to make reliable conclusions about this indication.
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In the last decades, several evidences point towards the presence of systemic pro-oxidative changes in all phases of BD, including euthymic states (Kuloglu et al. 2002; Kunz et al. 2008; Steckert et al. 2010). Also, markers of oxidative damage have been consistently found in post-mortem brain of BD patients, mainly in mood-regulated brain areas (GAWRYLUK et al. 2011). In the context of DA dysregulation hypothesis, the hyperdopaminergic state involved in mania compose a strong basis for the oxidative imbalance (Berk et al. 2007). Indeed, dopamine is metabolized by monoamine oxidase
oo
f
to hydrogen peroxide (H2 O2 ). In the presence of Fe2+ and H2O2, the formation of 6hydroxydopamine (6-OHDA) takes place (Stokes et al. 1999; Yamato et al. 2010). 6-
e-
pro-apoptotic pathways (Blum et al. 2001).
pr
OHDA is a potent neurotoxin capable to impair mitochondrial functioning and activate
Pr
Here, as expected, AMPH treatment caused a marked increase in lipid peroxidation and compromised the endogenous antioxidant GSH in all studied brain
al
areas. Regarding AMPH mania model, several authors have already demonstrated
rn
oxidative damage and impairment in endogenous antioxidants in limbic brain areas
Jo u
(Frey et al. 2006b; Macêdo et al. 2012; Macedo et al. 2013). TBARS levels in BD patients presented the larger effect size compared to the other enzymatic and non-enzymatic markers of oxidative stress (Andreazza et al. 2008). Still in BD patients, increased lipid peroxidation also negatively correlates with cognitive performance, mainly executive functioning (Newton et al. 2015). Serum TBARS levels are increased in all BD phases, but reaches the higher levels in the manic episodes, indicating the occurrence of a greatest oxidative stress in mania (Kunz et al., 2008). GSH is the main non-protein thiol in cells acting as a cofactor for antioxidant and detoxifying reactions (Marí et al. 2009; Ribas et al. 2014). Reduced GSH levels and
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abnormalities in GSH synthesizing enzymes were reported in brain and serum of BD patients. Indeed, mice with genetic deletion of glutamate cysteine ligase modulatory (GCLM) gene (a key GSH synthesizing enzyme) (Yang et al., 2002) presents a complex behavior resembling features of schizophrenia and BD mania. These animals showed novelty-induced hyperlocomotion and an exacerbated response to low dose of AMPH (Kulak et al., 2012). Other models of GSH deficiency in adult mice resulted in marked cognitive alterations, such as impairment in object recognition and spatial working
oo
f
memory (Cabungcal et al. 2007; das Neves Duarte et al. 2012).
pr
The neuroprotective effects of mood stabilizing drugs have been strongly associated to its antioxidant properties (Niciu et al. 2013). Lithium at therapeutically
protein
oxidation
and
DNA
Pr
peroxidation,
e-
relevant concentrations prevented glutamate- and H2 O 2 -induced cell death, lipid fragmentation
(Shao
et
al.
2005).
Interestingly, in early-diagnosed BD patients, reduction in serum TBARS levels only
al
occurs in Li responder patients, thus, reinforcing the involvement of antioxidant
rn
properties to Li mood stabilizer efficacy (De Souza et al., 2014). In animal models of
Jo u
mania, Li protected against brain pro-oxidative changes (lipid peroxidation, DNA oxidative damage, GSH depletion) induced by AMPH (Frey et al. 2006b) and sleep deprivation (Valvassori et al. 2017). In our results, Li completely reversed the AMPH-induced increase in lipid peroxidation in all tested brain areas, as well as fully restored GSH contents in the PFC and amygdala. For the first time, we showed LIRA ability to counteract lipid peroxidation and GSH impairment induced by AMPH mania model. Interestingly, this antioxidant effect of LIRA showed a brain area-selective pattern, occurring in the PFC and hippocampus, and not in amygdala. Notably, Li+LIRA240 in combination showed
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an increased antioxidant potency, fully reversing lipid peroxidation and GSH depletion induced by AMPH in all brain areas. GLP-1Rs are molecularly coupled to cell antioxidant defenses. When activated, GLP-1Rs via Gα-protein stimulates adenyl cyclase, and subsequently Akt (also known as protein kinase B – PKB). Activated Akt phosphorylates multiple targets in the cell to regulate adaptive responses to diverse insults, including mitochondrial
GSK3β,
a
major
protein
kinase
involved
oo
inhibits
f
dysfunction and oxidative stress. Importantly, Akt phosphorylates and, consequently in
pro-apoptotic
and
pr
neurodegenerative events, as well as in mood stabilizing Li mechanism (Freyberg et al. 2010). Accordingly, Li directly and indirectly inhibits GSK3β (Freland et al.,
e-
2012).
Pr
The antioxidant effects of LIRA may be related to a stimulation of nuclear
al
factor erythroid 2-related factor (Nrf2) pathway (Caihong et al., 2018). When
involved
in
rn
activated, Nrf2 migrates to nucleus and induces the expression of several genes antioxidant
response,
including
superoxide
dismutase,
catalase
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glutathione peroxidase, glutathione reductase and γ-glutamate cysteine ligase (Hayes and Dinkova-Kostova 2014). Similarly, Li display neuroprotective actions against excitotoxicity and pro-oxidant insults through Nrf2 pathway induction (Rojo et al., 2008; Cunha et al., 2016, Pan et al., 2019). Brain-derived neurotrophic factor (BDNF) plays a major role in synaptic plasticity and
neurogenesis.
Plasma BDNF negatively correlates with symptoms
severity as well as increases with mood stabilizer treatment (Fernandes et al., 2015). Here, we observed that AMPH (for 14 days) caused a marked decrease in BDNF expression in the PFC and hippocampus, while increased its levels in the amygdala. Li induced a discrete rise in this neurotrophin levels in PFC and hippocampus, while
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LIRA restored BDNF levels in the hippocampus. The combination Li+LIRA240 markedly restored BDNF levels in the PFC and hippocampus. However, similar to Li alone, this association demonstrated just a modest effect in amygdala. BDNF impairment has been importantly associated to AMPH-induced deficits in working memory and spatial recognition in mice. In this regard, Fries et al. 2015 reported that AMPH caused time-related changes in BDNF expression (mRNA and
f
protein levels) in the PFC, hippocampus and amygdala of mice. In their study,
oo
chronic AMPH treatment (for 35 days) induced a progressive decrease in BDNF
pr
expression in the hippocampus, while increased BDNF mRNA levels in amygdala (Frey et al., 2015). Other authors also showed that repeated AMPH increased BDNF
e-
levels in the amygdala of rodents. Regarding PFC, there are discrepancies in the
Pr
literature with some studies reporting decreased BDNF in the PFC after AMPH treatment (Valvassori et al., 2018), while others reported increased (Shen et al., 2014)
rn
al
or even no change in this neurotrophin levels (Stertz et al., 2013). Amygdala is the central core for affective learning, ensuring the subject
Jo u
capacity to adequately respond to sources of danger (Paré et al., 2004). Neuroimaging studies have reported an overactivated amygdala in the brain of BD patients, especially in manic episodes (Bermpohl et al., 2009), with subsequent volume reduction with disease progression. These findings have been coupled to decrements in the activity of some PFC subregions, such as ventrolateral PFC (Altshuler et al., 2005). Further, the connectivity between amygdala and PFC, namely ventrolateral prefrontal-amygdala emotional pathway, which is responsible for tuning control of amygdala reactivity, is disrupted in BD and accounts for the abnormal responses to emotional cues and impaired attentional shift in these patients (Strakowski et al., 2011).
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Here, we found that AMPH treatment markedly impaired fear learning in mice as demonstrated by the premature stepdown latency. Interestingly, this cognitive profile was followed by increased markers of oxidative stress and abnormal increased levels of BDNF
in the amygdala. This result points to a possible ongoing
neurodegenerative process in the amygdala potentially responsible for the disrupted emotional learning induced by AMPH mania model. Lithium treatment efficiently prevented this impairment in affective memory, but only partially rescued AMPH-
oo
f
induced neurochemical changes.
pr
Here, LIRA showed a considerable ability to upregulate hippocampal BDNF expression, and when in combination with Li, also reversed PFC BDNF changes.
e-
This represents a potential mechanism for LIRA protective effects against AMPH-
Pr
induced cognitive dysfunction. In fact, LIRA and other GLP-1R agonists can increase the expression of BDNF and other neurotrophins in mice brain exposed to several
al
neuropathology conditions, such as aging (Bomba et al., 2018), epilepsy (De Souza et
rn
al., 2019) and diabetes (Gumuslu et al., 2016). This LIRA effect can be explained
Jo u
through its stimulatory effect on protein kinase A (PKA) and Akt (Deng et al. 2018). Each of these kinases can mutually phosphorylate and, thereby, activate cAMP response element-binding protein (CREB). CREB activation can subsequently upregulate BDNF expression (Yossifoff et al. 2008; Zheng et al. 2012). Recently, it was also demonstrated that LIRA can restore spine density and dendrite outgrowth in the hippocampus of dexamethasone-treated rats through activation of mTOR1 signaling and induction of BDNF expression (Park et al. 2018). These pathways represent potential underlying mechanisms for the observed pro-cognitive and BDNF upregulating effects observed here, but needs further investigation. Limitations and Perspectives
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This study has some limitations. First, no existing animal model can fully resemble the complex psychopathological expression of BD mania. Second, our study evaluated a correlation between intervention and outcome and was not delineated to clarify possible mechanistic pathways, and, with our results, we cannot propose the precise mechanisms involved in the LIRA behavioral effects observed here. Therefore, investigating the possible molecular targets of LIRA effects against neurobehavioral features of BD mania, such as Akt activation, GSK3β inhibition and induction of Nrf2
oo
f
antioxidant cell response, remains as an interesting question for future research in the field. Finally, we did not measure plasma glucose levels in animals treated with Li and
e-
pr
LIRA, this is important since both drugs can interfere with glucose metabolism.
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Conclusions
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The present study demonstrated the potential antimanic/mood stabilizing effects
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of the GLP-1R agonist LIRA in AMPH mania model. Of note, LIRA alone was hyperlocomotion, executive and spatial memory
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effective against AMPH-induced
dysfunction. This LIRA effect was associated to a notorious antioxidant action in hippocampus and PFC and up-regulating effects on hippocampal BDNF expression. Also, LIRA improved the profile of Li behavioral effects, thereby overcoming the inability of Li to reverse the main cognitive changes induced by AMPH, as well as augmenting its antioxidant and BNDF modulatory actions. Finally, LIRA prevented the weight gain associated to Li exposure in mice. Taking together, our results suggest the possible involvement of GLP-1 system in the pathophysiology of BD and highlight this system as a promising target to development of new therapies able to counteract both metabolic and cognitive changes involved in this disease. In this context, LIRA can
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represent useful adjunctive tool for BD treatment with a great potential for translation application.
Acknowledgements
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The authors thank the Brazilian Institutions CAPES, FUNCAP and CNPq for the
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financial support of this study.
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Author’s disclosures
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The authors declare no conflict of interests.
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Figure Legends Figure 1. Experimental design timeline. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium. Figure 2. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone and associated with Lithium (Li) in the reversal of amphetamine 2.0 mg/kg (AMPH)induced changes in open field test parameters: A- zone transition number (n); B- total
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distance (cm2 ), C- entries in central area (n), D- time in central area (s). N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01,
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***P< 0.001, ****P< 0.0001 according to one-way ANOVA followed by Tukey׳s post-
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hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li:
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lithium.
Figure 3. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone
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and associated with Lithium (Li) in the reversal of amphetamine 2.0 mg/kg (AMPH)-
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induced changes in elevated plus maze test parameters: A- total entries (n); B- open
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entries/total entries (%), C- time in open arms/total duration (%). N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 according to one-way ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium. Figure 4. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone and associated with Lithium (Li) in the reversal of amphetamine 2.0 mg/kg (AMPH)induced changes in mice cognitive performance on y-maze test (A - correct alternations %, B- total entries); novel object recognition test (C- recognition index %), and passive avoidance test (D-step-down latency in seconds). N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001
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according to one-way ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium. Figure 5. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) in Lithium (Li)-induced weight gain (grams) in the amphetamine 2.0 mg/kg (AMPH) mania model. The graphic plot represents the weight gain in percentage (%) comparing the last day of treatment (14th day) with the first one (1st day) of the experimental and
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control groups. N=8 animals per group. Bars represent mean ± standard error media
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(SEM). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 according to one-way
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liraglutide; AMPH: amphetamine; Li: lithium.
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ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA:
Figure 6. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone
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and associated with Lithium (Li) in the amphetamine 2.0 mg/kg (AMPH)-induced
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changes in oxidative stress markers in the mice brain. Graphic plots represent reduced glutathione (GSH) concentrations in the prefrontal cortex (PFC)- A, hippocampus (HC)
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– B and amygdala (AM) – C, and malondialdehyde (MDA) concentrations in the same
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brain areas: PFC – D, HC – E and AM – F. N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01, ***P< 0.001, ****P< 0.0001 according to one-way ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium; GSH: reduced glutathione; MDA: malondialdehyde; PFC: prefrontal cortex; HC: hippocampus; AM: amygdala. Figure 7. Effect of Liraglutide 120 µg/kg (LIRA120) or 240 µg/kg (LIRA240) alone and associated with Lithium (Li) in the amphetamine 2.0 mg/kg (AMPH)-induced changes in brain-derived neurotrophic factor (BDNF) concentrations in the mice brain: A - prefrontal cortex, B – hippocampus and C - amygdala. N=8 animals per group. Bars represent mean ± standard error media (SEM). *P<0.05, **P< 0.01, ***P< 0.001,
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****P< 0.0001 according to one-way ANOVA followed by Tukey׳s post-hoc test. Abbreviations: SAL: saline; LIRA: liraglutide; AMPH: amphetamine; Li: lithium; BDNF: brain-derived neurotrophic factor; PFC: prefrontal cortex; HC: hippocampus; AM: amygdala. Figure 8. Summarizing representation of the experimental findings and possible intracellular pathways underlying the antimanic effects of Liraglutide (LIRA) treatment.
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Blue arrows represent downstream stimulatory pathways and red lines represent
neurotrophic factor; GSH: reduced glutathione; GLP-1RA:
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BDNF: brain-derived
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downstream inhibitory pathways. Abbreviations: AMPH: amphetamine; Li: lithium;
glucagon-like peptide-1 receptor agonist ; AC: adenylyl cyclase; PKA: protein kinase
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A; p-CREB: phospho- cAMP response element-binding protein; PI3K: phosphoinositide
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3-kinase; p-AKT (PKB): phosphor-protein kinase B; GSK3β: glycogen synthase kinase
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related factor 2.
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3 beta; KEAP1: kelch-like ECH associated protein 1; NRF2: nuclear factor erythroid -
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AJMCF, DSM – designed the study and wrote the first draft of the manuscript; NLC, AGS, MVRS – performed the behavioral determinations; AJMCF, PMJ, TQ, JVSO – performed the neurochemical determinations; DSM, SSV, TB – performed the statistical analysis, JQ, DL – contributed to the study design and revised the last version of the manuscript.
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The authors declare no conflict of interests
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Highlights:
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AMPH induced risk-taking behavior and cognitive deficits that resemble mania LIRA reversed AMPH-induced hyperlocomotion, working and recognition memory impairments Li+LIRA240 rescued all behavioral changes induced by AMPH LIRA reversed AMPH-induced hippocampal oxidative and neurotrophic changes Li+LIRA240 augmented Li antioxidant effects and reversed AMPHinduced BDNF changes
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