Aripiprazole prevents stress-induced anxiety and social impairment, but impairs antipredatory behavior in zebrafish

Journal Pre-proof Aripiprazole prevents stress-induced anxiety and social impairment, but impairs antipredatory behavior in zebrafish

Heloísa Helena de Alcantara Barcellos, Aline Pompermaier, Suelen Mendonça Soares, Victoria Costa Maffi, Marina Fernandes, Gessi Koakoski, Karina Kirsten, Bernardo Baldisserotto, Leonardo José Gil Barcellos PII:

S0091-3057(19)30538-6

DOI:

https://doi.org/10.1016/j.pbb.2019.172841

Reference:

PBB 172841

To appear in:

Pharmacology, Biochemistry and Behavior

Received date:

31 October 2019

Revised date:

26 December 2019

Accepted date:

26 December 2019

Please cite this article as: H.H. de Alcantara Barcellos, A. Pompermaier, S.M. Soares, et al., Aripiprazole prevents stress-induced anxiety and social impairment, but impairs antipredatory behavior in zebrafish, Pharmacology, Biochemistry and Behavior (2019), https://doi.org/10.1016/j.pbb.2019.172841

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© 2019 Published by Elsevier.

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Aripiprazole prevents stress-induced anxiety and social impairment, but impairs antipredatory behavior in zebrafish

Heloísa Helena de Alcantara Barcellos1,2, Aline Pompermaier3, Suelen Mendonça Soares1, Victoria Costa Maffi2, Marina Fernandes4, Gessi Koakoski5, Karina Kirsten1, Bernardo

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Baldisserotto1, Leonardo José Gil Barcellos1,2,3,5*

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Programa de Pós-Graduação em Farmacologia, Universidade Federal de Santa Maria (UFSM), Av. Roraima, 1000, Cidade Universitária, Camobi, Santa Maria, RS, Brazil, 97105900 2 Curso de Medicina Veterinária, Universidade de Passo Fundo (UPF), BR 285, São José, Passo Fundo, RS, Brazil, 99052-900 3 Programa de Pós-Graduação em Ciências Ambientais, Universidade de Passo Fundo, (UPF), BR 285, São José, Passo Fundo, RS, Brazil, 99052-900 4 Centro de Ensino Médio Integrado da Universidade de Passo Fundo, BR 285, São José, Passo Fundo, RS, Brazil, 99052-900 5 Programa de Pós-Graduação em Bioexperimentação, Universidade de Passo Fundo (UPF), BR 285, São José, Passo Fundo, RS, Brazil, 99052-900

*Please address correspondence to [email protected] (ORCID 0000-0002-4637-3377). Tel: +55 54 33168608 Fax: +55 54 33168485

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Abstract Environmental pollution caused by antipsychotic residues is a relevant ecological problem. Studies revealed that residues of these drugs are present in a wide range of different ecosystems and can have adverse effects on non-target organisms even in low environmental concentrations. Among these antipsychotic drugs, aripiprazole (APPZ) is a second-generation atypical antipsychotic that is a partial agonist of dopaminergic and serotoninergic receptors. APPZ is used to treat schizophrenia, bipolar disorder, autism, obsessive-compulsive disorder,

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and anxiety or panic disorders. Thus, in this study we posed the following question: “What

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will be the behavioral effects of waterborne APPZ on fish?” To answer this question, we

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exposed adult zebrafish to different APPZ concentrations (0.556, 5.56, and 556 ng/L) for 15

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minutes and evaluated their exploratory, anxiety-like, social, and anti-predatory behaviors. Our results showed that, despite the apparent beneficial reversal of stress-induced social

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impairment and anxiety-like behavior, APPZ exposure impaired the anti-predatory reaction of

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adult zebrafish. Taken altogether, our results show that APPZ-exposed zebrafish may have a decreased perception of predators, even at concentrations lower than those already detected in

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the environment. A failure to exhibit an antipredatory response may favor the predator, decrease the fitness of the prey species, and, consequently, affect the food chain. Our results highlight the risks and consequences associated with APPZ residues in water, which may affect aquatic life and endanger species that depend on appropriate behavioral responses for survival.

Keywords: Danio rerio, prey-predator relationship, social behavior, stress, environmental contamination, drug residues

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1. Introduction Environmental pollution caused by antipsychotic residues is a relevant ecological problem (Heberer 2002; Boxall 2004; Küstler and Adler 2014; Martin et al. 2017; Ford and Herrera 2018). Studies revealed that pharmaceutical drug residues are present in a wide range of different ecosystems (Arnold et al. 2014; Martin et al. 2017; Mazzitelli et al. 2018; LópezGarcía et al. 2018) even at low environmental concentrations (Cabeza et al. 2012; Subedi and Kannan 2015). These drug residues in the water can have adverse effects on non-target

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organisms (Kostich and Lazorchak 2007); these effects include endocrine disruption (Sabir

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and Akhtar 2018) and behavioral disturbance (Küstler and Adler 2014). Among the different

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drug classes, psychotropic drugs evoke major environmental concerns (Mazzitelli et al. 2018;

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López-García et al. 2018); studies have reported the effects of these drugs, prescribed for human psychiatric disorders, on behavioral and/or neuroendocrinological parameters in fish

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(Magno et al. 2015; Alvarenga et al. 2017; Kalichak et al. 2017; Barcellos et al. 2016).

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Aripiprazole (APPZ) is a second generation atypical antipsychotic that is a partial agonist of dopaminergic (Tamminga and Carlsson 2002; Hirose and Kikuchi 2005; Mamo et

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al. 2007) and serotoninergic (Hirose and Kikuchi 2005) receptors. This drug is used to treat schizophrenia, bipolar disorder (Burris et al. 2002; Tessler and Goldberg 2006; Biojone et al. 2011), autism (Accordino et al. 2016; Goel et al. 2018), obsessive compulsive disorder (Sahraian et al. 2018), and anxiety or panic disorders (Pignon et al. 2017). Among the most prominent characteristics of this drug is its ability to control the positive and negative effects of schizophrenia (Mailman and Murthy 2010) and irritability, hyperactivity, and stereotyped behaviors in autism (Accordino et al. 2016; Ichikawa et al. 2017). In mammals, with the use of this drug, anxiolytic, panicolytic, and anti-aversive effects have also been observed (Koener et al. 2012; Kling et al. 2014). Rodent studies clearly show that the effects of APPZ are dose dependent (Biojone et al. 2011), however there are no studies examining the effects

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of this drug on fish behavior. APPZ has a bioavailability of 87% (Molden et al. 2006) and an elimination half-life of up to 75 h (Diak and Metha 2008), both determined in humans. It has a 27% renal and a 60% fecal elimination rate and can be eliminated in its unmetabolized active form by up to 1% in urine and 18% in feces (Deleon et al. 2004). Through the metabolites excreted by users (Heberer 2002), the direct disposal of the product due to failures in the sanitary treatment of medicines used in hospitals (Lienert et al. 2011), or even by the disposal of empty packaging directly in water (Halling-Sorensen et al. 1998), this drug can

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contaminate the soil and the aquatic environment. It has been detected at an approximate

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concentration of 33 ng/L (Yuan et al. 2013) in affluents and 5.56 ng/L in effluent from water

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treatment plants in China (Subedi and Kannan 2015).

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The zebrafish (Danio rerio) is a well-known research fish model. This fish is an easyto-handle teleost with lower maintenance costs than other animal models (Saverino and Gerlai

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2009) and has a high potential for translational studies (Kalueff et al. 2013). Detailed

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knowledge of its hypothalamus-pituitary-interrenal axis (Ramsay et al. 2006; Ramsay et al. 2009) and of its behavior (Gerlai 2014; Kysil et al. 2017) also favor this species as a research

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model in aquatic toxicology, among other scientific disciplines (Howe et al. 2013). At a behavioral level, other psychoactive drugs such as risperidone (Idalencio et al. 2015) and methylphenidate (Endres et al. 2017) cause anxiolytic-like effects to fish. This anxiolytic-like effect verified in fish is similar to the anxiolytic effect seen in humans (Wong et al. 1995). Although this is the desired effect in human prescriptions, from an environmental/ecological perspective, an anxiolytic-like reaction may compromise antipredatory behavior since prey fish become more visible to predators (Martin et al. 2017). Anxiety and fear are important emotions associated with risk-assessment behaviors, crucial to the maintenance of species in the environment (Maximino et al. 2010). Stress responses and risk-assessment behaviors are beneficial to animals, as they allow individuals to

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restore their physiological and behavioral homeostasis, despite fluctuating risk conditions in the environment (McEwen and Wingfield 2003). These risk-assessment and stress-induced behaviors of any animal species require intact brain and body functions, as well as an intact behavioral repertoire (Karatsoreos and Mcewen 2011; Sterling 2012). However, aquatic pollutants can break this allostatic state and adversely affect fish behavior, foraging, or reproduction (Graeff and Junior 2010). Considering that APPZ acts on the central nervous system as a partial agonist of dopaminergic (Tamminga and Carlsson

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2002) and serotoninergic (Hirose and Kikuchi 2005) receptors and it is considered as a

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regulator drug of these systems, it can directly affect the neural basis of behavioral

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phenotypes (Burda et al. 2011).

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Thus, here we posed the following question: “What will be the behavioral effects of waterborne APPZ in fish? Can APPZ prevent stress-induced behavioral changes?” To answer

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this question, we exposed adult zebrafish to stress and to different APPZ concentrations

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[based on the concentration previously detected in the environment and the concentrations that blunt the cortisol response to acute stress stimuli as examined in Barcellos et al. (2016)]

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and evaluated their exploratory, anxiety-like, social, and anti-predatory behaviors, as these behaviors are crucial to the prey-predator relationship (Stewart et al. 2013; Colwill and Creton 2011).

2. Materials and Methods 2.1. Study strategy We aimed to examine the potential effects of APPZ on zebrafish behavior by exposing zebrafish to different APPZ concentrations in the water and subjecting them to three behavioral tasks: the social preference test (SPT), the novel tank test (NTT), and the preypredator test (PPT). APPZ-exposed fish, with and without stress, were subjected to the SPT

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and NNT tests in order to examine if this drug affects the well-known stress-induced behavioral phenotype (Gerlai et al. 2000; Kalueff et al. 2013). In addition, APPZ-exposed fish were subjected to the PPT test in order to examine if the drug affects the predator (Gerlai et al. 2000) and no predator (Oliveira et al. 2013) perception in zebrafish.

2.2. Ethical approval This study was approved by the Ethics Commission for Animal Use Committee

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(CEUA) of the University of Passo Fundo, UPF, Passo Fundo, RS, Brazil (Protocol

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#020/2016 - CEUA) and complied with the guidelines of the National Council for Animal

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Experimentation Control (CONCEA). In addition, this research was registered in SisGen

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(Sistema Nacional de Patrimônio Genético e do Conhecimento Tradiocional Associado) and

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complied with their guidelines (registration code A14E252).

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2.3. Animals and housing conditions

A population of 624 mixed-sex, 8-month old, and short-fin zebrafish, weighing 0.3 to 0.5 g

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and with a body length of 1.5 to 3 cm, from a heterogeneous wild-type breeding population from the Passo Fundo University, Brazil, was kept at a density of 1 fish/L. The total fish number included the 288 fish used to form the triplets of the PPT test, however the latter were not used in data collection. The number of fish used in behavioral testing was 336. To confirm the sex ratio, we killed 30 fish from the experimental population in ice-cold water and after dissecting them, we examined their gonads in a stereomicroscope (Nova Instruments, Piracicaba, Brazil); the sex ratio was confirmed as 16 males and 14 females. The maintenance glass tanks were equipped with biological filters, were under constant aeration, and the fish were submitted to a photoperiod of 14 h light and 10 h dark.

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We distributed the fish randomly in each experiment using the app RANDOM.ORG. Fish were acclimatized for 5 days in 24 4-L glass aquaria (20 × 15 × 14 cm, width × depth × height, respectively) (n=4 fish in each aquarium). In all the experiments, the experimenters and the data analysts were blinded to the treatments. Water temperature was maintained at 27.4 ± 1.1 °C, pH at 7.2 ± 0.5, dissolved oxygen at 5.7 ± 0.8 mg/L, non-ionized ammonia at  0.03 mg/L, total hardness at 39 ± 5 mg CaCO3/L, and alkalinity at 35.2 ± 7.3 mg CaCO3/L. We used a 12 cm long goldfish (Carassius auratus) as a non-predator stimulus fish due to

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its peaceful and friendly temperament (Kottelat et al. 1996) and a 20 cm long adult tiger oscar

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(Astronotus ocellatus), a Cichlid fish with strong predatory behavior (Smith 1981). Both fish

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came from a local aquarium market and were acclimatized for 5 days in a specific

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compartment for the stimulus fish in order to prepare them for the prey-predator test. This compartment had a 50 L capacity (56 × 30 × 30 cm). Fish were then acclimatized for 5 days

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in 4 L glass aquaria (20 × 15 × 14 cm, width × depth × height, respectively) where the water

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temperature was maintained at 27 °C, the pH at 7.2, the dissolved oxygen at 6.7 mg/L, the non-ionized ammonia at  0.03 mg/L, the total hardness at 39 mg CaCO3/L, and the alkalinity

zebrafish.

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at 37 mg CaCO3/L. The other water quality driving conditions were the same as in the

We fed zebrafish and goldfish a diet containing 48% crude protein (SUPERVIT®; Tropical, Chorzow, Poland) ad libitum and twice daily. The oscar fish was fed with one live zebrafish twice a day throughout the entire experimental period. However, during the day of the experiment, the zebrafish were fasted, with all fish receiving food 12 h before the behavioral trials in order to prevent the food from interfering with the behavior (Dametto et al. 2018). This feeding regimen is depicted in the approved ethical protocol.

2.4. Experimental procedures

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2.4.1. Drug and concentrations tested In each behavioral test, we exposed fish to three APPZ (Aristab®; Aché, Guarulhos, Brazil) concentrations. We set a concentration previously detected in the environment (5.56 ng/L) (Subedi and Kannan 2015) and the concentrations that blunt the cortisol response to acute stress stimuli (0.556 and 556 ng/L) (Barcellos et al. 2016). APPZ was dissolved in dimethylsulfoxide DMSO (Sigma-Aldrich, St Louis, MO, USA) to generate a stock solution (as described in Lee et al. 2011). Since previous evaluations in our laboratory have

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demonstrated no adverse biological effect of DMSO at this low concentration (less than

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0.01% v:v), we did not performed a DMSO control. In addition, no effects were reported for

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adult (Horie et al., 2013), even larvae zebrafish at this low concentration (Maes et at., 2012).

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We used nominal concentrations since the products used are new and they have concentrations guaranteed by the manufacturer and by the law. Thus, we did not measure the

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real concentrations of APPZ in the water the fish were exposed to. Another point to consider

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was the very short exposure time (15 min) that prevented any concentration decrease that originally spiked. Additionally, the reported recovery time of water samples spiked with

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APPZ within a day, ranges from 99 to 101.21% (Sastry et al. 2009; Kalaichelvi et al. 2010; Thakkar et al. 2011).

2.4.2. Behavioral testing

For SPT (96 fish) and NTT (96 fish), fish were left in 24 4-L glass aquaria (two experimental trials) in groups of four. From these aquaria, we captured all fish at the same time and placed them individually in four APPZ-treated 1-L beakers. Two stressed and non-stressed fish were subjected to SPT or NTT. We repeated this process 12 times to reach n = 12 per treatment (the water was renewed after each test). The acute stress stimulus was to chase fish with a hand net for 2 minutes after 15 minutes of exposure to APPZ and fish were immediately

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subjected to the behavior test. For PPT, we used a population of 432 fish; in order to avoid isolation effects (Giacomini et al. 2016b) we kept fish in triplets (Pagnussat et al. 2013) and subjected only one fish per aquarium to PPT in order to reach n = 12 per treatment (a total of 144 fish). At PPT, we subjected each zebrafish to the same APPZ concentrations and exposed them to a visual predator or no predator stimulus. To evaluate the behavioral parameters in each test, we videotaped fish using a Logitech Quick cam PRO 9000 camera (Logitech Co., Newark, USA). To avoid an effect of

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human activity, the operator exited the experimental room after the fish were released into the

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test aquaria in all behavioral tests. The videos were analyzed with the automated tracking

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software ANY-maze® (Stoelting Co., Wood Dale, USA).

2.4.2.1 SPT

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For the SPT, the tank test (30 × 15 × 10 cm, width × depth × height, respectively) (Savio et al.

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2012) was positioned between two equal-sized aquaria, one without fish and the other containing a group of 12 conspecifics with the same color pattern (Engeszer et al. 2004). After

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the treatment, fish were acclimated to the test tank individually for 30 s and their behavior was recorded for 60 s. We divided the tank virtually in three vertical segments to analyze data. The first segment was the nearest to the conspecifics‟ tank, while the third segment was next to the empty tank. The following parameters were analyzed: time spent in each segment (s), number of entries in the conspecifics‟ segment, and distance traveled (m) in this segment.

2.4.2.2 NTT In this test, we used rectangular glass aquaria (24 × 8 × 20 cm, width × depth × height, respectively) (Mocelin et al. 2015). We divided the tank test virtually in three horizontal segments to analyze data. Fish were recorded for 5 minutes and the following parameters

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were analyzed: time spent in different zones of the tank (top, middle, and bottom) (s), latency to first entry in the top zone (s), number of total crossings, time spent in freezing (s), and total distance traveled (m). Fish were considered to be in freezing when they ceased moving completely (except for their gills and eyes) while at the bottom of the tank (Kalueff et al. 2013).

2.4.2.3 PPT

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For PPT (n=144) we used a rectangular glass aquarium (104 × 30 × 30 cm, width × depth ×

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height, respectively), divided in three partitions with a glass and without allowing water to

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pass. The first partition (56 cm wide) was allocated to the predator, the second partition (24

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cm wide) was assigned to the tested zebrafish, and the third partition (24 cm wide) was left empty, containing only with water. After being exposed to APPZ, the zebrafish was

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introduced to the second partition and its activity was recorded for three minutes. We divided

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the second partition virtually in three segments to analyze data: high risk zone (zone 1, near the predator‟s partition), low risk zone (zone 3, far from the predator, near the empty

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partition), and intermediary neutral zone (zone 2). The analyzed parameter was the time spent in the high risk zone (s). Data were analyzed by 60 s stages [first stage (0 to 60 s), second stage (61 to 120 s), and third stage (121 to 180 s)], totaling 3 minutes of recording. We also filmed the behavior of stimulating fish (oscar and goldfish) while all the zebrafish were tested throughout the 180 s period.

2.5. Statistics To compare the SPT data, we used a two-way ANOVA followed by a post-hoc Dunnett‟s test. Using data from the same test, we aimed to determine the stress induced phenotype by comparing non-stressed and stressed fish by a Mann-Whitney test. To compare data from

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NTT, we also used a two-way ANOVA followed by Dunnett‟s multiple comparison test. Regarding PPT, first we compared all data using a two-way ANOVA; as we detected no apparent interaction or drug effect, we decomposed our interaction hypothesis and, for each concentration, we compared data from the non-stimulus and goldfish and oscar presence tests by a one-way ANOVA followed by Dunnett‟s post-hoc test. This type of analysis was used by Kalichak et al. (2019) to compare similar PPT tests. The alpha level was set at 0.05 in all

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analyses.

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3. Results

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3.1. SPT

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We confirmed the stress-induced behavioral phenotype, as stress reduced the time spent in the conspecific segment (P = 0.0008; Mann-Whitney U = 11; Fig. 1A) as well the number of

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entries (P = 0.0158; Mann-Whitney U = 29; Fig. 1B) and the distance traveled (P = 0.0017;

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Mann-Whitney U = 13; Fig. 1C) in this segment.

Figure 1. Social preference test of control (S-) and stressed (S+) zebrafish not exposed to APPZ. (A) Time spent at the conspecifics‟ segment (s); (B) number of entries in the conspecifics‟ segment; (C) distance traveled in the conspecifics‟ segment (m). Data were expressed as median ± interquartile range of 10-12 fish and compared by a Mann-Whitney test. (*p<0.05, **p<0.01, ***p<0.001).

The three APPZ concentrations prevented the social impairment induced by stress when it was considered the main SPT outcome; in fact, APPZ-exposed zebrafish spent a

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similar time in the conspecifics‟ segment (Fig. 2A). The two-way ANOVA of the time spent in the conspecifics‟ segment yielded significant drug (F3,88 = 6.043; P = 0.0009) and stress (F1,88 = 23.29; P < 0.0001) effects, while the interaction was non-significant (F3,88 = 0.8415; P = 0.4645) (Fig. 2A). APPZ-exposed zebrafish had similar entry results, with the exception of fish exposed to 556 ng/L of APPZ (Fig. 2B). Regarding the number of entries (Fig. 2B), the two-way ANOVA yielded a significant drug × stress interaction (F3,88 = 3.461; P = 0.0200), without significant isolated effects of the drug (F3,88 = 1.203; P = 0.3140) and stress (F1,88 =

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2.590; P = 0.1114). APPZ-exposed zebrafish swam similar distances in this segment (Fig. 2C)

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compared to non-stressed control fish. In this parameter, there were significant drug (F3,88 =

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3.124; P = 0.0299) and stress (F1,88 = 7.549; P = 0.0073) effects, while the interaction was

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non-significant (F3,88 = 0.8980; P = 0.4456). No differences were found between the APPZ concentrations. In addition, APPZ per se at the 0.556 ng/L concentration decreased the

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number of entries in the conspecifics‟ segment compared to non-exposed controls (Fig. 2B).

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Figure 2. Social preference test of control (S-) and stressed (S+) zebrafish exposed to different APPZ concentrations. (A) Time spent at the conspecifics‟ segment (s); (B) number of entries in the conspecifics‟ segment; (C) distance traveled in the conspecifics‟ segment (m). Data were expressed as mean ± S.E.M. of 10-12 fish and compared by a two-way ANOVA, followed by Dunnett‟s multiple range test. * indicates a significant difference against the nonstressed control fish (p<0.05).

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3.2. NTT The results of this test showed that at an 0.556 ng/L APPZ concentration the stressed fish increased the time they spent at the top zone (drug effect F3,88 = 1.454, P = 0.2326; stress effect F1,88 = 11.54, P = 0.0010; drug*stress interaction F3,88 = 1.429, P = 0.8214; Fig. 3A). Stress and APPZ exposure did not affect the crossings between zones (drug effect F3,88 = 0.5767, P = 0.6319; stress effect F1,88 = 45.50, P < 0.0001; drug*stress interaction F3,88 = 0.5590, P = 0.6435; Fig. 3B) and the latency to enter the top zone (drug effect F3,88 = 0.5503,

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P = 0.6492; stress effect F1,88 = 36.10, P<0.0001; drug*stress interaction F3,88 = 0.3723, P =

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0.7732; Fig. 3C); however, bottom dwelling increased in stressed zebrafish (time spent at

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bottom zone - drug effect F3,88 = 1.090, P = 0.3576; stress effect F1,88 = 200.1, P < 0.0001;

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drug*stress interaction F3,88 = 1.710, P = 0.1708; Fig. 3F). APPZ exposure did not prevent these alterations. The time spent frozen (drug effect F3,88 = 1.813, P = 0.1507; stress effect

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F1,88 = 0.5443, P = 0.4626 and drug*stress interaction F3,88 = 0.8408, P = 0.4751; Fig. 3D) and

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the distance traveled (drug effect F3,88 = 1.538, P = 0.2104; stress effect F1,88 = 0.0001, P = 0.9914 and drug*stress interaction F3,88 = 0.3597, P = 0.7822; Fig. 3E) were not different

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among groups at all APPZ concentrations tested.

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Figure 3. Novel tank test locomotor parameters of unstressed (S-) and stressed (S+) zebrafish exposed or not exposed to different APPZ concentrations. A) Time spent at the top zone (s); B) line crossings; C) latency to enter the top zone (s); D) time spent in freezing (s); E) total distance traveled (m); F) time spent at the bottom zone (m). Data were expressed as mean ± S.E.M. of 12 fish and compared by a two-way ANOVA followed by Dunnet´s multiple comparison test. The asterisks indicate significant differences against the control non-stressed fish (*p<0.05, **p<0.01, ****p<0.0001).

3.3. PPT At all APPZ concentrations and in all stages, there was not an interaction between the stimulus fish and APPZ exposure (stage 1, F6,132 = 0.5972, P = 0.7321; stage 2, F6,132 = 1.286, P = 0.2679; stage 3, F6,132 = 1.038, P = 0.4039). Thus, we compared the reaction of zebrafish to an empty segment (no stimulus), to a goldfish, and to an oscar fish in each APPZ concentration.

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At the 1st stage (0-60s), control fish spent less time in the high-risk zone when exposed to goldfish, while no differences were found in APPZ-exposed fish (no stimulus × goldfish × Oscar – Control – F2,33 = 3.395, P = 0.0456; APPZ 0.556 – F2,33 = 0.8449, P = 0.4387; APPZ 5.560 –F2,33 = 1.462, P = 0.2463; APPZ 556 – F2,33 = 2.048, P = 0.1451; fig. 4, stage 1). In control fish, this pattern was repeated in the second stage, while fish exposed to 0.556 and 5.56 ng/L of APPZ did not change the time they spent in the high-risk zone. However, zebrafish exposed to 556 ng/L of APPZ reduced the time spent in the high-risk zone when

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exposed to both the goldfish and the oscar fish (no stimulus × goldfish × Oscar – Control –

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F2,33 = 3.559, P = 0.0298; APPZ 0.556 – F2,33 = 1.139, P = 0.3323; APPZ 5.560 – F2,33 =

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0.746, P = 0.4821; APPZ 556 – F2,33 = 4.179, P = 0.0241; fig. 4, stage 2). In the 3rd stage

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(121-180s), the control zebrafish clearly reacted to the presence of both the goldfish and the oscar fish, as they spent less time in the high-risk zone; this reaction was abolished by the

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three APPZ concentrations (no stimulus × goldfish × Oscar – Control – F2,33 = 7.239, P =

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0.0025; APPZ 0.556 – F2,33 = 1.302, P = 0.2857; APPZ 5.560 – F2,33 = 2.092, P = 0.1395;

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APPZ 556 – F2,33 = 0.3540, P = 0.7045; fig. 4, stage 3).

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Figure 4. Prey-predator test of zebrafish exposed or not exposed to APPZ. Time spent at high-risk zone of fish exposed to 0.556, 5.56, and 556 ng/L of APPZ. (1st stage; 2nd stage; 3rd stage). Data were expressed as mean ± S.E.M. of 12 fish and compared by a one-way ANOVA followed by Dunnett‟s test in each APPZ concentration (*p<0.05; **p<0.01; ***p<0.001).

4. Discussion Here we show that, despite the apparent beneficial reversal of stress-related social impairment and absence of effect on stress-related bottom dwelling, APPZ exposure impairs

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the prey‟s reaction to a bigger stimulus fish that could be a predator. In fact, while analyzing the PPT data, we observed that APPZ decreases the perception of predator by the prey, even at concentrations lower than those detected in the environment (Fig. 4, stage 1 to stage 3). Mechanistically, this effect may be related to the APPZ panicolytic effect (Tamminga and Carlsson 2002; Biojone et al. 2011). The APPZ panicolytic effect is produced because APPZ is a partial agonist of 5-HT1A receptors and an antagonist of 5-HT2A serotoninergic receptors, producing an anti-aversive effect on fear or panic conditions

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(Tamminga and Carlsson 2002). This panicolytic-like mechanism could be the same as the

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one detected in rats (Biojone et al. 2011). Interestingly, the non-APPZ-exposed zebrafish

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perceived the goldfish before the oscar fish and distanced themselves further from the risk

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area. This first detection of the non-predatory stimulus fish may be because this fish was more active and spent more time at the bottom, thus it became more visible to zebrafish (Kelley and

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Magurran 2003). When exposed to APPZ, zebrafish lost this capacity except during the

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second minute of test, at the 556 ng/L APPZ exposure. Perhaps zebrafish perceive the absence of a potential risk only by visual cues (Kelley and Magurran 2003; Barcellos et al. 2010), but

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we cannot discard the possibility that another kind of behavior will be exhibited under a combination of visual and chemical cues (Coleman and Rosenthal 2006; Egan et al. 2009; Oliveira et al. 2017).

Another possible mechanism underlining the effects of APPZ on zebrafish behavior is the impairment of cortisol response to stress previously described in adult zebrafish (Barcellos et al. 2016). A link between psychotropic exposure with blunted cortisol response and altered behavior was previously found for risperidone (Idalencio et al. 2015), methylphenidate (Endres et al. 2017), bromazepam, fluoxetine, and nortriptyline (Marcon et al. 2016), all found to have an effect on adult zebrafish.

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In the SPTs, we showed that APPZ prevented stress-induced changes in zebrafish behavior. Data seemed to be biphasic, however more concentrations were needed to reach safe conclusions about pattern and concentration dependence that were found in rodents (Biojone et al. 2011). The main effect was the reversal of the stress-induced impairment in the social behavior, promoting social preference instead of isolation. In fact, this is a desired therapeutic effect in the treatment of pervasive developmental disorders in humans, improving the symptoms of isolation (Stigler et al. 2004; Erickson et al. 2010; Wink et al. 2010).

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Apparently, this effect on social behavior may be a positive factor in the environment,

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because the group preference (Gerlai et al. 2000) or shoaling (Miller and Gerlai 2011; Soares

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et al. 2018) reduce predation risk and facilitate foraging (Miller and Gerlai 2012; Kalueff et

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al. 2013). However, we did not test if APPZ influences the polarization or the dispersive ability of the shoal, which are necessary to detect or avoid a predator (Kalueff et al. 2013), so

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we cannot confirm if this is a positive reaction in fish.

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At the lower APPZ concentration (0.556 ng/L), an anxiolytic-like effect occurred in stressed zebrafish, similar to that observed in rats (Burda et al. 2011) and humans (Erickson et

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al. 2010) treated with APPZ. We verified that APPZ increased the time spent and reduced the latency of entry in the top zone in stressed fish at the first stage of the NTT (Fig. 2C). This effect could be caused by the partial agonistic effect that APPZ exerts on dopaminergic receptors, especially D2, thus reducing anxiety-like behavior (Burris et al. 2002; Hirose and Kikuchi 2005; Burda et al. 2011). However, this drug is not effective in modifying stressinduced dwelling (Fig. 2F), leading us to think that APPZ had an anxiolytic effect only in the initial stage of the NTT. It is also evident that APPZ did not affect the locomotion activity of fish (Fig. 2B and 2E), as was the case in rats (Kus et al. 2017). Interestingly, we observed that only the lowest APPZ concentration reduced the number of entries in the conspecifics‟ segment in the SPT (Fig. 2A) and increased the time

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spent at the top of the tank in the NTT (Fig. 3A). These U- and/or bell-shaped concentrationresponse curves suggest a hormetic effect (Calabrese and Baldwin 2001), similar to that observed in neuroendocrine and behavioral evaluations of fish exposed to APPZ (Barcellos et al. 2016) and to effects of other psychotropic drugs such as diazepam (Abreu et al. 2014; Giacomini et al. 2016a) and risperidone (Idalencio et al. 2015). A possible limitation of this study was our inability to investigate the sex effect on the behavioral parameters evaluated. However, this does not lessen the importance of this

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initial evaluation, since behavioral data regarding the effects of acute APPZ exposure in

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zebrafish are scarce.

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Taken altogether, our results show that APPZ-exposed zebrafish decreased their

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ability to perceive and recognize a predator, even at concentrations lower than those detected in the environment. A failure to exhibit all steps of the antipredatory response (in this case,

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predator perception and recognition) may favor the predator, decrease the fitness of the prey

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species, and, consequently, affect the food chain (Kelley and Magurran 2003; Stewart et al. 2013). Our results highlight the risks and consequences associated with APPZ residues in

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water, which may affect aquatic life and endanger species that depend on appropriate behavioral responses for survival.

Compliance with Ethical Standards: Conflict of Interest: The authors declare that there is no conflict of interest. Funding: This study was funded by Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES, by Conselho Nacional de Desenvolvimento Científico e Tecnológico (grants numbers 303263/2018-0 and 301156/2012-3), and by Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (grant number 18/2551-0000493-6).

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Aripiprazole prevented the stress-induced social impairment;

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Aripiprazole prevented the stress-induced bottom dwelling;

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Aripiprazole impairs the anti-predatory reaction of adult zebrafish;

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Aripiprazole may endanger fish species that depend of intact behavioral repertoire.

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