Efeitos do álcool e do enriquecimento ambiental na aprendizagem e no comportamento tipo ansioso em peixe Paulistinha

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(1)UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE BIOCIÊNCIAS PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA. RICARDO RODRIGUES AMORIM. EFEITOS DO ÁLCOOL E DO ENRIQUECIMENTO AMBIENTAL NA APRENDIZAGEM E NO COMPORTAMENTO TIPO ANSIOSO EM PEIXE PAULISTINHA. Natal 2017.

(2) RICARDO RODRIGUES AMORIM. EFEITOS DO ÁLCOOL E DO ENRIQUECIMENTO AMBIENTAL NA APRENDIZAGEM E NO COMPORTAMENTO TIPO ANSIOSO EM PEIXE PAULISTINHA. DEFESA DA DISSERTAÇÃO APRESENTADA AO PROGRAMA DE PÓS-GRADUAÇÃO EM PSICOBIOLOGIA DA UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE COMO REQUISITO PARA A OBTENÇÃO DO TÍTULO DE MESTRE EM PSICOBIOLOGIA (ÁREA: COMPORTAMENTO ANIMAL) Orientadora: Profa. Dra. Ana Carolina Luchiari Co-orientadora: Dra. Priscila Fernandes Silva. Natal 2017.

(3) Universidade Federal do Rio Grande do Norte - UFRN Sistema de Bibliotecas - SISBI Catalogação de Publicação na Fonte. UFRN - Biblioteca Setorial Prof. Leopoldo Nelson - -Centro de Biociências - CB. Amorim, Ricardo Rodrigues. Efeitos do álcool e do enriquecimento ambiental na aprendizagem e no comportamento tipo ansioso em peixe Paulistinha / Ricardo Rodrigues Amorim. - Natal, 2017. 79 f.: il. Dissertação (Mestrado) - Universidade Federal do Rio Grande do Norte. Centro de Biociências. Programa de Pós-Graduação em psicobiologia. Orientadora: Profa. Dra. Ana Carolina Luchiari. Coorientadora: Dra. Priscila Fernandes Silva. 1. Danio rerio - Dissertação. 2. Etanol - Dissertação. 3. Bemestar - Dissertação. 4. Estresse - Dissertação. 5. Ambiente enriquecido - Dissertação. I. Luchiari, Ana Carolina. II. Silva, Priscila Fernandes. III. Universidade Federal do Rio Grande do Norte. IV. Título. RN/UF/BSE-CB. CDU 639.34.

(4) EFEITOS DO ÁLCOOL NA APRENDIZAGEM E NO COMPORTAMENTO TIPO ANSIOSO EM PEIXE PAULISTINHA. RICARDO RODRIGUES AMORIM. Natal, 28 de abril de 2017 Banca Avaliadora. ________________________________ Profa. Dra. Ana Carolina Luchiari Departamento de Fisiologia – UFRN Orientadora. ________________________________ Dra. Priscila Fernandes Silva Departamento de Fisiologia – UFRN Co-orientadora ________________________________ Dra. Janine Inez Rossato Departamento de Fisiologia – UFRN Membro interno. ________________________________ Dr. Renato Hajenius Aché de Freitas Departamento de Ecologia e Zoologia – UFSC Membro externo. 3.

(5) AGRADECIMENTOS. Aos meus pais, Miguel e Célia, aos meus irmãos, Marina e Mateus e a grandes amigos de Vitória da Conquista. Obrigado por, mesmo de longe, sempre me transmitirem força e boas vibrações.. Às minhas orientadoras, Ana Carolina Luchiari e Priscila Fernandes, que durante todos os momentos deram luz aos diversos questionamentos que surgiram no decorrer do trabalho.. Às minhas colegas de trabalho que me passaram informações cruciais à sobrevivência no mestrado, e a todos que me ajudaram durante os protocolos experimentais, as análises de vídeos e as análises estatísticas. Muito obrigado, Mix, Luccas, Vanessa, Jéssica e Fulvio.. À minha companheira de lutas, Dayse Fernanda, por transmitir toda paciência do mundo em meio às diversas preocupações corriqueiras. Obrigado por ser meu ponto de equilíbrio, juntamente com nossas gatinhas queridas, Mumu e Fin.. Ao PPG Psicobiologia, por toda assistência institucional e burocrática e à Capes e ao CNPq, por financiarem este projeto.. 4.

(6) Dedico esse trabalho a todos os peixes que participaram dos protocolos experimentais aqui empregados.. 5.

(7) RESUMO. Bebidas alcoólicas são popularmente consumidas em diversas culturas humanas. Porém, o consumo alcoólico excessivo e a longo prazo pode promover danos sociais, físicos e psicológicos de difícil reversão. De fato, o álcool exerce diferentes efeitos no organismo. Baixas concentrações promovem euforia, relaxamento, e alívio do estresse/ansiedade (efeito ansiolítico), e a abstinência, após doses médias crônicas, aumentam o estresse/ansiedade (efeito ansiogênico), podendo interferir na aprendizagem. No entanto, há poucas informações sobre como essa droga altera aspectos cognitivos e psicológicos em situações de estresse/ansiedade que envolvam aprendizagem. O que demanda investigações aprofundadas com a proposição de modelos animais para uso translacional. Diante disso, características como: fácil manutenção e reprodução em laboratório, homologia genética superior a 70% à do genoma humano, e facilidade na administração de fármacos, tornam o peixe paulistinha (Danio rerio) um modelo ideal em pesquisas translacionais que envolvam drogas de abuso. Nesse sentido, o presente trabalho buscou investigar o efeito de diferentes tratamentos alcoólicos (crônico e agudo) no desdobramento da resposta de ansiedade e na aprendizagem associativa aversiva do peixe paulistinha. Para isso, foram testados os efeitos do álcool na aprendizagem associativa aversiva do paulistinha (artigo 1) e os efeitos do álcool e do enriquecimento ambiental (EE) na impossibilidade do peixe evitar um estressor (artigo 2). Os resultados indicaram que comportamentos tipo-ansiosos em paulistinha são alterados por mudanças nas concentrações alcoólicas, no regime de exposição ou no ambiente. Álcool agudo aumenta ansiedade e potencializa a percepção em relação ao eletrochoque. Já o EE promove efeito ansiolítico e diminui a percepção do eletrochoque. Por fim, sugerimos que esse trabalho serve de base para pesquisas neurofisiológicas genéticas e comportamentais sobre os efeitos da interação entre drogas de abuso e ambiente.. Palavras-chave: Danio rerio; etanol; bem-estar; estresse; ambiente enriquecido.. 6.

(8) Abstract. Alcoholic beverages are popularly consumed in various human cultures. However, excessive and long-term alcohol consumption can promote social, physical and psychological damages that are difficult to reverse. In fact, alcohol has different effects on the body. Low concentrations promote euphoria, relaxation, and stress/anxiety relief (anxiolytic effect), and abstinence after chronic medial doses increases stress / anxiety (anxiety) and may interfere with learning. However, there is little information on how this drug alters cognitive and psychological aspects in stress/anxiety situations involving learning. Therefore, in-depth investigations are required with the proposition of translational animal models. As a result, characteristics such as: easy maintenance and reproduction in the laboratory, genetic homology up to 70% to that of the human genome, and ease of administration of drugs, make zebrafish (Danio rerio) an ideal model in translational research involving drugs of abuse. In this sense, the present study sought to investigate the effect of different alcoholic treatments (chronic and acute) in the unfolding of the anxiety response and in the aversive associative learning of zebrafish.For this, the effects of alcohol in the aversive associative learning of zebrafish (manuscript 1) and the effects of alcohol and environmental enrichment (EE) on the impossibility of the fish to avoid a stressor were tested (manuscript 2). The results indicated that anxiety-like behaviors in zebrafish are altered by changes in alcoholic concentrations, in the exposure regime or in the environment. Acute alcohol increases anxiety and potentiates the electroshock perception. While EE promotes anxiolytic effect and decreases the perception of electroshock. Finally, we suggest that this work serves as a basis for genetic and behavioral neurophysiological research regarding the effects of the interaction between drugs of abuse and environment.. Keywords: Danio rerio; ethanol; welfare; stress; enriched environment.. 7.

(9) Sumário 1.INTRODUÇÃO GERAL............................................................................................. 1.1.Ansiedade.................................................................................................................. .................................................................................................... 1.2.Aprendizagem e memória.......................................................................................... 1.3.Álcool e ansiedade..................................................................................................... 1.4.Enriquecimento ambiental......................................................................................... 1.5.O paulistinha como modelo experimental................................................................. 2. OBJETIVOS DO ESTUDO........................................................................................ 2.1.Objetivo Geral........................................................................................................... 2.2.Objetivos Específicos................................................................................................ 3. ARTIGO 1……........................................................................................................... ABSTRACT...….............................................................................................................. INTRODUCTION........................................................................................................... MATERIALS AND METHODS.................................................................................... Animals and housing....................................................................................................... Alcohol exposure............................................................................................................. Inhibitory Avoidance Learning ...................................................................................... Statistical analysis........................................................................................................... RESULTS........................................................................................................................ DISCUSSION.................................................................................................................. ACKNOWLEDGEMENTS............................................................................................ REFERENCES................................................................................................................ 4. ARTIGO 2……........................................................................................................... ABSTRACT …............................................................................................................... INTRODUCTION........................................................................................................... MATERIALS AND METHODS.................................................................................... Animals and housing....................................................................................................... Anxiety induction............................................................................................................ Alcohol exposure and testing.......................................................................................... Behavior quantification................................................................................................... Statistical analysis........................................................................................................... RESULTS....................................................................................................................... DISCUSSION................................................................................................................. ACKNOWLEDGEMENTS............................................................................................ REFERENCES................................................................................................................ 5. CONSIDERAÇÕES FINAIS...................................................................................... 6. REFERÊNCIAS BIBLIOGRÁFICAS......................................................................... 9 9 10 12 14 15 19 19 19 20 20 21 22 22 23 25 27 27 29 34 35 43 43 44 45 45 46 47 48 49 49 53 58 58 67 68. 8.

(10) 1. INTRODUÇÃO GERAL 1.1. Ansiedade e Estresse A ansiedade é uma resposta emocional adaptativa presente em grande parte dos vertebrados (De-Grazia& Rowan, 1991). Tal resposta ocorre diante de ameaças potencialmente deletérias, sendo acompanhada por tensão, preocupação, inquietude e disforia (Barlow, 2004; NCHPEG, 2014). A ansiedade não pode ser confundida com o medo: uma resposta comportamental e fisiológica que ocorre perante ameaça eminente. A ansiedade pode ocorrer tanto na presença física quanto na expectativa de ameaça futura (APA, 2014). A resposta de ansiedade pode ser desencadeada apenas com a recordação do momento estressante previamente experienciado (Liotti et al., 2000). O que caracteriza os aspectos psicológicos marcantes de tal resposta. Embora a ansiedade seja uma resposta adaptativa às adversidades do meio no qual o animal está inserido, a reincidência de comportamentos tipo-ansiosos pode indicar a existência de algum transtorno de ansiedade (Etkin, 2009). Em 2015 cerca de 3,6% da população mundial sofria de transtornos de ansiedade, sendo a maioria mulheres: 4,6% comparado com 2,6% de homens (OMS, 2017). Ansiedade e medo em excesso caracterizam transtornos de ansiedade e promovem perturbações comportamentais decorrentes de aumento na sensibilidade à ameaça (Craske et al., 2009; APA, 2014). Dentre os principais transtornos de ansiedade na espécie humana se destacam: transtorno de ansiedade generalizada, transtorno obsessivo-compulsivo, pânico, agorafobia, fobia social e transtorno induzido por substância/medicamento (ABP, 2008; APA, 2014). Em condições normais, a ansiedade promove alterações neurofisiológicas que tem importante papel na regulação da homeostase do organismo em resposta às mudanças ambientais (Chang & Hsu, 2004; Serra, 2010). Mais especificamente, essas alterações ocorrem no funcionamento do eixo simpático-adrenomedular (Gouveia Jr et al., 2006) e do 9.

(11) eixo hipotálamo-pituitária-adrenal (HPA). A ativação desses eixos, no sistema nervoso simpático e medula adrenal, aumenta a atividade de enzimas sintetizadoras de catecolaminas: dopamina-P hydroxylase, tirosina hidroxilase, N-metiltransferase e feniletanolamina (Axelrod & Reisine, 1984). Tais hormônios promovem descarga de adrenalina no sangue. Isso prepara o organismo para situações imediatas de grande esforço físico, gerando, entre outras alterações, alerta, estimulação da força e frequência cardíaca, aumento da pressão arterial, broncodilatação e glicogenólise. A liberação de corticoides no sangue é resultado da ativação do eixo HPA em resposta ao estresse (Graeff, 2007). Nesse eixo, as respostas neuroendócrinas que culminam na resposta de ansiedade dependem da ativação cortical do núcleo basolateral da amígdala (Graeff, 2007) e, em seguida, da ativação do núcleo central da amígdala, a qual sinaliza aos neurônios do núcleo hipotalâmico paraventricular (Graeff, 2007). Esses secretam hormônio liberador de corticotropina (CRH) na circulação porta-hipofisária (Graeff, 2007). Consequentemente, hormônio adrenocortocotrópico (ACTH) é secretado pela adenohipófise na corrente sanguínea, atua sobre o córtex adrenal e promove a liberação de cortisol no sangue. Por fim, esse hormônio promove restituição dos níveis alostáticos do organismo (Serra, 2010). O desencadeamento dos processos supracitados, juntamente com a capacidade de memória associativa e aprendizagem dos animais, permite que indivíduos possam escapar de ameaças recorrentes (Barlow, 2004).. 1.2. Aprendizagem e memória A aprendizagem é o processo de aquisição de informações que são consolidadas na memória (Menzel & Byrne, 2008; Lent, 2010), resultando em mudanças estímuloespecíficas observadas mais tarde no comportamento (Bailey et al., 2008). Algumas formas 10.

(12) de aprendizagem como habituação e sensibilização são caracterizadas pela alteração da resposta neurofisiológica e comportamental frente a apresentação repetida de um estímulo, podendo haver diminuição (habituação) ou aumento (sensibilização) da força da resposta. Outra forma de aquisição de informações é a aprendizagem associativa que pode ser dividida em condicionamento clássico (associação entre um estímulo condicionado e um estímulo não condicionado) e condicionamento operante (associação entre determinada resposta comportamental e sua consequência) (Kirsch et al., 2004; Lent, 2010). A força na associação entre estímulo e resposta depende da experiência previa com estímulos e respostas relacionadas, e interfere na aquisição e recuperação da memória (Menzel & Byrne, 2008). A memória pode ser definida como a capacidade que os animais tem de armazenar informações que possam ser recuperadas e utilizadas posteriormente (Bailey et al., 2008; Lent, 2010). De modo resumido, os processos de memória podem ser classificados como: aquisição (armazenamento de informação do ambiente); consolidação (memorização de um evento/informação por longos períodos); retenção (disposição de informações para serem evocadas) e evocação: quando ocorre lembrança, acesso à informação armazenada (Menzel & Byrne, 2008; Lent, 2010). Em relação ao tempo de retenção a memória pode ser: ultrarrápida ou imediata (retenção dura poucos segundos); memória de curta duração (dura minutos ou horas), e memória de longa duração cujos engramas (unidades físicas da memória no cérebro) duram dias, semanas ou anos (Menzel & Byrne, 2008; Lent, 2010). A capacidade de aprender e utilizar as informações no futuro é essencial para o bom desempenho do indivíduo em diferentes atividades (Menzel & Byrne, 2008). Várias doenças que acometem a espécie humana estão associadas a prejuízos na aprendizagem e na retenção ou no armazenamento de memórias, como: doença de Parkinson, de Alzheimer ou transtornos de ansiedade (Sahakian et al., 1988; Park et al., 2001; Airaksinen et al., 11.

(13) 2005). Essas doenças afetam áreas cerebrais diretamente relacionadas à aprendizagem e memória. Tais como o hipocampo, a amígdala e áreas integradas (Squire, 2009; Lent, 2011). O hipocampo e a amígdala em mamíferos são estruturas anatomicamente homólogas ao pálio lateral (homólogo do hipocampo) e pálio medial (homólogo da amígdala) do cérebro de teleósteos (Rodrıguez et al., 2002; Broglio et al., 2005). Áreas paleais, hipocampo e amígdala possuem funções mnemônicas que por serem comparáveis, refletem o passado evolutivo em comum entre mamíferos e teleósteos (Rodrıguez et al., 2002; Broglio et al., 2005). O hipocampo e áreas associadas tem importante papel nos processos de formação, reorganização e consolidação da memória durante longos períodos pós-aprendizagem (Squire, 2009; Lent, 2011). Memórias com teor emocional e ou adquiridas em estado de alerta são mais bem consolidadas do que memórias de eventos pouco relevantes e ou adquiridas em estado de sonolência (Izquierdo, 1989; Squire, 2009). A amígdala e demais vias nervosas que regulam as emoções tem importante papel na consolidação de memórias com conteúdo emocional forte (Izquierdo et al., 2006; Squire, 2009). Por isso a exposição crônica a estressores provoca prejuízos na aprendizagem e na memória (Park al., 2001; Song et al., 2006). Tais prejuízos podem ser diminuídos por meio de alterações feitas no ambiente de inserção do indivíduo (Benaroya‐ Milshtein et al., 2004; Cirulli et al., 2010; Hüttenrauch et al., 2016) ou através da administração de substâncias com efeito ansiolítico (Gomez et al., 2013; Maximino et al., 2014).. 1.3. Álcool e ansiedade No ocidente, drogas com efeito ansiolítico (benzodiazepínicos, antidepressivos, buspirona, betabloqueadores e antipsicóticos), são bastante utilizadas no tratamento de transtornos de ansiedade (Andreatini et al., 2001; Altamura et al., 2013). Por outro lado, 12.

(14) bebidas alcoólicas também promovem efeito ansiolítico capaz de liberar angustias e tensões, sendo, por isso, consumidas entre diversas culturas humanas (Cabral, 2004; McGovern, 2009). Na atualidade, a facilidade em conseguir bebidas alcoólicas e a influência do contexto sociocultural aumentam o risco de consumo alcoólico indiscriminado entre diversas faixas etárias (Mandelbaum, 1965; Vieira et al., 2007; de Barros Jumqueira et al., 2016). Entre jovens o consumo vem crescendo e merece destaque, o que pode ocorrer tanto por influência de pessoas mais velhas (inclusive familiares), quanto pela necessidade de afirmação social no grupo de amigos, e/ou pela sensação de autonomia e maturidade provocada pela substância (Trindade & Correia, 1999; Bonito & Boné, 2016). Apesar de decrescer com o avançar da idade (Fuller, 2008), o consumo abusivo do álcool pode provocar, a longo prazo, problemas sociais (no trabalho e problemas na família), danos físicos (doenças hepáticas, cardíacas e cerebrais) e psicológicos (demência, dependência alcoólica e prejuízo na memória) (Holder & Griffith, 1995). Em 2012, 5,9% das mortes no mundo ocorreram por influência de bebidas alcoólicas: 33,4% por agravamento de doenças cardiovasculares, injúrias acidentais (17,1%), doenças gastrointestinais (16,2%) e canceres (12,5%; OMS, 2014). No Brasil, o consumo alcoólico esteve relacionado à incidência de cirrose hepática (63% em homens e 60% em mulheres) e de acidentes de trânsito (18% causados por homens e 5% por mulheres), 2,8% dos brasileiros possuem características que indicam possível dependência alcoólica (OMS, 2014). No Sistema Nervoso, o álcool exerce efeito na aprendizagem (Salamé, 1991; Santos et al. 2016), na memória (Scobie & Bliss, 1974; Kalev-Zylinska & During, 2007) na agressividade (Miczek et al., 1992; Gerlai et al., 2000), na percepção do meio (Santos et al. 2016) e na ansiedade (Gomez et al., 2013). O álcool agudo em baixas concentrações exerce 13.

(15) efeito redutor da ansiedade (Mottram, 1996; Mathur & Guo, 2011). Porém o álcool crônico seguido de abstinência pode gerar efeito ansiogênico (aumento da ansiedade), tolerância e dependência da substância (Kliethermes, 2005; Mathur & Guo, 2011). Apesar de estudos indicarem efeito ansiogênico na abstinência alcoólica e ansiolítico após ingestão de doses baixas de álcool, aspectos fisiológicos e comportamentais relativos ao consumo alcoólico na promoção e redução da resposta de ansiedade ainda não foram totalmente elucidados. Diante disso, é relevante o aprofundamento sobre o modo de ação do álcool no Sistema Nervoso Central, a fim de observar como esta droga altera aspectos psicológicos e cognitivos relativos ao enfrentamento de situações de estresse que envolvam ansiedade. Nesse sentido, estudos sobre o efeito dessa droga de abuso e diferentes contextos ambientais podem promover o entendimento de tais questões.. 1.4. Enriquecimento ambiental O enriquecimento ambiental pode ser definido como modificações no ambiente que resultam em melhorias no funcionamento biológico e no sucesso reprodutivo de indivíduos (Newberry, 1995). As modificações feitas no ambiente a fim de enriquecê-lo podem ser: sociais, quando se permite a interação com coespecíficos (Branchi et al., 2006; Cirulli et al., 2010); alimentares, com a adição de presas vivas ou alimentos escondidos (Carlstead et al., 1991; Brown et al., 2003); e sensoriais, com a adição de estímulos visuais, olfativos e táteis no ambiente (Mandairon et al., 2006; Salvanes et al., 2013). Um ambiente enriquecido, promove a melhoria do bem-estar animal em animais de cativeiro (Beattie et al., 1995; Newberry, 1995; Young, 2013), permite a reabilitação de animais para soltura em programas de conservação (Newberry, 1995), aumenta a propensão a interações sociais (Branchi et al., 2006) e melhora o desempenho de 14.

(16) predadores durante forrageamento (Brown et al., 2003). Tais melhorias são importantes principalmente quando se considera a possível existência de senciência – capacidade de interpretar estímulos ambientais externos e internos através de, ao menos, habilidades básicas de memória, julgamento e emoções (Silverman, 2008) – em vertebrados e diversos invertebrados (Chandroo et al., 2004; Broom, 2007). Nesse sentido, a melhoria no bemestar animal promovida pelo enriquecimento ambiental é indicada pelo progresso no desempenho em tarefas de aprendizagem (Tang et al., 2001; Falkenberg et al., 1992), pela redução da ansiedade e pela melhora na memória espacial de modelos animais (Hüttenrauch et al., 2016). Além disso, os efeitos do enriquecimento ambiental na melhoria da aprendizagem e na diminuição da ansiedade estão relacionadas a mudanças na plasticidade cerebral (Cirulli et al., 2010) e na redução de hormônios relacionados ao estresse e ansiedade (Benaroya‐ Milshtein et al., 2004). A exposição a ambiente enriquecido promove resultados positivos em animais submetidos a tratamentos com drogas de abuso: reduz preferência a etanol (Deehan et al., 2007), e auto-administração de anfetaminas em ratos (Bardo et al., 2001), Além disso, diminui sensibilidade à recompensa induzida pela morfina (Xu et al., 2007) e efeitos comportamentais, neuroquímicos e moleculares da cocaína em camundongos (Solinas et al., 2009). Desse modo, estudos com enriquecimento ambiental podem servir de base para o entendimento de mudanças na plasticidade (induzida por drogas de abuso) de estruturas neurológicas homólogas entre modelos animais (Cummins et al., 1977; Draganski & May, 2008).. 1.5. O paulistinha como modelo animal O peixe paulistinha (Danio rerio) é um ciprinídeo endêmico do nordeste da Índia, vivendo em torno das bacias dos rios Ganges e Brahmaputra, (Spence et al., 2008). 15.

(17) Atualmente este peixe é bastante utilizado como animal de estimação ou como modelo translacional para estudo em diversas áreas de biologia, fisiologia e medicina em laboratórios pelo mundo. Diversas características favorecem o uso do peixe paulistinha como modelo experimental: tamanho pequeno (entre 3 e 5 cm de comprimento); fácil manutenção e reprodução em laboratório com cerca de 300 ovos por desova, possibilitando que um pequeno estabelecimento possa comportar centenas de animais (Guo, 2004; Zon e Peterson, 2005; Norton & Bally-Cuif, 2010) Desse modo, o paulistinha tem sido apontado como alternativa válida em relação a modelos mamíferos, os quais demandam mais espaço, custos de manutenção e geram em média, apenas oito filhotes por ninhada. O paulistinha possui ainda mais de 70% de homologia genética com humanos e conservação de vias de sinalização e sistemas organizacionais e funcionais que possibilitam elevada translação para mamíferos (Barbazuk et al., 2000; Santoriello & Zon, 2012). Tais características aumentam a popularidade desse peixe como modelo experimental em pesquisas embriológicas e genéticas (Gerlai et al., 2006), bem como nas áreas de neurociências e comportamento animal (Kalueff et al., 2013), aprendizagem e memória (Roberts et al., 2013; Luchiari & Chacon, 2013), medo, estresse e ansiedade (Egan et al., 2009; Blaser & Rosemberg, 2012; Luca & Gerlai, 2012). Além disso, o paulistinha tem sido utilizado em pesquisas sobre os efeitos de drogas de abuso como cafeína (Egan et al., 2009), nicotina (Zimmermann et al., 2009; Sackerman et al., 2010), canabinóides (Stewart & Kalueff, 2014) e álcool (Gerlai et al., 2006; Echevarria et al., 2010; Mathur & Guo, 2011). Tais substâncias podem ser administradas diretamente na água do aquário, e o peixe pode absorvê-las passivamente pela superfície corporal e brânquias (Gerlai et al., 2006) sem que seja necessário a manipulação e administração invasiva das drogas (Iturriaga-Vásquez et al., 2012). Já a 16.

(18) maior complexidade do Sistema Nervoso de mamíferos demanda manipulação intensa para administração de fármacos/drogas o que dificulta o estudo de rotas bioquímicas, expressão proteica e áreas encefálicas específicas de ação isolada do álcool, por exemplo. A exposição a diferentes concentrações alcoólicas afeta paulistinha e humanos de modo semelhante: concentrações alcoólicas baixas (0.10 a 0.25%) promovem efeito facilitador (ansiolítico),. enquanto concentrações altas (0.75 a 1.00%) efeito inibidor,. semelhante aos anestésicos (movimentos mais lentos e pouco coordenados) (Gerlai et al., 2000). Além disso, paulistinhas sob efeito de abstinência alcoólica apresentam aumento da ansiedade (Mathur & Guo, 2011), O álcool exerce efeito semelhante em outros modelos animais: redução do comportamento tipo-ansioso em ratos Wistar (Momeni & Roman, 2014), aumento no consumo alcoólico correlacionado à intensidade do estresse e níveis de corticosteroides no plasma em macacos rhesus (Higley et al., 1991), e hiperexcitabilidade do sistema nervoso em ratos Long-Evans (Begleiter & Porjesz, 1977). Doses baixas de álcool podem ainda melhorar aprendizagem e memória, enquanto a abstinência alcoólica pode danificar tais parâmetros tanto em paulistinha quanto em modelos mamíferos (Alkana & Parker, 1979; Hernandez & Valentine, 1990; Chacon & Luchiari 2014; Luchiari et al., 2015; Santos et al. 2016). O álcohol apresenta potencial ansiolítico e ansiogêncio dependente de diversos fatores como: dose (Gerlai et al., 2000) tempo de consumo (Tran & Gerlai, 2013) além do contexto socioambiental (Echevarria et al., 2010) e estresse (Luca & Gerlai, 2012). Entretanto, alguns aspectos da ação e interação desses fatores na resposta de ansiedade ainda não estão claros. Nesse sentido, estudos sobre a relação do consumo de álcool com a subsequente resposta ansiolítica/ansiogênica necessitam de investigação mais profunda, com a proposição de modelos que ofereçam respostas translacionais. O modelo animal em questão deve: (1) apresentar características comportamentais similares àqueles de 17.

(19) mamíferos (validade facial), (2) possuir mecanismos controladores dos comportamentos semelhantes com humanos (validade construtiva), e (3) elementos (drogas, por exemplo) promotores de alterações fisiológicas e comportamentais exerçam os mesmos efeitos no modelo e em humanos (validade preditiva) (Willner, 1986). Além disso, após a escolha do modelo, é importante estabelecer condições padronizadas de reprodução e manutenção dos sujeitos experimentais para se garantir a validade preditiva entre o modelo e humanos. Isso pode ser conseguido por meio de protocolos de enriquecimento ambiental que, no peixe paulistinha, promovem tanto efeito ansiolítico quanto modificações na neuroplasticidade cerebral semelhantes às que ocorrem em modelos mamíferos (Benaroya‐ Milshtein et al., 2004; von Krogh, et al., 2010; Manuel et al., 2015). Apesar do peixe paulistinha apresentar resultados relevantes quanto à avaliação comportamental e responsividade a drogas em estudos psicopatológicos (Egan et al., 2009; Sackerman et al., 2010), as relações do álcool com aspectos cognitivos em situações de estresse e enriquecimento ambiental são pouco conhecidas. Nesse sentido, a necessidade de aspectos preditivos e construtivos à validação do paulistinha como modelo em estudos de ansiedade (Maximino et al., 2010) foi, em certo nível, considerada e discutida nesse trabalho.. 18.

(20) 2. OBJETIVOS DO ESTUDO 2.1. Objetivo geral Investigar o efeito do álcool (crônico e agudo) no desdobramento da resposta de ansiedade e na aprendizagem associativa aversiva do peixe paulistinha (Danio rerio).. 2.2. Objetivos específicos 1- Avaliar o desempenho do peixe paulistinha em tarefa de aprendizagem aversiva-ativa e exposto a diferentes tratamentos alcoólicos (Artigo 1); 2- Testar o efeito de tratamentos alcoólicos e do enriquecimento ambiental em situação de ansiedade estabelecida – impossibilidade de evitar o estressor – (Artigo 2).. 19.

(21) 1. Artigo 1: aceito para publicação na revista Zebrafish. 2. Effects of Alcohol on Inhibitory Avoidance Learning in Zebrafish (Danio rerio). 3. Ricardo Rodrigues Amorim, Prsicila Fernandes Silva, Ana Carolina Luchiari*. 4. Departamento de Fisiologia, Universidade Federal do Rio Grande do Norte, Natal, Brazil. 5. *Corresponding author: Departamento de Fisiologia, Centro de Biociências, Universidade. 6. Federal do Rio Grande do Norte, PO BOX 1510, 59078-970 Natal, Rio Grande do Norte,. 7. Brazil. Phone: +55 84 32153409, Fax: +55 84 32119206, E-mail:. 8. analuchiari@yahoo.com.br. 9 10. Abstract. 11. The zebrafish (Danio rerio) can be used in studies addressing the effects of drugs on. 12. learning, memory and anxiety. In the present study, we investigated the effect of different. 13. alcohol treatments (chronic and acute) on the learning and anxiety response of zebrafish in. 14. an inhibitory avoidance paradigm. Zebrafish were initially exposed to different alcohol. 15. treatments and submitted to an inhibitory avoidance protocol, where an electroshock was. 16. applied to the fish as it swam from the white to the black side of a shuttle box tank. 17. (naturally preferred environment of zebrafish). Animals from the control and 0.5% acute. 18. alcohol groups exhibited high latency to enter the black side of the tank after the first. 19. exposure to electroshock, in addition to higher freezing and a shorter distance from the. 20. bottom of the tank, suggesting acute alcohol exposure did not affect aversive learning in. 21. zebrafish. However, chronic submission and alcohol withdrawal impaired the fish’s. 22. capacity to properly respond to the aversive stimulus. Overall, our results show the harmful. 23. effects of chronic alcohol exposure, both continued intake and its cessation, but avoidance. 24. behavior persisted and anxiety increased following acute alcohol exposure.. 25. Keywords: zebrafish; ethanol; memory; electroshock; anxiety-like behavior 20.

(22) 26 27. Introduction Psychosocial problems resulting from the indiscriminate and excessive use of. 28. alcohol are challenges faced by modern medicine and public health.¹ Excessive. 29. consumption can lead to long-term physical damage (heart and liver disease), neurological. 30. impairment (impaired memory, dementia and development of addiction) and social issues. 31. (domestic violence and workplace problems).². 32. Alcohol intake leads to a biphasic and dose dependent effect, with acute low. 33. concentrations of alcohol inducing states of excitement and euphoria, while high doses. 34. have depressant effects.³ The initial sensation of relaxation and relief from stress and/or. 35. anxiety (anxiolytic effect) caused by the drug may trigger seeking behavior and the. 36. development of alcohol abuse.4,5 Chronic consumption may lead to tolerance,6,7,8 which. 37. increases the search for and use of the drug, contributing to the development of addiction.. 38. Cessation of alcohol intake, so-called withdrawal, may take place together with the. 39. withdrawal syndrome, whereby stress and anxiety are highly present.5 In this respect,. 40. alcoholics tend to consume more alcohol to avoid symptoms related to nervous system. 41. hyperactivity (anxiety, insomnia and tremors) during the withdrawal syndrome,9. 42. contributing to the development of addiction and dependency. Although knowledge. 43. regarding alcohol’s mechanism of action in the CNS has been increasing,10 little is known. 44. about how this drug alters psychological and cognitive aspects related to coping with. 45. stressful situations involving anxiety. In this regard, physiological and behavioral studies. 46. using animal models may contribute to the understanding of these issues.11. 47. Research on rodents and primates under the effect of alcohol facilitates translation. 48. to humans, but the complexity of mammalian neural connections hinders such studies.6 On. 49. the other hand, zebrafish presents a simple neural pathway which makes it an easier and. 50. more adequate model for studies on brain mechanisms of behavior.12 In this respect, the 21.

(23) 51. zebrafish (Danio rerio) is an alternative experimental model that has attracted attention due. 52. to its more than 70% genetic similarity to the human genome, conservation of signaling. 53. pathways and organizational and functional systems that allow high translation to. 54. mammals.13,14 As a result of the complex physiological and behavioral systems. 55. (comparable to mammals), simplicity of the biological model and drug administration in. 56. the water (which reduces handling stress), the zebrafish became one of the most promising. 57. models for the study of drug effects.15 Indeed, zebrafish learning has been validated in. 58. studies on memory,16,17,18 anxiety,5 stress19,20,21 and other subjects, with significant. 59. contribution to the understanding of mammals.. 60. Although alcohol is a potential anxiolytic/anxiogenic drug widely consumed in. 61. society and often addictive, there is a lack of knowledge regarding the induction of. 62. anxiety-like behavior related to its consumption. As such, the present study aimed to. 63. investigate the effect of different alcohol treatments (chronic and acute) on learning and. 64. anxiety response during inhibitory avoidance in zebrafish.. 65 66. Materials and Methods. 67. Animals and housing. 68. Adult zebrafish (n=56, mixed sex) were kept in 50 L tanks with a multistage. 69. filtration system. Temperature, pH, and oxygen were measured regularly (maintained at. 70. 28°C, pH~6.7, O2~6mg/L) and illumination set at a 12 h light and 12 h dark cycle. Fish. 71. were fed twice a day ad libitum with commercial pellets (Alcon Guppy, 44% protein; 5%. 72. fat) and frozen Artemia salina. Experimental fish were transferred to 4 glass home tanks. 73. (50 cm × 30 cm × 30 cm, width x depth x height, 30L, 14 individuals per tank) and the. 74. same water quality parameters were maintained for the tests. All the procedures were. 75. approved by the Animal Ethics Committee of the Federal University of Rio Grande do 22.

(24) 76. Norte (CEUA 007/2016).. 77. Alcohol exposure. 78. For alcohol exposure (99.8% absolute ethyl alcohol, Dinâmica, Química. 79. Contemporânea Ltda. Brazil), we used the 2x2 protocol (based on Gerlai et al.22), in which. 80. one of two chronic alcohol doses (0.0% or 0.5%) was paired with one of two acute alcohol. 81. doses (0.0% or 0.5%). Thus, four groups were formed (Fig. 1): Control (chronic 0.0% +. 82. acute 0.0%, n=14), Acute 0.5% alcohol (chronic 0.0% + acute 0.5%, n=11), Chronic 0.5%. 83. alcohol (chronic 0.5% + acute 0.5%, n=11) and Withdrawal (chronic 0.5% + acute 0.0%,. 84. n=10). The chronic alcohol dose of 0.5% was obtained by progressively increasing the. 85. alcohol concentration in the tank (dissolved in the home tanks). Alcohol exposure was. 86. continued for 24 h a day and the water in the tank was changed every 24 h to ensure. 87. alcohol concentration and water quality. For every manipulation procedure, fish were. 88. removed from its home tank to a smaller tank until water volume and alcohol dose were. 89. renewed. On the first 4 days, fish were exposed to 0.125% alcohol, from day 5 to 8,. 90. alcohol concentration was increased to 0.250%, and then increased again to 0.350% from. 91. day 9 to 12. Finally on day 13, alcohol concentration of 0.5% was administered to the fish. 92. and maintained for 10 consecutive days (day 13-23). This dose escalation procedure was. 93. applied to habituate the fish and reduce mortality. Both groups that received 0.0% alcohol. 94. during the chronic treatment (control group and acute 0.5%) underwent water changes. 95. every day so that manipulation was applied to all groups.. 23.

(25) 96. Fig.1. Time-line of Alcohol Exposure and Inhibitory Avoidance Learning in each. 97. group. In chronic treatment, zebrafish were exposed to 0.00% or 0.50% alcohol.. 98. Increasing doses of alcohol were given (0.00%, 0.125%, 0.225%, 0.325% and 0.50%) until. 99. the intended dose of 0.05% alcohol was reached. In acute treatment fish were exposed to. 100. 0.00% or 0.50% alcohol before the Avoidance Learning protocol. On the first day of this. 101. learning protocol (first training), fish that entered the black side of a shuttle box received. 102. 5s electroshock (6 V). On day 2 (second training), the same procedure was repeated. On. 103. day 3 (probe day), fish was allowed to move between compartments (black side / white. 104. side of the shuttle box) but no shock was applied to the fish. Time-line of the four alcoholic. 105. groups formed: Control, Acute 0.5% alcohol, Chronic 0.5% alcohol and Withdrawal.. 106. White bars represent the alcohol dose of 0.00%lightgraybars represent the alcohol dose of. 107. 0.125%, gray barsthe alcohol dose of 0.225%, dark gray barsthe alcohol dose of 0.325%. 108. and black bars represent the alcohol dose of 0.50%.. 109 110. Acute alcohol treatment took place on day 22, 23 and 24 (Fig. 1), whereby fish. 111. were exposed to alcohol for 60 min before and during the avoidance learning protocol (50. 112. min before and 10 min during the test). Alcohol was exposed using a 2 L tank (20 x 10 x. 113. 10 cm) containing 0.5% alcohol solution (10 ml alcohol + 1990 ml water) or only water. 114. (for the Control and Withdrawal groups). On days 22 and 23, after being tested, the fish. 115. were returned to their home tank, containing the alcohol dose corresponding to each group,. 116. until next training. This procedure ensures that the chronic group did not experience 24.

(26) 117. withdrawal, and the acute group did not decrease body alcohol concentration during the. 118. test.. 119. Inhibitory Avoidance Learning. 120. The testing apparatus used was a shuttle box tank (40 × 25 × 20 cm, Fig. 2), half. 121. white and half black, divided by an opaque lift-up partition. The aversive stimulus used. 122. was a 6 V electroshock applied for 5 s. To that end, steel gratings (24 cm × 10 cm, 5 mm. 123. mesh size, Fig. 2) were positioned on both sides of the opaque partition and on the opposite. 124. walls of the tank to conduct electricity through the water. The electric current was. 125. discharged only in the black side of the tank; the white side received the gratings to avoid. 126. visual differences between the two sides of the tank, other than tank color. The gratings on. 127. the black side were connected by wires to a 6 V stimulator that was activated for 5 s when. 128. the fish swam into the black side of the tank (adapted from Gorissen et al.23).. 129. Fig.2. Schematic view of the shuttle box used for inhibitory avoidance learning. The. 130. central partition is a lift-up door that, when raised, allowed fish to move between. 131. compartments. In both compartments, a pair of steel gratings was positioned, but only on. 132. the black side was it connected to an electric current discharger (6V).. 133 134. During inhibitory avoidance training (days 22 and 23), each fish was individually. 135. transferred from its respective tank to a 2 L tank for acute dose exposure, where it was kept. 136. for 50 min. The fish was then transferred to the white side of the shuttle box with the lift-. 137. up partition closed. The shuttle box had previously received water at the same alcohol 25.

(27) 138. concentration as in the 2 L tank. After 60 s acclimation in the white side, the opaque. 139. partition was raised 2 cm and the fish had up to 3 min to move to the black side of the tank.. 140. As soon as the fish entered the black side, the partition was closed and it received a 5 s. 141. electroshock. It was kept there for a further 30 s and then transferred to an individual tank. 142. (500 ml) where it received the same alcohol concentration as in the home tank (chronic. 143. dose corresponding to its group). Animals that did not enter the black side within the 3-min. 144. period were excluded from the tests. Training on day 23 was similar to that of day 22,. 145. except that on the former individuals that did not enter the black side within 3 minutes. 146. were gently guided to the black side with a net. Thus, all animals received the same. 147. number of electrical discharges.. 148. On the probe day (24), fish were exposed to the acute doses of alcohol (0.00 or. 149. 0.50% according to the group) for 50 min. They were then placed into the white side of the. 150. shuttle box (with the same alcohol concentration as before) for 60 s and the lift-up partition. 151. was raised. Fish behavior was recorded for 10 min, using a hand cam placed 50 cm in front. 152. of the tank. The front side of the tank, which was close to the camera, was free of. 153. white/black covering, but a half white/half black curtain was positioned behind the camera.. 154. Thus, even though the fish could see outside the front wall, its vision was limited to a few. 155. centimeters, where there was nothing else but the camera and an empty space. Behavior. 156. was analyzed by a tracking software (ZebTrack / UFRN,24) developed in MATLAB. 157. platform (R2014a, Math Works, Natick, MA). The parameters evaluated were latency to. 158. move from one compartment to the other, total distance traveled, average swimming speed,. 159. freezing (average immobility time) and distance from the bottom of the tank. To consider. 160. the fish is freezing, the software uses maximum speed default of 2 cm/s (or 2 px/s) and. 161. minimum time default of 1 s.. 162 26.

(28) 163. Statistical analysis. 164. All data analyses were performed using statistical software (Sigmastat 3.5; Systat. 165. Software, San Jose, CA, USA). After initial data analyses of normality and. 166. homoscedasticity, we conducted the Kruskal-Wallis test (followed by Dunn’s Multiple. 167. comparison post hoc test) to check for intergroup differences and the Wilcoxon Z test to. 168. evaluate differences between day 1 and 2, and between day 2 and 3 in terms of latency to. 169. reach the black side of the tank. Distance traveled, average speed, freezing, and distance. 170. from the bottom were analyzed using the Kruskal-Wallis test (followed by Dunn’s test). In. 171. all cases, the statistical significance was set at α <0.05.. 172 173. Results. 174. Figure 3 depicts the fish’s latency to swim from the white to the black side of the. 175. tank for each alcohol treatment on day 1 (first training), day 2 (second training) and day 3. 176. (probe day). On day 1, the Kruskal-Wallis test showed that individuals took longer to enter. 177. the black side when previously exposed to the chronic alcohol treatment or when under. 178. withdrawal from alcohol (H=22.48; p<0.001). On the 2nd day, after the first electroshock. 179. exposure, fish from the acute 0.5% alcohol group showed the highest latency to enter the. 180. black side (Kruska-Wallis, H=9.47; p=0.03). On the 3rd day, all groups showed high. 181. latency to enter the black side (Kruska-Wallis, H=6.21; p=0.10). From day 1 to day 2, the. 182. control (Wilcoxon Z test, Z=2.43; p=0.01) and acute 0.5% alcohol (Wilcoxon Z test,. 183. Z=1.54; p<0.01) groups showed an increase in latency to enter the black side, while the. 184. chronic 0.5% alcohol and withdrawal groups showed no statistical differences (Wilcoxon Z. 185. test, chronic: Z=0.88; p=0.18; withdrawal: Z=0.39; p=0.73). From day 2 to day 3, that is,. 186. after two electroshocks, none of the groups showed statistical differences (Wilcoxon Z test,. 187. control: Z=1.41; p=0.17; acute: Z=0.14; p=0.94; chronic: Z=0.01; p=1.00; withdrawal: 27.

(29) 188. Z=0.04; p=0.96).. 189. 190. Fig. 3. Zebrafish latency to reach the black side of the tank in the inhibitory. 191. avoidance learning protocol. After 1 min acclimation in the white side, the lift-up door. 192. was raised 2 cm from the bottom and allowed fish to move between compartments. On day. 193. 1 (first training), fish that entered the black side received 5s electroshock (6 V). On day 2. 194. (second training), the same procedure was repeated. On day 3 (probe day), fish was. 195. allowed to move between compartments but no shock was applied to the fish. Dark gray. 196. dots represent the control group (n=12), white dots represent the acute 0.5% group (n=11),. 197. light gray dots represent the chronic 0.5% group (n=11), and black dots represent the. 198. withdrawal group (n=14). For statistical differences see Results.. 199 200. Figure 4 shows locomotor parameters evaluated on the third day (second training).. 201. The Kruskal-Wallis test showed no significant differences in average speed (H=2.24;. 202. d.f.=3; p=0.52; Fig. 4a) and distance traveled between groups (H=1.41; d.f.=3; p=0.70;. 203. Fig. 4b). However, the parameters related to anxiety-like behavior differed between. 204. groups: the chronic 0.5% alcohol and withdrawal groups showed reduced freezing. 205. behavior compared to the control and acute 0.5% groups (Kruskal-Wallis, H=14.28; d.f.=3; 28.

(30) 206. p=0.003; Fig. 4c), and the acute 0.5% group showed the shortest distance from the bottom. 207. compared to the other groups (Kruskal-Wallis, H=20.79; d.f.=3; p<0.001; Fig. 4d).. 208. 209. Fig.4. Zebrafish locomotor parameters on the probe day of the inhibitory avoidance. 210. learning test. After 1 min acclimation in the white side, the lift-up door was raised 2 cm. 211. from the bottom and allowed fish to move between compartments. On day 3 (after fish. 212. have experienced 2 electroshocks on the days before), the fish behavior was recorded and. 213. analyzed in term of (a) average swimming speed, (b) total distance travelled, (c) freezing. 214. and (d) distance from the bottom of the tank. Groups tested were control (n=12), acute. 215. 0.5% alcohol exposure (n=11), chronic 0.5% alcohol exposure (n=11), and withdrawal. 216. from alcohol exposure (n=14). Different lower case letters indicate statistical differences. 217. between groups (Kruskal-Wallis, p<0.05).. 218 219 220 221. Discussion In this study, we demonstrated the zebrafish’s aversive learning ability, and that acute alcohol exposure did not affect zebrafish performance. However, chronic exposure to 29.

(31) 222. alcohol and alcohol withdrawal impair the fish’s proper response to the aversive stimulus.. 223. The inhibitory avoidance paradigm used here to assess learning and memory was shown to. 224. be a useful tool. In fact, it can be used to understand how drugs interferes on perception. 225. and avoidance of an aversive stimulus such as electroshock.. 226. Our data confirm previous studies in which zebrafish (in the absence of drugs). 227. showed inhibitory avoidance learning based on electroshock23,25,26,27,28,29,30 and other. 228. avoidance learning tasks.20,31,32,33 We also show that acute 0.5% alcohol-exposed animals. 229. were able to properly learn the task and remember it in the following tests, while the. 230. animals chronically treated with alcohol and those in alcohol withdrawal showed. 231. perception/learning impairment.. 232. Although anxiolytic and anxiogenic effects of alcohol have been investigated. 233. through different paradigms in zebrafish, few studies have related. 234. punishment/alcohol/learning. Horner et al.34 conducted one of the first avoidance learning. 235. studies using electroshock in fish (Goldfish, Carassius auratus), and later the avoidance. 236. task protocol was improved to better match those used in rodents, making studies in fish. 237. more translational.23,29,30,35 However, the present investigation is the first to test the effects. 238. of alcohol on inhibitory avoidance learning in zebrafish. In this protocol, zebrafish have to. 239. learn an association between specific environmental features with an aversive experience. 240. and then avoid entering the aversive environment when reexposed to the same context.27. 241. The aversive experience (in this case electroshock) is usually paired with a naturally. 242. preferred environment, which in our study was the dark background.36 Thus, the fish had to. 243. resist swimming from the light environment to the dark compartment in order to avoid. 244. being punished by an electric shock.. 245 246. In our study, fish in the control and acute alcohol exposure groups rapidly entered the black side of the shuttle box on the first day, indicating their natural preference. 30.

(32) 247. However, after the first shock experience, individuals avoided the dark chamber and/or. 248. took longer to enter it. This behavior indicates that fish learned the association between. 249. entering the black chamber and receiving the punishment, and remembered to avoid it.. 250. Alcohol is known to have a significant impact on physiology and cognition,37,38. 251. and is commonly referred to as a perception-attenuating drug. In zebrafish, as in humans,. 252. alcohol effects depend on the dose and exposure regime, presenting an inverted U curve as. 253. dose-response.7,15,39,40 Our data show that the group receiving an acute 0.5% dose of. 254. alcohol exhibited the same response as the control group, while anxiety-like behavior. 255. seems to increase in this group (i.e. high freezing and short distance from the bottom; Fig.. 256. 4c and 3d). These results corroborate those reported by Gulick & Gould 41, in which acute. 257. alcohol exposure caused anxiogenic effects on a fear conditioning paradigm in rats. It is. 258. suggested that a low alcohol dose induces an excitatory state, marked by enhanced. 259. functioning of inhibitory neurotransmitters and neuromodulators (such as GABA and. 260. glycine).42,43 This lead to increased sensitivity and a state of euphoria,44 which may have. 261. boosted zebrafish perception of the environment (thus moving quickly to the black. 262. chamber on the first day) and the aversive stimulus (taking longer to enter the black. 263. chamber and increasing anxiety-like behavior on days 2 and 3). In other studies, low. 264. alcohol doses were related to increased swimming activity,15,20 a behavior not observed. 265. here. The ingestion of low dose of alcohol was previously suggested to improve learning in. 266. fish6,45,46 and mammals.47,48,49 Besides improving the perception / attention when exposed. 267. to an aversive stimulus45,48. In our study, the acute low alcohol treatment induced a. 268. behavioral performance comparable to that exhibited by controls (0.0% alcohol).. 269. Moreover, the low dose used did not impair learning and allowed zebrafish to associate. 270. aversive stimulus with the black chamber, remembering it afterwards. While potentially. 271. threatening stimuli are expected to trigger fast adaptive responses50 and should be 31.

(33) 272. prioritized by the perception and attention systems in the face of positive or neutral stimuli,. 273. our results suggest that the mechanisms underlying avoidance learning were not affected. 274. by a low acute dose of alcohol, since they are extremely relevant to an animal’s survival.. 275. It is well documented that alcohol affects a number of zebrafish behaviors;7,15. 276. increased plasma alcohol concentration disturbs social behavior,22 shoaling,51 and. 277. aggressiveness,15 in addition to changing risk/danger perception.15,22 As a significant. 278. depressant of the CNS,52 alcohol inhibitory action decreases reflexive behavior,53,54. 279. compromising cognitive levels dependent on conditioned response. In contrast to what was. 280. observed for the acute alcohol treatment, chronic alcohol exposure and alcohol withdrawal. 281. disrupted the zebrafish response to electroshock, as observed for latency to enter the black. 282. chamber and the anxiety-like behavior expected after the shock (Figs. 3 and 4).. 283. Some common indicators of anxiety in zebrafish are freezing behavior,. 284. characterized by a lack of body movements except for the eyes and opercula, and distance. 285. from the bottom (geotaxis), a preference for the bottom in response to threat.55. 286. Examination of these behaviors (freezing and distance from the bottom) revealed that. 287. control and acute alcohol exposure lead to longer periods of freezing and geotaxis (Figure. 288. 4c), while chronic alcohol exposure and withdrawal showed low freezing and geotaxis. 289. behavior. Other studies have shown evidence of an alcohol dose-dependent effect on. 290. anxiety. For instance, Luca & Gerlai20 showed that 0.25% acute alcohol decreases freezing. 291. while 0.75% acute alcohol increases such behavior; Santos et al.18 pointed out that acute. 292. withdrawal from 0.50% alcohol and 1.00% acute alcohol increases freezing, and Tran &. 293. Gerlai7 demonstrated time-course changes in geotaxis following alcohol treatment, in. 294. which 0.50% acute alcohol decreases geotaxis, 1.00% acute alcohol increases it and 0.50%. 295. chronic alcohol exposure attenuates the effects of 0.50% acute alcohol.. 32.

(34) 296. Behavioral changes caused by alcohol are the result of its action in the brain. As a. 297. molecule, ethanol has nonspecific targets and can reach different receptors in different. 298. areas of the brain,56,57,58 affecting behavior and cognition at different levels depending on. 299. the amount.59 The higher the dose and longer the exposure regime, the larger the negative. 300. impact.60 Exposure to the chronic ethanol treatment can provoke deleterious effects in the. 301. brain, such as loss of dendritic spines,61 neuronal death56,62,63 and long-lasting memory and. 302. emotional deficits.64. 303. Indeed, worse than not learning to avoid punishment, zebrafish exposed to the. 304. chronic alcohol treatment and under alcohol withdrawal did not respond to the light-dark. 305. paradigm or the electric shock received in the dark chamber. Studies have shown that. 306. zebrafish have an innate tendency to seek darker environments and avoid lighted spaces,. 307. which are associated with anxiety.19,36,29 Thus, we expected that fish would rapidly cross to. 308. the black side of the tank on the first day in the light-dark shuttle box. Acute alcohol did. 309. not affect this response in zebrafish; however, the chronic and withdrawal groups took. 310. much longer to enter the dark chamber, suggesting that prolonged alcohol treatments may. 311. have prevented perception of what could be naturally aversive. It is generally agreed that. 312. long term use of alcohol leads to severe learning and memory deficits.65,66,67 This was. 313. corroborated in our study, whereby chronic alcohol exposure eliminated the electroshock. 314. association with a specific place (dark chamber), as can be seen by the decreased anxiety-. 315. like behavior and unchanged time to enter the electroshock compartment.. 316. In this respect, withdrawal from alcohol is always associated with significant. 317. negative changes in behavior and cognition.68,69 Other studies in zebrafish have reported. 318. discontinued alcohol use in association with decreased exploration,18 increased anxiety-like. 319. behavior,5 and increased plasma cortisol.68 In mammals, withdrawal induces other. 320. symptoms such as increased heart rate, profuse sweating, frequent nausea and headache, 33.

(35) 321. increased agitation and tremors, and delusions.70,71,72 In fish, our results suggest that after. 322. 22 intermittent days of alcohol exposure, withdrawal from alcohol induced inability to. 323. perceive the environment and form memory of a harmful experience. These data. 324. corroborate the behavioral parameters in zebrafish after discontinued alcohol exposure. 325. shown by Cachat et al.68, Tran & Gerlai7 and Mathur & Guo.5. 326. Although we did not investigate molecular mechanisms underlying learning and. 327. anxiety in zebrafish, the behavioral data presented here corroborate other studies (cited. 328. above). The present study raises a number of questions for investigation in future studies,. 329. as follows: when does the chronic deleterious effect of alcohol take place?; to what extent. 330. does alcohol influence learning?; which mechanisms are activated or affected in the CNS. 331. subjected to different stressors? Even though several questions have been raised, our study. 332. provides evidence that zebrafish exhibit anxiety-like behavior dependent on the alcohol. 333. dose and exposure regime, which resembles the effects of alcohol in humans. Due to the. 334. applicability of zebrafish to behavioral and genetic screening, we reinforce that studies. 335. focusing on genes and/or molecules that act in the zebrafish response to acute and chronic. 336. alcohol exposure may contribute to a better understanding of the effects of alcohol on. 337. anxiety states in humans.. 338 339 340. Acknowledgements The authors would like to thank Fúlvio Aurélio de Morais Freire for statistical. 341. support, Lisa Burger Garcia and Michael Germain for English revision, and Luccas. 342. Amorim and Maria Mixssaeyde Silva de Moura for technical assistance. The authors. 343. declare no competing interests.. 344 345 34.

(36) 346 347 348 349 350 351. References 1. Room R, Babor T, Rehm, J. Alcohol and public health. The lancet 2005;365:519530. 2. Holder HD, Griffith E. Alcohol and public policy: evidence and issues. Oxford University Press, 1995. 3. Peng J, Wagle M, Mueller T, Mathur P, Lockwood BL, Bretaud S, et al. Ethanol-. 352. modulated camouflage response screen in zebrafish uncovers a novel role for camp. 353. and extracellular signal-regulated kinase signaling in behavioral sensitivity to. 354. ethanol. Journal of Neuroscience 2009;29:8408-8418.. 355. 4. Mottram D. R. Drugs in sport, Routledge, 2005.. 356. 5. Mathur P, Guo S. Differences of acute versus chronic ethanol exposure on anxiety-. 357. like behavioral responses in zebrafish. Behavioural brain research 2011;219:234-. 358. 239.. 359. 6. Luchiari AC, Chacon DM, Oliveira JJ. Dose-dependent effects of alcohol on. 360. seeking behavior and memory in the fish Betta splendens. Psychology. 361. Neuroscience 2015;8:143.. 362. 7. Tran S, Gerlai R. Time-course of behavioural changes induced by ethanol in. 363. zebrafish (Danio rerio). Behavioural brain research 2013;252:204-213.. 364. 8. Tran S, Chatterjee D, Gerlai R. An integrative analysis of ethanol tolerance and. 365. withdrawal in zebrafish (Danio rerio). Behavioural brain research 2015;276:161-. 366. 170.. 367 368 369 370. 9. Finn DA, Crabbe JC. Exploring alcohol withdrawal syndrome. Alcohol Research and Health 1997;21:149. 10. Baggio S, Mussulini BH, de Oliveira DL, Zenki KC, da Silva ES, Rico EP. Embryonic alcohol exposure promotes long-term effects on cerebral glutamate 35.

(37) 371 372 373 374. transport of adult zebrafish. Neuroscience Letters 2017;636:265-269. 11. Becker HC. Animal models of alcohol withdrawal. Alcohol Research and Health 2000;24:105-114. 12. Souza BR, Romano-Silva MA, Tropepe V. Dopamine D2 receptor activity. 375. modulates Akt signaling and alters GABAergic neuron development and motor. 376. behavior in zebrafish larvae. Journal of Neuroscience 2011;31:5512-5525.. 377. 13. Barbazuk WB, Korf I, Kadavi C, Heyen J, Tate S, Wun E, et al. The syntenic. 378. relationship of the zebrafish and human genomes. Genome research 2000;10:1351-. 379. 1358.. 380 381. 14. Santoriello C, Zon, LI. Hooked! Modeling human disease in zebrafish. The Journal of clinical investigation 2012;122:2337-2343.. 382. 15. Gerlai R, Lahav M, Guo S, Rosenthal A. Drinks like a fish: zebra fish (Danio rerio). 383. as a behavior genetic model to study alcohol effects. Pharmacology biochemistry. 384. and behavior 2000;67:773-782.. 385. 16. Roberts AC, Bill BTR, Glanzman DL. Learning and memory in zebrafish larvae,. 386. The world according to zebrafish: How neural circuits generate behaviour, 2014.. 387 388 389. 17. Luchiari AC, Chacon DMM. Physical exercise improves learning in zebrafish, Danio rerio. Behavioural processes 2013;100:44-47. 18. Santos LC, Ruiz-Oliveira J, Oliveira JJ, Silva PF, Luchiari AC. Irish coffee: Effects. 390. of alcohol and caffeine on object discrimination in zebrafish. Pharmacology. 391. Biochemistry and Behavior 2016;143:34-43.. 392. 19. Blaser RE, Rosemberg DB. Measures of anxiety in zebrafish (Danio rerio):. 393. dissociation of black/white preference and novel tank test. PLoS One. 394. 2012;7:36931.. 395. 20. Luca, RM, Gerlai, R. Animated bird silhouette above the tank: acute alcohol 36.

(38) 396. diminishes fear responses in zebrafish. Behavioural brain research 2012;229:194-. 397. 201.. 398. 21. Egan RJ, Bergner CL, Hart PC, Cachat JM, Canavello PR, Elegante MF, et al.. 399. Understanding behavioral and physiological phenotypes of stress and anxiety in. 400. zebrafish. Behavioural brain research 2009;205:38-44.. 401. 22. Gerlai R, Chatterje D, Pereira T, Sawashima T, Krishnannair R. Acute and chronic. 402. alcohol dose: population differences in behavior and neurochemistry of zebrafish. 403. Genes. Brain and Behavior 2009;8:586-599.. 404. 23. Gorissen M, Manuel R, Pelgrim TN, Mes W, De Wolf MJ, Zethof J, et al.. 405. Differences in inhibitory avoidance, cortisol and brain gene expression in TL and. 406. AB zebrafish. Genes, Brain and Behavior 2015;14:428-438.. 407. 24. Pinheiro-da- Silva J, Silva PF, Nogueira MB, Luchiari AC. Sleep deprivation. 408. effects on object discrimination task in zebrafish (Danio rerio). Anim Cogn. 409. 2017;20:159-169.. 410. 25. Xu X, Scott-Scheiern T, Kempker L, Simons K, Active avoidance conditioning in. 411. zebrafish (Danio rerio). Neurobiology of learning and memory 2007;87:72-77.. 412. 26. Blank M, Guerim LD, Cordeiro RF, Vianna MR. A one-trial inhibitory avoidance. 413. task to zebrafish: rapid acquisition of an NMDA-dependent long-term memory.. 414. Neurobiology of learning and memory 2009;92:529-534.. 415. 27. Maximino C, de Brito TM, da Silva Batista AW, Herculano AM, Morato S,. 416. Gouveia A. Measuring anxiety in zebrafish: a critical review. Behavioural brain. 417. research 2010;214:157-171.. 418. 28. Ng MC, Hsu CP, Wu YJ, Wu SY, Yang YL, Lu KT. Effect of MK-801-induced. 419. impairment of inhibitory avoidance learning in zebrafish via inactivation of. 420. extracellular signal-regulated kinase (ERK) in telencephalon. Fish physiology and 37.

(39) 421. biochemistry2012;38:1099-1106.. 422. 29. Manuel R, Gorissen M, Piza Roca C, Zethof J, Vis HVD, Flik G, et al. Inhibitory. 423. avoidance learning in zebrafish (Danio rerio): effects of shock intensity and. 424. unraveling differences in task performance. Zebrafish 2014;11:341-352.. 425. 30. Manuel R, Gorissen M, Zethof J, Ebbesson LO, van de Vis H, Flik G, et al.. 426. Unpredictable chronic stress decreases inhibitory avoidance learning in Tuebingen. 427. long-fin zebrafish: stronger effects in the resting phase than in the active phase.. 428. Journal of Experimental Biology 2014;217:3919-3928.. 429. 31. Gerlai R, Lee V Blaser R. Effects of acute and chronic ethanol exposure on the. 430. behavior of adult zebrafish (Danio rerio). Pharmacology Biochemistry and. 431. Behavior 2006:85:752-761.. 432 433 434. 32. Pather S, Gerlai R. Shuttle box learning in zebrafish (Danio rerio). Behavioural brain research 2009;196:323-327. 33. Ruhl T, Zeymer M, von der Emde G. Cannabinoid modulation of zebrafish fear. 435. learning and its functional analysis investigated by c-Fos expression.Pharmacology. 436. Biochemistry and Behavior 2017;153:18-31.. 437. 34. Horner JL, Longo N, Bitterman ME. A shuttle box for fish and a control circuit of. 438. general applicability. The American journal of psychology 1961;74:114-120.. 439. 35. Manuel R, Gorissen M, Stokkermans M, Zethof J, Ebbesson LO, Vis HVD, et al.. 440. The effects of environmental enrichment and age-related differences on inhibitory. 441. avoidance in zebrafish (Danio rerio Hamilton). Zebrafish 2015;12:152-165.. 442. 36. Serra EL, Medalha CC, Mattioli R. Natural preference of zebrafish (Danio rerio). 443. for a dark environment. Brazilian Journal of Medical and Biological Research. 444. 1999;32:1551-1553.. 445. 37. Curtin JJ, Patrick CJ, Lang AR, Cacioppo JT, Birbaumer N. Alcohol affects 38.

(40) 446. emotion through cognition. Psychological Science 2001;12: 527-531.. 447. 38. WHO, Global status report on alcohol, 2004.. 448. 39. Bartholow BD, Pearson M, Sher KJ, Wieman LC, Fabiani M, Gratton G. Effects of. 449. alcohol consumption and alcohol susceptibility on cognition: a psychophysiological. 450. examination. Biological psychology 2003;64:167-190.. 451 452 453. 40. Salamé P. The effects of alcohol on learning as a function of drinking habits. Ergonomics 1991;34:1231-1241. 41. Gulick D, Gould TJ. Acute Ethanol Has Biphasic Effects on Short‐ and. 454. Long‐ Term Memory in Both Foreground and Background Contextual Fear. 455. Conditioning in C57BL/6 Mice. Alcohol Clin Exp Res 2007;31:1528-1537.. 456. 42. Celentano JJ, Gibbs TT, Farb DH. Ethanol potentiates GABA-and glycine-induced. 457. chloride currents in chick spinal cord neurons. Brain research 1988;455:377-380.. 458. 43. Wallner M, Hanchar HJ, Olsen RW. Low dose acute alcohol effects on GABA A. 459 460 461 462. receptor subtypes. Pharmacol Ther 2006;112:513-528. 44. Blaser RE, Chadwick L, McGinnis GC. Behavioral measures of anxiety in zebrafish (Danio rerio). Behavioural brain research 2010;208:56-62. 45. Scobie SR, Bliss DK. Ethyl alcohol: Relationships to memory for aversive learning. 463. in goldfish (Carassiusauratus). Journal of comparative and physiological. 464. psychology 1974;86:867.. 465. 46. Chacon DM, Luchiari AC. A dose for the wiser is enough: The alcohol benefits for. 466. associative learning in zebrafish, Progress in Neuro-Psychopharmacology and. 467. Biological Psychiatry 2014;53:109-115.. 468 469 470. 47. Alkana RL, Parker ES. Memory facilitation by post-training injection of ethanol. Psychopharmacology 1979;66:117-119. 48. Hernandez LL, Valentine JD. Mild ethanol intoxication may enhance Pavlovian 39.

(41) 471 472. conditioning. Drug Development Research 1990;20:155-167. 49. Vakili A, Tayebi K, Jafari MR, Zarrindast MR, Djahanguiri B. Effect of ethanol on. 473. morphine state-dependent learning in the mouse: involvement of GABAergic. 474. opioidergic and cholinergic systems. Alcohol and Alcoholism 2004;39:427-432.. 475. 50. Folyi T Liesefeld HR, Wentura D. Attentional enhancement for positive and. 476. negative tones at an early stage of auditory processing. Biological psychology. 477. 2016;114:23-32.. 478. 51. Buske C, Gerlai R. Early embryonic ethanol exposure impairs shoaling and the. 479. dopaminergic and serotoninergic systems in adult zebrafish. Neurotoxicology and. 480. teratology 2011;33:698-707.. 481 482 483. 52. Charness ME, Simon RP, Greenberg DA. Ethanol and the nervous system. New England Journal of Medicine 1989;321:442-454. 53. Campbell JA, Samartgis JR, Crowe SF. Impaired decision making on the Balloon. 484. Analogue Risk Task as a result of long-term alcohol use. Journal of clinical and. 485. experimental neuropsychology 2013;35:1071-1081.. 486 487 488. 54. Koike H, Sobue G. Alcoholic neuropathyCurrent opinion in neurology 2006;19:481-486. 55. Kalueff AV, Gebhardt M, Stewart AM, Cachat JM, Brimmer M, Chawla JS, et al.. 489. Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond. Zebrafish. 490. 2013;10:70-86.. 491. 56. Chandler LJ, Newsom H, Sumners C, Crews F. Chronic ethanol exposure. 492. potentiates NMDA excitotoxicity in cerebral cortical neurons. Journal of. 493. neurochemistry 1993;60:1578-1581.. 494 495. 57. Koob GF. A role for GABA mechanisms in the motivational effects of alcohol. Biochemical pharmacology 2004;68:1515-1525. 40.