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Universidade Federal do Rio Grande do Norte
Centro de Biociências
Programa de Pós-Graduação em Psicobiologia
Laboratório de Peixes Ornamentais
JAQUELINNE PINHEIRO-DA-SILVA
EFEITOS DA PRIVAÇÃO DE SONO EM TAREFAS COGNITIVAS
Natal – RN
2016
JAQUELINNE PINHEIRO-DA-SILVA
EFEITOS DA PRIVAÇÃO DE SONO EM TAREFAS COGNITIVAS
Natal – RN 2016
Dissertação apresentada ao Programa de Pós graduação em Psicobiologia, da Universidade Federal do Rio Grande do Norte para obtenção do título de Mestre em Psicobiologia. Área de concentração: Estudos do Comportamento Orientadora: Prof. Dra. Ana Carolina Luchiari
Catalogação da Publicação na Fonte. UFRN / Biblioteca Setorial do Centro de Biociências
Pinheiro-da-Silva, Jaquelinne.
Efeitos da privação do sono em tarefas cognitivas / Jaquelinne Pinheiro-da-Silva. – Natal, RN, 2016. 116 f.: il.
Orientadora: Profa. Dra. Ana Carolina Luchiari.
Dissertação (Mestrado) – Universidade Federal do Rio Grande do Norte. Centro de Biociências. Programa de Pós-Graduação em Psicobiologia. 1. Sono. – Dissertação. 2. Aprendizagem. – Dissertação. 3. Memória. – Dissertação. I. Luchiari, Ana Carolina. II. Universidade Federal do Rio Grande do Norte. III. Título.
RN/UF/BSE-CB CDU 159.963.2
Título: Efeitos da privação de sono em tarefas cognitivas
Autor: Jaquelinne Pinheiro-da-Silva
Data da defesa: 01 de março de 2016
Banca Examinadora:
Prof. Dra. Ana Carolina Luchiari Universidade Federal do Rio Grande do Norte, RN
Prof. Dr. Judney Cley Cavalcante Universidade Federal do Rio Grande do Norte, RN
Prof. Dra. Lia Rejane Müller Bevilaqua Instituto do Cérebro - UFRN, RN
“Aquele que é mestre na arte de viver faz pouca distinção entre o seu trabalho e o seu tempo livre, entre a sua mente e o seu corpo, entre a sua educação e a sua recreação, entre o seu amor e a sua religião. Distingue uma coisa da outra com dificuldade. Almeja, simplesmente, a excelência em qualquer coisa que faça, deixando aos demais a tarefa de decidir se está trabalhando ou se divertindo. Ele acredita que está sempre fazendo as duas coisas ao mesmo tempo”
Domenico de Masi
AGRADECIMENTOS
Aos meus pais, Ane e Natan, sem os quais nada disso seria possível. Por sempre respeitarem
minhas escolhas e pelo imenso amparo emocional e financeiro sempre presente, mesmo com
os quase 3000km de distância. Vocês são as principais inspirações da minha vontade de
crescer pessoal e profissionalmente.
À minha família que é meu principal equilíbrio psicológico, e mesmo toda espalhada por esse
mundo, sabe como me fazer sentir amada e querida.
À minha orientadora, prof. Ana Carolina Luchiari, por me receber tão bem no seu
laboratório, na sua casa e na sua família. Terá meu eterno agradecimento por toda dedicação,
paciência, orientação e, principalmente, o apoio mais que necessário desde antes do início do
mestrado.
Ao Vinícius, meu melhor amigo e companheiro de vida. Que está ao meu lado diariamente,
acompanhando (sempre com toda paciência possível) desde às pequenas conquistas e alegrias
até as noites mal dormidas e minhas crises. Eu não poderia ter parceria melhor.
Aos meus colegas de trabalho do Luchiari lab, pelas colaborações, trocas de ideias e
sugestões em cada reunião. Principalmente aos nossos estagiários, Adri, Lari, Helô, Rafa,
Elisa, Ian, Jessica, Vanessa e Mix pelas várias vezes que precisei de ajuda durante o percurso,
vocês foram fundamentais para a realização desse trabalho.
À Raíssa Nóbrega e Rômulo Almeida por me cederem espaço nos laboratórios de hormônios
e bioquímica, e pelo paciente auxílio com a homogeneização e análise das amostras.
Ao PPG Psicobiologia, por toda assistência institucional e burocrática e ao CNPq,
pelo financiamento deste projeto.
RESUMO
Aprendizagem e memória são processos importantes paras as espécies, pois permitem
o reconhecimento coespecífico, rotas e sítios de alimentação. Um dos comportamentos
conhecidos por facilitar à aprendizagem é o sono, fenômeno universal presente na maioria dos
vertebrados e altamente estudado sob vários aspectos. É sabido que a privação de sono altera
processos fisiológicos e comportamentais nos animais, no entanto, sua função no organismo
não é completamente compreendida. As hipóteses do papel do sono variam de conservação de
energia à consolidação de memória, com variadas funções durante a evolução dos animais. O
peixe paulistinha (Danio rerio) surgiu nos últimos anos como vertebrado modelo em genética
e biologia do desenvolvimento, e rapidamente se tornou popular em estudos do
comportamento, assim como aprendizagem e memória. Além de ser um animal de ritmo
circadiano diurno e possuir comportamento de sono bem caracterizado, o peixe paulistinha
ainda apresenta vantagens por seu tamanho pequeno e de baixo custo de manutenção, o que
estabelece essa espécie como modelo interessante para pesquisas sobre sono. No presente
estudo buscou-se analisar os efeitos da privação total ou parcial de sono sobre a aprendizagem,
e ainda os efeitos concomitantes com o uso de álcool e melatonina. Para isso, o projeto foi
dividido em 3 etapas, cada um com um tipo de condicionamento diferente: (1) Reconhecimento
de objetos, (2) Aprendizagem aversiva baseada em punição e (3) Aprendizagem apetitiva
baseada em reforço. Os resultados analisados mostraram que os peixes que foram parcialmente
privados de sono e os totalmente privados de sono + álcool conseguiram realizar as tarefas
igualmente aos grupos controle, no entanto, os peixes totalmente privados de sono e ainda os
totalmente privados + melatonina apresentaram memória e atenção prejudicadas durante os
testes. Por fim, nossos resultados sugerem que apenas uma noite de privação de sono é
suficiente para afetar o desempenho do peixe paulistinha em tarefas cognitivas. Ademais, a
exposição ao álcool na noite anterior ao teste parece suprimir os efeitos negativos da privação
de sono, enquanto a melatonina parece não ser eficiente para promover o estado de sono, ao
menos na metodologia aplicada aqui.
Palavras-chave: sono, aprendizagem, memória, álcool, paulistinha
ABSTRACT
Learning and memory are important mechanism for species, since its allows to
recognize conspecifics, routes and food place. Sleep is one of behaviors known by facilitate
learning, it is a widespread phenomenon, present in most of vertebrates lives and highly
investigated in many aspects. It is known that sleep deprivation modifies physiologic
behavioral processes in animals, however, sleep function in organism is still debatable.
Hypothesis range from energy conservation to memory consolidation, with different roles in
animal’s evolution. The zebrafish (Danio rerio) emerge in the last years as vertebrate model in
genetics and developmental biology and quickly become popular in behavioral studies, as
learning and memory. Despite the fact that zebrafish is a diurnal animal and have well
characterized sleep behavior, zebrafish fish still has advantages due to its small size and low
cost of maintenance, which establishes this species as interesting model for research on sleep.
In this study we aimed to analyze the effects of partial and total sleep deprivation on learning
acquisition, as well the concomitant administration of alcohol and melatonin. For this, the
research was divided in three phases, each one with a different kind of conditioning: (1) object
Recognition, (2) avoidance conditioning and (3) appetitive conditioning. The results showed
the fish partially sleep deprived and totally sleep deprived + ethanol could perform the tasks
just like the control group, however, fish totally sleep deprived and totally sleep deprived +
melatonin showed impairments in attention and memory during the tests. Our results suggest
that only one night of sleep deprivation is enough to harm the zebrafish performance in
cognitive tasks. In addition, ethanol exposure on the night previously the test seems to suppress
the negative effects of sleep deprivation, while the melatonin treatment seems not to be enough
to promote sleep state, at least on the protocol applied here.
Keywords: sleep, learning, memory, alcohol, zebrafish
SUMÁRIO
Introdução Geral 10
Objetivos 19
Capítulo 1: Sleep deprivation effects on objects discrimination task in zebrafish 20
1. Introdução 23
2. Material e Métodos 25
3. Resultados 29
4. Discussão 30
5. Referências 34
Figura 1 42
Figura 2 43
Figura 3 44
Legenda das Figuras 45
Material Suplementar
Capítulo 2: Sleep deprivation impairs learning: a matter of fact? 50
1. Introdução 52
2. Material e Métodos 53
3. Resultados 56
4. Discussão 57
5. Agradecimentos 61
6. Referências 62
Figura 1 69
Figura 2 69
Figura 3 70
Figura 4 70
Figura 5 71
Capítulo 3: Good night, sleep tight: the effects of sleep deprivation on spatial
associative learning in zebrafish
72
1. Introdução 74
2. Material e Métodos 76
3. Resultados 80
4. Discussão 83
5. Agradecimentos 89
6. Referências 89
Figura 1 96
Figura 2 97
Figura 3 98
Figura 4 99
Figura 5 100
Figura 6 101
Figura 7 102
Figura 8 103
Figura 9 104
Legenda das figuras 105
Tabela 1 106
Tabela 2 107
Conclusão Geral 108
Referências 109
10
INTRODUÇÃO GERAL
Aprendizagem é a modificação adaptativa do comportamento, relativamente
permanente, baseada na experiência do indivíduo (Alcock, 2013; Hilgard, 1948; Kolb &
Whishaw, 1998). A teoria da aprendizagem adaptativa considera cognição e
aprendizagem como o conjunto de habilidades que conferem, aliados à evolução de traços
particulares, vantajoso custo-benefício para espécies em condições ecológicas específicas
(Lefebvre, 1996).
Numa abordagem fisiológica, o processo de aprendizagem e memória é possível
devido às alterações na estrutura física do cérebro, através da criação ou alteração de
conexões sinápticas entre neurônios. Poucos minutos após uma experiência, atividades
neurais produzem mudanças no formato dos dendritos ou no número de conexões entre
os axônios de um neurônio e os dendritos de outro. Tais atividades neurais podem ser
induzidas por estimulação elétrica ou química do cérebro. A estimulação química pode
variar de hormônios a compostos neurotróficos e drogas psicoativas, enquanto que as
estimulações elétricas também podem ocasionar o fortalecimento de sinapses excitatórias,
causando alterações químicas em neurônios pré e pós-sinápticos (Kolb & Whishaw, 2001;
Mazur, 2002). Experiências diferentes levam a alterações em diferentes sistemas neurais
e, consequentemente, essas diferentes memórias podem ser armazenadas em partes
específicas do cérebro ou distribuídas de forma difusa entre várias partes (Thompson,
1991). A habilidade das sinapses fortalecerem ou enfraquecerem a comunicação entre
neurônios como resultado de experiências é chamada de plasticidade neuronal, e é o
mecanismo primordial para a adaptação e sobrevivência (Watson & Buzsáki, 2015).
Hipóteses sobre aprendizagem de tarefas específicas sugerem que a principal
vantagem da plasticidade é poder controlar e prever variações ambientais que são rápidas
demais para serem causadas por mudanças genéticas (Johnston, 1982). Assim, uma única
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exposição a determinado estímulo permite a modificação bioquímica do neurônio, em
consequência, na próxima exposição ao estímulo, a resposta neuronal já passa a ser
diferente. Neste sentido, o processo de aprendizagem oferece diversas vantagens para as
espécies uma vez que o indivíduo pode fazer ajustes adaptativos em seu comportamento
para obter benefícios do meio, como adquirir comida ou encontrar coespecífico, e ainda
antecipar ou alterar suas respostas comportamentais frente a presença de predadores ou
situações de risco (Kavaliers & Choleris, 2001; Sison & Gerlai, 2010).
A aprendizagem pode ocorrer por repetição ou por evento único. A habituação
ocorre quando o animal se acostuma a estímulos repetitivos inócuos e deixa de responder
a eles, a nível celular, a habituação de um reflexo ocorre devido à redução passageira da
eficácia de transmissão de informação pelos neurônios. Enquanto que na sensibilização
uma resposta é aumentada quando precedida de um estímulo muito forte, neste caso as
sinapses elevam a eficácia da transmissão, justamente o oposto da habituação. A
aprendizagem adquirida pode ser retida por curto ou longo período, dependendo do tempo
de estimulação.
A aprendizagem adquirida por meio de condicionamento pode ser operante ou
respondente e tem efeito de longo prazo, podendo persistir até por anos se não for
revertida por extinção, inibição latente ou novas associações (Moore, 2004). O
condicionamento clássico (ou respondente) ocorre quando o animal associa um sinal
anteriormente neutro (NS) (tornando-o o estímulo condicionado (CS)) com um estímulo
não condicionado (US) que o leva a apresentar a resposta não condicionada (UCR). O
estímulo não condicionado é, na maioria das vezes, como água ou comida, alteração
brusca de temperatura, dor, náusea, presença de coespecífico ou predador, etc. Após as
associações entre sinal e estímulo, a presença do estímulo neutro passa a desencadear
respostas semelhantes àquelas do estímulo não condicionado, de acordo com o repertório
típico de cada espécie (Moore, 1973).
12
O condicionamento pode ser dividido em duas categorias, considerando se o
estímulo não condicionado é apetitivo ou aversivo. O condicionamento apetitivo na
maioria das vezes é considerado prazeroso e o organismo tende a buscar o estímulo,
enquanto o condicionamento aversivo é considerado não prazeroso e o organismo
geralmente evita o estímulo, dependendo da operação estabelecedora (Powell, Honey, &
Symbaluk, 2016).
Ao passo que a aprendizagem é a alteração comportamental resultante de experiências, a
memória é a habilidade de recordar ou reconhecer experiências anteriores. A formação
de memória é característica importante em animais que vivem em ambiente estável, na
qual os elementos lembrados, lugares e rotas de experiência trazem vantagens para a
aptidão individual (Johnston, 1982). Além disso, os benefícios da função cognitiva
preditiva podem ser aumentados caso o indivíduo consiga armazenar a memória do que
será aprendido por longos períodos.
Dentre os comportamentos conhecidos por favorecer o processo de aprendizagem e
consolidação de memória, destaca-se o sono (Marshall & Born, 2007; Watson & Buzsáki,
2015). Este comportamento ocupa um terço de nossas vidas e, ainda que a comunidade
científica não tenha esclarecido plenamente o seu propósito ou finalidade, é senso comum
a necessidade básica vital que o estado comportamental do sono tem para a maioria dos
animais. Embora seja um fenômeno bastante difundido, presente em grande parte dos
vertebrados e também em alguns invertebrados, nem todos os animais apresentam o
comportamento de sono da mesma forma (Campbell & Tobler, 1984; Zimmerman,
Naidoo, Raizen, & Pack, 2008). A presença, a qualidade, a intensidade e as funções deste
estado em cada organismo são bem variadas.
O sono é geralmente relacionado com repouso, postura de descanso específica da
espécie, imobilidade reversível e redução da responsividade sensorial à estímulos
externos (Siegel, 2008). É um comportamento regulado tanto pelo relógio circadiano, que
13
realiza a sincronização, como por mecanismo homeostático, exemplificado pelo aumento
na intensidade e duração do sono após período de privação (Elbaz, Foulkes, Gothilf, &
Appelbaum, 2013). Contudo, nem todos os animais exibem os critérios de definição de
sono integralmente, alguns parecem poder reduzir o tempo de repouso ou até mesmo ficar
sem dormir por longos períodos, como acontece com aves migradoras e mamíferos
marinhos, sem apresentar riscos à saúde (Siegel, 2008).
Alguns estudos sugerem que mesmo um descanso tranquilo, embora consciente,
seria tão eficiente quanto o sono no sentido de recuperação das funções do corpo
(Kavanau, 1998). Além disso, esse tipo de “sono consciente” não apresentaria perigo ao
animal, uma vez que as respostas sensoriais não estariam desativadas para os estímulos
externos, como ocorre quando o animal dorme (Kavanau, 1998; Watson & Buzsáki,
2015). No entanto, do ponto de vista evolutivo, o sono inconsciente deve oferecer
algumas vantagens para o cérebro. Embora a função do sono permaneça desconhecida, as
hipóteses mais aceitas até o momento referem-se aos seus benefícios para as funções
cognitivas, tais como a consolidação de memória, restauração dos níveis de atividade
sináptica, redistribuição de energia e limpeza de metabólitos acumulados durante a vigília
(Herculano-Houzel, 2015; Marshall & Born, 2007; Schmidt, 2014; Stickgold & Walker,
2005; Tononi & Cirelli, 2006; Xie et al., 2013).
Tendo em vista que para a maioria das espécies o sono é um aspecto elementar
para as funções normais dos organismos, a privação deste estado causa grande impacto
em múltiplos processos fisiológicos, afetando parte essencial da vida e do bem-estar do
animal. Os efeitos da privação de sono têm sido relacionados com vários problemas de
saúde, incluindo diabetes, AVC e depressão, além de profundo impacto social e
econômico (Colten & Altevogt, 2006). Na sociedade moderna, o estado de vigília
prolongado é um fenômeno bastante difundido, seja ele alcançado por privação total de
14
sono aguda ou restrição de sono crônica. Embora o segundo tipo de privação seja a mais
comum no dia a dia, os efeitos da privação total são os mais abordados em pesquisas.
Trabalhos que utilizam neuroimagem, como ressonância magnética funcional
(fMRI) (Drummond et al., 2000) ou tomografia com emissão de pósitrons (PET) (Thomas
et al., 2000; Wu, Gillin, Buchsbaum, & Hershey, 1991) revelaram que a falta de sono
causa variações na ativação cerebral, e que tais alterações estão associadas a mudanças
no desempenho cognitivo. Pode-se considerar que as alterações comportamentais
observadas depois da privação de sono resultam de mudanças na funcionalidade de
neurotransmissores do Sistema Nervoso Central (SNC), como as catecolaminas,
acetilcolina, serotonina e GABA (Farooqui, Brock, & Zhou, 1996; Leibowitz, Lopes,
Andersen, & Kushida, 2006).
Complementando os estudos fisiológicos, análises comportamentais apontam que
a privação de sono causa danos para a atenção, aprendizagem e retenção de memórias
(Guzman-Marin et al., 2005; Leibowitz et al., 2006; Spiegel, 2004; Van Cauter, 2005;
Yu, Tucci, Kishi, & Zhdanova, 2006). A falta de sono por tempo prolongado, além de
prejudicar a aprendizagem, pode provocar estados de irrealidade semelhante ao sono,
perda do controle endócrino e autonômico, levando até mesmo à exaustão e morte (M. L.
Andersen et al., 2008).
Ainda que o primeiro trabalho com privação de sono seja datado de 1894, quando
de Manaceine mostrou que cachorros morrem após poucos dias de privação de sono, as
pesquisas nesta área são consideradas relativamente novas em comparação às outras áreas
da medicina. Além do mais, muitos foram os questionamentos sobre os resultados desses
estudos mais antigos, devido ao método usado para manter os animais continuamente
acordados terem possivelmente causado demasiado estresse e lesionado os indivíduos. O
primeiro estudo com privação de sono em humanos, através de análises psicológicas e
fisiológicas, já registrou prejuízos no tempo de resposta, déficit na habilidade motora e
15
capacidade de memorização (Patrick & Gilbert, 1896). Contudo, mesmo hoje em dia os
estudos com sono em humanos são muito difíceis de serem realizados, seja devido ao alto
custo, a dificuldade de se encontrar e aplicar testes em voluntários ou aprovações éticas.
Assim, modelos animais translacionais ainda configuram a melhor alternativa para a
pesquisa.
Muitos estudos tem sido realizados em animais envolvendo a privação de sono e
suas consequências (Kushida, 2004; Newman, Paletz, Rattenborg, Obermeyer, & Benca,
2008; Oleksenko, Mukhametov, Polyakova, Supin, & Kovalzon, 1992; Rechtschaffen,
Bergmann, & Bernard M, 2002; Sauer, Herrmann, & Kaiser, 2004). Embora
aprendizagem e memória tenham sido muito bem caracterizadas em espécies de roedores
tradicionais de laboratórios, algumas tarefas cognitivas têm sido empregadas também em
um novo e promissor modelo animal, o peixe paulistinha (Danio rerio).
O peixe paulistinha tem muitas vantagens na pesquisa comportamental visto que,
por ser um vertebrado, possui características organizacionais e funcionais do sistema
fisiológico semelhantes a aves e mamíferos (Miklósi & Andrew, 2006), além disso os
efeitos genéticos no cérebro e desenvolvimento de órgãos sensoriais são muito bem
compreendidos nesta espécie. O desenvolvimento de novos modelos comportamentais
visa, usualmente, focar em um modelo teórico específico que seja aplicável a qualquer
outro modelo animal estudado, levando em consideração os aspectos do repertório
comportamental específico da espécie.
Embora a literatura comportamental referente a estudos de aprendizagem ainda
seja limitada e dispersa para o peixe paulistinha, muitos trabalhos relevantes têm surgido
nas últimas décadas. Abordando, por exemplo, aprendizagem associativa e não-
associativa (Best et al., 2008; Chacon & Luchiari, 2014; Sison & Gerlai, 2011),
aprendizagem de esquiva ativa 16
16
(Xu, Scott-Scheiern, Kempker, & Simons, 2007), aprendizagem espacial baseada em
reforço (Williams, White, & Messer, 2002), aprendizagem de discriminação visual
(Colwill, Raymond, Ferreira, & Escudero, 2005), tarefa de reconhecimento de objetos
(Lucon-Xiccato & Dadda, 2014; Oliveira, Silveira, Chacon, & Luchiari, 2015) e
aprendizagem social (Engeszer, Ryan, & Parichy, 2004).
Por ser um vertebrado diurno, o peixe paulistinha oferece também muitas
vantagens para os estudos do relógio circadiano e a regulação do sono. Além de ser
favorável a experiências genéticas e comportamentais de alto rendimento, este modelo é
transparente em seus primeiros estágios de desenvolvimento, permitindo captura de
imagens neuronais in vivo (Elbaz et al., 2013). O estado comportamental do sono
caracterizado no peixe paulistinha por Zhdanova, Wang, Leclair, & Danilova (2001)
assemelha-se a outros grupos de vertebrados. Ademais, muitos pesquisadores observaram
que o peixe paulistinha contempla os principais agentes reguladores do sono presentes
em mamíferos, inclusive grupos de células colinérgicas, monoaminérgicas e
hipocretinérgicas, com a vantagem de ser um organismo de fisiologia muito mais simples.
Outra grande vantagem no uso do peixe paulistinha na pesquisa é que, devido a
sua capacidade de equilibrar as concentrações osmóticas em relação ao meio, a aplicação
de algumas drogas é muito mais simples e não-invasiva do que em outros animais. Drogas
solúveis em água podem ser misturadas diretamente no aquário em que o peixe se
encontra, e após cerca de 60 minutos, o animal atinge a mesma concentração do fármaco
no meio externo (Gerlai, Lahav, Guo, & Rosenthal, 2000). Esta característica favorece
também o uso de drogas relacionadas ao estado do sono, como álcool e melatonina.
Segundo Roehrs & Roth (2001), o álcool tem efeito sedativo e altera as
características do sono, o alerta durante a vigília, e ainda funções fisiológicas durante o
sono. A ingestão de álcool antes de dormir diminui a temperatura corporal, no entanto,
17
devido aos efeitos variados do álcool no organismo, é difícil especificar em que aspectos
o álcool prejudica o estado do sono.
Semelhante a outras drogas de abuso, o álcool interage com os sistema
dopaminérgico, serotonérgico e GABAérgicos em peixes, do mesmo modo que tem sido
mostrado em mamíferos (Kalueff, Stewart, & Gerlai, 2014; Langen, Dietze, & Fink,
2002). No peixe paulistinha, o álcool é a droga mais estudada, sendo descrito em vários
trabalhos por induzir mudanças comportamentais e prejudicar a aprendizagem e memória,
(Luchiari, Salajan, & Gerlai, 2015; Tran & Gerlai, 2013), bem como em humanos e outros
mamíferos (Beveridge, Smith, & Porrino, 2013; Obernier, White, Swartzwelder, &
Crews, 2002). Em pesquisas de desenvolvimento, a transparência deste animal permite
examinar alterações estruturais e anatômicas consequentes da exposição ao álcool no
desenvolvimento embrionário (Bilotta, Saszik, Givin, Hardesty, & Sutherland, 2002;
Carvan, Loucks, Weber, & Williams, 2004), além disso, o peixe paulistinha tem grande
similaridade psicofarmacológica com roedores e humanos (Collier, Khan, Caramillo,
Mohn, & Echevarria, 2014; Gerlai et al., 2000).
Assim como nos mamíferos, melatonina é o principal regulador neural do sistema
circadiano em zebrafish. A melatonina é um hormônio naturalmente produzido pela
glândula pineal, conhecido por promover o comportamento de sono. Em animais diurnos,
este hormônio está presente em baixas quantidades durante o dia e em alta quantidade
durante a noite e, mesmo sem pistas ambientais, a secreção de melatonina continua
apresentando ritmo circadiano (Scheer & Czeisler, 2005). A melatonina exógena tem sido
proposta por promover indiretamente o sono através de avanço de fase no relógio
circadiano (Arendt, 2003) ou por inibir características da vigília (Scheer & Czeisler,
2005). Nos últimos anos, a melatonina tem sido descrita por promover o sono em
vertebrados diurnos incluindo humanos (Brzezinski et al., 2005; Zhdanova, 2005),
primatas não humanos (Zhdanova et al., 2002), gatos domésticos (Goldstein & Pavel,
18
1981), pássaros (Mintz, Phillips, & Berger, 1998) e peixe paulistinha (Zhdanova et al.,
2001). O comportamento do peixe paulistinha influenciado pela melatonina tem resposta
dose dependente (Wang et al., 2014), podendo ser observadas alterações após 20 minutos
da aplicação do hormônio (Zhdanova et al., 2001).
Embora nos últimos anos alguns estudos tenham utilizado o peixe paulistinha
como modelo para aprofundar conhecimentos sobre o estado comportamental do sono,
até o presente, não temos conhecimento de nenhuma pesquisa com este modelo que
associasse o comportamento de sono com o processo de aprendizagem. Baseado nisso, e
na importância do sono (como regulador de processos fisiológicos e comportamentais) e
da aprendizagem e memória (como elementos críticos que conferem vantagens
adaptativas para a sobrevivência dos indivíduos) este estudo teve como objetivo avaliar
os efeitos da privação de sono aguda (por uma ou poucas noites seguidas) na
aprendizagem e memória do peixe paulistinha, através de diferentes tipos de testes
cognitivos. Ademais, abordamos também como a combinação de álcool e melatonina
(drogas ativas sobre o comportamento de sono) atuam no desempenho comportamental
quando associadas a privação de sono.
Conhecer a estrutura básica envolvida no comportamento do sono pode nos ajudar
a, não apenas compreender melhor esse fenômeno, mas também propor ajustes no período
de sono-vigília com mínimos efeitos na saúde. Desta forma, o peixe paulistinha representa
um potencial modelo para exploração dos mecanismos mais básicos da regulação do sono.
19
Objetivo Geral
Avaliar os efeitos da privação de sono no desempenho de diferentes tarefas cognitivas,
usando o peixe paulistinha como modelo animal.
Objetivos específicos
Estabelecer modelos experimentais de condicionamentos para o peixe paulistinha;
Testar os efeitos da privação de sono na aprendizagem e memória através da tarefa
de reconhecimento de objetos (Capítulo 1), de aprendizagem aversiva baseada em
punição (Capítulo 2); aprendizagem apetitiva baseada em reforço (Capítulo 3);
Investigar os efeitos do álcool e da melatonina na condição de privação de sono,
em tarefas de condicionamento aversivo e apetitivo.
20
Capítulo 1
Sleep deprivation effects on objects discrimination task in zebrafish (Danio rerio)
Submetido: Animal Cognition (Qualis: A1, FI: 2,58 – 2014)
RESUMO
Estudos anteriores têm mostrado que o peixe paulistinha é capaz de discriminar objetos
de diferentes cores e formas. À vista disso, utilizamos a tarefa de reconhecimento de
objetos para avaliar os efeitos da privação de sono na memória em peixe paulistinha
(Danio rerio). Quatro tratamentos foram testados: (1) controle, (2) privação parcial de
sono, (3) privação total de sono por pulsos de luz e (4) privação de sono por extensão do
período de luz. Os resultados obtidos mostraram que o grupo controle e o grupo
parcialmente privado de sono, exploraram mais o objeto novo que o objeto já conhecido,
indicando habilidade de discriminação. Contrariamente, ambos os grupos totalmente
privados de sono exploraram os dois objetos de forma similar, independentemente de sua
novidade. Essas respostas sugerem que apenas uma noite de privação de sono é suficiente
para afetar a discriminação de objetos em peixe paulistinha, indicando impactos negativos
em processos cognitivos.
21
Sleep deprivation effects on objects discrimination task in zebrafish (Danio rerio) 1
2
Jaquelinne Pinheiro-da-Silva, Priscila Fernandes Silva, Marcelo Borges Nogueira, Ana 3
Carolina Luchiari* 4
5
Departamento de Fisiologia, Centro de Biociências, Universidade Federal do Rio Grande 6
do Norte, Natal, RN, Brazil. 7
*Corresponding Author: Departamento de Fisiologia, Centro de Biociências, 8
Universidade Federal do Rio Grande do Norte, PO BOX 1511, 59078-970 Natal, Rio 9
Grande do Norte, Brazil. Phone: +55 84 32153409, Fax: +55 84 32119206, E-mail: 10
12
Abstract 13
Zebrafish is an ideal vertebrate model for neurobehavioral studies with translational 14
relevance to humans. The widespread phenomenon of sleep has been studied in many 15
aspects, but we still do not understand how and why sleep deprivation alters behavioral 16
and physiological processes. There are hypotheses suggesting its role in memory 17
consolidation. In this sense, the aim of this study was to analyze the effects of sleep 18
deprivation on memory in zebrafish (Danio rerio) using an objects discrimination 19
paradigm. Four treatments were tested: Control, Partial Sleep Deprivation, Total Sleep 20
Deprivation by light pulses and Total Sleep Deprivation by extended light. The control 21
group explored longer the new object than the known object, indicating clear 22
discrimination. The partial sleep deprived group explored the new object more than the 23
22
other object in the discrimination phase, which suggests some discriminative 24
performance. On the contrary, both total sleep deprivation groups equally explored all 25
objects, regardless its novelty. It seems that only a single night of sleep deprivation is 26
enough to affect discriminative response in zebrafish, indicating its negative impact for 27
cognitive processes. We suggest that this study could be a useful screening tool for 28
cognitive dysfunction and better understanding of sleep-wake cycles on cognition. 29
30
Key words: sleep; fish; perception; memory; discrimination. 31
32
33
Acknowledgments 34
The authors would like to thank Mr. and Mrs. Haghverdian for english review and Ms. 35
Nascimento for technical assistance. The authors declare no competing interests. 36
37
23
Introduction 38
Sleep is a naturally recurring condition characterized by rest, altered state of conscience, 39
and suspension of sensory, perceptual and motor voluntary activities (Schmidt 2014). 40
While it is a universal behavioral and physiological phenomenon present in the majority 41
of vertebrates, sleep state does not present the same characteristics in all animals (Lyamin 42
et al. 2007). The presence, quality, intensity and functions of sleep vary between species 43
and across the lifespan (Siegel 2008). However, it is unknown why exactly animals sleep. 44
Hypotheses range from energy allocation and conservation to synapses remodeling and 45
memory consolidation, with a myriad of possible functions throughout animal’s evolution 46
(Siegel 2005; Tononi and Cirelli 2006; Schmidt 2014; Herculano-Houzel 2015). 47
Regarding the purpose of sleeping, some studies suggest that the REM (rapid eye 48
movements) stage of sleep favors learning in some way, acting on important information 49
consolidation and irrelevant information elimination to avoid unnecessary overhead (Poe 50
et al. 2000; Louie and Wilson 2001; Stickgold and Walker 2005; Stickgold 2005). It is 51
also proposed that brain activity during the REM stage of sleep may facilitate 52
development and maintenance of memories by strengthening already formed circuits and 53
promoting new synapses connections (Roffwarg et al. 1966; Rasch et al. 2009; Blumberg 54
2010; Hobson and Steriade 2011; Schmidt 2014). Other studies also indicate that non-55
REM stage of sleep plays a large role in consolidating memories to brain cortical areas 56
(Euston et al. 2007; Prince and Abel 2013). Learning and memory are critical processes 57
that bring many advantages for the species, such as conspecific and mates recognition, 58
routes and places remembering and feeding time-place identification, which are 59
important treats for the animal’s fitness (Johnston 1982; Sison and Gerlai 2010). 60
24
While sleep is suggested to have imperative role in the animals’ life, sleep 61
deprivation (SD) has a significant impact in neurological and physiological processes; 62
many studies suggest that SD is adverse to neurogenesis, attention, learning and memory 63
retention (Spiegel 2004; Van Cauter 2005; Guzman-Marin et al. 2005; Leibowitz et al. 64
2006; Yu et al. 2006). Prolonged sleep deprivation causes attention and memory 65
problems, state of unreality similar to sleep, loss of autonomic and endocrine control, and 66
even can lead to exhaustion and death (Andersen et al. 2008). However, to which extent 67
SD could affect learning and memory processes still need to be identified for future 68
genetic or drug screens. 69
Recently, a novel memory paradigm based upon the principles of one-trial learning 70
was developed specifically for the zebrafish (Oliveira et al. 2015). In this paradigm, the 71
fish explored a dyad of objects without any reinforcement, and then it is tested for a new-72
object recognition. Objects discrimination protocols were previously tested in several 73
animals models, such as rats (Bevins and Besheer 2006), pigeons (Koban and Cook 74
2009), and fishes (Siebeck et al. 2009; Schluessel et al. 2012; Schluessel et al. 2014; 75
Lucon-Xiccato and Dadda 2014). The paradigm is simple and requires short time 76
experimentation, thus it is potentially high throughput. However, this paradigm has not 77
been used for behavioral brain research. In the current study, we investigate the effect of 78
sleep deprivation on the behavioral performance of zebrafish in the objects discrimination 79
paradigm. Thus, we tested zebrafish (Danio rerio), a valuable model for sleep research 80
(Zhdanova 2011), to attempt to address the following questions: Is one night of SD 81
enough to alter performance in a one-trial learning task? Does partial SD affect memory 82
in the same way as total SD? 83
25
84
Material and Methods 85
Stock conditions 86
Adult zebrafish (Danio rerio, ± 3 month of age) obtained from a local fish farm were 87
transferred to a storage system (50 L tanks) at the Ornamental Fish Vivarium, 88
Department of Physiology – UFRN. Each four 50 L-tanks formed a recirculating system 89
with multi-stage filtration including a mechanical filter, a biological filter, an activated 90
carbon filter, and a UV light sterilizing unit. The animals were kept in the tanks (one 91
fish/L), with aerated and filtered water, at a temperature of ± 26.5 °C, and pH (7.1) and 92
oxygen (5-10 mg/L) measured regularly. Photoperiod was set on 12:12 light:dark cycle, 93
with zeitgeber time (ZT) 0 corresponding to lights-on at 7am, and light intensity during 94
the light phase set at 250 lx. Feeding frequency was twice a day, with brine shrimp and 95
flake food diet (60% protein and 15% fat). The Ethical Committee for Animal Use of 96
Federal University of Rio Grande do Norte gave permission for all animal procedures 97
(CEUA 022/2012). 98
99
Experimental Treatments 100
In order to compare the effects of sleep deprivation on the performance of zebrafish in a 101
memory test, 44 fish were divided into four distinct light:dark conditions. 102
Sleep deprivation was achieved by (1) exposing fish to brief light pulses during 103
dark phase or (2) extending the light phase of the cycle. According to Yokogawa et al. 104
(2007), light has powerful suppressive effect on sleep in zebrafish, with no evidence for 105
sleep rebound. Sigurgeirsson et al. (2013) compared extended period of light and 106
26
electroshock as promoters of sleep deprivation and confirmed that both light and shock 107
are sleep and wakefulness modulators, but light-induced deprivation causes less deviation 108
from normal sleep–wake bouts. Moreover, circadian rhythms are maintained under 109
constant light by regular feeding at lights-on and lights-off (Sigurgeirsson et al. 2013) and 110
it is unlikely that only one night deprivation with lights-on could disrupt the circadian 111
clock (Yokogawa et al. 2007). Therefore, we used light instead of the electroshock 112
protocol to cause one night only deprivation in zebrafish and tested its effects on 113
memory. 114
We applied the following treatments: Control Group (12L:12D; n=11), Partial sleep 115
deprivation (18L:06D; n=11), Total sleep deprivation by light pulses (18L:06D+pulses; 116
n=11) and Total sleep deprivation by extended light (24L:00D; n=11). For pulses of light 117
deprivation, 1 minute of light pulse were administered every 5 min (thus, 4 min light + 1 118
min dark), during the whole 6 h-period of the dark phase, preventing the fish from more 119
than 1 min resting in dark. The above light:dark conditions were imposed only during the 120
night after memorization phase of the task (see below). 121
122
Objects Discrimination Task 123
The task was performed from ZT0-ZT6. For the memory test, we used a one-trial objects 124
discrimination procedure in which fish were not allowed to learn a pattern (more than one 125
trial for association). This procedure was adapted from Siebeck et al. (2009); Schluessel 126
et al. (2014); Lucon-Xiccato and Dadda (2014) and already tested and validated for 127
zebrafish by Oliveira et al. (2015). Thus, the objects discrimination test took place in 128
three phases: (1) acclimation in tank, (2) memorization phase and (3) discrimination 129
27
phase. During the period between memorization phase (2) and discrimination phase (3), 130
we exposed fish to the light:dark conditions described above. All phases occurred in 15 L 131
tank (40x25x20 cm) with all walls covered in white to avoid external interferences. The 132
objects used were plastic cubes (4x4x4 cm) and, to avoid color preferences, the color of 133
the objects (yellow, green, pink, orange, blue and purple) were totally randomized 134
between animals and treatments. 135
The acclimation phase (1) lasted five days. Fish were allowed to explore the test 136
tank for 15 min per day, without objects, to acclimatize to the new arena and reduce 137
novelty stress. To reduce isolation stress, since the zebrafish is a highly social animal, 11 138
fish explored the test tank together on the first day, half of the group on second day, and 139
so forth, so that on the 5th day each fish explored the tank alone for 15 min. After the 15-140
min period in test tank, fish was transferred to their home tank. 141
Memorization Phase (2) occurred on the 6th day. Two 3D objects (named A and B) 142
with same colors, size and shape, were introduced in the tank, each one positioned next to 143
each smaller wall and around 30 cm away from each other. Fish were individually 144
allowed to explore the tank with the two objects for 15 min. Behavior was recorded from 145
above using a handy cam (Sony Digital Video Camera Recorder; DCR-SX45) (Fig. 1). 146
After that, fish returned to its home tank. In both phases, we considered that animal’s 147
permanence in a 3 cm area around the objects, characterizes exploration behavior 148
(Lucon-Xiccato and Dadda 2014). 149
On the night following the memorization phase, each group was exposed to one of 150
the light:dark conditions: 12L:12D (control group), 18L:06D (partial sleep deprivation), 151
18L:06D+pulses (light pulses deprivation) and 24L:00D (extended light deprivation). 152
28
On the next day, the Discrimination Phase (3) took place. For this, object B of 153
memorization phase was replaced by a unfamiliar object (named object C), with same 154
size and shape but different color (Fig. 1). Fish were able to explore the objects in the 155
tank for 15 min and behavior was recorded. 156
157
Behavioral Analysis 158
Video frames from each trial were analyzed using a new custom-made multi-target 159
tracking software (named ZebTrack/UFRN) developed in MATLAB (R2014a; 160
MathWorks, Natick, MA). This software was designed by our laboratory as an alternative 161
to other costly existing tracking system, and is able to approach and quantify several 162
swimming path patterns, including speed, distance traveled, and time spent in specific 163
areas of the tank, being more appropriate than a manual recording method. Details of the 164
tracking software are available in the Online Resource. 165
166
Statistical Analysis 167
The parameters analyzed were time spent around each object, average and maximum 168
swimming speed and total distance traveled. The time fish spent around the objects (up to 169
3 cm far away from each side of the objects) was used to estimate exploration (Lucon-170
Xiccato and Dadda 2014), and we compared the objects exploration time on 171
memorization and discrimination phases, and also between the two phases. The time 172
spent close to the objects at memorization and discrimination phase were statistically 173
compared using Student t test. Total distance traveled, Maximum and Average Speed 174
29
were analyzed by one-way ANOVA followed by post-hoc comparisons using Student-175
Newman-Keuls (SNK). For all comparisons, the significance level was set to p<0.05. 176
177
Results 178
During the objects memorization phase (6th day) neither groups showed statistical 179
differences between objects A and B exploration time (Student t test: control group: 180
t=0.43 p=0.67; partial SD group: t=0.29 p=0.78; total SD with light pulses: t=0.75 181
p=0.47; total SD with extended light: t=-1.92 p=0.08) (Fig. 2). 182
The control, as well the partial SD and total SD with extended light groups showed 183
similar exploration of the object A between the memorization and discrimination phases 184
(6th vs. 7th days; Student t test: control group: t=0.60 p=0.56; partial SD group: t=0.59 185
p=0.56; total SD with extended light: t=-1.79 p=0.10). However, there were differences 186
in exploration time between object A on the 6th and 7th days for the totally deprived group 187
(Student t test: t=-2.94 p=0.01), and this group spent significant more time exploring 188
object A in the memorization phase (6th day). 189
For the control group, there were significant differences in exploration between 190
objects A and C in the discrimination phase (Student t test: t=-3.75 p=0.005) and also 191
between object B on the memorization and C in the discrimination phase (Student t test: 192
t=-2.22 p=0.05), indicating higher exploration of object C (Fig. 2a). 193
The partial SD group showed similar exploration of object B on the 6th day and C 194
on the 7th day (Student t test: t=-1.63 p=0.13). However, fish showed higher exploration 195
of object C than object A on the 7th day (Student t test: t=-2.26 p=0.04) (Fig. 2b). 196
For the total SD with light pulses (18L:6D+pulses) and total SD with extended light 197
30
(24L:00D) groups, there were no differences between object B exploration in the 198
memorization phase and object C in the discrimination phase (Student t test: SD with 199
light pulses: t=1.15 p=0.28; SD with extended light: t=-1.26 p=0.23). Fish also showed 200
similar exploration of objects A and C in the discrimination phase (Student t test: SD 201
with light pulses: t=-0.09 p=0.93; SD with extended light: t=-1.23 p=0.24) (Fig. 2c and 202
2d). 203
The maximum swimming speed was similar among the four groups in the 204
memorization phase (ANOVA, F=0.44 p=0.72), but fish from the partial and total sleep 205
deprived groups showed higher maximum speed than the control group in the 206
discrimination phase (ANOVA, F=4.73 p=0.008). The comparison between the phases 207
(6th vs. 7th days) for each group showed that the control group and the total SD with 208
extended light had similar maximum speed between the two days (Student t test: Control: 209
t=0.39 p=0.69; SD with extended light: t=0.90 p=0.37), while the other groups increased 210
speed on the 7th day (Student t test: partial SD: t=-2.44 p=0.02; SD with light pulses: t=-211
3.42 p=0.003 t=-3.38 p=0.007) (Fig. 3a). 212
The total distance traveled did not differ among the groups in the memorization 213
phase (ANOVA, F=1.46 p=0.24) or in the discrimination phase (ANOVA, F=1.44 214
p=0.26). Likewise, the comparison between the phases showed none of the groups 215
differed in terms of distance traveled on the 6th and 7th days (Student t test: Control: 216
t=0.13 p=0.90; partial SD: t=-0.33 p=0.74; SD with light pulses: t=-0.3 p=0.77; SD with 217
extended light: t=0.99 p=0.33) (Fig. 3b). 218
219
Discussion 220
31
In this study, we observed that total sleep deprivation prevents memory of a unique event 221
in zebrafish, while restricted sleep still allows memory formation. Adding to other studies 222
on the sleep role on memory and learning tasks, the current study approached a recently 223
validated protocol on objects discrimination (Oliveira et al. 2015) and showed its 224
relevance for sleep investigations. Our results confirm that zebrafish is able to 225
discriminate visual stimuli based on colors, corroborating other authors’ findings (Fetsko 226
2002; Colwill et al. 2005; Oliveira et al. 2015). Moreover, we show here that animals 227
partially sleep deprived were able to present some discrimination of the objects but only 228
one night of sleep deprivation is sufficient to abolish discriminative response. 229
Exploration is an important behavioral response to environmental changes and its 230
novelties (Kalueff and Zimbardo 2007), and the zebrafish’s behavioral repertoire includes 231
it. Our control group (12 h dark phase) explored both objects equally on the first day of 232
test, but on the following day when a new object was introduced, fish explored the 233
novelty (object C) more than the known object (object A) and also more than the former 234
object (object B on day 6) that was located on the same place (Fig. 2a). On the other 235
hand, even if we were to consider that the partially sleep deprived group explored the 236
new object more and was able to form a memory from the previous day, this group did 237
not show the same exploration pattern observed in the control group (Fig. 2a vs. 2b). It is 238
possible that six hours in the dark, after the SD period, allowed for sleep recovery and did 239
not affect the performance of the subjects in the discrimination task. A longer scheme of 240
restricted sleeping nights (for instance more than 3 days) may promote some cumulative 241
effect and produce higher losses on the cognitive function. 242
Even though SD promotes varied effects, which are widely distinct between 243
32
species, a common argument among authors is its huge impairment on memory 244
consolidation (McGaugh 2000; Andersen et al. 2008; Killgore 2010; Rasch and Born 245
2013; Watson and Buzsáki 2015). In this study, all groups had the opportunity to interact 246
with the objects on the first testing day, however only the total sleep deprived groups 247
were unable to discriminate the objects on the following day. These fish did not 248
recognize object A from the previous experience (day 6), and responded to both objects 249
as novelties, which indicates impairment on memory formation (Fig. 2c and 2d). Other 250
authors have also shown that sleep loss prevents the consolidation of acquired memory 251
(Leconte et al. 1974; Linden et al. 1975; Prince and Abel 2013). According to Marshall 252
and Born (2007), memory consolidation seems to occur mostly during periods of sleep or 253
inactivity. Additionally, zebrafish has directly light responsiveness cells (Weger et al. 254
2011) and the use of light as a method to avoid sleep probably reduced the melatonin 255
levels. In this concern, the discrimination impairment in our results are consistent with 256
studies that has shown abnormalities in melatonin rhythms in humans leads to changes in 257
cognition and behavior (Melke et al. 2008). 258
Although the partial sleep deprivation group did not present abnormalities in 259
memory in the discrimination phase of the present study, both the partial and total sleep 260
deprived animals showed higher maximum speed during the second testing day (Fig. 3). 261
It is well known that SD is a stressful condition that causes distinguished agitation and 262
anxiety behavior (Meerlo et al. 2002; Andersen et al. 2004; Mueller et al. 2008; 263
Mashoodh et al. 2008). The hyperactive behavior observed in our study supports this 264
idea. Furthermore, agitation and impairment in working memory and attention were 265
related to prolonged wakefulness (Harrison et al. 2000; Thomas et al. 2000), due to the 266
33
effect of vulnerability of the cognitive performance in brain after sleep deprivation 267
(Alhola and Polo-Kantola 2007). 268
Our test required memory of a single episode, which is much weaker than the 269
memory based on repetition, and thus, more vulnerable to the restless brain. In addition, 270
visual tasks would be especially susceptible to non-sleepers because iconic memory has a 271
short duration and limited capacity (Raidy and Scharff 2005). Other authors suggest that 272
animals exposed to a to-be-remembered stimulus hold it in memory and present faster 273
response on its reappearance when no stressful situations are imposed (Wilkie 1983; Kim 274
and Diamond 2002; Shettleworth and Westwood 2002). Therefore, the one trial-learning 275
paradigm used here appears to be an easy and effective test, which could be a useful 276
screening tool for cognitive dysfunction and better understanding of sleep deprivation 277
effects. 278
Despite the fact that zebrafish present resembling cognitive performance to 279
mammals and allows translational interpretation, our study still needs others approaches 280
in order to cover some limitations. For instance, in future studies one should investigate 281
chronic sleep deprivation scheme in order to understand how the cumulative effects of 282
restless affect behavior. Binks et al. (1999) report that the effects of sleep loss do not 283
become apparent until about 36-40 hours. While our results clearly show zebrafish 284
memory impairment upon only one night of deprivation, it would be important to test 285
cognition after longer periods. Also, the circadian aspect seems to be related to sleep 286
deprivation feedback (Prince and Abel 2013), which means that the time of day the task 287
is applied may interfere on memory formation. Thus, testing the animals closer to its 288
resting time may improve its performance. 289
34
Finally, our study has some practical implications. Zebrafish have become an 290
appropriate model to understanding the relationship between sleep deprivation and 291
memory consolidation with reliable translational relevance. On this basis, our cognitive 292
protocol can be later used in sleep deprivation studies focusing on techniques that show 293
changes in the brain (neurotransmitters, proteins, neuroplasticity), as sleep deprivation 294
trials with humans are too expensive, hard to be realized and limited (Alhola and Polo-295
Kantola 2007). While partial sleep deprivation did not cause immediate memory decline, 296
total sleep deprivation for a single night was shown to be highly damaging. Overall, 297
studies focusing on sleep and sleep deprivation are still needed and the zebrafish paves 298
the way for better understanding of sleep disorders and its cognitive relationship. 299
300
Online Resource 301
Supplementary data associated with this article can be found in the online version. 302
303
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42
Figure 1
20 cm
40 cm
Memorization Phase Discrimination Phase
Top-view camera
43
Figure 2
0
30
60
90
120
150
A B A C
Memorization phase Discrimination phase
Tim
e (
s)
(a) Control
**
*
A B A C
Memorization phase Discrimination phase
(b) Partial SD *
0
30
60
90
120
150
A B A C
Memorization phase Discrimination phase
Tim
e (
s)
(c) Total SD (light pulses)
A B A C
Memorization phase Discrimination phase
(d) Total SD (extended light)
44
Figure 3
0
20
40
60
80
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120
140
Control Partial SD Total SD(Pulses)
Total SD (Light)
Maxim
um
sp
eed
(cm
/s)
memorization phase
discrimination phase
a)
0
2
4
6
8
10
12
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16
Control Partial SD Total SD(Pulses)
Total SD (Light)
Av
era
ge
sp
eed
(cm
/s)
b)
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100
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250
300
Control Partial SD Total SD(Pulses)
Total SD(Light)
To
tal d
ista
nce t
rav
elled
(cm
)
c)
a
ab
a
b
a
b ab ab
*
45
Figure Captions
Fig. 1 Schematic view of the one-trial objects discrimination paradigm (40x25x20 cm3) at
memorization phase (with objects A and B in same colors) and discrimination phase (with
object C in different color). The tank was all covered in white self-adhesive plastic film. The
objects colors were randomized among all animals and treatments. For both phases, fish were
able to explore the objects for 15 min and behavior was registered with a top-view camera
Fig. 2 Sleep deprivation impairs objects discrimination in zebrafish. Zebrafish preference
for objects A x B, or A x C for the four groups: (a) Control, (b) Partial SD, (c) Total SD with
light pulses and (d) Total SD with extended light (n=11/each group). Bars means exploration
time in each object, in memorization and discrimination phases. Fish were observed for 15
min and analyzed using video-tracking software (ZebTrack).
(*) indicates statistical difference between fish exploration in each object (Student t Test, p <
0.05)
Fig. 3 Behavioral analysis during memorization phase and discrimination phase of a
one-trial learning paradigm. One-way ANOVA applied to compare (a) maximum speed
swimming + SD, (b) mean speed swimming + SD and total distance traveled by the fish +
SD, between the four groups: Control, Partial SD, Total SD with light pulses and Total SD
with extended light. Data corresponds to 15 min of behavioral observation during the test,
both in memorization phase as discrimination phase, analyzed using video-tracking software
(ZebTrack).
Different letters indicate statistical difference between fish maximum speed (One-way
ANOVA, p < 0.05)
Supplementary Material: Sleep
deprivation e�ects on objects
discrimination task in zebra�sh (Danio
rerio)
ZebTrack Software
The data used in this paper was generated by the software Zebtrack, shown inFigure 1, which was developed in Matlab with the help of its Image ProcessingToolbox. ZebTrack was created to track �sh, especially the Zebra�sh, howeverit can be used to track any other animal. It is capable of tracking one ormore animals (the user must specify how many animals it wants to track),producing data such as position and velocities. The produced data can bemanipulated, afterwards, in Matlab environment, or exported to Excel format.For now, ZebTrack does not work at real time. A video of the experiment mustbe previously recorded and then given as input to the software. The softwareallows the de�nition of polygonal areas such as a processing area (movementsoutside this area are ignored), excluding areas (movements inside this area areignored) and areas of interest (at the end of the experiment user will receivedata regarding each one of these areas, such as number of times each animalentered the area, time spent inside the area, etc... ).
The tracking algorithm is based on background subtraction (Piccardi 2004).We use the background subtraction method presented in (Wren et al. 1997),which computes temporal statistics of individual pixels . This method consistsof creating a background model, IBG, associated with its variance Iσ2 (eachpixel at position (x, y) of the background model will have a value, IBG(x, y),and an associated variance Iσ2(x, y)). After this, by comparing the backgroundmodel and the current frame we can determine foreground objects. ZebTrackcan work with colored or gray-scale images.
Often it is not possible to record the environment without any animals be-fore the experiment starts (as this would stress the animal), the backgroundis created using footage of the experiment itself. In order to do so, the userchooses an initial and �nal instant, and a sample period. By averaging the nsampled frames fs of the video in the chosen interval, it is possible to computethe background image along with its variance, as shown in Equations 1 and 2(Kaehler & Bradski 2015). If the image is colored, this is done for each color
1
2
Fig. 1: ZebTrack software. ZebTrack user interface showing starting and end-ing frames (top left), estimated background model (bottom left), andprocessing area - green - and areas of interest - blue - (right).
channel, generating a background and variance matrix for each channel. It isimportant that the user chooses a high number of frames n to average, as wellas a part of the video and sample period such that the animals do not stay inthe same position for several sampled frames, as this would cause the animal tobecame part of the background. By inspecting the background created the usercan check if it was successfully created.
IBG(x, y) =1
n
∑fs(x, y) (1)
Iσ2(x, y) =1
n
∑fs(x, y)2 − IBG(x, y) (2)
It is also possible to use a dynamic background, as presented in (Kolleret al. 1994). In this case, the background is updated using only the regions of thecurrent frame that are not classi�ed as foreground. We also tried the backgroundsubtraction method proposed by (Stau�er & Grimson 1999), however, besidesbeing computationally more costly, it did not improve the results.
The segmentation process consists of subtracting the background image fromthe current frame fk, and thresholding the resulting image, as shown in Equation3 (V is 255 for an 8 bit image). This results in a black and white image, dstk,where white regions represent foreground candidates. The threshold value, T ,can be determined by the user or can be computed automatically (more on thislater). Once again, if the image is colored, this is done for each color channel,and the �nal result dstk is obtained by taking an OR of each channel result.
dstk(x, y) =
{V if |IBG(x, y)− fk(x, y)| > T
√Iσ2(x, y)
0 otherwise(3)
3
The next step is to remove small noises (small white regions) by applyingmorphological operations on the image. First we apply an erosion followed by adilation, using the same structuring element for both operations. After this, weclassify connected regions (blobs), computing for each one its area and center ofmass. After discarding blobs below a certain minimum area and above a certainmaximum area (both values are user de�ned), we will have two possibilities:1) more (or equal) valid blobs than the number of animals we are currentlytracking or; 2) the opposite. In the �rst case, we will associate each animal tothe blob which is closest to its last tracking position (or next predicted position,depending on the kind of �lter being used, as will be commented later), ignoringthe remaining blobs. In the second case, we associate each blob to the closestanimal last tracking position (or next predicted position). Notice that this willcause some animals not being associated with a blob. In this case, we considerthe animals to be stationary or following a predicted trajectory (once again thiswill depend on the kind of �lter being used). As said before, the thresholdvalue T can be computed automatically if the user chooses so. This is done bycomparing the number of valid blobs, b, found, and the number of animals, a,selected by the user. During the tracking process, if b < a then the value of Tis decremented, if b > a, it is incremented.
To perform the �ltering of the tracking information the user can choose twotypes of �lters: moving average or Kalman Filter. For slow and predictableswimming style �sh, such as the Siamese �ghting �sh, the Kalman �lter givesgood results. However, for fast swimming and turning �sh, such as the Zebra�sh,the moving average is recommended. When using the Kalman �lter, the statevector X, at time instant k, of each tracked animal i, is composed of Xi[k] =[xi yi xi yi]
T , where (xi, yi) represents the 2D position, in pixels, of the animal,and (xi, yi) represents its 2D velocity in pixels/s. The system is modeled asusual (Kalman 1960) : Xi[k + 1] = AXi[k], where the state transition modelmatrix A is given by
A =
1 0 ∆t 00 1 0 ∆t0 0 Bx 00 0 0 By
, (4)
where ∆t is the time di�erence, in seconds, between the current frame beingprocessed and the last one processed by the software. The terms Bx < 1 andBy < 1 are slowdown factors for the velocity of the animal. They were usedso that the animals would come to a gentle stop while using the predictedstate estimate after a few iterations, avoiding the animal to leave (or come toa sudden stop at the edge of) the processing area. The measurement model,Zi[k] = HXi[k], consist of two-dimensional pixel measurement of the center ofmass of each animal i, hence H = [1 0 0 0; 0 1 0 0].
In order to validate the software developed, we compared it with EthoVision,a well known and widely used tracking software. We compared the resultsobtained in a 2 minute experiment with a single �sh, and they were nearlyidentical, as shown in Figure 2. Also, ZebTrack has been used successfully in
4
Fig. 2: Validation of the software. Comparison of the results obtained by Zeb-Track (on the left) with the results of EthoVision (on the right) of anexperiment with a single �sh observed for 2 minutes. The camera is lo-cated as the top of the aquarium. Notice that the tracked trajectories ofboth softwares look nearly identical.
dozens of experiments.
References
Kaehler, A. & Bradski, G. (2015), Learning OpenCV: Computer Vision in C++
with the OpenCV Library, second edition edition edn, O'Reilly Media, Farn-ham.
Kalman, R. E. (1960), `A new approach to linear �ltering and prediction prob-lems', Transactions of the ASME�Journal of Basic Engineering 82(SeriesD), 35�45.
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Piccardi, M. (2004), Background subtraction techniques: a review, in `2004IEEE International Conference on Systems, Man and Cybernetics', Vol. 4,pp. 3099�3104 vol.4.
Stau�er, C. & Grimson, W. (1999), Adaptive background mixture models forreal-time tracking, in `Computer Vision and Pattern Recognition, 1999. IEEEComputer Society Conference on.', Vol. 2, pp. �252 Vol. 2.
Wren, C., Azarbayejani, A., Darrell, T. & Pentland, A. (1997), `P�nder: real-time tracking of the human body', IEEE Transactions on Pattern Analysis
and Machine Intelligence 19(7), 780�785.
50
Capítulo 2
Sleep deprivation impairs cognitive performance: a matter of fact?
A ser submetido: Zebrafish Journal (Qualis: B1, FI: 1,94 – 2014)
RESUMO
Neste capítulo, nós testamos os efeitos da privação de sono no peixe paulistinha através
de um paradigma de condicionamento aversivo. Além disso, acrescentamos duas drogas
que possuem respostas comportamentais já estudadas nesta espécie (álcool e melatonina)
e são conhecidas por alterar padrões do estado de sono. Para isso, nós dividimos os
animais em 5 tratamentos de sono diferentes: controle (12C:12E), privação parcial de
sono: (18C:06E), privação total de sono (18C:06E+ pulsos de luz), privação total de sono
+ álcool e privação total de sono + melatonina. Os resultados sugerem que a privação
parcial de sono não prejudicou a performance do animal. Por outro lado, a privação total
de sono prejudicou a habilidade cognitiva em peixe paulistinha, enquanto os peixes que
receberam álcool na noite anterior ao teste, ainda que privados de sono, responderam ao
estímulo como o grupo controle. Tratamento com melatonina parece não induzir o estado
de sono, ao menos no protocolo aplicado aqui.
51
Sleep deprivation impairs cognitive performance: a matter of fact? 1
Jaquelinne Pinheiro-da-Silva1, Steven Tran2 and Ana Carolina Luchiari1* 2
1Departamento de Fisiologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, 3
Brazil 4
2University of Toronto, Department of Cell and Systems Biology, Canada 5
6
Abstract 7
The zebrafish (Danio rerio) has become a valuable organism for behavioral studies 8
examining learning and memory. The diurnal circadian rhythm in addition to sleep-like state 9
characteristics of zebrafish similar to mammals, has established this small vertebrate as a 10
translational model for sleep research too. Despite sleep being an evolutionarily conserved 11
phenomenon, its functions in the body are still debatable. The lack of sleep is commonly 12
associated with decreased attention, changes in responsiveness, locomotor activity and 13
impaired performance on cognitive tasks. In the current study, we examined the effect of 14
sleep deprivation on zebrafish learning performance in an avoidance conditioning paradigm. 15
In addition, we also examined the effects of two drugs known to alter sleep (alcohol and 16
melatonin), which have already been well characterized in this species. We divided the 17
animals into 5 different sleep treatment groups: control (12L:12D), partial sleep deprivation 18
(18L:06D), total sleep deprivation (12L:18D+light pulses), total sleep deprivation + ethanol 19
and total sleep deprivation + melatonin. Our results suggest that partial sleep deprivation did 20
not alter learning performance. In contrast, total sleep deprivation impaired learning 21
performance in zebrafish. However, fish that received a 1 hour acute exposure to alcohol on 22
the night before the learning task, performed similarly to the control group. Melatonin 23
treatment did not improve learning performance. 24
25
Key words: zebrafish, sleep deprivation, learning, memory 26
52
1. Introduction 27
The zebrafish (Danio rerio) has emerged as a vertebrate model in genetics and 28
developmental biology over the past several decades, and is rapidly becoming popular for 29
behavioral studies examining learning and memory.1,2 This small fish has a well 30
characterized sleep-like state distinguished by circadian regulation, periods of inactivity, 31
resting place preference and rebound homeostasis.3,4 These features summed with the 32
zebrafish diurnal circadian rhythm5 has establish this species as a relevant and translational 33
model for sleep research. 34
Sleeping behavior characteristics, intensity and function varies over the lifetime of a 35
given animal6 despite it being a worldwide physiological phenomenon present in most 36
vertebrates.7,8 Although the function of sleep is still unknown, it is clearly important for 37
learning and memory consolidadtion.9–15Although the specific function of sleep remains 38
unclear, it is a common biological need. The deficits associated with altered sleep behavior 39
such as loss of attention and health-related problems have been demonstrated both clinical 40
and experimentally.16–20 It is estimated that in the United States, 50 to 70 million people 41
suffer from sleep disorders with the majority not being properly diagnosed or treated.21 42
Sleep deprivation (SD) has a negative impact on the quality of life and regular 43
physiological functions.11 Research on the learning process has already demonstrated that 44
sleep deprivation impairs memory consolidation13–15,18,22,23, as memory seems to be 45
particularly sensitive to SD when it takes place during acquisition. Sleep stages affect 46
memory consolidation in different ways. For example, hippocampus-dependent memories 47
such as declarative and spatial memories are supported by slow-wave sleep and are severely 48
affected by SD.9,12 Zhdanova et al.24 also showed deficits in associative learning behavior 49
after SD in zebrafish. However, in this paper we focused on investigating SD from a different 50
perspective, that is, the effects of SD prior to learning a new task. 51
53
The conditioning process is highly dependent with the nature of the stimuli utilized. In 52
classical conditioning protocols, animals learn to associate a conditioned stimulus (CS) with 53
an unconditioned stimulus (US), independent of its appetitive or aversive properties. In a 54
situation where the unconditioned stimulus causes an instinctive avoidance reaction 55
(unconditioned response), the animal responds by escaping the aversive stimulus, thereby 56
preserving its safety.25,26 Learned responses using this specific type of conditioning are 57
valuable due to its profound adaptive value. It is known that stressful situations including 58
avoidance conditioning affect the quality of sleep27 as well as subsequent reactions to 59
unpleasant stimuli in zebrafish.28 However, the extent to which SD prior to training affects 60
learning performance remains unknown. 61
To examine the basic mechanisms of learning and memory in zebrafish, we used the 62
avoidance conditioning paradigm similar to previous studies.29–32 In this experiment, we 63
examined the effects of SD on associative learning using an aversive stimulus. Whereas 64
studies on exogenous agents that enhance or reduce sleep-like behavior in both fish and 65
mammals have been examined33–38, the effects of sleep altering drugs (e.g. alcohol and 66
melatonin) on sleep deprived fish remain unknown. Therefore, we hypothesized that SD 67
impairs learning behavioral performance in an aversive conditioning paradigm, whereas sleep 68
promoting drugs will enhance performance. 69
70
2. Materials and Methods 71
2.1. Animals and Stock Conditions 72
This study used adult wild-type zebrafish (Danio rerio, months old, mixed sexes) 73
obtained from a local fish farm and transferred to a storage system (50L tanks) at the 74
Ornamental Fish Vivarium, Department of Physiology – UFRN. Four 50-L tanks formed a 75
stock unit in a closed recirculation system with mechanical, biological, and chemical 76
54
filtration and UV disinfection. Animals were housed (one fish/L) with aerated and filtered 77
water, and temperature (28°C), pH (7.1) and oxygen measured regularly. Fish were kept on a 78
12h light-dark cycle, with zeitgeber time (ZT) 0 corresponding to lights on time (07am – 79
19pm), light intensity was 250lx. Zebrafish were fed twice a day with brine shrimp and a 80
commercial diet (60% protein and 15% fat, Nutricom Pet). Experiments were performed in 81
accordance with the Ethics Committee for Animal Use of the Federal University of Rio 82
Grande do Norte (application number: CEUA 022/2012). 83
2.2. Sleeping conditions and drug treatments 84
Light suppresses sleep in zebrafish with no evidence for rebound during the light 85
phase3, thus, we used two different light:dark cycles to induced SD. The sleep deprivation 86
was achieved by extending the light phase of the cycle and by exposing fish to light pulses 87
during the dark phase. The partial sleep deprivation consisted of 18h of light followed by 88
only 6h of dark (18L:06D), while the total sleep deprivation was achieved by 18h of light 89
followed by 6h of light pulses (4-min light and 1-min dark). The pulses of light were 90
presented to prevent fish from resting for more than 1 min in the dark. The control, partial 91
and total sleep deprivation protocols were conducted for 3 consecutive days prior to the 92
learning test. 93
Two sleep altering drugs were used concomitantly with the total sleep deprivation 94
condition, one expected to suppress the deleterious effects of sleep deprivation: melatonin, 95
and the other expected to exacerbate the negative effects deprivation: alcohol.35 Melatonin 96
was administered directly to the housing tank water at a final concentration of 100nM. 97
Melatonin was administered once a day, with 30% of the tank water being replaced daily for 98
10 consecutive days prior to the learning test, adapted from Zhdanova et al.24 Note that since 99
the melatonin treatment lasted 10 days, the sleep deprivation protocol was inducted on day 8 100
(the last 3 days of treatment). On the last night od sleep deprivation, zebrafish were exposed 101
55
to 0.5% alcohol for 1 hour before the beginning of the dark phase in a 30 L tank, and then 102
returned to their housing tank.39 103
Therefore, the treatments used in the present study were: Partial Sleep Deprivation 104
(18L:06D, n=32), Total Sleep Deprivation(18L:06D+pulses, n=30), Total Sleep Deprivation 105
+ 0.5% Ethanol (Eth 18L:06D+pulses, n=26), Total Sleep Deprivation +Melatonin (Mel 106
18L:06D+pulses, n=28), and Control (12L:12D, n=24). 107
2.3. Aversive Learning Task 108
An aversive conditioning protocol modified from Blank et al.32 and Xu et al.31was 109
administered to each group described above and was performed during the daytime (ZT1 – 110
ZT8). Zebrafish were individually trained in a 15L shuttle box tank (40x25x20cm) divided by 111
an opaque wall with a 2cm opening at the bottom allowing the fish to swim to both sides. The 112
walls of the tank were completely covered with opaque plastic self-adhesive white films but 113
different visual cues were present on the bottom of each side of the tank (Fig. 1) (white 114
background vs. back and white checkered pattern). 115
In the current study, we used an electroshock apparatus which has been successfully 116
utilized for cognitive avoidance tasks29 to demonstrate learning31 and memory retention32 in 117
zebrafish. The electroshock apparatus consisted of 2 manual shock machines, one for each 118
compartment of the shuttle box, which delivered an electroshock to serve as an aversive 119
stimulus. Each machine had two electrodes positioned through the wall and placed on each 120
side of the tank. The electroshock (6V) was administered: (a) consistently on one side of the 121
tank (either the white or checkered pattern side) or (b) on random sides. Animals were tested 122
individually in the shuttle box. After 2 minutes of habituation to the tank, the fish received a 123
2 second electroshock followed by 60 seconds without shocks, this was repeated for a total of 124
20 trials. Behavior responses were recorded during the 20 minutes following habituation and 125
analyzed using an automated video tracking software, ZebTrack. The video tracking software 126
56
has been previously validated by Pinheiro-da-Silva et al. ( unpublished data), capable of 127
quantifying swimming patterns, including speed, distance travelled, and time spent in 128
predefined areas. To evaluate learning performance in zebrafish, we quantified the time that 129
fish spent on each side of the tank, their average speed, maximum speed, freezing and total 130
distance traveled during each trial. The data was analyzed using paired Student’s t test to 131
compare time spent on each side of the tank and One-Way Analysis of Variance (ANOVA) 132
with Tukey’s Honest Significant Difference (HSD) tests to compare average speed, 133
maximum speed, total distance traveled, and freezing. The data was tested and shown to be 134
normally distributed and different groups exhibited equal variance. Significance was reported 135
at p < 0.05. 136
3. Results 137
During the test, groups that received electroshocks on random sides showed no 138
significant differences in the time spent on each side of the shuttle box (Fig. 2) (Student t test: 139
Control group: t(24) = 0.99 p =0.33; Partial SD group: t(20) = -0.81 p= 0.42; Total SD group: 140
t(26) = -1.37 p = 0.18; Total SD + Ethanol: t(28) = -1.49 p= 0.15; Total SD + Melatonin: 141
t(20) = 0.71 p= 0.48). When fish received shocks consistently on one side of the tank, the 142
Control, Partial SD and Total SD + Ethanol groups spent more time on the side without the 143
shock (Student t test: Control group: t(22) = -2.05 p= 0.05; Partial SD group: t(30) = -3.03 p 144
= 0.005; Total SD + Ethanol: t(23) = -4.19 p= <0.001). However, there were no significant 145
differences in the time spent on each side of the tank for the Total SD and Total SD + 146
Melatonin groups (Student t test: Total SD: t(28) = -1.13 p= 0.27; Total SD + Melatonin: 147
t(26) = -0.62 p= 0.54) (Fig. 3). 148
While not quantified, visual observation during the test suggested that all animals 149
showed normal swimming behavior. One-Way ANOVA did not detect significant differences 150
in the average speed among the five treatment groups (F(4,65)= 0.27 p= 0.89) (Fig.4a), but 151
57
the Control group exhibited a higher maximum speed in comparison to the Total SD + 152
Melatonin group (F(4,70)= 3.18 p = 0.019) (Tukey’s HSD test, p<0.05). However, the 153
maximum speed of the all other groups was not significantly different from each other (p> 154
0.05) (Fig.4b). 155
We also observed some episodes of freezing behavior, as expected due to the aversive 156
nature of the stimulus. Fish from the control group spent the most time freezing, while fish 157
from the Total SD + Melatonin group spent the least (One-Way ANOVA, F(4, 66)=3.34 158
p=0.015). Differences in freezing among the other groups were non-significant (p> 0.05) 159
(Fig. 5a). The total distance fish travelled during the test was also analyzed. Using One-way 160
ANOVA and Tukey’s HSD test, we found that the Total SD group travelled a significantly 161
shorter distance compared to all other groups (One-Way ANOVA, F(4, 70) =14.97 p<0.001) 162
(Fig. 5b). 163
164
4. Discussion 165
In the current study, we found that total sleep deprivation (SD) impaired avoidance 166
learning in zebrafish, D. rerio, while acute alcohol exposure on the last night of SD increased 167
performance in the learning task. Sleep is an important behavioral state associated with 168
energy allocation and conservation, synapses remodeling and memory consolidation.8,40,41 It 169
should also be noted that sleep deprivation induces sleep rebound, which occurs in the dark 170
phase during the first few days of SD or even during the light phase following prolonged 171
SD.8,42 Sleep rebound is related to a homeostatic sleep response, which is a process through 172
which partially sleep deprived fish can recover during the dark phase43,44 and may explain 173
why these fish were able to perform well on the learning task. 174
As with most animals, zebrafish exhibit a natural tendency to explore new 175
environments in search of food, mates or shelter.45–47 Considering that associating an aversive 176
58
unconditioned stimulus with the environment that it is paired with is an important cognitive 177
ability that can increase an animal’s chances for survival24,48, we expected zebrafish to avoid 178
the compartment associated with the electroshock even after three nights of SD. However, 179
fish in the total sleep deprivation condition did not learn to avoid the aversive stimulus, 180
whereas an acute dose of alcohol increased learning performance while melatonin treatment 181
did not. Our findings are in accordance with previous studies examining the deleterious 182
effects of sleep deprivation on cognition.18,20,49 183
In our study, fish exposed to electroshocks on only one side of the tank learned to avoid 184
the stimulus when they were not sleep deprived. These results are in line with previous 185
studies on avoidance learning29,31,32,50 and corroborates the ability of zebrafish to learn and 186
respond to an aversive stimulus. Likewise, partially sleep deprived fish also learned the 187
association similar to controls. The performance of partially sleep deprived fish may be 188
attributed to sleep rebound during the shortened dark phase. According to Yokogawa et al.3, 189
sleep rebound is a common homeostatic process that follows a sleep deprivation period. 190
Thus, although partial sleep deprivation reduced maximum speed and there was a trend 191
towards decreased freezing behavior, 6 hours of darkness may have been enough to allow 192
zebrafish to recover and perform well in the aversive conditioning paradigm (Fig. 3). 193
Conversely, totally sleep deprived fish receiving electroshocks consistently on one side 194
of the tank did not learn to avoid the compartment associated with the shock and spent an 195
equal amount of time on both sides of the shuttle box (Fig.3). Studies examining rodent and 196
human behavior following sleep deprivation have highlighted the resistance of emotional 197
memory to the effects of SD51,52. However, negative stimuli have been shown to be more 198
reinforcing than positive stimuli for associative learning tasks.53 In a previous study, our 199
group has observed that sleep deprivation impairs the discrimination of a novel object 200
(unpublished data). Therefore, the inability to avoid electroshocks after three nights of total 201
59
SD is in accordance with previous studies examining the effects of sleep deprivation on 202
learning and memory. 203
Although we showed that sleep deprivation impaired learning performance in zebrafish, 204
one could argue that SD may have impaired general locomotor activity and zebrafish simply 205
spent more time on the same side of the tank, independent of the stimulus. Alternatively, fish 206
may have exhibited a place preference and simply avoided the non-preferred compartment. 207
To test these alternative possibilities, fish from each treatment group also received 208
electroshocks on random sides of the tank (Fig 2). As expected, fish receiving electroshocks 209
on random sides of the tank spent an equal amount of time on both sides of tank, showing 210
that zebrafish did not exhibit a place preference. 211
Behavioral parameters including average and maximum swimming speed, freezing 212
behavior and total distance traveled were also analyzed. Although the average speed did not 213
differ between groups, the control group showed a higher maximum speed compared to all 214
other groups. However, totally sleep deprived fish exhibited the lowest total distance 215
traveled. This finding is in accordance with a study by Zhdanova et al.44, which showed that 216
sleep deprivation decreases daytime locomotor activity in zebrafish. Examination of freezing 217
behavior revealed that the control group froze for a longer period of time compared to all 218
other groups which may be related to changes in the light:dark cycle. Other studies have also 219
shown that sleep deprivation reduces response time and impairs sustained attention.18,54 Thus, 220
sleep deprivation in our study may have affected the fish’s ability to properly respond to the 221
stimulus. Freezing behavior is a complete suspension of movement resulting from increased 222
stress/anxiety and may reflect submissive behavior.55 In this study, we expected to observe 223
freezing behavior elicited by the avoidance conditioning paradigm. 224
Alcohol is one of the most studied pharmacological agent in zebrafish research and has 225
been shown to alter behavioral responses as well as impair learning.2,56,57 This substance 226
60
when added to the water can easily be absorbed by the fish with blood alcohol levels quickly 227
reaching equilibrium with the external alcohol concentration.58 In this study, fish were 228
exposed to an acute alcohol dose (0.5%) on the last night of sleep deprivation and 229
subsequently tested for avoidance learning on the following day. The sleep deprivation plus 230
alcohol group was able to acquire the association between the US and CS similar to the 231
control group, suggesting unimpaired learning performance. 232
Alcohol consumption is most often associated with sleep disruptions.37,59 However, the 233
effect of alcohol is dose-dependent with stimulant and sedative effects.37,57,60,61 Based on our 234
results, alcohol exposure seems to have increased learning performance in sleep deprived 235
fish. Alcohol’s sedative effects may have promoted sleep onset similar to a previous a study 236
by Roehrs and Roth,35 which reported that alcohol has sedative and sleep-promoting effects. 237
In addition, studies have found that alcohol increases the time spent in slow waves sleep 238
(SWS).62,63 A similar pattern was also observed in sleep deprived elderly subjects; elderly 239
people showed an increase in SWS during the recovery night following sleep deprivation 240
after consuming an alcohol beverage.64Alcohol is known to reduce core body temperature, 241
contribute to the entrainment of sleep,65 and low doses have been reported to be beneficial for 242
treating insomnia.66 However, the development of alcohol tolerance/dependence should be 243
taken into consideration. For example, tolerance to alcohol’s sedative effects can be 244
established after only a few days of consumption.67Moreover, the use of alcohol to improve 245
sleep may lead to excessive intake. Thus, the relationship between alcohol and sleep requires 246
greater attention, with a focus on identifying the physiological/neural mechanisms underlying 247
alcohol’s sleep-promoting properties. 248
In contrast to alcohol’s effect on avoidance learning following sleep deprivation, 249
exposure to melatonin did not alter learning performance. Melatonin is the main sleep-250
promoting hormone in zebrafish and is produced in the pineal gland.71 Administration of 251
61
melatonin has been shown to increase sleep under light:dark conditions44 and has dose 252
dependent effects on locomotor behavior72 which can be observed within 20 minutes of drug 253
exposure.44 In the current study, melatonin administration did not increase learning 254
performance in sleep deprived fish, and this could be related to the exposure regimen. 255
Moreover, our sleep deprivation protocol (light pulses) may have inhibited melatonin’s action 256
in the brain. For example, sleep disorders cannot be properly treated with melatonin if users 257
does not have the proper conditions for sleep induction.73,74 Additionally, Rawashdeh et al.73 258
reported that melatonin’s action in zebrafish is inhibited under constant light in an active-259
avoidance conditioning paradigm. These authors showed that pinealectomy or interfering 260
with melatonin signaling improved memory consolidation, suggesting a role for melatonin in 261
memory formation at night. 262
Finally, our results reinforce the zebrafish as a valuable model organism for high 263
throughput screening of sleep-related drugs. Sleep is an essential behavioral phenomenon and 264
our data support the negative effects of sleep deprivation on cognition. We found a 265
significant learning impairment in zebrafish following total sleep deprivation. Although 266
melatonin did not alter learning performance, acute alcohol exposure improved learning 267
performance in sleep deprived fish. The simplicity of this protocol due to the lack of a time-268
consuming training phase will make it a useful tool for future behavioral and brain tissue 269
analyses. It will also allow investigators to examine how sleep deprivation affects cognitive 270
function and whether different drugs can be used for the treatment of sleep disorders. 271
272
5. Acknowledgments 273
The authors are grateful to Ms. Adrielly Nascimento and Mr. Rômulo Almeida for their 274
assistance with data collection, and Mrs. Priscila Fernandes for her valuable suggestions and 275
insights. 276
62
277
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Fig. 1
Fig.1: Schematic view of the avoidance conditioning paradigm. Tanks (15L, 40x25x20 cm)
divided by an opaque wall with a 2cm opening at the bottom allowing the fish to swim to
both sides. The walls of the tank were completely covered with opaque plastic self-adhesive
white films but different visual cues were present on the bottom of each side of the tank
(white background vs. black and white checkered pattern).
Fig. 2
Fig. 2: Adults zebrafish performance in Avoidance Conditioning following 3 nights of partial
sleep deprivation, total sleep deprivation and total SD + Ethanol or + Melatonin
administration. Electroshock (12mV, 0.2sec, every 1 min) administered in random sides. n =
24-32 fish/group, *p<0.05.
0
200
400
600
800
1000
b/w white b/w white b/w white b/w white b/w white
Control Partial SD Total SD Total SD + EtOH Total SD + Mel
Tim
e sp
ent
in e
ach
sid
e (s
)
70
Fig. 3
Fig.3: Adults zebrafish performance in Avoidance Conditioning following 3 nights of partial
sleep deprivation, total sleep deprivation and total SD + Ethanol or + Melatonin administration.
Electroshock (12mV, 0.2sec, every 1 min) administered in one side only (white side or
black/white side). n = 24-32 fish/group, *p<0.05.
Fig. 4
Fig.4: We applied one-Way ANOVA to compare (a) average speed and (b) maximum speed
during the avoidance conditioning test, among the five groups: Control, Partial SD, Total SD,
Total SD + Ethanol, Total SD + Melatonin. Data corresponds to 20 min of behavior recorded
during the test and analyzed using video-tracking software (ZebTrack). n = 11-16 fish/group,
(*) indicates statistical difference between groups, p<0.05.
0
200
400
600
800
1000
Shock Blank Shock Blank Shock Blank Shock Blank Shock Blank
Control Partial SD Total SD Total SD + EtOH Total SD + Mel
Tim
e sp
ent
in e
ach
sid
e (s
)
* * *
0
50
100
150
200
250
300
Control Partial SD Total SD Total SD +EtOH
Total SD +Mel
Ma
xim
um
Sp
eed
(cm
/s)
0
1
2
3
4
5
6
Control Partial SD Total SD Total SD +EtOH
Total SD +Mel
Av
era
ge
Sp
eed
(cm
/s) *
a) b)
71
Fig.5
Fig. 3
Fig.5: We applied one-Way ANOVA applied to compare (a) freezing behavior and (b) total
distance the animal traveled during the avoidance learning experiment, for the five groups:
Control, Partial SD, Total SD, Total SD + Ethanol, Total SD + Melatonin. Fish were observed
for 20 min and analyzed using video-tracking software (ZebTrack). Different letters and (*)
indicates statistical differences between the groups. n = 11-16 fish/group, p<0.05.
0
100
200
300
400
500
600
Control Partial SD Total SD Total SD +EtOH
Total SD +Mel
0
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Control Partial SD Total SD Total SD +EtOH
Total SD +Mel
To
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72
Capítulo 3
Good night, sleep tight: the effects of sleep deprivation on spatial associative
learning in zebrafish
À ser submetido: Behavioural Brain Research (Qualis: A1, FI: 3,02 – 2014)
RESUMO
Utilizamos um teste simples de aprendizagem especial associativa para investigar os
efeitos da privação de sono em peixe paulistinha, utilizando um cardume da mesma
espécie como estímulo positivo. Os animais testados após noite de sono normal
rapidamente aprenderam à associar o estímulo ao local apresentado no aquário,
reforçando o peixe paulistinha como modelo válido e confiável para tarefas de
condicionamento. Embora a privação parcial de sono não tenha sido suficiente para
prejudicar a habilidade cognitiva, a privação total de sono afetou a performance do peixe
em todas as condições de apresentação do estímulo. Nossos resultados sugerem que o
sono é elemento importante para a memória, prejudicando a aquisição da aprendizagem
em performance associativa.
73
Good night, sleep tight: the effects of sleep deprivation on spatial associative learning in 1
zebrafish 2
3
Jaquelinne Pinheiro-da-Silvaa, Steven Tranb, Priscila Fernandes Silvaa, Ana Carolina 4
Luchiaria,* 5 a Departamento de Fisiologia, Centro de Biociências, Universidade Federal do Rio Grande do 6
Norte, Natal, Brazil. 7 b University of Toronto, Department of Cell and Systems Biology, Canada 8
10
Abstract 11
Learning and memory processes confer important advantages to an animal’s survival. A 12
number of endogenous and exogenous factors can interfere with cognitive ability. Sleep 13
is a worldwide physiological phenomenon known to contribute to the consolidation of 14
learning and memory. The zebrafish has emerged as a powerful model organism, sharing 15
organizational and functional characteristics with other vertebrates, providing great 16
translational relevance. In this paper, a simple spatial associative learning test was used to 17
investigate the effects of sleep deprivation on learning performance in zebrafish, using a 18
conspecific shoal as a positive stimulus. Control animals on a regular light:dark cycle 19
were able to learned the association between the unconditioned stimulus and the 20
conditioned stimulus, reinforcing zebrafish as valid and reliable model for appetitive 21
conditioning tasks. Although partial sleep deprivation was not enough to impair cognitive 22
ability, total sleep deprivation significantly impaired zebrafish learning performance in 23
all conditions. Our results suggest that sleep is important for learning and memory and 24
that the effects of sleep deprivation can be investigated using zebrafish. 25
Keywords: learning, memory, associative learning task, sleep 26
74
27
1. Introduction 28
Learning is an important process that allows for the acquisition of new skills or 29
concepts from past experiences [1–4]. The ability to modify behavioral patterns based on 30
past experiences confers several advantages such as finding food and mates [5], as well 31
as avoiding predators [6]. Moreover, the benefits associated with the predictive nature of 32
learning may be enhanced when individuals retain long-term memories. However, a 33
number of endogenous and exogenous factors can enhance and/or impair learning [7–9]. 34
Among the number of factors that favor learning and memory consolidation, sleep is 35
one of the most studied. Sleep behavior is defined as a resting state in which 36
consciousness is partially or completely lost, and there is a decreased response to external 37
stimuli as well as voluntary motor activities[10,11]. During sleep, the brain exhibits two 38
types of electrical activity: 1) slow wave activity (NREM sleep, non-rapid eye 39
movement) divided into four stages, and 2) desynchronized brain wave activity (REM 40
sleep, rapid eye movement) represented by muscle atonia and wake-like brain activity 41
[12,13]. While only endothermic animals exhibit REM sleep, ectothermic vertebrates also 42
show sleep-like behavior that confers adaptive advantages[11,13]. 43
Even though the specific function of sleep in animals is still unknown, the cumulative 44
effects of sleep deprivation have been associated with negative health consequences 45
including obesity, diabetes, stroke, and depression, along with a profound economic and 46
societal impact[14]. Research on sleep and sleep disorders have been increasing, 47
however, diagnoses and treatments are still limited. Current research on sleep requires an 48
interdisciplinary approach to thoroughly understand how sleep affects human health. 49
75
Research on the effects of sleep on learning and memory has been a focus of 50
numerous studies in the field of behavioral neuroscience and psychobiology. Research in 51
this area of study have focused on rodents and primates[15–19]. However, research on 52
phylogenetically distant animals may identify evolutionarily conserved mechanisms 53
regulating sleep behavior and the development of learning and memory. Zebrafish have 54
been a focus of attention because it is a tractable genetic model that shares organizational 55
and functional characteristics with other vertebrates[20–23]. The main neurotransmitter 56
systems regulating sleep in mammals are widely conserved in zebrafish, including 57
monoaminergic, cholinergic and hypocretinergic cell groups[24–32]. A sleep-like state 58
has been characterized in zebrafish by Zhdanova[33,34] and is similar to other 59
vertebrates including mammals. One of the major advantages of the zebrafish model is 60
the non-invasive nature of drug administration. Water soluble drugs can be added directly 61
to the water which is then taken up by the immersed by through its skin and gills. This 62
property allows us to test the effects of drugs that are known to alter sleep-like behavior 63
such as alcohol and melatonin, substances with known sedative and sleep promoting 64
effects [35–38]. 65
Although zebrafish have been increasingly used in learning and memory studies [39–66
44], the effects of sleep deprivation on learning performance is unclear. In the present 67
study, we determined the effects of partial and total sleep deprivation and administration 68
of alcohol or melatonin in sleep deprived fish on learning performance in an appetitive 69
conditioning task. To examine learning and memory, we used a spatial associative 70
learning paradigm, with a conspecific image as a reward due to the highly social nature of 71
this species [45,46]. 72
76
73
2. Material and methods 74
2.1.Animals and housing 75
Zebrafish (Danio rerio) were obtained from a local fish farm (Natal, Rio Grande do 76
Norte, Brazil) and acclimatized for one month before the behavioral experiments. Adult 77
zebrafish (3 months, mixed sexes) were transferred to 50 L tanks in a recirculating 78
system with multistage filtration, including a mechanical filter, a biological filter, an 79
activated carbon filter and a UV light sterilizing unit. Temperature, pH, and oxygen were 80
measured regularly with fish density maintained at one animal/liter. 81
Fish were kept on a light:dark cycle of 12L:12D (12 hours light:12 hours dark), with a 82
light intensity of 250 lx. They were fed twice daily with brine shrimp and a commercial 83
diet. Protocols were reviewed and approved by the Ethical Committee for Animal Use of 84
Federal University of Rio Grande do Norte (CEUA 022/2012). 85
86
2.2. Sleep deprivation and drug treatments 87
To determine the effect of sleep deprivation (SD) on learning performance, 210 88
zebrafish were randomly assigned to five different experimental groups. To induce sleep 89
deprivation, we altered the light:dark cycles which consisted of extending the light phase 90
and exposing fish to pulses of light during the dark phase. 91
The (1) Control group was kept on a 12L:12D cycle (n=30). The (2) Partial sleep 92
deprivation group was kept on a 18L:06D cycle (n=45), which is 18h of light and only 6h 93
of dark. The (3) Total sleep deprivation group was also maintained on 18h of light and 94
06h of dark, however, during the dark phase, light pulses were applied for 1 min every 4 95
77
min (n=45). Two other groups were also maintained under total sleep deprivation 96
conditions and received additional drug treatments: (4) Total sleep deprivation + Ethanol 97
group (acute dose of 0.5% alcohol, for 1 hour exposure; n=45) and (5) Total sleep 98
deprivation + Melatonin group (10 days of chronic exposure to 100nM melatonin; n=45). 99
The light:dark cycle manipulations were maintained for 72h prior to the learning test. 100
The ethanol concentration was achieved by diluting ethanol (99,8% P.A– ACS, 101
Dinâmica) in the tank water to a final concentration of 0.5% in a 15L tank, a dose 102
previously shown to be stimulatory [47]. Ethanol exposure took place 1-hour prior to the 103
onset of the dark phase. Melatonin (cat#M5250, Sigma-Aldrich) was also diluted directly 104
in the tank water at a final concentration of 100nM. The melatonin treatment continued 105
for 10 continuous days, 24h per day, with the tank water and drug concentration being 106
replaced every day(adapted from Zhdanova et al. [48]). 107
108
2.3.Conditioning test 109
The appetitive conditioning protocol was modified from a study by Pather and Gerlai 110
[43]. Zebrafish from each group described above were transferred from their home tank 111
to a testing tank (40 x 25 x 20 cm, width x depth x height) and tested once individually. 112
The testing tank was divided in half by a white partition with a 2cm opening at the 113
bottom which allowed the fish to swim from one side to the other (Fig. 1). Two monitors 114
(LG-Flatron E2011P-BN, 20”) were placed on opposite sides of the testing tank on the 115
outside. The monitors were connected to a desktop computer (Intel Pentium G3220 116
3.00GHZ) that ran a software application that presented animated zebrafish images at 117
varying intervals on each monitor. Zebrafish are a highly social species and prefer to stay 118
78
in close proximity to their conspecifics both in nature and in the laboratory[45]. Zebrafish 119
also respond in a similar manner to computer-animated conspecific images as to they do 120
to live conspecifics [46,49]. Ruhl and McRobert [50] have shown differences in sex and 121
body size preferences during shoal formation in zebrafish, thus, we used an image of six 122
zebrafish of mixed sexes and similar body sizes compared to the experimental fish to 123
simulate naturally occurring shoals of zebrafish. 124
The animated conspecific image was presented on: (a) one side only, (b) alternating 125
sides, and (c) random sides. 15 fish from each of the 4 groups were presented images as 126
described above (random side presentations were not applied to the control group). For 127
the one side only presentations, half of the experimental fish received the stimulus on the 128
left side and half on the right side of the tank. We expected that when images were 129
presented only on one side, zebrafish would prefer to stay on the stimulus side. In 130
contrast, when images were presented on alternating sides, zebrafish were expected to 131
learn to shuttle back and forth due to the rewarding nature of the stimulus, allowing the 132
quantification of learning performance. 133
After being introduced to the testing tank, experimental fish were shown a blank 134
screen for 2 min (habituation). An image of conspecifics was presented for 30s always 135
starting on the left side of the tank followed by an interval of 60s without the stimulus 136
(both screens blank) this was repeated 20 times, with additional images presented either 137
on the same side, alternating sides, or random sides. Therefore, we had 20 stimulus 138
presentations and 20 inter stimulus intervals (ISI). Fish were tested individually and their 139
behavior was recorded using a handycam (Sony Digital Video Camera Recorder; DCR-140
79
SX45) positioned 1.5 m away in front of the testing tank. The tests were conducted 141
between 9am and 4pm. 142
143
2.4.Behavioral analysis 144
The video recordings were analyzed using ZebTrack, a video tracking software 145
developed in MatLab, previously described by Pinheiro-da-Silva et al. (Unpublished 146
results). We quantified the amount of time zebrafish spent on each side of the tank during 147
the 30s of stimulus presentation and during the 60s of inter stimulus interval (ISI) for all 148
20 sessions. We also analyzed other behavioral parameters such as average and 149
maximum speed, total distance traveled and freezing. 150
151
2.5.Statistics 152
To apply inferential statistics, we first evaluated the data by an exploratory 153
analysis due to potential problems with outliers, homogeneity, normality, zero 154
trouble, collinearity and variables independency, as suggested by Zuur et al. [51]. 155
The average time zebrafish spent on the stimulus side was averaged into separate 156
blocks consisting of 5 stimulus trials and were compared by Repeated Measures 157
Analysis of Variance (RM ANOVA) for each group depending on whether the 158
stimulus was presented on one side only, alternating sides or random sides. The time 159
fish spent on the stimulus to-be side during the 20 ISI was also averaged into blocks 160
(every four ISI) and different groups were compared using RM ANOVA depending 161
on the presentation scheme. 162
80
Average and maximum speed, freezing and distance travelled were also compared 163
between the groups after pooling data from the 3 different presentation schemes using 164
One-way ANOVA. For all comparison, the probability level considered for 165
significance was p≤0.05. 166
To develop a model for the time spent on the correct side of the tank (response 167
variable) and the explanatory variable (stimulus or ISI trials and treatments), we used 168
a mixed effects model analysis for longitudinal data. The term longitudinal is related 169
to repeated measures of the response variable through time [51]. The mixed models 170
presented random effect factors (represented by the variation within our zebrafish 171
behavior), fixed effect factors (represented by the influence of the explicative 172
variables: stimulus trials and treatments) and error. 173
To develop the mixed model, we used the glmmPQL command from the MASS 174
package [52] of the R program [53]. We decided to use this algorithm on the 175
glmmPQL command due to the abnormal distribution and overdispersed 176
characteristics presented by the residuals of the response variable during the 177
exploratory analysis. Moreover, the response variable was discrete quantitative data 178
that varied between 0 and 30 (stimulus trials) or 0 and 60 (ISI), which may present a 179
binomial distribution error with logit link function (according to Zurr et al. [51]). The 180
glmmPQL command was effective because it presents mixed generalized models with 181
a ‘quasi’ distribution, suggesting the data was overdispersed. 182
183
3. Results 184
81
Here we analyzed two types of behavior: (1) response to the stimulus, and (2) 185
learning performance. 186
For the groups that received 20 trials of stimulus on only one side of the tank, the 187
mixed models comparison showed that the total SD group differed from all other groups 188
in the amount of time spent on the stimulus side of the tank (Table 1). Comparing the 189
means of 5 stimulus trials (blocks) by RM ANOVA revealed significant differences in 190
the time spent near the stimulus between different groups. The control and the total SD + 191
Eth groups spent more time on the stimulus side starting from the 6th stimulus 192
presentation (Control group: F=6.725 p=0.007, Fig. 2b; total SD + Eth: F=3.379 p=0.054, 193
Fig. 2h). The partial SD and the total SD + Mel groups spent more time near the stimulus 194
side by the second half of the stimulus presentations (RM Anova: partial SD group: 195
F=5.842 p<0.011, Fig. 2d; total SD + Mel: F=5.48 p=0.013, Fig. 2j). The total SD group 196
did not show a significant difference in the time spent on the stimulus side of the tank 197
even after 20 stimulus presentations (RM Anova: total SD group: F=2.988 p=0.073, Fig. 198
2f). 199
When the group image was presented on opposite sides, the mixed models 200
comparison showed there were no differences in the time spent on the stimulus side 201
between the control group and any of the other groups (Table 1). The RM ANOVA for 202
the blocks of each 4 stimulus trial showed significant differences for the control, partial 203
SD and total SD + Eth groups (Control group: F=6.649 p=0.023, Fig. 3b; partial SD 204
group: F= 3.89 p=0.037, Fig. 3d; total SD + Eth: F=4.451 p=0.0477, Fig. 3h). Both the 205
total SD and total SD + Mel groups did not show differences between the different blocks 206
82
(RM Anova, total SD group: F=2.308 p=0.128, Fig. 3f; total SD + Mel: F=1.006 207
p=0.424, Fig. 3j). 208
When the stimulus was presented on random sides, the mixed models did not show 209
any significant differences in the time spent on the stimulus side (Table 1). RM ANOVA 210
for the blocks (5 stimulus trials) did not show any differences between any groups (RM 211
Anova, partial SD group: F=3.601 p=0.046, Fig. 4b; total SD group: F=0.573 p=0.644, 212
Fig. 4d; total SD + Eth: F=1.201 p=0.351, Fig. 4f; total SD + Mel: F=0.711 p=0.564, Fig. 213
4h). 214
Figures 5, 6 and 7 showed that the time spent next to the stimulus presentation side 215
during the ISI for the groups that received the stimulus presentation scheme on one side 216
only, opposite sides and random sides, respectively. In Fig. 5, when the conspecific 217
image was presented on the same side only, zebrafish in the control, partial SD, and SD + 218
Eth groups increased the time spent on the side where the stimulus would appear next 219
(stimulus-to-be side) over time. However, no such increase was observed in the total SD 220
and total SD + Mel groups. Fig. 6 shows the amount of time zebrafish spent on the 221
stimulus-to-be side during the ISI when the conspecific image was presented on 222
alternating sides. Zebrafish in the control, partial SD and total SD + Eth groups spent 223
more time on the stimulus-to-be side during the ISI near the end of the testing session. 224
Fig. 7 shows that when the conspecific image was presented on random sides of the tank, 225
zebrafish did not spend more time on the stimulus-to-be side over time. 226
A comparison of time spent on the stimulus-to-be side across the 20 ISI using the 227
mixed model is shown in Table 2. We found that when conspecific images were 228
presented on one side only or on alternating sides, the total sleep deprived group differed 229
83
from the control group, and the total SD group differed from the partial SD group when 230
conspecific images were presented on random sides. When we compared the mean time 231
spent in the stimulus-to-be side for each 4 ISI blocks, RM ANOVA indicated that in the 232
one side only presentation condition, only the total SD and total SD + Mel group showed 233
no differences between trials (RM ANOVA, control: F=12.669 p<0.001; partial SD 234
group: F=11.756 p<0.001; total SD group: F=0.258 p=0.899; total SD + Eth: F=26.682 235
p<0.001; total SD + Mel: F=0.371 p=0.825, Fig. 8). Fig. 8b shows that the control, partial 236
SD and total SD + Eth groups increased the time spent on the stimulus-to-be side across 237
trials during the ISI (RM ANOVA, control: F=12.196 p<0.001; partial SD group: F= 238
4.468 p=0.019; total SD group: 0.080 p=0.987; total SD + Eth: F=4.406 p=0.02; total SD 239
+ Mel: F=0.503 p=0.734). When conspecific images were presented on random sides, 240
only the partial SD group showed significant differences between blocks (RM Anova, 241
partial SD group: F=3.755 p=0.033; total SD group: F=2.383 p=0.11; total SD + ethanol: 242
F=2.584 p=0.091; total SD + melatonin: F=0.176 p=0.947, Fig. 8c). 243
Analysis of locomotor parameters revealed that maximum speed was the highest for 244
the partial SD group (One way ANOVA, F= 11.28 p<0.001, Fig. 9a), while average 245
speed was higher for partial SD and total SD + Eth and lower for the control group (One 246
way Anova, F=12.31 p<0.001, Fig. 9b). One way ANOVA found no significant 247
differences in freezing between the different groups (F=1.85 p=0.12, Fig. 9c). The total 248
distance travelled was higher for the control, partial SD and total SD + Eth and lower for 249
total SD and total SD + Mel groups (One way Anova, F=11.71 p<0.001, Fig. 9d). 250
251
4. Discussion 252
84
In the current study, we demonstrate that sleep deprivation impairs learning 253
performance in a spatial associative learning task in zebrafish (Danio rerio). Sleep 254
deprivation for 72 consecutive hours was shown to interfere with stimuli perception, as 255
well as impair fish’s performance in the learning task. While partial sleep deprivation did 256
not impair learning performance, total sleep deprivation combined with ethanol exposure 257
increased learning performance, whereas total sleep deprivation combined with melatonin 258
did not. 259
Our findings confirm previous studies where zebrafish (controlled sleep and absence 260
of drugs) showed appetitive reinforcement-based learning in a spatial alternation task 261
[40,43]and also in other associative learning tasks [54–63]. We also show here that sleep 262
deprived animals were unable to properly respond to the stimuli, even when receiving 263
melatonin treatment (commonly referred to as a ‘sleep hormone’). However, fish that 264
were sleep deprived and exposed to alcohol responded to the stimulus in a similar manner 265
as the control group. 266
The stimulus we used in the learning task proposed herein was a computer animated 267
zebrafish group image. Qin et al.[49]have previously shown that live conspecifics (inside 268
or outside the tank) or computer images (2D or 3D) are equally effective to induce robust 269
shoaling behavior in zebrafish. The zebrafish is a highly social species that prefers 270
swimming in groups [64], as other shoaling fish species [65,66]. Zebrafish can recognize 271
conspecifics and exhibit preference for groups with similar characteristics. Shoaling may 272
reduce predation risks, facilitate foraging and boost reproductive success [46,67]. 273
However, a recurrent lack of sleep leads to a sleep debt that has physical and 274
psychological implications: simple mental tasks may become more difficult and 275
85
perception, attention and vigilance may become distorted [68]. 276
While there is a general agreement that an inadequate amount of sleep results in a 277
slower reaction time, there is little consensus on the effects of sleep deprivation on higher 278
cognitive abilities [55,68–71], including learning and memory. In this study, when the 279
stimulus was presented on only one side of the tank or on alternating sides, it was 280
expected to attract the experimental fish to the specific side where the conspecifics image 281
was shown (random sides was used as a control for possible place preference response). 282
Although there was a more robust shoaling response when the stimulus was presented on 283
one side only compared to when the stimulus was presented on alternating sides, our 284
results show that fish from the control (12L:12D), partial SD and total SD+Eth groups 285
responded to the stimulus in a similar manner and exhibited a preference for the stimulus 286
presentation (Fig.2 and Fig.3). In contrast, the total SD and total SD+Mel groups did not 287
exhibit a preference for the stimulus, at least within the 30 s of stimulus presentation. 288
Although sleep deprivation impaired behavioral responses to conspecific images 289
(Fig.2 and Fig.3), this may be due to the effect of sleep deprivation on locomotion. 290
However, this is unlikely the case since average and maximum swimming speeds were 291
not significantly different between the total SD and the control group (Figs. 9a and Fig. 292
9b). Similar findings have been reported by Yokogawa et al. [32] who found that several 293
hours of sleep deprivation reduced activity levels in adult zebrafish. 294
The spatial associative task in this study consisted of a 30 s stimulus presentation 295
followed by a 60 s inter stimulus interval (ISI). Despite the fact that zebrafish spent time 296
near the conspecific image during the presentation period, we expected zebrafish to learn 297
the pattern of presentation and anticipate the next side where the stimulus would appear, 298
86
and respond by moving to that place during the next ISI. When the stimulus was 299
presented on one side only, our results indicate that the control, partial SD and total 300
SD+Eth groups increased the time on the stimulus presentation side, even when the 301
stimulus was not there (Figs. 5a, b, d and 8a), whereas total SD and total SD+Mel groups 302
did not exhibit this response (Figs. 5c, e and 8a). 303
By presenting the stimulus on alternating sides, we examined the fish’s ability to 304
learn a slightly more complex presentation pattern. Due to the task design, the animals 305
had to choose between right or left side of the tank, thus, it was expected that by random 306
chance, 50% of choices during all trials would be correct. Fig. 6 shows that animals were 307
making random choices in the first half of the trials during the ISI, but significantly 308
increased the time on stimulus-to-be side by the second half of the trials, indicating that 309
the control, partial SD and total SD+Eth groups learned to wait for the presentation of the 310
stimulus on the correct side of the tank (Fig. 6a, b, d and 8b) while the total SD and total 311
SD+Mel groups did not (Fig. 6c, e and 8b). 312
By presenting the stimulus on random sides, we confirmed that zebrafish did not 313
simply exhibit a side bias. As expected, since the unpredictable presentation of the 314
stimulus does not predict the location of the next stimulus presentation, none of the 315
groups exhibited a preference during the ISI (Fig. 7 and 8c). 316
Overall, our results are in line with Pather and Gerlai [43], which suggests associative 317
learning performance in this task is driven by an animal's motivation to join groups. 318
However, the total SD group did not exhibit a preference for the stimulus suggesting this 319
group may not perceive the conspecific image as rewarding. In this case, the stimulus 320
may not have been rewarding enough to reinforce learning. Moreover, we observed that 321
87
SD decreased locomotor behavior in zebrafish, as shown in Fig. 9, similar to the effects 322
of SD effects on rest-activity rhythm [72]. Sleep deprivation was also shown to cause 323
changes in daytime locomotor activity as well as enhance arousal thresholds on the 324
following day [36]. 325
In addition to the negative effects of SD on learning performance, we also examined 326
the effects of two drugs known to affect sleep: ethanol and melatonin. Ethanol has been 327
shown to induced behavioral changes in zebrafish [73], impaired coordination and 328
swimming, as well as alter fear and anxiety responses [47]. Melatonin is a pineal-329
produced hormone shown to promote sleep and entrain circadian rhythmicity [36,74]. 330
In our tests, the total SD+Eth group exhibited a preference for the stimulus and 331
learned to anticipate the presentation of the stimulus when it presented on one side only 332
and on alternating sides similar to the control group. Ethanol is classified as a depressant 333
[75] with sedative effects [76] and exposure on the last night of sleep deprivation may 334
have promoted sleep-like behavior. This hypothesis is supported by Roehrs and Roth 335
[77] and Williams and Salamy [35] who found that ethanol changes sleep structure, in 336
addition to its sedative and sleep-promoting effects. 337
Studies in humans have shown that an ethanol dose of 0.16% reduced sleep latency 338
and increased sleep time [77,78]. While zebrafish and humans are phylogenetically 339
distant, it is worth exploring potential links between sleep and ethanol consumption 340
However, we should take into account that ethanol is still a drug and (1) may cause 341
tolerance and dependence [59,79,80], (2) under uncontrolled use results in the disruption 342
of sleep architecture and continuity [81], (3) chronic heavy consumption leads to neural 343
damage [59], (4) there is no effective treatment for alcoholism [82] and (5) sleep 344
88
disorders may persist even after the cessation of ethanol consumption [83]. Therefore, 345
even though we have found improved learning performance in the total SD + Eth group, 346
additional research still needs to be conducted. 347
Another sleep deprived group in our study was treated with melatonin for 10 days 348
before the test (total SD+Mel). Although this group showed a slightly different response 349
pattern, we noticed that fish responded to the stimulus presentation when images were 350
presented on one side only (Fig. 2j). However, learning performance was impaired for 351
this group compared to controls. Studies have already shown that exogenous melatonin 352
facilitates daytime and nighttime sleep, without changing sleep structure and duration 353
[37,84–86]. Our findings suggest that exogenous melatonin administration may have 354
promoted sleep in fish in the total SD condition which may have contributed to the 355
perception of the conspecific stimulus when presented on one side only (Fig. 2d), but not 356
when images were presented on alternating sides (Fig. 3d). 357
In contrast, behavioral parameters such as average speed, freezing and total distance 358
traveled were similar between total SD and total SD + Mel groups (Fig. 9), suggesting 359
that melatonin treatment was not effective in altering these behavioral measures. Light is 360
the most important environmental factor influencing melatonin levels [87], which favors 361
sleep behavior in appropriate sleeping conditions (silence, dim light or dark)[88,89]. 362
Rawashdeh et al. [90] have shown that melatonin suppresses memory formation and 363
associated melatonin with poor performance in diurnal animals during nocturnal learning 364
tasks which may explain our results. 365
Although zebrafish have recently been used as an effective model in learning and 366
memory tests, data the effect of sleep deprivation on cognitive task performance has not 367
89
been published to date. In this paper we utilized a previously validated protocol to test the 368
effects of SD on a spatial associative learning task. We found that sleep deprived animals 369
exhibited poor cognitive performance. 370
Behavioral studies represent an important method to identify neuropathology. The 371
finding that SD impairs learning performance implies that sleep deprivation affects brain 372
function in fish, as previously seen in mammals [91–93]. Therefore, the zebrafish 373
represents a useful vertebrate model to investigate the molecular mechanisms regulating 374
sleep, learning, and their interaction. Although research on the effect of sleep deprivation 375
on cognitive function using zebrafish is still in its infancy, we presented robust results 376
showing the negative effects of SD on a simple and complex learning task, with alcohol 377
exposure increasing learning performance in sleep deprived fish. Furthermore, our results 378
reinforce the utility of zebrafish as an appropriate model for the proposed analysis. 379
380
5. Acknowledgements 381
We would like to thank Mr Rafael Revorêdo, Ms Heloysa Araújo and Ms Maria 382
Elisa Leite for skillful technical assistance. The authors declare no competing 383
interests. 384
385
6. References 386
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Figure 6
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0102030405060
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2 4 6 8 10 12 14 16 18 20Tim
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Figure 7
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Figure 8
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Figure 9
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Total SD +Mel
a) Maximum speed (cm/s)
*
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Total SD +Mel
b) Average speed (cm/s)
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Total SD +Mel
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Total SD +Mel
d) Total distance traveled (cm)
bb
aa
b
105
Figure captions
Fig. 1.Schematic view of the spatial associative task. Two monitors presented the stimulus
image placed flush against the two sidewalls of the experimental tank (40x25x20 cm).
Fig. 2.Time spent on each side of the test tank (graphs on the left) and on the stimulus side
(graphs on the right), during the 20 trials when the stimulus was presented on one side only.
Five groups were tested: Control (a,b), Partial SD (c, d), Total SD (e, f), Total SD + Eth (g, h)
and Total SD + Mel (i, j). Behavior was analyzed using a tracking software (ZebTrack). Means+
SEM are shown for blocks of 5min. (*) and different letters indicates statistical significance at p
< 0.05.
Fig. 3.Time spent on each side of the test tank (graphs on the left) and on the stimulus side
(graphs on the right), during the 20 trials when the stimulus was presented on alternating sides.
Five groups were tested: Control (a, b), Partial SD (c, d), Total SD (e, f), Total SD + Eth (g, h)
and Total SD + Mel (i, j). Behavior was analyzed using a tracking software (ZebTrack). Means
+ SEM are shown for blocks of 5 min. Different letters indicate statistical significance at p <
0.05.
Fig. 4.Time spent on each side of the test tank (graphs on the left) and on the stimulus side
(graphs on the right), during the 20 trials when the stimulus was presented on random sides.
Four groups were tested: Partial SD (a, b), Total SD (c, d),Total SD + Eth (e, f) and Total SD +
Mel (g, h). Behavior was analyzed using a tracking software (ZebTrack). Means + SEM are
shown for blocks of 5 min. Different letters indicate statistical significance at p < 0.05.
Fig. 5.Analysis of zebrafish response during the 20 ISI when the stimulus was presented on one
side only. The (a) control, (b) partial SD and (d) total SD + Eth groups increased the time spent
on the stimulus-to-be side, while the total SD and total SD + Mel groups spent a similar amount
of time on both sides of the tank. Means± SEM are shown.
Fig. 6.Analysis of zebrafish response during the 20 ISI when the stimulus was presented on
alternating sides. The (a) control, (b) partial SD and (d) total SD + Eth groups spent more time
on the stimulus-to-be side, while the total SD and total SD + Mel groups spent a similar amount
of time on both sides of the tank. Means ± SEM are shown.
Fig. 7.Analysis of zebrafish responses during the 20 ISI when the stimulus was presented on
random sides. None of the groups showed a side preference, spending a similar amount of time
on both sides of the tank. Means ± SEM are shown.
Fig. 8.Comparison of time spent on the stimulus to-be side, for the types of stimulus
presentation: (a) One side only, (b) Alternating sides, (c) Random sides. Trials were divided in 5
blocks and analyzed for the five groups: control, partial SD, total SD, total SD + Eth and total
SD + Mel. Means + SEM are shown. Different letters indicate statistical significance at p <
0.05.
Fig. 9.Behavioral parameters analyzed for (a) maximum speed swimming, (b) average
speed swimming, (c) freezing behavior and (d) total distance traveled by the fish,
between the five groups: control, partial SD, total SD, total SD + Eth and total SD +
Mel. Data corresponds to 30 min of testing, analyzed by tracking software (ZebTrack).
(*) and different letters indicate statistical significance at p< 0.05).
106
Table 1: Estimates of mixed effect model for time spent in the stimulus side during the stimulus presentation. Stimulus presentation scheme
One side only Opposite sides Random sides
Fixed effects Value±sem t-value p-value Value±sem t-value p-value Value ±sem t-value p-value
Intercept 0.16±0.16 -1.02 0.30 -0.76±0.06 -12.91 0.00* -0.97±0.14 -6.91 0.00* Stimulus trials 0.04±0.01 5.45 0.00* 0.02±0.003 4.84 0.00* 0.01±0.006 1.76 0.07
Control 1 1 -
Partial SD -0.30±0.18 -1.63 0.11 -0.07±0.06 -1.10 0.27 1
Total SD 0.39±0.18 -2.15 0.03* -0.10±0.06 -1.68 0.09 -0.33±0.17 -1.95 0.06 Total SD + Eth -0.03±0.18 -0.16 0.87 -0.10±0.06 -1.60 0.11 0.10±0.18 0.58 0.56
Total SD + Mel -0.26±0.18 -1.41 0.16 -0.002±0.07 -0.04 0.97 0.05±0.17 0.27 0.79
Random effects Variance St Dev Variance St Dev Variance St Dev
Intercept 0.39 0.63 5.95 2.44 0.26 0.51 Stimuli 0.00009 0.03 1.17 1.08 0.0004 0.02
Residual 14.52 3.81 5.90 2.43 6.71 2.59 SD: sleep deprivation, sem: Standard error of the mean, St Dev: Standard Deviation.
107
Table 2: Estimates of mixed effect model for time spent in the stimulus-to-be side during the inter stimulus interval (ISI).
SD: sleep deprivation, sem: Standard error of the mean, St Dev: Standard Deviation.
Stimulus presentation scheme
One side only Opposite sides Random sides
Fixed effects Value±sem t-value p-value Value±sem t-value p-value Value ±sem t-value p-value
Intercept -1.01±0.09 -10.85 0.00* -0.69±0.05 -13.22 0.00* -0.95±0.12 -7.41 0.00*
Stimulus trials 0.03±0.004 6.25 0.00* 0.01±0.00 5.56 0.00* 0.01±0.00 2.08 0.03*
Control 1 1 -
Partial SD 0.00±0.09 0.01 0.98 -0.05±0.05 -1.01 0.31 1 Total SD -0.33±0.09 -3.57 0.00* -0.23±0.05 -4.19 0.00* -0.37±0.14 -2.5 0.01*
Total SD + Eth 0.13±0.08 1.50 0.13 -0.09±0.05 -1.74 0.08 0.14±0.15 0.95 0.34
Total SD + Mel -0.13±0.09 -1.47 0.14 -0.10±0.06 -1.69 0.09 0.04±0.14 0.31 0.75
Random effects Variance St Dev Variance St Dev Variance St Dev
Intercept 0.26 0.51 2.99 1.73 0.28 0.53
Stimuli 0.0004 0.02 3.46 1.86 0.0004 0.02
Residual 8.82 2.97 9.61 3.10 10.56 3.25
108
CONCLUSÃO GERAL
O sono é um fenômeno biológico e fisiológico de grande importância no
organismo, porém insuficientemente entendido. Pesquisas sobre o comportamento de
sono em humanos são de difícil aplicabilidade devido ao alto custo, e a dificuldade em
encontrar voluntários e controlar experimentos em laboratórios. Nesse sentido, o peixe
paulistinha aparece como vantajoso modelo animal, pois apresenta controle do sono por
ciclo circadiano, alteração comportamental em condição de privação de sono e
responsividade a drogas relacionadas à indução/privação de sono, além de seu relevante
aspecto translacional em pesquisas com mamíferos.
Em termos de desempenho cognitivo, nosso estudo apresentou resultados
significativos do peixe paulistinha nos três paradigmas testados: discriminação de
objetos, aprendizagem aversiva baseada em punição e aprendizagem apetitiva baseada
em reforço. Nas três tarefas aplicadas, a privação parcial de sono não prejudicou a
performance dos animais, no entanto, a privação total de sono afetou negativamente a
percepção do estimulo, a aprendizagem e a formação/resgate de memória. Além disso, a
exposição ao álcool na noite anterior ao teste parece favorecer o descanso do animal, que
responde ao teste cognitivo posterior com boa performance. Ademais, a melatonina
exógena não melhorou o desempenho do animal nas tarefas cognitivas, seja porque não
permitiu o sono ou porque interferiu diretamente na aprendizagem.
109
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